Contemporary Cardiology

Series Editor: Peter P. Toth

Karam Kostner
Gerhard M. Kostner
Peter P. Toth Editors

Lipoprotein(a)
Contemporary Cardiology
Series Editor
Peter P. Toth, Ciccarone Center for the Prevention of Cardiovascular Disease
Johns Hopkins University School of Medicine
Baltimore, MD, USA
For more than a decade, cardiologists have relied on the Contemporary Cardiology
series to provide them with forefront medical references on all aspects of cardiology.
Each title is carefully crafted by world-renown cardiologists who comprehensively
cover the most important topics in this rapidly advancing field. With more than 75
titles in print covering everything from diabetes and cardiovascular disease to the
management of acute coronary syndromes, the Contemporary Cardiology series has
become the leading reference source for the practice of cardiac care.
Karam Kostner • Gerhard M. Kostner
Peter P. Toth
Editors

Lipoprotein(a)
Editors
Karam Kostner Gerhard M. Kostner
Mater Hospital Medical University of Graz
University of Queensland Graz, Austria
Brisbane, QLD, Australia

Peter P. Toth
Ciccarone Center for the Prevention of
Cardiovascular Disease
Johns Hopkins University School of
Medicine
Baltimore, MD, USA

ISSN 2196-8969     ISSN 2196-8977 (electronic)

Contemporary Cardiology

ISBN 978-3-031-24574-9    ISBN 978-3-031-24575-6 (eBook)

https://doi.org/10.1007/978-3-031-24575-6

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Preface

Lipoprotein metabolism embodies great biochemical complexity and broad-­

spectrum functionality within serum and tissues. At first glimpse, one assumes that
the role of a lipoprotein is to distribute lipids and sterols to systemic tissues and
foster intermediary metabolism. Over the past five decades, we have come to learn
that lipoproteins are highly active polymolecular supersystems that are extraordi-
narily responsive to prevailing metabolic conditions, undergo continuous modifica-
tion in serum, can undergo chemical alteration when taken up into tissues, and have
both beneficial and deleterious roles in health and disease. The functionality of a
lipoprotein is impacted not only by its cargo of apoproteins, but also the specific
constituents of its lipidome, proteome, and capacity to interact with cell surface
receptors, enzymes, and intracellular signaling pathways.
Lipoprotein(a) [Lp(a)] was discovered 60 years ago and has been a biochemical
and physiological enigma. It is unique among lipoproteins in that it represents a
low-density lipoprotein (LDL) particle with a covalently linked apoprotein(a) moi-
ety bound to its apoprotein B scaffold. The kringle IV repeats of the apoprotein(a)
create a whole family of molecules that are genetically determined and also impact
its metabolism, level in serum, and many of its molecular behaviors. A large number
of clinical, epidemiological, and basic scientific investigations identify Lp(a) as
highly pathogenic. Elevated levels of Lp(a) correlate with increased risk for athero-
sclerotic disease as well as aortic valve calcification. Like its lipoprotein cousin,
LDL, it can induce endothelial cell dysfunction, potentiate adhesion molecule
expression, promote the influx of inflammatory white cells into the subendothelial
space of arteries, activate pro-inflammatory nuclear transcription factors, promote
smooth muscle cell migration, and foam cell formation. Lp(a) activates calcium
deposition proteins which can induce both aortic valve and arterial calcification.
Lp(a) may also be prothrombotic. Lp(a) is an important transport vehicle of oxi-
dized phospholipids, which can be proinflammatory, proatherogenic, and stimulate
osteogenesis in various cell types.

v
vi Preface

Somewhat contrapuntal to such a diverse array of potentially injurious activity

are the observations that Lp(a) participates in wound healing and angiogenesis,
impacts the mortality associated with various types of cancer, participates in immu-
nity and complement activation, is an acute phase reactant, and can modulate sys-
temic inflammatory tone as well as risk for autoimmune disease, among other
effects. Unlike other lipoproteins whose clearance from plasma is well understood,
our understanding of how Lp(a) is cleared from the systemic circulation is remark-
ably incomplete. We do not know which receptors along the hepatocyte surface
drive this process. Interestingly, although high levels of Lp(a) are predictive of
heightened risk for coronary artery disease and risk of myocardial infarction, mul-
tiple longitudinal cohort studies also suggest that elevated Lp(a) levels are protec-
tive against the development of diabetes mellitus. The mechanistic basis for this
finding also remains to be elucidated. Insight into the genetics of Lp(a) is progress-
ing rapidly as is our characterization of the many Kringle IV isoforms and how their
functions vary.
Lipoprotein(a) is now recognized as an important risk factor for the development
of atherosclerotic disease and aortic valve stenosis. It is generally recommended
that Lp(a) be measured at least once in one’s lifetime for overall risk assessment.
Lp(a) levels are genetically determined and, unlike the levels of other lipoproteins,
generally unresponsive to lifestyle modification. Lp(a) levels are also poorly respon-
sive to such drugs as statins, ezetimibe, fibrates, and bile acid-binding resins.
Although responsive to high-dose niacin therapy, multiple trials failed to show any
clinical benefit from Lp(a) reduction with this drug. Two recent trials with the use
of proprotein convertase subtilisin: kexin type 9 antibodies did show that Lp(a)
reduction with these molecules contributed to overall risk reduction in patients with
established cardiovascular disease. The apheresis of Lp(a) also demonstrates car-
diovascular benefit with reduced risk for acute coronary syndromes and death in
patients with elevated Lp(a). With the dawn of ribonucleic acid therapeutics, we
now have both RNA oligonucleotide and antisense technology directed against
hepatic Lp(a) production. These are being tested in large prospective, randomized
clinical trials to evaluate their efficacy and safety. We must also resolve how best to
measure Lp(a) levels and adopt a uniform means of expressing its measured value.
This is important not only for reproducible quantification, but also to make com-
parison between studies done in different parts of the world more feasible. Although
relatively unimportant for other lipoproteins, the kidney plays a major role in Lp(a)
metabolism. In the settings of chronic kidney disease and nephrotic syndrome,
Lp(a) can become markedly elevated. In this volume, these issues are discussed in
considerable detail.
Given all that we know and do not know about Lp(a), we thought it was time to
produce a book which synthesizes what we do know about this still highly enig-
matic lipoprotein, both positive and negative. We also explore unanswered
Preface vii

questions. While the book is highly scientific throughout, we emphasize clinical

aspects whenever possible. Chapters were prepared by leading experts in the field of
Lp(a) research. We anticipate that Lp(a) will emerge as a treatment target in the
clinical arena and hope that this volume provides both context and knowledge that
helps to ensure that clinicians will evaluate patients for Lp(a), incorporate it into
cardiovascular risk stratification, and treat it as appropriate.

Brisbane, Australia Karam Kostner

Graz, Austria  Gerhard M. Kostner
Baltimore, MD, USA  Peter P. Toth
Contents

1 60 Years of Lp(a) Research: From Ouchterlonys Double

Diffusion to Copy Number Variation and a Significant
Risk Factor for CHD��������������������������������������������������������������������������������    1
Gerd Utermann
2 
Lp(a) Biochemistry, Composition, and Structure ��������������������������������   39
Gerhard M. Kostner
3 Genetics of Lipoprotein(a)����������������������������������������������������������������������   55
Gerd Utermann
4 Lp(a) Metabolism������������������������������������������������������������������������������������   75
John S. Millar and Daniel J. Rader
5 Contemporary Aspects of Lp(a) Metabolism and
Therapies Based on Tracer Kinetic Studies in Humans ����������������������   91
Dick C Chan, Jing Pang, and Gerald F Watts
6 
Role of Proprotein Convertase Subtilisin Kexin Type 9
in Lipoprotein(a) Metabolism���������������������������������������������������������������� 113
Antonio Gallo, Kévin Chemello, Romuald Techer, Ali Jaafar,
and Gilles Lambert
7 
The Role of Cell Surface Receptors in Lp(a) Catabolism�������������������� 125
Lamia Ismail, Déanna Shea, and Sally McCormick
8 
Physiological Roles and Functions of Lipoprotein(a) �������������������������� 135
Zaid N. Safiullah, Thorsten Leucker, Steven R. Jones, and
Peter P. Toth
9 
The Role of Lp(a) in Atherosclerosis: An Overview ���������������������������� 159
Anastasiya Matveyenko, Marianna Pavlyha, and Gissette
Reyes-Soffer

ix
x Contents

10 Molecular Mechanisms of Lipoprotein(a) Pathogenicity:

Tantalizing Clues and Unanswered Questions�������������������������������������� 173
Michael B. Boffa and Marlys L. Koschinsky
11 Thrombosis, Inflammation, and Lipoprotein(a):
Clinical Implications�������������������������������������������������������������������������������� 189
Maya S. Safarova and Patrick M. Moriarty
12 The Kidney Is the Heart of the Organs:
Its Role in Lp(a) Physiology and Pathophysiology ������������������������������ 207
Hans Dieplinger
13 
Lp(a) as a Cardiovascular Risk Factor�������������������������������������������������� 231
Angela Pirillo and Alberico Luigi Catapano
14 Lp(a) and Aortic Valve Stenosis, Stroke, and Other
Noncoronary Cardiovascular Diseases�������������������������������������������������� 241
Anne Langsted and Pia R. Kamstrup
15 Lipoprotein(a) in Cardiovascular Disease: Evidence
from Large Epidemiological Studies������������������������������������������������������ 251
Peter Engel Thomas, Signe Vedel-Krogh,
and Børge G. Nordestgaard
16 Lipoprotein(a) and Immunity ���������������������������������������������������������������� 261
O. I. Afanasieva, T. I. Arefieva, M. V. Ezhov, and S. N. Pokrovsky
17 
When Should We Measure Lipoprotein(a)?������������������������������������������ 275
Karam Kostner
18 
Measurement of Lipoprotein(a) in the Clinical Laboratory���������������� 281
David Sullivan, Catherine Woolnough, Nimalie Perera,
Jay Ramanathan, and Tony Badrick
19 Standardization of Analytical Methods for the
Measurement of Lipoprotein(a): Bridging Past and Future
Initiatives�������������������������������������������������������������������������������������������������� 297
Noemie Clouet-Foraison, Tomas Vaisar, and Santica M. Marcovina
20 On the Way to a Next-Generation Lp(a) Reference
Measurement System Based on Quantitative Protein
Mass Spectrometry and Molar Units ���������������������������������������������������� 325
Christa Cobbaert, Liesbet Deprez, and Renee Ruhaak
21 
Therapy of Elevated Lipoprotein(a)������������������������������������������������������ 347
S. Ibrahim and Erik S. G. Stroes
22 Antisense Oligonucleotide Therapy to Treat
Elevated Lipoprotein(a)�������������������������������������������������������������������������� 359
Sotirios Tsimikas
Contents xi

23 
Lipoprotein Apheresis for Reduction of Lipoprotein(a)���������������������� 377
Ulrich Julius and Sergey Tselmin
24 Elevated Lp(a): Why Should I Test For It, If I Cannot Treat It?
A Patient’s Perspective���������������������������������������������������������������������������� 409
Sandra Revill Tremulis
25 Unresolved Questions������������������������������������������������������������������������������ 425
Gerhard M. Kostner and Karam Kostner
Index������������������������������������������������������������������������������������������������������������������ 437
Contributors

O. I. Afanasieva National Medical Research Center of Cardiology named after

E. I. Chazov MOH of Russian Federation, Moscow, Russia
T. I. Arefieva National Medical Research Center of Cardiology named after
E. I. Chazov MOH of Russian Federation, Moscow, Russia
Tony Badrick Royal College of Pathologists of Australasia Quality Assurance
Program, St Leonards, NSW, Australia
Michael B. Boffa Robarts Research Institute, Schulich School of Medicine and
Dentistry, The University of Western Ontario, London, ON, Canada
Department of Biochemistry, Schulich School of Medicine and Dentistry, The
University of Western Ontario, London, ON, Canada
Alberico Luigi Catapano IRCCS MultiMedica, Milan, Italy
Department of Pharmacological and Biomolecular Sciences, Università degli Studi
di Milano, Milan, Italy
Dick C Chan Medical School, University of Western Australia, Perth, WA,
Australia
Kévin Chemello Laboratoire Inserm UMR 1188 DéTROI, Université de La
Réunion, Saint-Pierre (Ile de La Réunion), France
Noemie Clouet-Foraison Division of Metabolism, Endocrinology, and Nutrition,
University of Washington, Seattle, WA, USA
Christa Cobbaert Leiden University Medical Center, Leiden, The Netherlands
Liesbet Deprez European Commission Joint Research Center (JRC), Geel, Belgium
Hans Dieplinger Department of Genetics, Institute of Genetic Epidemiology,
Medical University of Innsbruck, Innsbruck, Austria
M. V. Ezhov National Medical Research Center of Cardiology named after
E. I. Chazov MOH of Russian Federation, Moscow, Russia

xiii
xiv Contributors

Antonio Gallo Laboratoire Inserm UMR 1188 DéTROI, Université de La Réunion,

Saint-Pierre (Ile de La Réunion), France
S. Ibrahim Department of Vascular Medicine, Amsterdam UMC, University of
Amsterdam, Amsterdam, The Netherlands
Lamia Ismail Department of Biochemistry, School of Biomedical Sciences,
University of Otago, Dunedin, New Zealand
Ali Jaafar Laboratoire Inserm UMR 1188 DéTROI, Université de La Réunion,
Saint-Pierre (Ile de La Réunion), France
Steven R. Jones Ciccarone Center for the Prevention of Cardiovascular Disease,
Johns Hopkins University School of Medicine, Baltimore, MD, USA
Ulrich Julius Department of Internal Medicine III, Lipidology and Center for
Extracorporeal Treatment, University Hospital Carl Gustav Carus at the Technische
Universität Dresden, Dresden, Germany
Pia R. Kamstrup Department of Clinical Biochemistry, Copenhagen University
Hospital-Herlev and Gentofte, Herlev, Denmark
The Copenhagen General Population Study, Copenhagen University Hospital-­
Herlev and Gentofte, Herlev, Denmark
Marlys L. Koschinsky Robarts Research Institute, Schulich School of Medicine
and Dentistry, The University of Western Ontario, London, ON, Canada
Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The
University of Western Ontario, London, ON, Canada
Gerhard M. Kostner Institute of Molecular Biology and Biochemistry, Medical
University of Graz, Graz, Austria
Karam Kostner Department of Cardiology, Mater Hospital and University of
Queensland, Brisbane, Australia
Gilles Lambert Laboratoire Inserm UMR 1188 DéTROI, Université de La
Réunion, Saint-Pierre (Ile de La Réunion), France
Anne Langsted Department of Clinical Biochemistry, Copenhagen University
Hospital-Herlev and Gentofte, Herlev, Denmark
Department of Clinical Medicine, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen, Denmark
Thorsten Leucker Ciccarone Center for the Prevention of Cardiovascular Disease,
Johns Hopkins University School of Medicine, Baltimore, MD, USA
Santica M. Marcovina Medpace Reference Laboratories, Cincinnati, OH, USA
Anastasiya Matveyenko Division of Preventive Medicine and Nutrition,
Department of Medicine, Vagelos College of Physicians and Surgeons, Columbia
University, New York, NY, USA
Contributors xv

Sally McCormick Department of Biochemistry, School of Biomedical Sciences,

University of Otago, Dunedin, New Zealand
John S. Millar Division of Translational Medicine and Human Genetics,
Department of Medicine, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA, USA
Institute for Diabetes Obesity and Metabolism, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
Patrick M. Moriarty Division of Endocrinology, Diabetes and Clinical
Pharmacology, Department of Internal Medicine, University of Kansas Medical
Center, Kansas City, KS, USA
Atherosclerosis and Lipoprotein Apheresis Center, University of Kansas Medical
Center, Kansas City, KS, USA
Børge G. Nordestgaard Department of Clinical Biochemistry, Herlev and
Gentofte Hospital, Copenhagen University Hospital, University of Copenhagen,
Copenhagen, Denmark
Jing Pang Medical School, University of Western Australia, Perth, WA, Australia
Marianna Pavlyha Division of Preventive Medicine and Nutrition, Department of
Medicine, Vagelos College of Physicians and Surgeons, Columbia University, New
York, NY, USA
Nimalie Perera Department of Chemical Pathology, Royal Prince Alfred Hospital,
Sydney Local Health District, NSW Health Pathology, University of Sydney,
Sydney, NSW, Australia
Angela Pirillo Center for the Study of Atherosclerosis, E. Bassini Hospital,
Milan, Italy
IRCCS MultiMedica, Milan, Italy
S. N. Pokrovsky National Medical Research Center of Cardiology named after
E. I. Chazov MOH of Russian Federation, Moscow, Russia
Daniel J. Rader Division of Translational Medicine and Human Genetics,
Department of Medicine, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA, USA
Institute for Diabetes Obesity and Metabolism, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
Department of Genetics, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA, USA
Institute for Translational Medicine and Therapeutics, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
Smilow Center for Translational Research, University of Pennsylvania,
Philadelphia, PA, USA
xvi Contributors

Jay Ramanathan Department of Chemical Pathology, Royal Prince Alfred

Hospital, Sydney Local Health District, NSW Health Pathology, University of
Sydney, Sydney, NSW, Australia
Department of Medicine, Liverpool Hospital, Sydney South West Local Health
District, Liverpool, NSW, Australia
Gissette Reyes-Soffer Division of Preventive Medicine and Nutrition, Department
of Medicine, Vagelos College of Physicians and Surgeons, Columbia University,
New York, NY, USA
Renee Ruhaak Leiden University Medical Center, Leiden, The Netherlands
Maya S. Safarova Atherosclerosis and Lipid Genomics Laboratory, Mayo Clinic,
Rochester, MN, USA
Department of Cardiovascular Medicine, University of Kansas Hospital and Medical
Center, Kansas City, KS, USA
Zaid N. Safiullah Osler Medical House Staff, The Johns Hopkins Hospital,
Baltimore, MD, USA
Déanna Shea Department of Biochemistry, School of Biomedical Sciences,
University of Otago, Dunedin, New Zealand
Erik S. G. Stroes Department of Vascular Medicine, Amsterdam UMC, University
of Amsterdam, Amsterdam, The Netherlands
David Sullivan Department of Chemical Pathology, Royal Prince Alfred Hospital,
Sydney Local Health District, NSW Health Pathology, University of Sydney,
Sydney, NSW, Australia
Romuald Techer Laboratoire Inserm UMR 1188 DéTROI, Université de La
Réunion, Saint-Pierre (Ile de La Réunion), France
Peter Engel Thomas Department of Clinical Biochemistry, Herlev and Gentofte
Hospital, Copenhagen University Hospital, University of Copenhagen,
Copenhagen, Denmark
Peter P. Toth Ciccarone Center for the Prevention of Cardiovascular Disease,
Johns Hopkins University School of Medicine, Baltimore, MD, USA
Sandra Revill Tremulis, MBA Redwood City, CA, USA
Sergey Tselmin Department of Internal Medicine III, Lipidology and Center for
Extracorporeal Treatment, University Hospital Carl Gustav Carus at the Technische
Universität Dresden, Dresden, Germany
Sotirios Tsimikas Division of Cardiology, Sulpizio Cardiovascular Center,
University of California San Diego, La Jolla, CA, USA
Gerd Utermann Institute for Human Genetics, Medical University of Innsbruck,
Innsbruck, Austria
Contributors xvii

Institute for Genetic Epidemiology, Medical University of Innsbruck,

Innsbruck, Austria
Tomas Vaisar Division of Metabolism, Endocrinology, and Nutrition, , University
of Washington, Seattle, WA, USA
Signe Vedel-Krogh Department of Clinical Biochemistry, Herlev and Gentofte
Hospital, Copenhagen University Hospital, University of Copenhagen,
Copenhagen, Denmark
Gerald F Watts Medical School, University of Western Australia, Perth, WA,
Australia
Lipid Disorders Clinic, Department of Cardiology and Internal Medicine, Royal
Perth Hospital, Perth, WA, Australia
Catherine Woolnough Department of Chemical Pathology, Royal Prince Alfred
Hospital, Sydney Local Health District, NSW Health Pathology, University of
Sydney, Sydney, NSW, Australia
Chapter 1
60 Years of Lp(a) Research:
From Ouchterlonys Double Diffusion
to Copy Number Variation
and a Significant Risk Factor for CHD

Gerd Utermann

A Historical Review

At the beginning, a note of caution. A historical review by a non-historian by neces-

sity is subjective and biased reflecting how developments in a field are perceived in
retrospect by a time witness. A PubMed search for Lp(a) in March 2022 resulted in
10.330 hits. Citations therefore have to be selective. In this historical review, the
author has tried to cite the first original work on a specific topic instead of a recent
review but in some instance may have failed. For a comprehensive review of the
Lp(a) literature until 2001, the reader is referred to Utermann (2001).

The Discovery of Lp(a)

Lipoprotein(a) was first described in 1963 by the Norwegian Physician Kåre Berg
(1963) (Fig. 1.1). As frequently in science, the history of Lp(a) started with a smart
idea, but ended with an unexpected result. In 1961/1962, Allison and Blumberg
(Allison and Blumberg 1961; Blumberg et al. 1962) described a polymorphism of
beta-lipoproteins, which they designated the Ag-system. They had observed that
some sera from polytransfused patients with thalassemia contained antibodies,
which distinguished between Ag-positive and Ag-negative sera from normal indi-
viduals in a test called Ouchterlonys double diffusion (Fig. 1.2). The availability of
anti-Ag sera depended on luck, required testing of many patients and quality was

G. Utermann (*)
Institute for Human Genetics, Medical University of Innsbruck, Innsbruck, Austria
Institute for Genetic Epidemiology, Medical University of Innsbruck, Innsbruck, Austria
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 1

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_1
2 G. Utermann

a b

Fig. 1.1 Panel a: Kåre Berg and the author at the first “International Lp workshop” 1967 in
Marburg/Lahn (Germany). Panel b: Title page of Kåre Berg’s first publication on Lp(a) with dedi-
cation to the organizer of the workshop Gerhard G. Wendt

Fig. 1.2 Double diffusion

in agarose gel according to
Ouchterlony to test the
purity of lipoprotein
fractions. In wells A, B,
and C, different antisera
and in wells 1–4
lipoprotein fractions were
applied. (A: anti-beta-­
lipoprotein; B: anti-­
lipoprotein(a); C:
anti-human serum; 1:
Lp(a), 2: beta-lipoprotein;
3: Lp(a); 4:
beta-lipoprotein)

difficult to control. Because antisera from polytransfused patients were not readily
available, limited in quantity, and could not be reproduced in the laboratory Kåre
Berg, at the time at the Institute of Forensic Medicine, Rikshospitalet, University of
Oslo/Norway had an idea to overcome these limitations. If an antigen elicited an
immune response in humans, it should do so also in rabbits. He started a series of
experiments, in which he immunized rabbits with individual human sera or beta-­
lipoproteins. The rabbit sera were then “absorbed” with different individual human
sera to remove antibodies against foreign antigens present in all human sera.
Subsequently, the absorbed rabbit sera were tested against a panel of human sera for
antibodies recognizing individual human sera. Indeed, the plan worked. Some rab-
bit antisera reacted positive with some human sera and negative with others in
Ouchterlonys double diffusion (Fig. 1.2). Moreover, Berg could show that the
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 3

antigen in fact was a beta-lipoprotein. However, it was not identical with the
Ag-antigen (Berg 1964). Therefore, Berg introduced the name “Lp-System,” which
was later changed in Lp(a) to distinguish Lp(a) from other “Lp” Antigens. One was
“Lp(x)” which was detected by antisera produced in horse but turned out to be an
artifact. Berg distinguished between Lp(a+) and Lp(a−) individuals and showed by
family studies that the Lp(a) trait seemed to follow an autosomal dominant mode of
inheritance (Berg and Mohr 1963). Soon following the breakthrough discovery of
Berg, several laboratories tried to reproduce his finding, but with mixed results. In
principle, all confirmed Berg’s findings, but several researchers noticed that with
their antibodies the immune reaction was not an all or none. Instead, they observed
strong reactions (Bergs positives), no reactions (Bergs negatives), but also weak and
very weak reactions. It followed a discussion on whether the weak reactions were
true Lp(a) reactions or whether the antisera which recognized weak reactions were
unspecific containing antibodies to other components.
To clarify the situation and exchange latest research results, the human geneticist
Gerhard G. Wendt initiated the first “International Lp workshop” in Marburg/Lahn,
Germany (Wendt 1967). In preparation of the conference researchers from six dif-
ferent laboratories, including Kåre Berg’s sent in 17 antisera, which were tested
against a standard panel of 71 individual sera and analyzed for identity. The result
was that all antisera recognized the same antigen Lp(a), but confirmed the existence
of weak and very weak reactions which occurred to different degrees depending on
the antiserum. The issue was only resolved when researchers developed methods to
semi-quantify and finally quantify Lp(a), which demonstrated that Lp(a) in fact is a
quantitative trait (Harvie and Schultz 1970; Ehnholm et al. 1971). Methods to quan-
tify Lp(a) demonstrated large differences in median Lp(a) levels between and within
major human ethnic groups. The distributions of Lp(a) levels were highly skewed in
European and East-Asian populations but less so in sub-Saharan Africans (Fig. 1.3).
Mean and median Lp(a) levels were two to fourfold higher in Africans than
Europeans. The distributions in Asian populations were heterogenous with higher
Lp(a) levels in South-East Asia (Sandholzer et al. 1991; Parra et al. 1987a; Helmhold
et al. 1991).
4 G. Utermann

a
30
25 Khoi San
20 (N = 173)
15
10
5
0
30
Black Africans
25
(N = 193)
20
15
Relative Frequency (%)

10
5
0
30
Caucasians
25
(N = 224)
20
15
10
5
0
30
25 Chinese
(N = 198)
20
15
10
5
0
0 25 50 75 100 125 150 175
Lp(a) Concentration (mg/dl)

Fig. 1.3 Histograms showing: Panel (a) the distribution of plasma Lp(a) levels in four popula-
tions. Panel (b) the frequency distribution of the KIV-2 VNTR alleles in the same populations. The
total number of KIV repeats including the “unique” kringles is given. Panel (c) the inverse correla-
tion of KIV-2 repeats with Lp(a) concentration in the four populations. The Black Africans in this
study were from South Africa and represent different ethnicities. The Chinese samples were from
Hongkong and the “Caucasians” from Austria. (Figure reproduced from Kraft et al. 1996b with
permission)
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 5

b 12 Khoi San
(N = 346)
8

0
12
Black
Africans
8
(N = 386)
Relative Frequency (%)

0
12
Caucasians
(N = 448)
8

0
12
Chinese
(N = 396)
8

0
11 15 20 25 30 35 40 45 50
Number of Kringle-IV Repeats

Fig. 1.3 (continued)

6 G. Utermann

c
150 Khoi San
(N = 173)
100

50

0
Black Africans
Lipoprotein(a) Concentration [mg/dl]

150
(N = 193)
100

50

150 Caucasians
(N = 224)
100

50

0
150 Chinese
(N = 198)
100

50

0
8 18 28 38 48 58
Sum of Kringle-IV Repeats

Fig. 1.3 (continued)

Isolation and Characterization of Lp(a)

Beginning in 1968, first attempts were made to isolate Lp(a) from plasma and it was
shown that the antigenic property of Lp(a) is associated with a lipoprotein distinct
from LDL (Wiegandt et al. 1968; Utermann and Wiegandt 1969; Schultz et al. 1968).
A major breakthrough in Lp(a) research was the purification and characterization
of Lp(a) in 1970 by Christian Ehnholm (Fig. 1.4) in Kai Simons group in Helsinki,
Finland. They purified Lp(a) by a combination of preparative ultracentrifugation
and gel filtration on Sepharose 2B/4B columns and determined the physicochemical
properties of the particle (Ehnholm et al. 1971; Simons et al. 1970). Characteristics
of Lp(a) were a hydrated density of 1.09 g/mL, a molecular weight estimated by gel
filtration of 4.8 MDa and by electron microscopy of 5.6 MDa. Lp(a) had pre-beta
mobility in agarose gel electrophoresis and appeared as a spherical particle upon
electron microscopy. Notably it differed from LDL in amino acid composition and
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 7

a b

Fig. 1.4 Panel (a) Christian Ehnholm during a visit in Marburg/Lahn 1971 with the author. Panel
(b) laboratory equipment with Sepharose 4B column (red arrows) for final purification of Lp(a)
according to Ehnholm et al. (Ehnholm et al. 1971; Simons et al. 1970)

contained a very high amount of protein-bound carbohydrate. In further work, they

characterized the carbohydrates in more detail and found that Lp(a) contains an
about six times higher amount of sialic acid, a three times higher content of hexos-
amines and twice as much hexoses than LDL (Ehnholm et al. 1972). The antigenic
property of Lp(a) and the high carbohydrate content were associated with a protein
which occurred as a separate band in polyacrylamide gel electrophoresis, when
Lp(a) disaggregated spontaneously upon storage at 0 °C (Ehnholm et al. 1972;
Utermann et al. 1972). The final purification of Lp(a) by Sepharose 4B column
chromatography required near-acrobatic skills. Christian Ehnholm had introduced
us into the handling of the column when he visited our lab in Marburg in 1971
(Fig. 1.4). The dimension of the glass column which had to be filled with the
Sephadex slurry by hand was 2.5 cm (diameter) × >160 cm (height). We used a lad-
der to fill the column and apply the lipoprotein sample.
The availability of a standardized reproducible method to purify Lp(a) allowed its
further characterization. By SDS-polyacrylamide gel electrophoresis under reducing
and non-reducing conditions and immunochemical methods, it was shown that Lp(a)
contains two high molecular weight proteins, apolipoprotein B [apoB] identical with
apoB in LDL (MW about 500 kDa) and a glycoprotein with a MW (about 600 kDa)
which appeared to be even larger than apoB. Both proteins were hold together by
one or more disulfide bonds (Utermann and Weber 1983; Gaubatz et al. 1983).
8 G. Utermann

Lp(a) is usually depicted in cartoons as a global particle with an LDL in its center
and apo(a) wrapped around. Studies by electron microscopy of negatively stained
Lp(a) (Sines et al. 1994) and small-angel X-ray scattering (Prassl et al. 1995) sup-
ported this model but other studies suggested that apo(a) may protrude as a “tail”
from the particle depending on the environment (Weisel et al. 2001) and that Lp(a)
can switch between globular and “flexible tail” structures (Becker et al. 2004).

Metabolism

Studies of the metabolism of Lp(a) started in 1979 when Gerhard Kostner’s group
in Graz/Austria initiated a series of in vivo turnover studies in humans (Krempler
et al. 1979, 1980, 1983). Healthy individuals with different concentrations of Lp(a)
were injected with radioiodinated Lp(a) and in subsequent experiments with radio-
iodinated LDL. They demonstrated that (1) Lp(a) is not a metabolic product of other
apoB-containing lipoproteins, (2) that Lp(a) concentrations in plasma are deter-
mined by the rate of synthesis rather than by its catabolism, and (3) that Lp(a) is
catabolized at a slower rate than LDL.
Binding studies of radioactively labeled Lp(a) to human fibroblasts in compari-
son with LDL confirmed results of Havekes et al. (1981) and demonstrated that
Lp(a) binds with high affinity to the same cell surface receptor as LDL. However,
binding capacity for Lp(a) was lower than for LDL (Krempler et al. 1983). Lp(a) did
not bind to fibroblasts from patients with homozygous FH. These findings are in line
with later binding studies in fibroblasts and experiments in transgenic mice by
Goldstein and Brown (Hofmann et al. 1990) which indicated that Lp(a) is removed
from plasma by the LDL receptor pathway and with the observation that patients
with FH due to LDL receptor mutations or apoB100 mutations have elevated Lp(a)
in plasma (Seed et al. 1990; Utermann et al. 1989; Lingenhel et al. 1998; Van der
Hoek et al. 1997; Kraft et al. 2000). Together, these findings give a consistent pic-
ture. However, later studies on these topics were highly controversial and neither
confirmed the binding of Lp(a) to fibroblasts, nor the in vivo turnover studies in
humans and transgenic mice or the family studies (Soutar et al. 1991; Knight et al.
1991; Cain et al. 2005; Rader et al. 1995). Other receptors and pathways were impli-
cated to be involved in the removal of Lp(a) from plasma (reviewed in McCormick
and Schneider 2019). The liver (Cain et al. 2005) and the kidney (Kronenberg et al.
1997) both have been suggested as major sites of Lp(a) clearance from plasma. A
role for the kidney in Lp(a) clearance from the circulation was championed by the
group of Florian Kronenberg and Hans Dieplinger in Innsbruck and is supported by
several lines of evidence. Turnover studies demonstrated a reduced clearance of
Lp(a) in patients with kidney disease (Frischmann et al. 2007). Large arteriovenous
differences between Lp(a) concentrations were observed in the renovascular system
(Kronenberg et al. 1997). A problem with this study is that it requires the assump-
tion of unreasonably high synthesis rates of Lp(a) to compensate for the loss in the
kidneys. Further, Lp(a) binds with high affinity to megalin/gp330, a member of the
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 9

LDLR family expressed preferentially in kidneys (Niemeier et al. 1999) and frag-
ments of apo(a) were found in human urine (Oida et al. 1992; Mooser et al. 1996;
Kostner et al. 1996). None of this is direct evidence and at present the tissue(s) and
pathways of Lp(a) removal from the circulation remain unresolved. In contrast, the
liver as the site of synthesis and secretion of Lp(a) is undisputed. Hans-Georg Kraft
and colleagues from Innsbruck determined apo(a) isoform phenotypes (see below)
in plasma from patients undergoing liver transplantation and their organ donors.
They observed that genetic isoform phenotypes changed completely from recipients
to the donors phenotype following transplantation (Kraft et al. 1989). Apo(a) mRNA
was also most abundant in the liver from rhesus baboons and cynomolgus monkeys
(Tomlinson et al. 1989; Hixson et al. 1989; Azrolan et al. 1991).

The LPA Gene and Apolipoprotein(a)

Two important discoveries were made in 1987, the unique structure of apo(a)
(McLean et al. 1987) and the isoform polymorphism of apo(a) (Utermann et al. 1987).
The sequence of apo(a) had remained elusive for a long time and the reason
became clear when the sequence was finally resolved. Attempts to determine the
amino acids sequence of apo(a) by protein sequencing resulted in partial amino acids
sequences which demonstrated a high homology to plasminogen (Eaton et al. 1987;
Kratzin et al. 1987). Only by the breakthrough work of Richard Lawn and col-
leagues, at that time working at Genentech, the full sequence was elucidated. As a
pioneer in cloning technologies and DNA sequencing, Richard Lawn (Fig. 1.5) who
had previously sequenced hemoglobin loci from thalassemia patients started cloning
and cDNA sequencing of the LPA gene. This turned out to be much more compli-
cated than previous work. The result was unanticipated and astonishing. The deduced
amino acid sequence of apo(a) consisted of an array of so-called kringle domains
with a high internal homology and homology to kringle 4 from plasminogen. Ten

a b *
hedgehog 3’
3 3 3 3 3 3 3 3 3 3 3 3 3
apo (a)
72-75%
5’ 3’
plasminogen S P 1 2 3 4 5 PRO

75-85%
human 5’ 3’
a S 4 4 4 4 4 4 4 4 4 4 4 4 4 5 PRO
apo(a)

Fig. 1.5 Panel (a) Richard M. Lawn who published the first cDNA sequence of apo(a) (McLean
et al. 1987). Panel (b) Illustration of the convergent evolution of primate and insectivore apo(a).
The cDNA structures of plasminogen and human and hedgehog apo(a) are shown. Kringle types
are denoted by numbers and the protease domain by PRO. The stars indicate the sites of the
unpaired Cys residues which form the disulfide bridge with apoB in LDL. The percentages give the
degree of homology between plasminogen and apo(a). (Reproduced from Lawn et al. 1995b with
permission)
10 G. Utermann

kringle type IV different in sequence was identified, nine of them in single copy
(KIV-1, KIV-3 to KIV-10) whereas one (KIV-2) occurred in six identical copies in
the sequenced DNA. In addition, the protein contained one kringle with homology to
KV from PLG, a signal sequence and a plasminogen-like protease domain. The latter
was predicted to be inactive toward plasmin substrates due to mutation in the cata-
lytic triad. The findings showed that the LPA gene had been derived from the PLG
gene during evolution by a number of changes including duplication of PLG, dele-
tions and expansions of domains and mutations. The structure of all kringles is sta-
bilized by three internal disulfide bridges, which results in the typical appearance of
a Danish bretzel called “kringle.” One kringle (KIV-9) in addition contains one
unpaired cys residue, which turned out to be responsible for the covalent binding to
apoB of the LDL particle (Koschinsky et al. 1993; Brunner et al. 1993).
The sequence was so unusual that in an accompanying “News and Views” article
in Nature Joseph Goldstein and Michael Brown wrote that “the…. finding chal-
lenges the notion that evolution makes sense” (Brown and Goldstein 1987). The
enormous challenge which sequencing of LPA posed at the time becomes evident
when one considers that a successful search for mutations in the KIV-2 repeats of
LPA became possible only very recently (Coassin et al. 2019). It was certainly the
most heroic undertaking in Lp(a) research and opened new unexpected avenues.
Sequence analysis of PLG and human and rhesus LPA (Tomlinson et al. 1989)
had shown that LPA had evolved from PLG after the split of Old-world from New-­
world monkeys some 40 million years ago and that Lp(a) existed only in Old-world
monkeys. Therefore, it was a surprise when Laplaud et al. in France reported pres-
ence of Lp(a) in the plasma of a hibernator, the hedgehog (Laplaud et al. 1988). The
surprise became even bigger when Lawns group sequenced the LPA of hedgehog.
Instead of KIV repeats, it contained multiple copies of KIII as the sole kringle type
and lacked the protease domain (Lawn et al. 1995a, 1997), but like the primate
counterpart hedgehog apo(a) formed a Lp(a) particle with LDL. Reports on the
presence of Lp(a) in guinea pigs (Rath and Pauling 1990) and the marmoset (a New-­
world monkey) (Guo et al. 1991) were not confirmed. Hence, the occurrence of a
Lp(a)-like particle in the hedgehog by convergent evolution apparently remained a
solitary act (Lawn et al. 1995a, 1997).

Functional Studies

As shown by the work of Lawn and colleagues, the LPA gene had evolved from PLG
during primate evolution suggesting that the function of Lp(a) might be related to
the function of plasminogen and blood clotting. Already in the “News and Views”
article mentioned above, Goldstein and Brown had put forward the hypothesis that
Lp(a) through binding to fibrinogen might be involved in wound healing (Brown
and Goldstein 1987). The hypothesis—which still appears attractive, but was never
rigorously tested though—considers that Lp(a) is a macromolecular complex con-
taining two very different components, apo(a) and LDL. For most functions assigned
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 11

to Lp(a), the apo(a) alone is sufficient which leaves open the question, why the
particle exists. Beginning with the work of Harpel et al. (1989, 1995), numerous
studies demonstrated effects of Lp(a) on the blood clotting cascade and connected
thrombosis to atherosclerosis (Nachman 1992). Lp(a) was described as “an inter-
loper into the fibrinolytic system” (Miles and Plow 1990). Several interactions of
apo(a) with diverse ligands have been reported (Fig. 1.6) but whether any is of
physiological or pathophysiological relevance in humans remains unclear. A promi-
nent hypothesis explaining the atherogenic potential of Lp(a) was derived from the
finding that it is a “sink” for oxidized phospholipids (Tsimikas et al. 2005; Kiechl
et al. 2007; Bergmark et al. 2008). Lack in understanding of the function and patho-
physiological properties have recently been reviewed by an NHLBI working group
(Tsimikas et al. 2018). Existence of Lp(a) and the apo(a) size polymorphism in Old-­
world monkeys implies that Lp(a) may have, or had, a function beyond one species.
The detection of many null mutations in the LPA gene (see below) may, however,
indicate that this function has been lost in modern humans with the possible excep-
tion of Africans.

Thrombolysis

Cells
PLG Matrix Platelets
A
tTP
Fibrin(ogen)
SMC Plasmin
OxPL
Tetranectin
C
TGFβ
N C
N Fibronectin
LDL
DANCE
Messangial
cell β2GP-I

Monocyte/ Decorin
Macrophage

Megalln/gp330
Anglogenesis
Endothelial cells VLDL-R
(LDL-R)
VCAM-1 PAI-1
MCA
E-selectin

Fig. 1.6 Model of Lp(a) and reported interactions of Lp(a)/apo(a) with components of the blood
clotting system, cell receptors, and other binding proteins. The binding of oxidized phospholipids
(OxPL in red) is considered as crucial for the pathogenicity of Lp(a). For explanation, see text and
Schmidt et al. (2016). (Modified from Utermann 1989, 2001 with permission)
12 G. Utermann

Animal Studies and the Era of Transgenics

With the exception of the hedgehog, Lp(a) exists only in Old-world monkeys and
humans (Makino et al. 1989). The availability of natural animal models to study the
metabolism and pathophysiology of Lp(a) is limited. Rainwater and colleagues ana-
lyzed the genetics of Lp(a) extensively in baboons (Rainwater et al. 1986; White
et al. 1994a) and few studies were performed in rhesus monkeys (Rudel et al. 1977;
Williams-Blangero and Rainwater 1991; Enkhmaa et al. 2015) and more recently in
chimpanzees (Noureen et al. 2017). The isoform polymorphism and the inverse cor-
relation between isoform size and Lp(a) levels in plasma existed in all these pri-
mates. Chimpanzees from different West-African and Central-African habitats had
significantly different Lp(a) levels and isoform distributions in plasma (Noureen
et al. 2017). Experimental studies with these species are not allowed and unethical.
Therefore researchers started to generate mice transgenic for apo(a) immediately
following the cloning of apo(a) cDNA (Chiesa et al. 1992). The first animals gener-
ated had apo(a) free in plasma because mouse LDL apparently lacked the structural
requirement for binding apo(a) and forming the Lp(a) complex. Different approaches
were used to overcome this. Infusion of human LDL into apo(a) transgenic mice
resulted in the association of secreted apo(a) with circulating LDL and formation of
Lp(a) which could only be resolved by disulfide reduction (Chiesa et al. 1992). In
another study, human Lp(a) was infused into mice transgenic for the human LDL
receptor which confirmed cell culture studies which had shown high-­affinity bind-
ing of Lp(a) to the receptor (Hofmann et al. 1990). With such short-­term experi-
ments, it was not possible to investigate the pathophysiology and in particular the
atherogenic potential of Lp(a). This became possible when apo(a) transgenic mice
were crossed with mice strains transgenic for human apoB (Linton et al. 1993;
Callow et al. 1994). These mice strains were used to study the assembly (Callow
et al. 1994; Callow and Rubin 1995) and the atherogenic potential of Lp(a) (Callow
et al. 1995; Mancini et al. 1995a). To identify sequence elements that regulate liver-
specific tissue expression, sex hormone and diet response mice transgenic for yeast
artificial chromosomes (YACs) containing entire human apo(a) alleles were pro-
duced (Frazer et al. 1995; Acquati et al. 1999).

Unraveling the Genetics of Lp(a)

The second important finding in 1987 was the discovery of the size polymorphism
of apo(a). Beginning with its detection, it was clear that Lp(a) was a genetic trait.
Family and twin studies had shown that heritability of the trait is high. Morton et al.
(1985) concluded from a large family study that Lp(a) levels are controlled by one
major dominant gene and a residual heritable component. The gene(s) controlling
Lp(a) levels were unknown. This started to change when a group in Innsbruck/
Austria demonstrated that several genetic isoforms of apo(a), which differ in size,
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 13

occur in the population and that the size of isoforms correlated inversely with
plasma Lp(a) concentrations (Utermann et al. 1987; Utermann 1989), suggesting
that Lp(a) concentrations might be controlled by the LPA locus, which was con-
firmed by subsequent sib–pair linkage studies in European and North-American
White families (Boerwinkle et al. 1992; Kraft et al. 1992; Demeester et al. 1995;
Scholz et al. 1999) and in African Americans (Mooser et al. 1997). Sib–pair linkage
studies in families from South Africa and from Gabon demonstrated that the LPA
locus is the major locus determining Lp(a) levels also in autochthonous populations
from sub-Saharan Africa (Scholz et al. 1999; Schmidt et al. 2006). Compared to
populations of European descent, the KIV-2 VNTR explained less of the variation
in Lp(a) levels in Africans.
The size polymorphism was detected with the at that time new technique of
Western blotting. This method had, however, a drawback. The intensity of isoforms
varied widely depending on the associated Lp(a) concentrations. Many individuals
exhibited only one isoform upon Western blotting. For those individuals, it was
unclear whether they were homozygotes, i.e., expressed two isoforms of identical
size or whether one isoform was below detection limit or due to a none-expressed
allele (so-called null alleles). DNA technology was the way to overcome the prob-
lem. Already, the DNA sequence data demonstrating multiple identical copies of
kringle IV-2 had Lawn and colleagues led to speculate that differences in repeat
number might underlay the size polymorphism of apo(a). Semiquantitative data
from Southern blotting using a KIV-2-specific sequence as probe (Utermann 1989;
Lindahl et al. 1990) and differences in length of apo(a) mRNA from liver (Koschinsky
et al. 1990) supported this. The application of pulse-field gel electrophoresis/
Southern blotting, which had started as a collaboration and ended in a race, finally
allowed the group of Helen Hobbs in Dallas (Boerwinkle et al. 1992; Lackner et al.
1991) and Hans-Georg Kraft and colleagues in Innsbruck (Kraft et al. 1992) to
demonstrate the size polymorphism at the DNA level. By using appropriate nucle-
ases (e.g., KpnI), which cut the DNA only outside the KIV-2 sequence, allowed to
retain the entire repeat block in large DNA fragments of 20 to >200 kb. Its size
could be finally determined by PFGE/Southern blotting. It turned out that the pro-
tein size polymorphism resulted from a transcribed and translated copy number
variation. The genomic size of one KIV-2 copy was 5.6 kb. Today only a few protein
coding VNTRs have been characterized including in the PMU genes (Swallow et al.
1987), human proline-rich protein (Lyons et al. 1988), and the gene coding for
length variation in the keratin 10 chain (Korge et al. 1992). Very recently, these
transcribed and translated genes including LPA were identified in a genome- and
exomewide search (Mukamel et al. 2021). LPA is the most extensively studied with
a large impact on human health (Schmidt et al. 2016; Kronenberg 2016). In particu-
lar, Helen Hobbs and colleagues in Dallas characterized the LPA locus in detail at
the molecular level (Lackner et al. 1991, 1993).
The analysis by PFGE/Southern blotting alone, however, also resulted in an
incomplete picture. Whether and to which extend an allele was transcribed and
translated into protein could not be seen. Only the simultaneous application of
PFGE/Southern blotting of DNA and Western blotting of plasma allowed a
14 G. Utermann

comprehensive characterization of LPA alleles by KIV-2 copy number, isoform size,

and associated Lp(a) concentration (allele-associated Lp(a) concentration.
It remained however unclear whether the association of the KIV-2 VNTR with
Lp(a) concentrations reflected a causal relationship and—if so—what the mecha-
nism might be. Studies in transfected liver cell cultures from humans and in primary
liver cells from baboons demonstrated that post-translational processing of apo(a) is
the major determinant of Lp(a) concentrations (White et al. 1994a, b; Brunner et al.
1996; Lobentanz et al. 1998). No apo(a)-apoB complexes were detected inside the
cells, but only in the cell culture media indicating that Lp(a) assembly takes place
outside cells at the plasma membrane, in the space of Disse or in plasma following
the separate secretion of apo(a) and LDL. This view is supported by transfusion of
LDL into the plasma of apo(a) transgenic mice (Chiesa et al. 1992) and studies by
Marlys Koschinsky’s group who demonstrated extracellular formation of the disul-
fide bond between cys4326 of apoB in LDL (Callow and Rubin 1995) and the free
cys residue in KIV-9 of apo(a) (Koschinsky et al. 1993; Becker et al. 2006).
By sib-pair analysis using genotypes defined by PFGE/Southern blotting, it was
demonstrated that 70–95% of the variability in Lp(a) concentrations in the popula-
tion is determined by the LPA locus. Together with earlier observations that iso-
forms of the same size are associated with a wide range of Lp(a) concentrations, this
implied that sequence variation in LPA in addition to the CNV determines Lp(a)
concentration. Such variation was identified in the form of restriction site polymor-
phisms in the KIV-2 repeat (Mancini et al. 1995b) a pentanucleotide polymorphism
(PNRP) (Mooser et al. 1995; Trommsdorff et al. 1995) and a +93 C/T polymor-
phism (Zysow et al. 1995; Kraft et al. 1998), which explained some of the variation
in Lp(a) concentrations independent from the KIV-2 VNTR. Hence, at the end of
the 1990th the genetics of the Lp(a) trait was in principle clarified. The LPA locus
was identified as the major locus for Lp(a). The two alleles at the locus defined by
KIV-2 copy number and sequence variation determine Lp(a) levels in an individual
and the frequency distribution of alleles determine the distribution of Lp(a) concen-
trations in a population (Utermann 1999). However, the details of the genetic archi-
tecture of the Lp(a) trait remained to be solved, i.e., the types of sequence variation,
frequencies of SNPs, LDs with copy numbers, effect size on Lp(a) levels, etc., had
to be determined. Sequence variation described to this point with one possible
exception (Zysow et al. 1995; Kraft et al. 1998) had no proven direct causal effect
on Lp(a). In a next step, an attempt was made to find likely causal variation in LPA
by improved mutation screening and sequencing techniques. Some variants were
detected most of which were silent and only one, a Thr>Pro substitution in position
12 of KIV-8 (identical with KIV-8 T23>P in Ogorelkova et al. 2001) was associated
with Lp(a) levels but functional studies supporting causality were lacking (Prins
et al. 1997, 1999). Ogorelkova and colleagues in Innsbruck analyzed the “unique”
kringles 6–10 in LPA in different ethnic groups by the mutation screening technol-
ogy denaturing gradient gel electrophoresis (DGGE) and subsequent Sanger
sequencing of aberrant fragments. They were the first to identify several single
nucleotide polymorphisms (SNPs) in the unique kringles of LPA, which resulted in
amino acid substitutions and splice site variation which were strongly associated
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 15

with Lp(a) levels (Ogorelkova et al. 1999, 2001). With one exception, the SNPs
were not shared between populations (Fig. 1.7). The splice site SNP (Ogorelkova
et al. 1999) was shown by expression experiments in cell culture to result in a trun-
cated apo(a) protein unable to form the Lp(a) complex. This null allele had a fre-
quency of 0.053 in Tyrolians from Austria and 0.0635 in the Finnish population. It
was rediscovered in a large population genetic study in Finns (Lim et al. 2014)
without reference to the previous work. A high number of homozygotes was identi-
fied which had no associated clinical symptoms which led the authors to conclude
that Lp(a) has no essential function in vivo which is amazing for results from a
study analyzing an isolated population. Parson et al. (2004) identified a mutation in
the KIV-2 region of LPA which resulted in a stop codon (R21X) and extremely low
allele-associated Lp(a) levels. Later, large-scale studies showed that this variant has
a carrier frequency between 1.6% and 2.1% in European populations; 1000 Genome
data found that the R21X variant mostly occurs in Europeans and South Asians, is
absent in Africans, and shows varying frequencies in South American populations
(Di Maio et al. 2020).
A resequencing study of LPA was performed by the group of Crawford in indi-
viduals of non-Hispanic black and white ancestry from North America (Crawford
et al. 2008). Nineteen of the identified SNPs were then analyzed for an association
with plasma Lp(a) levels in >7000 participants of a population-based survey which

M75T

P71T W72R

S37F G17R R18W

- 1231 +93
PNR C>T K-IV-2 VNTR V91A R45Q T66M
(n = 5-11) (n = 2 - 43)

K-IV K-IV K-IV K-IV K-IV K-IV K-IV K-IV K-IV K-IV K-V Protease
1 2 3 4 5 6 7 8 9 10

Dra III (+/-) P52L Y2F

R60S
-772 G>A S6I
L87V T23P
+121 G>A

L101V
Khoi San donor splice
Africans (Khoi San and Blacks)
Europeans site mutation
Blacks and Europeans +1 G>A

Fig. 1.7 Figures illustrating differences in the genetic architecture of the Lp(a) trait between
human ethnic groups. The structure of the LPA gene with variants known until 2001 is shown. Data
compiled from Ogorelkova et al. (2001), Prins et al. (1999), Scanu et al. (1994). The color code is
only used for variants detected in the study including all four populations (Ogorelkova et al. 2001)
and illustrates that many variants in LPA occur only in one or few populations which is in agree-
ment with later larger studies (Mukamel et al. 2021; Dumitrescu et al. 2011). (Modified from
Utermann 1999 with permission)
16 G. Utermann

included three ethnic groups: Mexican Americans and non-Hispanic blacks and
whites (Dumitrescu et al. 2011). They found 15 SNPs which were associated with
Lp(a) levels in at least one ethnic group but none in all groups. They were not in
strong LD with the KIV-2 VNTR and explained from 7% to 11% of the variance of
Lp(a) levels in the respective ethnic group. Four of the variants were predicted by
PolyPhen to be possibly or probably damaging, but no functional studies were per-
formed. One variant KIV-8 Thr>Pro had also been described by Ogorelkova et al.
(2001). Hence, this large study extended and confirmed previous work. Very
recently, Sally McCormick’s group in New Zeeland performed a detailed functional
analysis of two non-synonymous variants R990Q and R1771C, which had been
detected in GWAS. They showed that both are causative for null Lp(a) phenotypes
and occur in positions homologous to positions in PLG which when mutated result
in PLG deficiency (Morgan et al. 2020). These are the first functionally character-
ized non-synonymous null mutations in the LPA gene.
With the exception of the one identified deleterious SNP R21X (Parson et al.
2004), the KIV-2 VNTR remained a black box for mutation detection until very
recently, when Asma Noureen and colleagues specifically amplified the KIV-2 tar-
get region by PCR from 90 PFGE-separated alleles from Asian, European, and four
different African populations and identified several SNPs in populations of African,
Asian, and European ancestry by Sanger sequencing (Noureen et al. 2015). As
reported for many other genes and from genome sequencing, they observed a higher
frequency of variable sites in Africans. Two previously unreported splice site vari-
ants were detected. One was a true null allele with no detectable Lp(a) associated
and the other had a high frequency (10–40%) in Africans. Their approach had the
advantage that SNPs could be assigned to KIV-2 copy number, but the disadvantage
that sensitivity was low and mutation detection was limited and depended on copy
number and on the number of KIV-2 repeats carrying the variant (intra-allelic fre-
quency). These problems were overcome when Stefan Coassin and colleagues in
Florian Kronenberg’s group in Innsbruck developed deep sequencing protocols,
which allowed systematic high-throughput mutation analysis of the KIV-2 VNTR
(Coassin et al. 2019). They identified a variety of new variants in LPA and analyzed
the effects on Lp(a) and associations with CHD of previously known (Di Maio et al.
2020) and newly identified variants (Coassin et al. 2017, 2020; Schachtl-Riess et al.
2021) in great detail. Their work and very recent genomic analysis (Mukamel et al.
2021) are presently at the cutting edge of Lp(a) genetics research. In particular by
the genomic analysis of Mukamel et al. (2021), many gaps in our knowledge of the
genetic architecture of the Lp(a) trait have been filled. They estimated KIV-2 VNTR
length from whole-genome sequencing data and defined VNTR alleles by imputa-
tion of SNP data which allowed to estimate frequencies and effects of VNTR hap-
lotypes on Lp(a) levels in populations of African, Asian, and European ancestry. A
total of 17 protein-altering variants each of which reduced Lp(a) levels significantly
as well as variants in the 5′ UTR which increased Lp(a) levels were observed.
Previously, the variants responsible for inter-population differences were largely
unknown. SNPs which had been claimed to explain level differences between ethnic
groups (Deo et al. 2011; Chretien et al. 2006) do this in a statistical sense only with
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 17

a few exceptions (Coassin et al. 2017; Schachtl-Riess et al. 2021). Mukamel et al.
(2021) now reported highly significant differences in the frequencies of variants
with causal effects between major human ethnic groups. This explains much of the
inter-ethnic differences in the genetic architecture of the Lp(a) trait between
these groups.
In addition to the major LPA locus, other genes have been identified which make
minor contributions to the variability of Lp(a) level variation including APOE (De
Knijff et al. 1991; Klausen et al. 1996) and APOH/ß2GPI (Hoekstra et al. 2021).
Genetic variation, which is restricted to an ethnic group, may also contribute. An
example is PCSK9. Loss-of-function mutations in this gene lower Lp(a) levels in
American blacks (Mefford et al. 2019).
A further category are genetic variants, which are rare or very rare, but have large
effects in carriers. Known examples are the genes for FH (Utermann et al. 1989; Van
der Hoek et al. 1997; Kraft et al. 2000), abetalipoproteinemia (Menzel et al. 1990),
lipoprotein lipase deficiency (Sandholzer et al. 1992a), and LCAT deficiency
(Steyrer et al. 1994).

Lp(a), CHD, and Mendelian Randomization

The role of Lp(a) in cardiovascular disease has long been debated and the debate
followed an up and down parkour. The very first study reporting an association
observed a higher frequency of “Lp+” among patients with myocardial infarction
compared to controls (Renninger et al. 1965). This study of poor quality was pub-
lished in German language and largely ignored. The field started with the publica-
tions of Dahlen in Sweden (1974), who reported an association of
“pre-beta1-lipoprotein/Lp(a)” with CHD (Frick et al. 1974; Berg et al. 1974; Dahlén
et al. 1975). In a highly cited paper, Gerhard Kostner and colleagues reported
increased Lp(a) levels in patients with CHD over controls and defined 30 mg/dL as
the threshold for elevated Lp(a) in plasma (Kostner et al. 1981), a value which was
used in practice until recently. Histological demonstration and quantification of
Lp(a)/apo(a) in the aortic wall and atherosclerotic plaques strengthened the idea that
Lp(a) is a risk factor for cardiovascular disease (Költringer and Jürgens 1985; Rath
et al. 1989; Niendorf et al. 1990; Beisiegel et al. 1990). Further strong support
evolved from the homology of apo(a) with plasminogen (McLean et al. 1987) and
the functional studies based on this finding which assigned a dual role in the patho-
genesis of cardiovascular disease to Lp(a). As a particle composed of LDL and
apo(a), it was believed to be atherothrombotic (Loscalzo 1990). At the beginning of
the 1990th, the view of most researchers in the field was that Lp(a) is a risk factor
which was summarized in a popular paper by Richard Lawn in “Scientific American”
(Lawn 1992) with the title “Lipoprotein(a) in Heart Disease.”
Until then, all epidemiological studies relating Lp(a) to coronary risk were retro-
spective case–control studies. Circumstantial evidence for the pathogenicity of
Lp(a) was in addition deduced from functional studies and histology. To gain further
18 G. Utermann

insights into the metabolism, function, and pathophysiology of Lp(a), research

groups started to generate transgenic mice first for apo(a) (Lawn et al. 1992) fol-
lowed by double transgenics for apo(a) and LDL (Linton et al. 1993; Callow et al.
1994, 1995). These studies ended with an unresolved controversy. The double trans-
genics indeed had Lp(a) in plasma. Results on the development of atherosclerosis in
these animals were however controversial. Atherosclerotic plaques were reported
for transgenics expressing apo(a), suggesting that apo(a) unbound to LDL is athero-
genic (Lawn et al. 1992). The same group reported that plaque formation is signifi-
cantly (eight-fold) increased in apo(a)/human apo B double transgenics (Callow
et al. 1995). These results were not confirmed by another study: neither mice
expressing apo(a) alone nor double transgenics for apo(a) and human apoB devel-
oped significant aortic fatty lesions (Mancini et al. 1995a). Taken together, these
animal models did not provide additional strong evidence that Lp(a) is a risk factor
for atherothrombotic disease.
Studies in humans had the potential to change this when the concept of Mendelian
randomization was applied in human epidemiologic studies—though the term had
not been coined at the time. Numerous association studies starting end of the 1970th
had investigated the relation between genetic polymorphisms and lipid levels, apo-
lipoproteins, or the sequelae of atherothrombotic disease. The effect of the apoE
polymorphism on lipid, lipoproteins, hyperlipidemia, and CHD was the first of this
kind (Utermann et al. 1977, 1979, 1984; Menzel et al. 1983). These studies were not
performed to answer the question whether the respective intermediate was likely a
causal factor in the pathogenesis of the disease and did not follow the principle of
Mendelian randomization. The questions were rather whether genetic variation con-
tributes to the variation of the intermediate, e.g., LDL-C and if so what the mecha-
nism might be. The observation that high LDL levels or low HDL levels were risk
factors for CHD was accepted knowledge at the time (Humphries et al. 1992;
Paulweber et al. 1988). Another question of these association studies was whether
apolipoprotein genetic variation could be used as predictive markers for CHD
(Hegele et al. 1986; Hegele and Breslow 1987). Katan had first formulated the prin-
ciple of Mendelian randomization in a letter in Lancet (Katan 1986) following dis-
cussions with Gerd Assmann and the author of this article at a European Lipoprotein
Club meeting. At the meeting, Katan had reported on an epidemiological study
showing that patients with ovarian cancer had low plasma cholesterol. The question
what was first, the hen or the egg, was unanswered and it was suggested to Katan to
determine apoE isoforms in the patients and controls. Given the effect of the apoE
polymorphism on cholesterol levels, this should result in differences in apoE allele
frequencies between the groups in case that low cholesterol is causal for the disease.
This discussion marked the birth of the concept of Mendelian randomization studies
(Katan 2004; Davey Smith and Ebrahim 2004) (Fig. 1.8).
The very first study which applied a Mendelian randomization approach investi-
gated the possible contribution of Lp(a) to coronary risk in patients with familial
hypercholesterolemia (FH) using apo(a) isoforms for stratification (Seed et al.
1990). In this study, the principle was applied but not clearly defined. This was fol-
lowed by two studies by Christoph Sandholzer and colleagues from Innsbruck in
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 19

Fig. 1.8 Panel (a) Histogram showing the distribution of binned short and long apo(a) isoform
frequencies in patients with CHD and controls in six populations (Data from Sandholzer et al.
1992c converted into graphic form. Adopted from Schmidt et al. 2016 with permission). Panel (b)
Schematic illustration of the principle of Mendelian Randomization as first applied for Lp(a) in the
studies of Sandholzer et al. (1992b, c). (From Kronenberg and Utermann 2013, used with
permission)
20 G. Utermann

which the principle of Mendelian randomization was clearly described. To cite from
these papers: “This is the first study which firmly establish a relationship between
genetic apo(a) isoforms, Lp(a) levels and CHD” and “The data demonstrate that
alleles at the apo(a) locus determine the risk for CHD through their effects on Lp(a)
levels and firmly establish the role of Lp(a) as a primary genetic risk factor”
(Sandholzer et al. 1992b).
For the time these studies were performed, they were large including 355 CHD
patients and 399 controls from China (Sandholzer et al. 1992b). The second study
(Sandholzer et al. 1992c) was even larger with more than 1.000 patients and con-
trols from six ethnic groups in which Lp(a) concentrations and apo(a) isoforms
were determined. In both studies, small isoforms (i.e., isoforms with fewer KIV-2
repeats) which determine higher Lp(a) levels were significantly more frequent in
CHD patients than in controls (Sandholzer et al. 1992c) (Fig. 1.8). In a further
smaller study, apo(a) KIV-2 genotypes were determined by PFGE/Southern blotting
together with apo(a) isoforms, Lp(a) levels, and disease status in patients that had
undergone coronary angiography. The results confirmed that apo(a) alleles with low
KIV-2 copy number and high associated Lp(a) concentration were significantly
overrepresented in the patients (Kraft et al. 1996a). Despite the small sample size,
the highly significant results reflected the fact that genotypes and expression level
of each allele was known.
These studies apparently were premature and at odds with some prospective
studies, which were the gold standard at the time. Though a first small prospective
study by Rosengren et al. (1990) reported serum Lp(a) as independent risk factor for
myocardial infarction in middle aged Swedish men, two subsequent studies, one
from Finland (Jauhiainen et al. 1991) and a large study from the US (Ridker et al.
1993), failed to find significant associations and concluded that Lp(a) is not an inde-
pendent risk factor. This provoked editorials in leading medical journals titled “Has
Lipoprotein ‘little’(a) Shrunk?” (Barnathan 1993). Subsequent prospective studies
(Schaefer et al. 1994; Cremer et al. 1994) and meta-analysis of a large number of
prospective studies published over the following years showed that these studies
were clearly outliers and found a strong association of Lp(a) concentration with
myocardial infarction and related phenotypes (Danesh et al. 2000; Bennet et al.
2008; Erqou et al. 2009). Today, it is known that the large influential study by Ridker
et al. (1993) was flawed by problems with Lp(a) quantification which was clarified
by the group in a later less prominently published paper (Suk et al. 2006). The
approach to relate apo(a) isoforms to CHD was also taken up by several groups and
meta-analysis of a series of 40 studies including 58.000 participants confirmed the
seminal studies of Sandholzer et al. (1992b, c) on the association of isoform size
with CHD (Erqou et al. 2010). Even this did not convince the entire community.
Only by the large Mendelian randomization studies of groups in Copenhagen/
Denmark (Kamstrup et al. 2009) and Oxford (Clarke et al. 2009) Lp(a) was finally
“…resurrected by genetics” (Kronenberg and Utermann 2013). Borge Nordestgaard’s
group determined Lp(a) levels and the sum of KIV-type-2 repeats from both apo(a)
alleles by quantitative PCR (qPCR) in relation to CHD in participants from the
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 21

Copenhagen City Heart Study. They demonstrated a strong relation between Lp(a)
levels, repeat number, and disease risk which was highest for individuals with high
Lp(a) concentration and low sum of repeat numbers (Kamstrup et al. 2009). The
conclusion from this study: “These data are consistent with a causal association
between elevated Lp(a) levels and increased risk of MI” (Kamstrup et al. 2009) was
almost identical with the one from the early isoform studies (Sandholzer et al.
1992b,c). Resurrection had happened twice but unlike in the bible, one was not
enough. The second resurrection again changed headlines in journals, e.g., in
“Lipoprotein(a): There’s life in the old dog yet” (Kronenberg 2014a) or
“Lipoprotein(a): the underestimated cardiovascular risk factor” (Thompson and
Seed 2014) and finally triggered the development of drugs to lower Lp(a) in people
with increased risk.
If genetically elevated Lp(a) levels increase risk for CVD as shown, geneti-
cally lowered Lp(a) should result in the opposite, i.e., risk reduction. This was in
fact shown in population-based Mendelian randomization study from Finland
(Lim et al. 2014) by the PROCARDIS study in Germany (Kyriakou et al. 2014)
and by Stefan Coassin, Florian Kronenberg, and colleagues in Innsbruck who
tested this hypothesis in two large studies (Coassin et al. 2017; Schachtl-Riess
et al. 2021): they discovered two common splice site variants (4925G>A and
4733G>A) newly detected by deep sequencing in the KIV-2 repeat which both
decreased Lp(a) concentrations tremendously (Coassin et al. 2017; Schachtl-
Riess et al. 2021). The 4925G>A variant is observed in about 22% of European
populations and is associated with smaller isoforms (mainly 19–25 K-IV repeats)
and decreases Lp(a) concentrations by roughly 30 mg/dL. Carriers of these
smaller isoforms who carry at the same time the 4925G>A splice site variant have
a decreased risk for CHD (Coassin et al. 2017) which has also been confirmed by
an Icelandic study (Gudbjartsson et al. 2019). The other splice site variant
4733G>A is with 38% even more frequent and occurs over a wide apo(a) isoform
range and lowers Lp(a) by 13.6 mg/dL and also the risk for CHD (Schachtl-Riess
et al. 2021). Using data from more than 440.000 participants from the UK Biobank
revealed that carriers of both variants have low Lp(a) concentrations and a 12%
decreased risk for CHD compared to non-carriers of the two mutations (Schachtl-
Riess et al. 2021).
Data from an Icelandic study confirmed that Lp(a) levels are associated in a
dose-dependent manner with risk for CAD, PVD, aortic valve stenosis, heart fail-
ure, and lifespan (Gudbjartsson et al. 2019). Short apo(a) alleles were also associ-
ated with risk but no additional residual association beyond the association with
Lp(a) levels was observed for the KIV-2 polymorphism when Lp(a) was at the same
time in the statistical model (Gudbjartsson et al. 2019). This can be explained by the
fact that the Lp(a) concentration is the measured biological exposure which is only
explained partially by the K-IV repeat polymorphism or other genetic variants.
The Mendelian randomization approach was further used to estimate the magni-
tude of a drug effect on Lp(a) to achieve a desired reduction of the risk for CHD
(Burgess et al. 2018; Lamina and Kronenberg 2019; Madsen et al. 2020).
22 G. Utermann

Non-Genetic Effects, Renal Disease, and Type 2 Diabetes

Estimates of the magnitude of the effect of the LPA locus on Lp(a) levels may be
misleading. The studies from which the estimates were derived were performed on
random population samples and healthy sib–pairs and families. The high heritability
of more than 90% does therefore not exclude that rare and common conditions, e.g.,
diseases not represented in the sample may have significant effects on Lp(a) in
affected individuals and groups and add to their health problems. Early studies on
the effects of environment and various disease states were small, definition, treat-
ments, and subtypes of disease differed between studies, and with few exceptions,
they were controversial.
It was early recognized that Lp(a) levels in an individual may not be stable over
time. Hormones and particularly changes in hormone levels during puberty and
pregnancy were recognized as a cause (reviewed in Kostner and Kostner 2004) but
slight fluctuations without apparent reason seem to be normal. Nutrition and physi-
cal activity have no effects.
Of clinical relevance are two associations of Lp(a) beyond the one with CAD. One
is the association with chronic kidney disease (CKD) and the other with type 2 dia-
betes mellitus (T2D). The relationship of Lp(a) with renal disease including CHD in
CKD is particularly complex. The first observation on elevated Lp(a) in hemodialy-
sis patients probably is by Papadopoulos et al. (1980) who described a high fre-
quency of an additional “pre-beta-lipoprotein” band visible in agarose gel
electrophoresis in the patients. This “pre-beta-lipoprotein” appears identical with
the “pre-beta1-lipoprotein” described by Dahlen to be associated with CAD (Dahlen
1974; Frick et al. 1974) and identified as Lp(a) (Berg et al. 1974). H. Parra in Jean-­
Luis Fruchart’s group in Lille/France first reported Lp(a) elevation in patients with
chronic renal failure (Parra et al. 1987b). Numerous studies followed which con-
firmed the observation and further revealed that the extend of Lp(a) increase depends
on the type and treatment and that the increase in Lp(a) may have different causes,
i.e., impaired removal from the circulation (Frischmann et al. 2007) or increased
synthesis (Kronenberg et al. 1996). The increase is higher in patients with end-stage
renal disease treated by continuous ambulatory peritoneal dialysis compared to
those under hemodialysis (Kronenberg et al. 1996; Kronenberg 2014b). Patients
with nephrotic syndrome develop excessive elevations of Lp(a) (Kronenberg et al.
1996, 2004; Takegoshi et al. 1991; Wanner et al. 1993). Following renal transplanta-
tion Lp(a) levels decrease to almost normal concentrations (Kronenberg et al.
1994a) demonstrating that the increased Lp(a) levels are secondary to disease
(Kronenberg et al. 1994a; Black and Wilcken 1992).
Patients with ESRD have an increased risk for arterial vascular disease. Therefore,
the report of Cressman and colleagues (1992) describing elevated Lp(a) as indepen-
dent risk factor for CHD was noted with interest and triggered several follow-up
studies. Though confirmed by most studies, it turned out that it was not the full truth
but matters were more complicated. Apo(a) isoforms also play a role as demon-
strated by Florian Kronenberg, Hans Dieplinger, and colleagues in Innsbruck who
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 23

unrevealed the complex interplay between renal disease, Lp(a) levels, and athero-
sclerotic vascular disease (Kronenberg et al. 1994b, 1999a, b; Koch et al. 1997).
Despite higher Lp(a) levels in the patients, isoform frequencies were not different
from controls (Dieplinger et al. 1993; Kronenberg et al. 1995). The pronounced
increases in levels in patients were associated mainly with the longer isoforms and
much less so with the short isoforms (Kronenberg et al. 1995, 1996; Dieplinger
et al. 1993). The extend of Lp(a) increase with renal disease is mirrored by the rapid
reduction following renal transplantation which is also dependent on the genetic
phenotype (Kronenberg et al. 1994a). In heterozygous patients with one short and
one long isoform which were analyzed before and following transplantation the
change in concentration of the long, but not short isoform was impressively demon-
strated by Western blotting (Kronenberg et al. 2003).
As in the general population, apo(a) phenotypes predicted the risk for atheroscle-
rotic vascular disease in ESDR patients (Kronenberg et al. 1994b, 1999a) but sur-
prisingly and in contrast to the work of Cressman and colleagues (1992) Lp(a)
levels were found to be poor predictors. This paradoxical situation was explained by
the different timelines of events. The increase of Lp(a) in patients with high molecu-
lar weight phenotypes starts only with the onset of disease and therefore does not
last long enough for a significant pathogenic effect. In contrast, the exposure to high
Lp(a) is lifelong in patients with low molecular phenotypes resulting in more prein-
jury and rapid development of atherothrombotic vascular disease. Lp(a) levels lose
their predictive power whereas apo(a) types retain it. Kronenberg coined the term
“galloping” atherosclerosis for the rapidly progressive form of vascular disease in
ESDR patients with small apo(a) isoforms (Kronenberg et al. 1994b). As for most
disease associations in the Lp(a) field, the reported association of apo(a) isoforms
with ASVD in CKD patients was not confirmed by all and is controversial. In a
recent review (Hopewell et al. 2018), it was concluded that CKD patients with high
Lp(a) levels are at increased risk whereas it is unclear whether apo(a) isoforms are
predictive for ASVD.
The situation on the role of Lp(a) in T2D is far from clear and illustrates how in
some areas of Lp(a) research there was little progress over longer periods. Early
small case-control studies from the 1990s were controversial. Some reported ele-
vated (Bruckert et al. 1990; Heller et al. 1993), some lower (Rainwater et al. 1994),
and some no change in Lp(a) levels (Császár et al. 1993) compared to controls.
Rainwater et al. first reported decreased Lp(a) levels and larger apo(a) isoforms
(Rainwater et al. 1994). Prospective studies investigating the role of Lp(a) as risk
factor for incident or prevalent T2D observed an association of very low Lp(a) levels
with disease (Mora et al. 2010; Ye et al. 2014; Paige et al. 2017) but it was unclear
whether low Lp(a) is causally related to disease. To clarify this several groups initi-
ated Mendelian randomization studies. These were, however, controversial. In a
Mendelian randomization study from Copenhagen (Kamstrup and Nordestgaard
2013), no causal relation of low Lp(a) with T2D was observed and the role of long
KIV-2 repeats remained unclear. A study in Chinese patients with CHD in which
Lp(a) levels and KIV-2 repeats were determined, both low Lp(a) and high repeat
number were associated with T2D. This resulted in the conclusion that low Lp(a)
24 G. Utermann

predisposes to T2D (Mu-Han-Ha-Li et al. 2018). This view was challenged by the
work of Tolbus et al. (2017) who used SNPs tagging Lp(a) levels or KIV-2 repeat
number. They found high KIV-2 repeat numbers but not Lp(a) levels associated with
T2D. In the very large population-based Iceland study, Kari Steffansson’s group
(Gudbjartsson et al. 2019) analyzed the association of loss-of-function (LOF) muta-
tions in the LPA gene for a Mendelian randomization approach. These included
known “null” mutations in LPA which are associated with very low or no Lp(a) in
plasma. Presence of these mutations increased the risk for T2D in the Icelandic pop-
ulation. This to date is the most convincing evidence that very low Lp(a) predisposes
to T2D. The mechanism underlying this relation is unknown but if known could shed
new light on the still unresolved question for what reason evolution created Lp(a).

The Road to Therapy

The recommendations for individuals with a high risk for CHD or which suffered
already from MI, angina pectoris, and related phenotypes and had elevated Lp(a) in
plasma was for a long time to reduce other risk factors more rigorously. There were
simply no drugs available. Niacin was the first recognized to lower Lp(a), but the
effect was small the drug affected also LDL and HDL metabolism and had unde-
sired side effects. An unexpected disappointment was that HMG-CoA-reductase
inhibitors which effectively lower LDL by increasing LDL receptors at the cell
surface did not lower Lp(a) (Kostner et al. 1989). The next surprise was that PCSK9
inhibitors, which also exert their effect on LDL by increasing LDL receptor-­
mediated uptake of LDL did also decrease Lp(a) concentrations (Raal et al. 2014)
and are particularly effective in patients with familial hypercholesterolemia and
elevated Lp(a) (Vuorio et al. 2020). The Lp(a)-lowering effect of PCSK9 inhibitors
was shown in cell culture experiments to be mediated through an overexpression of
LDL receptors and increased internalization of Lp(a) by the receptors (Romagnuolo
et al. 2015). This is at odds with the lack of an effect of HMG-CoA-reductase inhib-
itors and presently unexplained. PCSK9 inhibitors are now a choice for treatment of
at-risk patients with high Lp(a), but do not selectively lower Lp(a) in patients with
very high Lp(a). Only recently, Lp(a) has been specifically targeted by antisense
therapy against apo(a) (Tsimikas et al. 2015; Graham et al. 2016) with ongoing
phase-III trials. A method, which has been very effective in reducing the risk for
MACE in patients with severe clinical CHD, is lipoprotein apheresis (Jaeger et al.
2009; Roeseler et al. 2016; Pottle et al. 2019; Schettler et al. 2017). This therapy had
been developed by the groups of Walter Stoffel in Cologne, which used antibody
columns (Stoffel et al. 1981) and Dietrich Seidel in Göttingen who used heparin
linked to columns (Eisenhauer et al. 1987; Armstrong et al. 1989) to absorb LDL
and Lp(a) from plasma. This therapy is presently still the only one with a proven
significant risk reduction in patients with severe CHD.
1 60 Years of Lp(a) Research: From Ouchterlonys Double Diffusion to Copy Number… 25

Outlook

A historical article describes the development of a scientific field from the past until
the present day but not beyond. But can we learn from history for the future? What
we certainly can learn is where the gaps in our knowledge are and what the impor-
tant questions for the future might be.
In Lp(a) research, one major open question is what the physiological function of
Lp(a) may be, a question which to the authors mind is tightly linked to evolutionary
genetic aspects. The rhetorical question of Brown and Goldstein “does evolution
make sense” in their News and Views article in Nature accompanying the cloning
and sequencing of the apo(a) cDNA (Brown and Goldstein 1987) may well be
extended to Lp(a).
It has been concluded that Lp(a) has no significant function since a large num-
ber of Europeans is heterozygous or compound homozygous for “null mutations”
in the LPA gene and consequently Lp(a) is absent in plasma; these individuals are
healthy and without any signs of disease (Schmidt et al. 2006). This conclusion is
not warranted. First, the conclusion can only be that Lp(a) has no health-related
function under present day conditions in the respective population (i.e., Finns).
Second and more importantly, it seems highly unlikely that a macromolecular
complex the assembly of which requires specific sites for non-covalent interac-
tion between two extremely different large components, a lipoprotein and a gly-
coprotein, and the formation of a covalent disulfide bridge between them being
present in all Old-world monkeys has survived for 40 Mio years when it has no
function.
Considering that Lp(a) concentrations on average are higher in South-East
Asians and severalfold higher in Africans compared to Europeans and that “null
mutations” are significantly less frequent in Africans though Africans had longer
time to accumulate deleterious mutations in none-essential genes, it may be specu-
lated that Lp(a) has a significant role in Africa and further Old-world tropical popu-
lations. If Lp(a) has an unknown health-related function, e.g., in Africans, lowering
Lp(a) by drugs for prevention of coronary disease may have undesired side effects.
Large-scale studies of the Lp(a)/apo(a) trait in relation to disease, e.g., infectious
disease in African populations may therefore elucidate the physiological role of
Lp(a) and also have practical consequences for Lp(a) lowering drug regimes in
these populations.

Acknowledgments I thank Anita Neuner for help with the literature, Eugen Preuss for preparing
figures, and Florian Kronenberg for his support and for critically reading the manuscript.
26 G. Utermann

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Weisel JW, Nagaswami C, Woodhead JL, Higazi AA, Cain WJ, Marcovina SM, Koschinsky
ML, Cines DB, Bdeir K. The structure of lipoprotein(a) and ligand-induced conformational
changes. Biochemistry. 2001;40:10424–35.
Wendt GG. International Lp-workshop. Humangenetik. 1967;3:269–72.
White AL, Hixson JE, Rainwater DL, Lanford RE. Molecular basis for null lipoprotein(a) pheno-
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White AL, Rainwater DL, Hixson JE, Estlack LE, Lanford RE. Intracellular processing of Apo(A)
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Williams-Blangero S, Rainwater DL. Variation in Lp(a) levels and apo(a) isoform frequencies in
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Ye Z, Haycock PC, Gurdasani D, Pomilla C, Boekholdt SM, Tsimikas S, Khaw KT, Wareham NJ,
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Chapter 2
Lp(a) Biochemistry, Composition,
and Structure

Gerhard M. Kostner

Abbreviations

Apo(a) Specific antigen of Lp(a)

ASGPR Asialo-glycoprotein receptor
LCAT Lecithin:cholesterol acyl transferase
LDL Low-density lipoprotein
Lp Lipoprotein
Lp(a) Lipoprotein(a)

Historical Developments

In the early days, atherosclerosis research was dominated among others by two
patho-mechanisms, one related to lipids and lipoproteins and the other to hemostasis
and fibrinolysis. At that time, no one knew that Lp(a) constitutes a connection
between them. Since lipids are mostly water insoluble, they have to be transported in
blood complexed with amphipathic compounds such as phospholipids and apo-­
lipoproteins. The qualitative and quantitative separation of lipoproteins (Lp) was
performed by (1) electrophoresis, (2) ultracentrifugation, and (3) by immune-­affinity
methods such as ELISA or immune-specific adsorbers. The nomenclature of lipo-
proteins reflected the separation methods and there were basically three classifica-
tion systems: (1) based on the electrophoretic mobility yielding alpha-, ß-, and pre-ß
Lp; (2) density classes with the main fractions VLDL, LDL, and HDL; and (3) Lp
families with the main fractions Lp-A, Lp-B, and Lp-C (reviewed in Kostner 1983).

G. M. Kostner (*)
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 39

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_2
40 G. M. Kostner

The nomenclature relating to electrophoretic separation was used by the “East-Coast

Lipid Laboratories” represented by Don Fredrickson and his collaborators (1967),
the density fractions were propagated by John Gofman and Frank Lindgren from the
“West-Coast group at the Donner Laboratories” (Gofman et al. 1949), and the ABC
concept was pushed by Petar Alaupovic from Oklahoma City (Alaupovic et al. 1972).
The major component of VLDL and LDL, apo-LpB was recognized as a rather poly-
morphic apo-Lp with numerous allotypes and polymorphic form, that were described in
several publications by Allison and Blumberg (1961). Allison and Blumberg tested sera
from multi-transfused patients by immune diffusion (Ouchterlony test) for the presence
of iso-antibodies against LDL. Many of these antibodies turned out to be unique, as they
showed no cross-reactivity in Ouchterlony tests. The different polymorphic forms of
apoB detected in this exercise were classified in the “Ag-system” where Ag stands for
“antigen.” The iso-precipitins described by Allison and Blumberg were not readily
accessible by the rest of the scientific community and in order to be independent from
the laboratory of Allison and Blumberg, Kare Berg, a geneticist from the university of
Oslo, Norway, took another line by using xeno-antibodies against apoB (Berg 1963).
He set out to hyper-immunize rabbits with LDL isolated from 20 healthy arbitrarily
chosen donors. Although these antisera could not distinguish between the LDL from the
donor individuals, K. Berg cross-absorbed the antisera with individual LDLs and finally
came up with an immune serum that recognized an unique “Lp antigen” that he called
Lpa. Lpa was present in some, but not in all sera testes from his patients. Sera that were
positive for this factor were called Lpa+ and those negative were called Lpa−. In a panel
of 314 sera from healthy adult donors, 34% were Lpa+ and the remaining ones Lpa-
negative. Notably, in our own studies, we quantified Lp(a) in a group of 107 healthy and
76 myocardial infarction (MI) patients from Venice and found that 35% of them had
Lp(a) levels of >30 mg/dL, the value that had been adopted as the cut-off for coronary
heart disease (CHD) and MI in numerous subsequent studies (Kostner et al. 1981). In
early days, Lp(a) was considered to be a qualitative genetic trait and a gene frequency
of 0.1881 was calculated in the Norwegian population (Berg 1963). In the Ag system
mentioned above, some 14 different alleles were characterized that obviously reflected
some sequence variations in the APOB gene or possibly variations in the sugar moiety
(reviewed in Kostner 1976). Additional independent polymorphisms of apoB that might
not be related to the Ag system, called the Tl system (from “trypsin-treated Lps”) and
the El system (from “electrophoresis”), have been described, but did not get much atten-
tion in the following years (reviewed in Kostner 1976). Other suggested polymor-
phisms, the Ld system and the Lt system, turned out to be in fact Ag alleles (Utermann
1989). For completeness, it is noteworthy to mention “Lp(x)” that was described by
Bundschuh and Vogt (1965) as a factor distinct from Lp(a). Lp(x) was identified with
xeno-antibodies from horse, but not from rabbits, and was believed it to be a heterolo-
gous form of Lp(a). All these polymorphisms were more or less forgotten in later years,
as they apparently had no relevance for atherosclerosis or cardiovascular diseases.
The current view of the buildup of Lp(a) emerged from several subsequent stud-
ies carried out in the laboratories of A. Scanu (Fless et al. 1985), our own, and sev-
eral others. In fact, it is mostly believed that there exists in human plasma one rather
homogenous fraction of Lp(a) consisting of a bona fide LDL and one apo(a) glyco-
protein covalently linked by a disulfide bridge. Whether or not this reflects the true
in vivo situation under all circumstances remains to be established.
2 Lp(a) Biochemistry, Composition, and Structure 41

Lipo- Area Conc.

protein % mg/dl

1
HDL 26 36

4 Lp(a) 31 42

VLDL 4 5
2
LDL 39 52

TChol 135

TG 121

100

50

0
0 5 10 mm 15

Fig. 2.1 Separation of Lp(a) by electrophoresis: Lp(a) migrates in gel electrophoresis as extra
pre-ß1 band and may be quantitated either after staining for lipids with Sudan black, or by
staining with a cholesterol reagent. Here, Lp(a) is separated by the Helena® Electrophoretic
system https://www.helena.com/. The concentration in mg/dL refers to Lp(a)-cholesterol as
staining was performed with a cholesterol dye. Since Lp(a) consists of some 25–30% of cho-
lesterol, Lp(a) mass in mg/dL may be calculated by multiplication with a factor of 3–4. (1 and
2): Plasma with Lp(a) of <30 mg/dL; (4) plasma with a Lp(a) concentration of 140 mg/dL. (From:
Kostner, K.M.; Kostner, G.M. Lp(a) and the Risk for Cardiovascular Disease: Focus on the
Lp(a) Paradox in Diabetes Mellitus. Int. J. Mol. Sci. 2022, 23, 3584. https://doi.org/10.3390/
ijms23073584)

Purification and Composition of Lp(a)

In the first reports by K. Berg and his collaborators, Lp(a) was denominated “pre-­
ß1” and/or “sinking pre-ß lipoprotein” (Berg 1963). In paper or agarose gel electro-
phoresis, Lp(a) migrated somewhat faster than pre-ß-Lp (VLDL) but slower than
alpha-Lp (HDL). This is depicted in Fig. 2.1. It must, however, be mentioned at this
point that the actual position of Lp(a) by electrophoretic methods depends on the
carrier material, the type, and pH of the electrophoresis buffer and the presence of
anti-coagulants such as heparin.
42 G. M. Kostner

The purification of Lp(a) succeeds by a combination of methods including ultra-

centrifugation, poly-anion precipitation, size exclusion chromatography, and affin-
ity chromatography. In the case of density gradient ultracentrifugation, most of the
Lp(a) from fasting plasma of healthy individuals is found between LDL and HDL2
as shown in Fig. 2.2. These properties of Lp(a) in electrophoresis and ultracentrifu-
gation led to its term “sinking pre-ß lipoprotein” in early publications.
As will be detailed later, apo(a) is characterized by a unique size polymorphism
with great differences in their molecular mass. It is therefore evident that different
isoforms of apo(a) cause significant different hydrated densities of the correspond-
ing Lp(a) that may be found at variable positions in the density gradient. In the case
of heterozygous individuals with striking differences of the apo(a) mass, even two
distinct Lp(a) bands may be found in the density gradient.
It must be emphasized here that apo(a) is not only found in a single distinct frac-
tion by ultracentrifugation, but there is rather a distribution over the whole density
gradient in most of the plasma samples of blood donors. This has been emphasized
particularly in a review article published by Fless (1990).
In the plasma sample shown in Fig. 2.2, only some 75% of apo(a) was found
between LDL and HDL, and there were appreciable amounts also found in the
VLDL, HDL, and bottom fraction. If one separates post-prandial plasma or plasma
from hypertriglyceridemic patients by density gradient ultracentrifugation, the

% Distribution of Lp(a)
Density Gradient Ultracentrifugation

%
VLDL 5,8

2,7

LDL
10,9
20,1
Lp(a) 44,6

HDL2 3,2
1,0

8,2
HDL3

BOTTOM 3,7

Fig. 2.2 Separation of Lp(a) by density gradient ultracentrifugation that highlights the heteroge-
nous nature of Lp(a). After prestaining all serum constituents with Coomassie blue, lipoproteins
were separated by ultracentrifugation in the SW-41 Rotor, Beckmann® for 24 h at 40,000 rpm. In
the particular plasma, some 75% of Lp(a) was found in the HDL1 region, the rest distributed
between the top up to the bottom fraction. (From: Kostner, K.M.; Kostner, G.M. Lp(a) and the Risk
for Cardiovascular Disease: Focus on the Lp(a) Paradox in Diabetes Mellitus. Int. J. Mol. Sci.
2022, 23, 3584. https://doi.org/10.3390/ijms23073584)
2 Lp(a) Biochemistry, Composition, and Structure 43

St. Apo(a) Alb.

94
67
43
30
20.1
14.4

Fig. 2.3 Western blot of Apo(a) isolated from plasma or urine. Plasma Lp(a) was purified as
described in Kostner et al. (1999) from a donor containing 90 mg/dL of Lp(a). The urine of the
same individual was concentrated 50-fold and both fractions were separated by SDS–polyacryl-
amide gel electrophoresis followed by W-blotting. St: protein molecular weight standard, the num-
bers indicating the mass in kDa. P refers to plasma and U refers to urine. Alb: Plasma albumin used
as a reference. (From: Kostner, K.M.; Kostner, G.M. Lp(a) and the Risk for Cardiovascular
Disease: Focus on the Lp(a) Paradox in Diabetes Mellitus. Int. J. Mol. Sci. 2022, 23, 3584. https://
doi.org/10.3390/ijms23073584)

situation is even more complex and much larger amounts of apo(a) are found in
VLDL or IDL. The exact morphology of Lp(a) outside of HDL1 has not been eluci-
dated in detail. Whether they are artifacts or true metabolic entities is not fully clear.
We characterized the apo(a) immune reactivity found in the bottom fraction of
human plasma and found that they consist of fragments created by Ca2+-dependent
proteases that are abundant on cell surfaces from several organs (Frank et al. 2001;
Gries et al. 1987). These fragments are not bound to lipoproteins and have masses
of some 50–150 kDa. Similar fragments of apo(a) are found in urine despite their
rather large size (Kostner et al. 2001) (Fig. 2.3). The amount of apo(a) fragments in
urine correlates significantly with the parent Lp(a) plasma concentration and we
therefore proposed the use of urinary apo(a) as a clinical chemical risk parameter
for atherosclerotic diseases (Kostner et al. 1996).

Preparation of Pure Lp(a)

The preparation of pure Lp(a) with high yield from plasma with low concentra-
tion—mostly corresponding to large apo(a) isoforms is not an easy task. Therefore,
most investigators use plasma from donors with high Lp(a) values, that usually
contain small apo(a) isoforms. These Lp(a) specimens have a density not much dif-
ferent from that of LDL and therefore by ultracentrifugation used mostly as a first
step, large amounts of Lp(a) may be contaminated with LDL or may be lost in the
LDL fraction. There are two more hassles that must be considered by purification of
Lp(a) from various donors: (1) Lp(a) with large apo(a) isoforms in pure form are
prone to spontaneous precipitation and (2) Lp(a) associates with numerous other
44 G. M. Kostner

apo-Lp such as for example apoE, apoH (ß2-glycoprotein), and many kinds of other
serum constituents. These Lp(a) complexes may be dissociated by the addition of
amino acids such as Lys, Pro, hydroxy-Pro, and others. We therefore elaborated a
purification procedure that yielded Lp(a) with a purity of some 98%. It was sug-
gested that this material might be suitable for use as a “gold standard” in value
assignment for clinical chemical analyses (Kostner et al. 1999).
In short, blood is harvested from donors with Lp(a) concentrations of >30 mg/dL
and in the first step either citrate plasma or serum is prepared after coagulation.
After adding some preservatives (EDTA, BHT, PMSF, thiomersal), all lipoproteins
with d < 1.060 g/mL are separated by ultracentrifugation. Next, the density fraction
>1.060 < 1.125 g/mL is obtained by ultracentrifugation. After concentration to
approx. 10–20 mg/mL Lp(a)-cholesterol, proline at a final concentration of
0.1 mol/L is added. This step is essential as it dissociates all proteins from Lp(a)
other than apo(a) and apoB. In the next step, lipoproteins are separated by size
exclusion chromatography over Biogel A-15 m and the Lp(a) peak is harvested and
concentrated by pressure dialysis. For storage over a longer time period, pure Lp(a)
may be frozen at −20 to −70 °C in the presence of stabilizers. We tried several ones
including saccharose, polyethylene glycol, and others and at the end found out that
the 1:1 admixture of pure glycerol gave the best results. If prepared according to this
procedure, Lp(a) shows one band in agarose gel electrophoresis and is also virtually
pure by SDS–PAGE.

Chemical Composition of Purified Lp(a)

There is quite some variation in the composition of purified Lp(a) that is donor spe-
cific reflecting the isoform size, the lipid status of the plasma, and more. In Table 2.1.,
some average values are shown of Lp(a) isolated from fasting healthy donors in
comparison with LDL.
It must be emphasized here that the composition of Lp(a) shown in Table 2.1. is
just a snapshot of a fraction isolated from fasting plasma of normolipemic healthy
individuals. Lp(a) outside of the HDL1 density fraction or Lp(a) isolated from dys-
or hyperlipemic plasma may have quite significantly deviant structures and
compositions.

Table 2.1. Chemical composition of Lp(a) in comparison with LDL

Compound Lp(a) % w/w LDL % w/w
Protein 26–30 21.0
Carbohydrates 4–8 1.3
Cholesteryl ester 31–37 42.0
Free cholesterol 7–8 9.0
Phospholipids 16–20 20.7
Triglycerides 4–6 6.0
2 Lp(a) Biochemistry, Composition, and Structure 45

The Protein Structures of Lp(a) and Apo(a)

As mentioned above, the chemical composition of Lp(a) depends on the individual

donor, its nutritional and health status, and the preparation procedure. An idealized
Lp(a) particle separated from plasma at d 1.060–1.125 g/mL is composed of an
LDL-like core lipoprotein with a lipid composition close to that of the LDL fraction
of d 1.030–1.060 g/mL. To this core lipoprotein, the specific apo(a) glycoprotein is
covalently linked by a disulfide bridge (Fig. 2.4). The disulfide bridge links Cys
4326 in apoB-100 with the only free Cys 4057 in apo(a), that is located in kringle
four (K-IV) Type-9. Due to the size polymorphism, there is a great variation in the
molecular mass of apo(a) that reaches from 350 to >800 kDa. There are only a few
reports on the morphology and structure of Lp(a) revealed by physico-chemical
methods and electron microscopy. An interesting view of Lp(a) has been published
by Weisel et al. (2001) who studied Lp(a) architecture by rotary-shadowing electron
microscopy. They proposed that the protein components of Lp(a) after exposure of
glycerol consist of rings made up of dense nodules of various size. After exposure
to tranexamic acid, apo(a) and apoB dissociated and apo(a) formed a long tail with
distinct kringle units but still linked to the LDL core.

Fig. 2.4 Hypothetical model of Lp(a) showing the LDL core with apoB-100 as the major surface
protein and apo(a). 41 refers to K-IVT-1, 42 to K-IV-T2, and so one. 5 = kringle V and P = the
protease domain. (The figure was drawn by Timo Speer, Med. University of Homburg/Saar,
Germany. From: Kostner, K.M.; Kostner, G.M. Lp(a) and the Risk for Cardiovascular Disease:
Focus on the Lp(a) Paradox in Diabetes Mellitus.Int. J. Mol. Sci. 2022, 23, 3584. https://doi.
org/10.3390/ijms23073584)
46 G. M. Kostner

The structure of apo(a), the characteristic glycoprotein component of Lp(a),

has a unique structure as summarized by Morrisett in 1990 (Morrisett et al. 1990).
It consists of repetitive protein segments, so-called kringles (K), that are highly
homologous to K-IV in plasminogen. K-IV’s contain approximately 110 amino
acids forming a secondary structure, which resembles “Danish kringles” (McLean
et al. 1987). The N-terminal part of apo(a) consists of various numbers of unique
or repetitive copies of these kringle-IV’s. In addition, apo(a) has one copy of a
K-V like kringle and a non-functional protease-like domain, both highly homog-
enous to that of plasminogen. A cartoon of the apo(a) structure is shown in
Fig. 2.5. In humans, there exist more than 30 genetic size polymorphisms of
apo(a). The smallest apo(a) isoform consists of the protease domain, one copy of
K-V and 11 K-IV’s of which K-IV Type-1 (T-1) and T-(3–10) are unique in their
primary structure, whereas K-IV T-2 is present in 2 identical copies. Larger iso-
forms differ by the number of K-IV T-2’s; the largest apo(a)’s described so far had
52–54 K-IV’s.

Fig. 2.5 cDNA structure of apo(a) in comparison to plasminogen. In apo(a), kringle-IV’s (K-IV)
homologous to plasminogen are repeated several times. There is also one K-V like domain and a
protease domain in apo(a) (P) with a homology of 94 to that of plasminogen. The Arg of plasmino-
gen is replaced in apo(a) by Ser and thus the protease cannot by activated in Lp(a) by t-PA or
urokinase. (From: Kostner, K.M.; Kostner, G.M. Lp(a) and the Risk for Cardiovascular Disease:
Focus on the Lp(a) Paradox in Diabetes Mellitus.Int. J. Mol. Sci. 2022, 23, 3584. https://doi.
org/10.3390/ijms23073584)
2 Lp(a) Biochemistry, Composition, and Structure 47

High-resolution structures of whole apo(a) have not been published, but there
exist a few crystal structures of recombinant apo(a)-kringle-IV’s (Ye et al. 2001).
The crystal structure refined to a resolution of 1.45 Å revealed important structural
features of kringle-IV-T-7 that are postulated to be responsible for the interaction
with Lysine groups.

The Carbohydrate Moiety of Apo(a)

The K-IV domains are connected by linker regions that are highly glycosylated by
N- and O-linked sugars. The best characterization of the apo(a) carbohydrate
arrangement has been published by Garner et al. (2001) who demonstrated that
approx. 20% of the oligosaccharide structures consist of two major Asp-linked
N-oligosaccharides. N-glycans are complex biantennary structures in either a mono-
or disialylated state. The remaining 80% of the sugars are Ser/Thr O-linked oligo-
saccharides and are present in all apo(a) isoforms. The majority consist of the
mono-sialylated core type-I structure, NeuNAcα2-3Galβ1-3GalNAc, and the
remaining consist of disialylated and non-sialylated O-glycans. The latter finding
prompted us to elucidate the possibility that Lp(a) might bind to the asialo-­
glycoprotein receptor (ASGPR) on liver cells.

 he Role of Structural Apo(a) Features for the

T
Lp(a) Metabolism

It is well known that the sialic acid content of glycoproteins regulates their fate in
circulating blood. Aged protein may be devoid of their terminal sialic acid due to the
action of specific sialidases. There are specific asialo-glycoprotein receptors on liver
cells that very effectively bind and catabolize such asialo-glycoproteins. This is sche-
matically displayed in Fig. 2.6. ASGPR may be specific for galactose or mannose.
In a study we published in 2003 (Hrzenjak et al. 2003), it was important that
antimicrobial agents, among other preservatives, were added immediately after
drawing blood and also throughout the different isolation steps, in order to obtain
“native Lp(a)” as far as possible. The metabolic experiments were carried out in
hedgehogs, the only animal species with the exception of old-world monkeys that
synthesize an Lp(a) like lipoprotein. In vivo experiments in these animals revealed
that desialylated Lp(a) is catabolized 25 times faster than native Lp(a) and is almost
exclusively taken up by the liver. Concomitant injection of asialo-Lp(a) with asialo-­
orosomucoide to hedgehogs reduced the half-life of asialo-Lp(a) to values observed
for native Lp(a). Mannan, the competitive inhibitor for the mannose-specific
ASGPR, had no effect. Similar results were observed in wild-type mice where desi-
alylated Lp(a) is catabolized 50–100 times faster than sialylated Lp(a). Here also,
only asialo-orosomucoide, but not mannan, acted as a competitive inhibitor.
48 G. M. Kostner

Fig. 2.6 The sugar moiety Does not Binds to ASGPR receptor
of glycoproteins consists bind to ASGPR on liver cells
of complex antennary and is internalized
structures, many of them
containing sialic acid (Sia)
as terminal sugar. Aged
proteins may be targets of
sialidases that cleave sialic
acid off, and in turn,
Gal Gal
galactose (Gal) or
mannoses may be exposed
to the surface. These latter
sugars are avidly bound to GlcNAc GlcNAc
asialo-glycoprotein
receptors (ASGPR) on core sugars core sugars
liver cells and catabolized

protein protein

Sialoglycoprotein Asialoglycoprotein

Whether or not the results of these experiments might be transferable to the situ-
ation in humans cannot be answered from these experiments. Any Lp(a) preparation
isolated from human plasma may contain only a small fraction of the non-sialylated
Lp(a), because of its very short half-life in the circulating blood of the donor.
Whether such putative non-sialylated Lp(a) is directly secreted from human liver or
is generated during circulation in blood or might be an artifact generated during
Lp(a) preparation remains to be investigated. It is, however, important to note that
we also carried out similar metabolic studies with native Lp(a) in wild-type mice
and in ASGPR knockout mice. In the ko-mice, the HL-2 subunit of the ASGPR was
absent. When native freshly isolated Lp(a) was injected in either mice, we observed
a measurable retardation of the Lp(a) catabolism in the KO-mice as compared to
wild-type mice. The amount of Lp(a) labeled with the non-degradable isotope
[125I]
tyramine cellobiose accumulating in the liver of knockout mice was signifi-
cantly lower compared with wild-type mice (Hrzenjak et al. 2003). We believe this
is a strong argument for the ASGPR pathway being indeed involved in Lp(a) catab-
olism in humans.
We also observed that some 87% of intravenously injected asialo-Lp(a) was
cleared by ASGPR-negative mice within 1 h. Previous studies by Roos et al. (1983)
demonstrated two galactose-specific receptors in rat liver: The Kupffer cell-specific
glycoprotein receptor readily interacts with galactose-exposing particles of the size
of LDL (Fadden et al. 2003). We assume that this receptor might be responsible for
the clearance of asialo-Lp(a) in our ASGPR ko mouse model. Yet, the Kupffer cell-­
specific glycoprotein receptor is not expressed in humans, as human genome analy-
sis reveals a pseudogene that is not translated into a protein (Van Berkel et al. 1985).
Taken together, the role of the ASGPR for the catabolism of Lp(a) in humans is far
from being clear and requires further investigation.
2 Lp(a) Biochemistry, Composition, and Structure 49

Impact of Gene Variants on the Apo(a) Structure

This topic is thoroughly covered in the article by G. Utermann in this book, and
thus, I mention in this paragraph only a few aspects that might be relevant for struc-
tural considerations of Lp(a). Among the numerous polymorphisms and mutations
described elsewhere in this book, there is in particular one mutation relevant here,
the truncated form of apo(a) expressed by the so-called null allele. The first report
on truncated apo(a) was published by M. Ogorelkova from the laboratory of
G. Utermann in Innsbruck (Ogorelkova et al. 1999). Gene sequencing of APOA in
Caucasian individuals with almost zero Lp(a) levels revealed a G→A substitution at
the 1+ donor splice site of K-IV type 8 introns. This nonsense mutation led to the
expression of a truncated form of apo(a) that consisted of a N-terminal fragment
lacking all entities after kringle-IV-T7. Since K-IV T9 in apo(a) contains the single
free –SH group that is necessary for the covalent binding to apoB-100, such trun-
cated apo(a) are not able to stably assemble with LDL. Interestingly, there are small
amounts of free truncated apo(a) found in plasma indicating that the liver secretes
such apo(a) mutants, but it seems that they are very rapidly catabolized. This opens
up the question whether plasma Lp(a) levels might be drastically reduced in general
by inhibition of Lp(a) assembly.

Impact of the Assembly on Plasma Concentrations of Lp(a)

We addressed this question in in vivo and in vitro studies using tranexamic acid for
the inhibition of Lp(a) assembly (Frank et al. 1999). In vivo studies were performed
with single transgenic apo(a) mice or double transgenic apo(a):apoB mice both of
them carrying the relevant human genes. The assembly of apo(a) in the test tube
may be fully inhibited by Lys or analogs thereof such as delta-amino valeric d-AVA
acid or tranexamic acid TXA, the latter being the strongest inhibitor that is also
known to inhibit fibrinolysis by plasmin (displayed in a cartoon in Fig. 2.7). The
mice were fed 150 mg/dL of d-AVA or TXA for 1–2 weeks and the plasma apo(a)
and Lp(a) concentrations were followed over time. In the double transgenic Lp(a)
mice, in contrast to what we expected, the concentration of Lp(a) rose after 1 week
of feeding to almost twice the value observed in the absence of d-AVA or TNX feed-
ing. This was a transient effect since after omitting the inhibitors from the chow, the
Lp(a) concentration returned to the pretreatment values. We also revealed that the
increase of plasma Lp(a) was fully accounted for by the presence of genuine Lp(a)
and not by free apo(a). When single transgenic apo(a) mice were treated in a similar
protocol apo(a) rose after 1 week by 57% and returned to pretreatment values as
well. Similar results were obtained by d-AVA feeding, yet they were less pro-
nounced. We then harvested the livers of mice treated with TNX or d-AVA and
found that their concentration in the liver was significantly lower than without
treatment.
50 G. M. Kostner

Fig. 2.7 Possible models of Lp(a) assembly and inhibition by tranexamic acid. There are currently
two models of Lp(a) assembly discussed: (1) apo(a) is biosynthesized in the liver and after passage
through the Golgi apparatus it binds to the surface of liver cells. Bypassing LDL associate with
apo(a) and both components are covalently linked by a disulfide bridge. The first step of assembly,
the interaction of kringles-4 with Lys groups of apoB-100 may be competed for by Lys analogs
such as Tranexamic acid. Free apo(a) not complexed to LDL might be degraded by hydrolytic
enzymes. Alternatively, the assembly may take place intracellularly in the liver cells. (From:
Kostner, K.M. and Kostner, K.M. Lipoprotein(a): a historical appraisal. J Lipid Res. 2017, 58,
1–14. https://doi.org/10.1194/jlr.R071571)

In turnover studies carried out with TNX or d-AVA or without, we demonstrated

that half-lives of Lp(a) were prolonged by approximately 33% in the former.
Moreover, in vitro experiments with McA-RH 7777-XL rat liver cells stably trans-
fected with apo(a) helped to get insight in the possible mechanism of our findings.
Cells treated with TNX or d-AVA showed lower amounts of expressed apo(a) in cell
extracts, yet the amount of apo(a) in the medium was significantly increased. Our
findings are compatible with the interpretation that apo(a) after biosynthesis and
cell excretion is bound to liver cell surfaces where Lys groups at the surface are
essentially involved. Surface bound apo(a) might be catabolized more rapidly,
whereas dissociation from the surface by Lys analogs shuttles apo(a) into the circu-
lation or into the incubation medium where its concentration increases. We believe
that these findings have a profound impact on the individual plasma levels of Lp(a)
in humans: The stronger the binding of newly synthesized apo(a) on the cell surface
is, the lower is the final Lp(a) concentration in circulation. This might be reflected
by the fact, that large apo(a) isoforms with a greater number of K-IV’s bind stronger
2 Lp(a) Biochemistry, Composition, and Structure 51

to liver cell surfaces and are partly degraded before assembling with LDL to intact
circulating Lp(a).

Impact of Apo(a) Mutations on Coronary Artery Disease (CAD)

Given the fact that Lp(a) is a strong independent risk factor for atherosclerosis and
CAD it was speculated that individuals with apo(a) mutations or polymorphisms
that cause reduced plasma Lp(a) levels might be at a lower risk for CAD. This was
addressed in a cohort of the PROCARDIS study published by Kyriakou (Kyriakou
et al. 2014). Indeed, it was found that the LPA null allele as identified by the
rs41272114 SNP not only is associated with reduced plasma Lp(a) concentrations
but also with a significantly reduced CAD risk.
Among all the mutations in the whole apo(a) gene including the promoter region
and regulatory cis-acting regions described in the literature, there are two publica-
tions from the Innsbruck laboratory that need attention (Noureen et al. 2015;
Coassin et al. 2019). It was known since the first published report on the LPA gene
sequence by McLean and Lawn (McLean et al. 1987) that there are silent mutations
in the repetitive gene region coding for the K-IV T2. These variations were called
K-IV2 A and K-IV2 B. Despite of the difference in the gene sequence, the A and B
alleles translated into the same apo(a) protein sequence. Sequencing the apo(a) gene
is not an easy task because of the repetitive structure caused by K-IV T2 and the
homology of the non-repetitive kringles K-IV-1 and K-IV-3 to K-IV 10. Nevertheless,
Stefan Coassin and his colleagues succeeded to elaborate a sophisticated protocol
that allowed apo(a) sequencing in larger quantities. In these studies, gene variants in
the K-IV-T2 region were identified that translated drastically into plasma Lp(a) con-
centrations. The exact mechanisms on a molecular level are not fully clear, but the
results highlight the importance of the polymorphic APOA gene sequence for the
apo(a) protein expression and plasma concentration of Lp(a).

Impact of the LDL Structure on the Lp(a) Assembly

The reason why apo(a) only assembles with LDL and not with other serum proteins
that may have Lys groups exposed to the surface has never been fully explored. As
a matter of fact, the greatest portion of full length apo(a)—if not all is found on
apoB-100 containing lipoproteins in human plasma. This led us to assume that the
composition and morphology of LDL have just the right prerequisites for this
assembly. Two observations published earlier by our laboratory strongly support
this concept.
Numerous reports in the literature demonstrate that Lp(a) hardly binds directly to
the LDL receptor (Hofer et al. 1997). This is at a first consideration surprising, as
some 50% of the Lp(a) moiety consist of apoB-100. The most plausible explanation
that Lp(a) is not bound to the LDL-R would be that the large glycoprotein apo(a)
52 G. M. Kostner

masks the epitope in apoB-100 responsible for LDL receptor binding. This concept
is strongly supported by our studies of patients suffering from familial defective
apoB-100 (FDB) (Durovic et al. 1994). FDB patients express a mutant apoB-100
where Arg at position 3500 is substituted by Gln. This substitution causes the bio-
synthesis of an LDL particle that has a strongly reduced binding affinity to the LDL
receptor and in turn patients with FDB are hypercholesterolemic.
In the investigations published by Ernst Steyrer et al. (1994), we studied the in vitro
assembly of Lp(a) by mixing purified LDL with recombinant apo(a) and followed the
covalent linkage of both components. Whereas wild-type LDL mixed with apo(a)
complexed under the given experimental conditions between 15% and 44% with
apo(a), LDL from a homozygous FDB patient showed only 2–16% association. The
corresponding figure using LDL from heterozygous FDB individuals was 2–30%.
Moreover, we found that in heterozygous FDB patients the ratio of defective to wild-
type apoB100 in Lp(a) is significantly lower than in LDL from the same patients.
These results strongly suggest that the epitope in apoB-100 that is involved in LDL
receptor binding is also highly relevant for apo(a) binding and covalent linkage.
Another example for the importance of the right morphology of LDL to warrant
an ideal assembly has been published in 1994 by our group. It is known that patients
suffering from LCAT deficiency (LCAT-D) have a grossly altered plasma lipopro-
tein pattern. We made also the observation that 9 heterozygous LCAT-D patients
had only 2–13 mg/dL of Lp(a) whereas the Lp(a) concentration in the non-affected
siblings was significantly higher (Ernst Steyrer et al. 1994). Eleven of the studied
homozygous LCAT-D patients exhibited plasma Lp(a) levels of virtually zero. The
morphology of lipoproteins in the LDL region isolated from homozygous LCAT-D
patients was grossly altered with large vesicles and small spheres and there was an
almost complete lack of cholesteryl esters (Fig. 2.8). When LCAT-D LDL were
incubated with recombinant apo(a) for 20 h at 37 °C, no complex of

Fig. 2.8 Negative-stain electron microscopy of LDL (density 1.030–1.063 g/mL) and Lp(a) iso-
lated from a healthy control individual and from a homozygous patient suffering from LCAT
deficiency. (From: Kostner, K.M.; Kostner, G.M. Lp(a) and the Risk for Cardiovascular Disease:
Focus on the Lp(a) Paradox in Diabetes Mellitus. Int. J. Mol. Sci. 2022, 23, 3584. https://doi.
org/10.3390/ijms23073584)
2 Lp(a) Biochemistry, Composition, and Structure 53

apoB100:r-­apo(a) had been formed (i.e., no assembly to Lp(a) took place). Normal
LDL under the same conditions showed complete assembly to Lp(a). We concluded
that the integrity of the LDL structure is a prerequisite for the biosynthesis of genu-
ine Lp(a). Furthermore, it appears that the lack of complexing apo(a) to bona fide
LDL leads to a fast catabolism or degradation of the expressed apo(a). Thus, not
only is an intact LCAT activity necessary, but there must also be an abundance of
“normal” native LDL. These factors substantially regulate plasma Lp(a) metabo-
lism and serum Lp(a) levels.

Disclosures I have nothing to disclose.

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Chapter 3
Genetics of Lipoprotein(a)

Gerd Utermann

Introduction

The genetics of lipoprotein(a) [Lp(a] is both, simple and complex at the same time.
Initially described as a dominant trait (Berg 1963; Berg and Mohr 1963) with two
immunologically defined phenotypes, Lp+ and Lp−, where Lp+ is dominant over
Lp−, it is now well established that Lp(a) is a quantitative trait with a very broad
distribution in all studied populations (Schmidt et al. 2016). Lp(a) concentrations
vary more than 1000-fold between individuals in the same population and range
from undetectable to >200 mg/dL in healthy individuals (Utermann 1989). In all
populations, the distribution of Lp(a) levels is skewed and far from Gaussian. In
Europeans, most individuals have low and few have very high Lp(a) concentrations.
Mean and median Lp(a) concentrations, the distribution of Lp(a) concentrations
(e.g., skewness) vary widely between human ethnic groups. Sub-Saharan Africans
have by far the highest levels and lowest skewness. Compared to Europeans, they
are two to fourfold higher. The highest concentrations were reported for Black
Sudanese (Sandholzer et al. 1991) and Gabonese Bantu (Schmidt et al. 2006).
Differences exist also within populations from major ethnic groups. In Europe,
Finns have the lowest reported concentrations (Erhart et al. 2018; Waldeyer et al.
2017). Asian populations are even more heterogeneous. Most studies report very
low levels and highly skewed Lp(a) distributions in East Asians (Japanese and
Chinese). South-East Asians, e.g., Indians and Thais, have concentrations between
Europeans and East Asians and Africans (Schmidt et al. 2016; Sandholzer et al.
1991; Helmhold et al. 1991; Enkhmaa et al. 2016) (Fig. 3.1). Notably different
Lp(a) concentrations have also been reported for the same or similar population,
e.g., Chinese (Sandholzer et al. 1991; Helmhold et al. 1991; Enkhmaa et al. 2016).

G. Utermann (*)
Institute for Genetic Epidemiology, Medical University of Innsbruck, Innsbruck, Austria
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 55

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_3
56 G. Utermann

Gabonese
Khoisan
Egyptians
Black South Africans
Asian Indians North
Asian Indians South
Chinese
Inuits
Thai
Indonesians
Russians
Austrians
Japanese
Danes
Finns

-2,0 0,0 2,0 4,0

In Lp(a) concentraion

Fig. 3.1 Distribution of Lp(a) concentrations in 15 populations. Median, range, and 95% confi-
dence intervals are given (in ln mg/dL). Colors denote continental groups. (Data from Sandholzer
et al. 1991; Schmidt et al. 2006; Kraft et al. 1996; Khalifa et al. 2015; Scholz et al. 1999;
Trommsdorff et al. 1995)

It is unclear whether this reflects heterogeneity in large populations (e.g., Chinese

from Singapore vs Hongkong) or differences in assay methods.
In healthy individuals, Lp(a) concentrations remain rather constant over time
though they may fluctuate moderately. One reason is the effect of hormones which
result in changes during puberty and pregnancy (Kostner and Kostner 2004) but
environmental factors like nutrition have little effects and can be neglected here.
Twin studies resulted in heritability estimates of h2 > 90% in Europeans (Austin
et al. 1992; Boomsma et al. 1993). How could this trait be viewed as a simple domi-
nant and how is the quantitative trait genetically controlled? Both questions can be
answered today. The answer to the first question is simple. The presence of Lp(a) in
human serum or plasma was initially shown by an immunological test. The low
sensitivity of the test resulted in a Lp(a)-positive reaction only in individuals with
higher Lp(a) concentrations in plasma. The high Lp(a) concentrations were inher-
ited in families in a dominant fashion. Depending on the sensitivity of the antise-
rum, more or less individuals were tested “positive” which explains why different
frequencies of Lp(a)+ were reported by researchers at that time (Wendt 1967). The
answer to the second question will be the major topic of this review.
3 Genetics of Lipoprotein(a) 57

Structure of Lp(a) and LPA Gene

For understanding the genetics of the Lp(a) trait, a brief description of the structure
of Lp(a) is necessary. Lp(a) is a complex, assembled from one low-density lipopro-
tein (LDL) and the high molecular weight glycoprotein apolipoprotein(a) [apo(a)]
which confers the immunological specificity to the particle. Both are held together
by non-covalent binding of domains in apo(a) to apoB in LDL and by covalent bind-
ing through a single disulfide bridge between apo(a) and apoB (Schmidt et al. 2016;
Brunner et al. 1993; Koschinsky et al. 1993; McCormick et al. 1995; Callow and
Rubin 1995; Ernst et al. 1995; Gabel and Koschinsky 1998). Apo(a) is highly gly-
cosylated, does not bind lipids, and is not a true apolipoprotein. The protein has a
high homology to plasminogen from which it has evolved during primate evolution
by a series of gene duplication, deletions, domain duplications, and point mutations
(McLean et al. 1987; Tomlinson et al. 1989). The result is an odd protein consisting
of a signal sequence, ten different domains with homology to PLG kringle type IV
(KIV-1 to KIV-10), one PLG-derived kringle type V(KV), and a protease domain
with AA-substitutions rendering it inactive toward plasmin substrates.

LPA KIV-2 VNTR and Lp(a) Concentration

One of the kringles, KIV-2 occurs in multiple identical copies and varying copy
numbers in the gene (Utermann 1989; Lackner et al. 1991, 1993; Boerwinkle et al.
1992; Kraft et al. 1992) (Fig. 3.2). The genomic size of one KIV-2 unit is 5.6 kb.
This variable number of repeats (VNTR) is translated and transcribed into protein
resulting in a size polymorphism of apo(a) (Schmidt et al. 2016; Utermann 1989;
Utermann et al. 1987). The size polymorphism of LPA/apo(a) has been demon-
strated at the protein level by polyacrylamide- or agarose-gel-electrophoresis/Western
blotting of plasma using antibodies against apo(a)/Lp(a) (Fig. 3.3a) or at the DNA
level by enzymatic digestion of genomic DNA using appropriate DNAses followed
by pulsed-field-gel electrophoresis and Southern blotting with KIV-2-specific
probes (Fig. 3.3b). Using enzymes, e.g., KpnI that cut the genomic DNA only out-
side the KIV-2 repeats (Fig. 3.3), the complete block of DNA can be cut out and the
number of repeats determined from its size (Lackner et al. 1991; Boerwinkle et al.
1992; Kraft et al. 1992). A technique, which has been used only in one single pub-
lication and needs special skills in molecular cytogenetics, is fiber-FISH. This
enabled to visualize and count the number of KIV-2 repeats of single alleles under
the microscope (Erdel et al. 1999) (Fig. 3.3). The frequency of KIV-2 alleles varies
significantly between different ethnic groups (Fig. 3.4a).
The KIV-2 VNTR held the key to the understanding of the genetics of the Lp(a)-
trait. The size of apo(a) isoforms was shown to be inversely correlated with Lp(a)
concentration in plasma (Fig. 3.4b). On average, small isoforms corresponding to
low KIV-2 copy number were associated with high Lp(a) in plasma and large
58 G. Utermann

Fig. 3.2 Panel (a): Exon-Intron structure of the human LPA gene. Domains are represented in dif-
ferent colors (KIV-2 = red; KIV-1 and KIV-3 to KIV-10 = black; KV = green; protease domain
purple). Indicated are the KIV-2 VNTR, cutting sites for KpnI (Lackner et al. 1991) and rs3798200
(Clarke et al. 2009) (Adapted from Noureen et al. 2015 with permission). Panel (b): Fibre-FISH
image of LPA alleles with four and nineteen KIV-2 repeats (colored in red-yellow; count yellow
dots flanked by red). (Copy of Fig. 1b in Erdel M et al. Nat. Genet 1999; 21:357–358)

isoforms (high KIV-2 copy numbers) with low concentrations (Schmidt et al. 2016;
Utermann et al. 1987). There is, however, wide variation within alleles defined by
copy number (allele-associated Lp(a) levels) especially for low copy number alleles
(Fig. 3.5).
The analytical techniques agarose-gel-electrophoresis/Western blotting and
PFGE/Southern blotting achieve a similar resolution and with both >30 alleles of
different sizes have been demonstrated (Schmidt et al. 2016). They are, however, not
equivalent but rather complement each other. By PFGE/Southern blotting the KIV-2
genotype can be precisely determined and >95% of individuals were found to be
heterozygotes. It can, however, not be measured which concentration of Lp(a) is
associated with each allele. By contrast, agarose-gel-electrophoresis/Western blot-
ting allows assignment of Lp(a) concentration to both alleles if total Lp(a) concentra-
tion is known. Due to the extremely wide range of concentrations and the sensitivity
limits of Western blotting, apo(a) alleles associated with very low or absent Lp(a) in
plasma cannot be seen (so-called “null” alleles). Hence, in a considerable number of
samples, only one isoform is seen on the blot. Further for such samples, it cannot be
distinguished whether they are from a rare homozygote or from individuals with one
“null” allele. The frequency of “null” alleles has been estimated from 1% to 29%
depending on the population and sensitivity of Western blotting (Kraft et al. 1996;
Marcovina et al. 1996; Gaw et al. 1994) which is interesting in view of the frequency
of true “null” alleles defined today at the molecular level (Ogorelkova et al. 1999; Di
Maio et al. 2020; Morgan et al. 2020; Mukamel et al. 2021).
3 Genetics of Lipoprotein(a) 59

a b

35

27
26
23
22
20

19

15

14

K5 K5 marker
Southern blot Western blot

Fig. 3.3 Determination of the Size of Alleles of the KIV-2 VNTR by PFGE/Southern Blotting
(Panel a) and Western Blotting (Panel b). The same four samples from one family were analyzed.
The allele with 15 KIV repeats (corresponding to 6 KIV-2 repeats) in the lane denoted K5 in the
Southern blot by a black arrow is not expressed (=null allele). The corresponding isoform is miss-
ing in the Western blot (red arrow). (Modified from Noureen et al. 2015 with permission)

Only the simultaneous application of both analytical techniques allows for a

complete picture of the KIV-2 size polymorphism and its association with Lp(a)
concentrations in plasma and the identification of “null alleles” (Fig. 3.3). Few such
studies have been performed to date because they require carefully prepared intact
DNA and the application of the laborious technique of PFGE/Southern blotting for
large samples (Kraft et al. 1996; Gaw et al. 1994). These studies demonstrated sig-
nificant differences in Lp(a) levels and KIV-2 allele and isoform frequencies and the
relation of KIV-2 alleles with Lp(a) concentrations between major human ethnic
groups. Importantly differences in KIV-2 allele frequencies did not explain the large
differences in Lp(a) levels especially the much higher Lp(a) levels in Africans
which was consistently observed in all studies (Schmidt et al. 2016; Kraft et al.
1996; Gaw et al. 1994). To circumvent the obstacles of the laborious DNA typing by
PFGE-Southern blotting, researchers have used proxy values in epidemiological
studies. In some, the total number of KIV-2 repeats was determined by quantitative
60 G. Utermann

Fig. 3.4 Panel (a): Frequency distribution of binned KIV-2 VNTR alleles (numbers denote KIV
repeats including KIV-1 and KIV-3 to 10) in pooled data from three continental groups (Adapted
from Schmidt et al. 2016 with permission). Panel (b): Lp(a) concentrations by ancestry associated
with binned KIV-2 VNTR alleles. Note large differences in concentrations of Lp(a) associated with
KIV-2 alleles of the same binned size category between the major continental groups. (Data from
Schmidt et al. 2006; Kraft et al. 1996; Scholz et al. 1999)

PCR (qPCR) (Kamstrup et al. 2009). This allowed neither identification of the gen-
otype nor assignment of allele-associated Lp(a) concentrations. Second, SNPs in
the LPA gene (see below) in LD with the KIV-2 repeats were used as proxies (Clarke
et al. 2009). Both approaches allow to detect strong associations. A serious caveat
is, however, that LDs of SNPs with KIV-2 alleles may differ significantly between
ethnic groups and use as proxy for Lp(a) concentration may lead to grossly false
3 Genetics of Lipoprotein(a) 61

150
allele associated Lp(a) [mg/dl]

125

100

75

50

25

0
10 15 20 25 30 35 40 45
KIV-CNV size [number of KIV repeats]

Fig. 3.5 Illustration of the inverse correlation of KIV-2 VNTR allele size with Lp(a) concentration
(allele-associated Lp(a) concentration) in Gabonese Bantu. (Data from Schmidt et al. 2006.
Adapted from Schmidt et al. 2016 with permission)

results. This has been demonstrated for rs3798220 which is associated with low
KIV-2 copy number and high Lp(a) in Europeans (Clarke et al. 2009) and median/
high copy numbers and low Lp(a) in East and South East Asians (Khalifa et al.
2015) (Fig. 3.6).
The apo(a) VNTR explains about 40–70% of the heritability of the quantitative
Lp(a) trait depending on study design and population (Schmidt et al. 2006;
Boerwinkle et al. 1992; Kraft et al. 1992). The KIV-2 VNTR was further used in
family and sib-pair linkage studies to estimate the heritability of Lp(a) explained by
the LPA locus. This demonstrated that heritability ranged from about 70% to >95%
in populations of European descent (Boerwinkle et al. 1992; Kraft et al. 1992). LPA
is also the major locus determining Lp(a) levels in Africans but explained heritabil-
ity is lower in Africans than in populations of European descent (Schmidt et al.
2006; Mooser et al. 1997; Scholz et al. 1999; Enkhmaa et al. 2019). Hence, it
appears that both the KIV-2 VNTR and the LPA locus explain less of the genetic
variability of Lp(a) in Africans suggesting that other loci or environmental factors
have a larger impact on Lp(a) levels in Africans. Several GWAS confirmed that LPA
is the major locus determining Lp(a) levels in Europeans (Mack et al. 2017; Li et al.
2015; Lu et al. 2015; Ober et al. 2009). Minor loci detected by GWAS are the genes
coding for apo E (Mack et al. 2017) and apo H (Hoekstra et al. 2021). ApoH codes
for beta-2 glycoprotein 1 which has been shown to physically interact with apo(a) in
human plasma (Köchl et al. 1997) and which has been implicated in the pathogen-
esis of anti-phospholipid syndrome. A candidate association study implicated TLR2
as a gene modulating Lp(a) levels (Mack et al. 2017). The genomic heritability, i.e.,
the heritability explained by the measured genetic variation in a GWAS was esti-
mated to account for 49.5% of the total variability of Lp(a) levels (Mack et al. 2017).
62 G. Utermann

China
rs3798220
TT TC

55 55

50 50

45 45

40 40

35 35

KIV
KIV

30 30

25 25

20 20

15 15

10 10

50 45 40 35 30 25 20 15 10 5 0 5 10 15 20 25 30 35 40 45 50

Japan
rs3798220
TT TC

50 50

45 45

40 40

35 35
KIV
KIV

30 30

25 25

20 20

15 15

10 10

50 45 40 35 30 25 20 15 10 5 0 5 10 15 20 25 30 35 40 45 50

Frequency Frequency

Fig. 3.6 Graphic representation of the distribution of SNP rs3798220 which is associated with
short KIV-2 alleles in Europeans (Clarke et al. 2009) in Chinese and Japanese were it is associated
with long KIV-2 alleles. (Calculated from the data of Khalifa et al. 2015)
3 Genetics of Lipoprotein(a) 63

Causal Effect of the KIV-2 VNTR on Lp(a) Concentration

Genetic studies, e.g., family/sib–pair linkage and analysis by a variance compo-

nents model can provide estimates on the magnitude of the heritability explained by
a variant or locus but cannot provide evidence that the variation itself is causal. For
the KIV-2 VNTR causality was demonstrated in cell culture experiments. Transient
and stable expression in the human hepatocarcinoma cell line HepG2 of recombi-
nant apo(a) which differed only in the number of identical KIV-2 domains showed
that the amount of apo(a) secreted into the culture media correlated inversely with
the number of KIV-2 repeats in the recombinant isoform mimicking the situation in
plasma (Brunner et al. 1996; Lobentanz et al. 1998). This was due to differences in
processing of the translated proteins in the endoplasmic reticulum (ER). For large
isoforms, a predominant apo(a) precursor protein was retained in the ER and little
mature protein secreted from the cells. For small isoforms, most protein was secreted
into the cell media. The mature form was present only in low levels in the Golgi
apparatus. Temperature blocking experiments showed that no apo(a)/apoB com-
plexes can be demonstrated inside the cells (Lobentanz et al. 1998). Studies in pri-
mary baboon hepatocytes came to the same conclusion (White et al. 1993, 1994).
Steady-state labeling and pulse chase experiments demonstrated that the residence
time of an isoform in the ER is determined by its size. Together the experiments in
human and baboon cell cultures have shown that the efficiency of post-translational
processing of apo(a) is a major determinant of Lp(a) plasma levels and that the con-
tribution of the KIV-2 repeat is causal. Considering that the structure of each kringle
in mature apo(a) is stabilized by three internal disulfide bonds and one N-linked
glycosylation site, it is reasonable to assume that the more kringles are present in an
isoform the more difficult it becomes for a cell to fold correctly. White et al. (1994)
also studied processing and secretion of so-called transcript positive null alleles in
the primary baboon hepatocytes and observed retention and degradation of the pro-
tein in the cell. Together with in-vivo turnover experiments in humans which had
demonstrated that Lp(a) plasma concentrations are determined by apo(a) synthesis
(Krempler et al. 1980; Rader et al. 1993) and that apo(a) isoforms of different size
are synthesized at different rates this clearly establishes apo(a) synthesis as the criti-
cal process determining the quantitative Lp(a) polymorphism.

 imple Repeats, RSPs, and SNPs in LPA and Their Effects

S
on Lp(a) Levels

The discrepancy between the heritability explained by the VNTR and by the locus
which exists in all studied populations needed an explanation and suggested further
genetic variation at the LPA locus or nearby beyond the KIV-2 VNTR with effects
on Lp(a) levels. Several polymorphisms including SNPs, simple repeats, or restric-
tion site polymorphisms in LPA were shown to explain some of the “missing
64 G. Utermann

heritability” of Lp(a) levels. A pentanucleotide repeat (PNRP) at the 5′ at −1374 of

the LPA locus explained 15% of the variability of Lp(a) levels in Europeans inde-
pendent from the KIV-2 VNTR but none in Africans (Trommsdorff et al. 1995).
Expression of 105 kb 5′-flanking fragments containing the LPA promoter and the
PNRP from 10 different alleles with 8 or 9 PNRs in HepG2 cells found equal pro-
moter activity for all tested allelic fragments regardless whether they were from alleles
associated with high or low Lp(a) in plasma (Bopp et al. 1995). A G>A polymorphism
at −914 is also located in the tested fragments and had no effect on promoter activity.
Variation in linkage disequilibrium with the PNRP and the −914G/A polymorphism
has therefore to be implicated as responsible for the effects on Lp(a) levels.
Several non-synonymous SNPs were detected in the “unique” kringles KIV-3 to
10 by sequencing of exons and flanking regions (Ogorelkova et al. 2001; Prins et al.
1997, 1999; Crawford et al. 2008). Some had dose-dependent effects and some, e.g.,
KIV-8 T23P (also called T12P) were predicted by bioinformatic tools like PolyPhen
to have effects on Lp(a) levels (Crawford et al. 2008). They were detected in popula-
tions from Africa, Europe, and North Americans of African, Mexican, and European
descent. Most were present in only one ethnic group and none in all (Ogorelkova
et al. 2001; Dumitrescu et al. 2011).
This is clear evidence that the genetic architecture of the Lp(a) trait differs sub-
stantially between human ethnic groups, a conclusion consistent with recent
genomic analysis (Mukamel et al. 2021).
Only for few SNPs functional data were provided. One is the donor splice site
variant KIV-8+1G>A (rs41272114) which has a carrier frequency of 6% in
Europeans (Ogorelkova et al. 1999). The variant codes for a truncated form of
apo(a) which lacks the site for formation of the covalent disulfide bridge with apoB
in LDL and prevents assembly of the Lp(a) complex. The free truncated form of
apo(a) is fragmented in plasma resulting in Lp(a) deficiency in homozygous carriers
(Ogorelkova et al. 1999). The variant was observed in similar frequency in Austrians
and Finns (Ogorelkova et al. 1999; Lim et al. 2014) and was used in Mendelian
randomization approaches to demonstrate that genetically reduced Lp(a) levels
result in a reduced risk for CHD (Lim et al. 2014; Kyriakou et al. 2014).
A nonsense mutation (R21X) was detected by cloning and sequencing of KIV-2
(Parson et al. 2004) from members of a family segregating a “null” allele and its
carrier frequency determined by a sophisticated PCR protocol. Western blotting of
plasma from family members carrying the variant demonstrated a truncated iso-
form. The variant had a low carrier frequency of 2% in Austrians. Recently, Di Maio
et al. (2020) investigated the R21X variant in >10.000 individuals from three
European population samples and determined carrier frequencies from 1.6% to
2.1%. The variant was associated with KIV-2 alleles of medium copy number and
mean Lp(a) levels in carriers were −11.7 mg/dL lower than in non-carriers. The
frequency distribution differed between world populations. According to data from
3 Genetics of Lipoprotein(a) 65

the 1000 Genomes project, the variant is present in Europeans and South-East
Asians, occurs with varying frequency in South Americans, and is absent in Africans
(Di Maio et al. 2020).
Surprisingly, the R21X variant was found to be present on the same allele as the
null mutation KIV-8+1G>A (rs41272114). All alleles carrying the R21X mutation
also carried the null mutation KIV-8+1G>A but not vice versa suggesting that
KIV-8+1G>A is the older mutation and R21X occurred on an allele with the splic-
ing defect in KIV-8 generating a “double null” variant (Di Maio et al. 2020).
Only three non-synonymous variants have been demonstrated by functional
studies to have effects on plasma Lp(a) levels. The variant I4399M (rs3798220) in
the protease domain of LPA has been associated with elevated Lp(a) and CHD risk
(Shiffman et al. 2008; Luke et al. 2007) and a benefit from aspirin therapy was
reported (Chasman et al. 2009). An effect of the variant on fibrin clot architecture
and fibrinolysis has been suggested (Shiffman et al. 2008; Luke et al. 2007) but this
was not confirmed in all populations and a dependency from ethnicity was postu-
lated (Rowland et al. 2014). McCormick and coworkers (Morgan et al. 2020) care-
fully characterized two non-synonymous SNPs R990Q and R1771C, which both
result in a null phenotype. They occur in positions of LPA which are homologue to
positions in PLG where mutations result in PLG deficiency. The positions are
important for proper folding of the protein and variants poorly transit to the Golgi
and are not secreted (Morgan et al. 2020).
With the exception of the R21X variant, which had been detected by analysis of
a single family, the KIV-2 VNTR had remained a black box for mutation detection
and was not accessible by standard sequencing nor next-generation sequencing
(NGS). Depending on the number of identical KIV-2 repeats, this region can include
up to 70% of the coding sequence of the LPA gene. This region therefore may sig-
nificantly contribute to functionally relevant variation in the gene. A single study
using a laborious cloning- and a protocol for specific batch-wise PCR-amplification
of KIV-2 repeats from alleles separated by PFGE detected several previously unre-
ported variants in the KIV-2 repeats including a donor splice site mutation desig-
nated K421+1G>A which was associated with a “null allele” (Noureen et al. 2015)
(Fig. 3.3a). This variant occurred in two African and one European alleles. A puta-
tive acceptor splice site variant K422-6T>G associated with short alleles was pres-
ent with high frequency (10% in Khoi San to 40% in Egyptians) only in African
samples. Due to the small total number of only 90 alleles from six ethnic groups, the
study was limited and exact population frequencies and data on effects of variants
on Lp(a) levels in populations were not provided.
Coassin et al. (2017) used the batch amplification of KIV-2 in combination with
NGS. Starting with a discovery set of samples from individuals with discordance
between KIV-2 copy number and Lp(a) levels, they identified a novel frequent
splice site variant G4925A. The variant results in a reduction in splicing activity in
66 G. Utermann

an in vitro assay but not a “null allele.” 4925G>A has a carrier frequency of 21% in
Europeans is associated with short repeats (mainly 19–25 K-IV repeats). It reduced
Lp(a) levels by 31.8 mg/dL and coronary risk significantly (Coassin et al. 2017).
Only recently, a pipeline for ultradeep sequencing of the KIV-2 repeat domain of
LPA (Coassin et al. 2019) and methods to measure KIV2 VNTR length from whole-­
exome sequencing data has been developed and allowed for the systematic investi-
gation of variation in this genomic region. The effect of a splice site variant
4733G>A detected in this study on Lp(a) levels and CVD was studied in detail
(Schachtl-Riess et al. 2021) together with the previously reported splice site variant
4925G>A (Coassin et al. 2017). The 4733G>A allele had a high carrier frequency
of 38% and occurred on KIV-2 repeats of all sizes. Overall, it reduced Lp(a) levels
by 13.6 mg/mL. The two splice site variants cooperate in their effect on Lp(a) levels
and CHD risk reduction (Schachtl-Riess et al. 2021).
A further possible level of complexity of the genetic architecture of the Lp(a)
trait is the presence of cis-epistatic effects of variants on Lp(a) levels. A GWAS of
DNA methylation identified a novel association signal associated with elevated
Lp(a) levels in the LPA promoter (Coassin et al. 2020). The effect turned out to be
caused by a non-methylated SNP (rs10455872) which is in LD with short KIV-2
alleles (Coassin et al. 2020). A cis-epistatic effect on Lp(a) levels and coronary risk
was recently demonstrated for variants rs1800769 and rs9458001 which are jointly
associated with elevated Lp(a) levels and with risk for CHD (OR 1.37). Most of this
effect was however explained by rs140570886 (Zeng et al. 2022) known to be asso-
ciated with Lp(a) levels (Mack et al. 2017).
An epistatic effect of two SNPs and the KIV-2 VNTR was also noted in the study
by Mukamel et al. (2021). These authors estimated KIV-2 VNTR copy number from
whole genome sequencing data. Fusing these data by imputation with SNP data,
they were able to define KIV-2 haplotypes and estimate their effects on Lp(a) con-
centrations. They identified 17 protein altering variants. Six of the variants abol-
ished splice sites totally or partially and six were missense variants, all of which
greatly reduced Lp(a) levels. Most of these variants were detected in the KIV-2
VNTR. Variants resulting in increased Lp(a) concentration were found in the 5′UTR
of the LPA gene (Mukamel et al. 2021).
The work of Mukamel et al. (2021) also provided new insights into the genetic
basis underlying the differences in Lp(a) levels between human ethnic groups in
particular between sub-Saharan Africans and Europeans. Analysis by ancestry dem-
onstrated that these differences are largely explained by a significantly lower fre-
quency of Lp(a) decreasing variants and higher frequency of Lp(a) increasing SNPs
in Africans compared to Europeans (Fig. 3.7).
3 Genetics of Lipoprotein(a) 67

Fig. 3.7 Illustration

showing the association of
loss-of-function (LoFs)
SNPs and Lp(a) increasing
SNP rs1800769 in the
5′UTR of the LPA gene
with KIV-2 VNTR alleles
by ancestry. Note the
excess of LoFs and
decrease of rs1800769 in
Europeans. (From
Mukamel et al. 2021 with
permission)

Summary

The presently available knowledge of the genetic determination of Lp(a) levels in

plasma is summarized in Fig. 3.8. The two LPA alleles in an individual determine
Lp(a) concentration in a codominant manner. The concentration conferred by each
allele (allele-specific concentration) depends on the number of KIV-2 repeats (KIV-2
VNTR allele) which determine secretion rates of apo(a) from liver cells and SNPs
effecting Lp(a) concentration in the LPA allele. Most functionally characterized
SNPs with causal effects described to date are loss of function or nonsense variants
which reduce Lp(a) concentration in carriers. Most SNPs with strong effects on
Lp(a) are restricted to one or few ancestries. Cis-acting epistatic effects between
SNPs and between SNPs and alleles of the KIV-2 VNTR have also been described.
The high numbers of KIV-2 VNTR alleles and SNPs effecting Lp(a) concentra-
tion which occur in different allelic associations result in an allelic series with
numerous alleles where each allele has an individual effect on Lp(a). The frequency
distributions of the LPA alleles determine Lp(a) level distributions in populations.
The up to fourfold differences in median Lp(a) concentrations and of the distribu-
tions of Lp(a) levels between ethnic groups are not explained by the KIV-2 VNTR
68 G. Utermann

Fig. 3.8 Illustration of the genetic determination of Lp(a) concentrations in plasma by the com-
bined effects of the KIV-2 VNTR and SNPs in the LPA gene. The number of KIV-2 repeats deter-
mines apo(a) isoform size and correlates inversely with the rate of synthesis and with Lp(a)
concentration in plasma. Allele 1 in subject A codes for a long isoform and moderately low Lp(a)
and allele 3 in subject B for a short isoform and high Lp(a). This basic situation is modulated by
SNPs. As examples allele 2 in subject A carries the Lp(a) decreasing SNPs KIV-8 IVS+1G>A
(Ogorelkova et al. 1999) and KIV-2 R21X (Parson et al. 2004) which are in strong LD (Di Maio
et al. 2020) and result in a null allele and allele 4 in subject B which codes for an isoform of inter-
mediate size and carries the variant 4733G>A in KIV-2 which affects splicing and moderately
decreases Lp(a) (Schachtl-Riess et al. 2021). The total plasma Lp(a) concentration in a subject is
the sum of the two allele-associated concentrations (cis-epistatic effects are not considered). Other
loci may have minor effects by unknown mechanisms
3 Genetics of Lipoprotein(a) 69

but rather by different types, allele distributions, and LDs with KIV-2 alleles
between them. Other gene loci beyond LPA, i.e., APOE and APOH have only small
effects on Lp(a) concentrations.

Acknowledgments I thank Anita Neuner for help with the literature, Eugen Preuss for preparing
figures, and Florian Kronenberg for his support and for critically reading the manuscript.

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Chapter 4
Lp(a) Metabolism

John S. Millar and Daniel J. Rader

Introduction

Lp(a) is a lipoprotein of unknown function that is an important causal factor in ath-

erosclerotic cardiovascular disease (ASCVD) and aortic valvular stenosis. Plasma
levels of Lp(a) are highly genetically determined, and the distribution of plasma
levels of Lp(a) in the general population is skewed to the left with about one quarter
of individuals having elevated levels that put them at increased risk of cardiovascu-
lar disease (Varvel et al. 2016).
Lp(a) consists of an LDL-like lipoprotein containing apolipoprotein (apo) B100
that is bound to apo(a), a highly glycosylated protein of variable length (Schmidt

J. S. Millar
Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman
School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Institute for Diabetes Obesity and Metabolism, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA, USA
e-mail: [email protected]
D. J. Rader (*)
Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman
School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Institute for Diabetes Obesity and Metabolism, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA, USA
Department of Genetics, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA, USA
Institute for Translational Medicine and Therapeutics, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
Smilow Center for Translational Research, University of Pennsylvania,
Philadelphia, PA, USA
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 75

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_4
76 J. S. Millar and D. J. Rader

et al. 2016). The apo(a) peptide consists of a series of domains that are highly
homologous to several domains of plasminogen (McLean et al. 1987). The
N-terminal portion of apo(a) consists of a variable number of repeating domains
that are homologous to kringle IV of plasminogen. This is followed by a non-­
variable portion that consists of two additional domains that are homologous to the
kringle V and the serine protease domains of plasminogen. The number of kringle
IV repeats in the variable portion of apo(a) range from 2 to more than 40 resulting
in over 30 different isoforms ranging in size from approximately 300 to 800 kDa
(Kronenberg and Utermann 2013).
While the vast majority of apo(a) in blood is found covalently bound to apoB100
on Lp(a) particles that overlap the LDL–HDL density range (Rainwater et al. 1995),
it has been noted that a proportion of apo(a) can be found non-covalently associated
with triglyceride-rich lipoproteins (Bersot et al. 1986). It is unclear if these com-
plexes develop into mature Lp(a) particles. Such a model suggests that mature Lp(a)
particles are formed extracellularly in plasma consistent with findings from some
in vitro studies examining apo(a) secretion from hepatocytes (Koschinsky et al.
1991; White et al. 1993). However, there is a report showing evidence for apo(a)
binding to apoB-containing lipoproteins intracellularly (Bonen et al. 1997) leading
to an alternative model whereby Lp(a) can be formed intracellularly and secreted as
an intact particle.
In addition to the apoB100 and apo(a) components, proteomic analysis of highly
purified Lp(a) has identified 35 additional proteins that are associated with Lp(a)
(von Zychlinski et al. 2011). In addition to proteins known to be involved in lipid
metabolism (such as apoE and apoC-III), other proteins found associated with Lp(a)
include those involved in wound healing (coagulation [fibrinogen], complement
activation [complement C3 and C4A]), and inflammatory response (platelet activat-
ing factor acetyl hydrolase). However, biological significance of these additional
proteins associated with Lp(a) is unknown.
While the Lp(a) resembles LDL containing a covalently linked apo(a) peptide,
analysis of the lipid portion of Lp(a) has revealed that unlike LDL, Lp(a) is enriched
in oxidized phospholipids (Tsimikas and Witztum 2008), both in the lipoprotein por-
tion of the particle as well as being bound to the apo(a) peptide (Leibundgut et al.
2013). It has been shown that oxidized phospholipids are transferred from LDL to
Lp(a) in vitro and proposed that Lp(a) is the preferential carrier of oxidized phospho-
lipids in plasma (Bergmark et al. 2008). The presence of large amounts of oxidized
lipids on Lp(a) could contribute to the atherogenicity of this lipoprotein. A genome-
wide association study designed to identify risk factors for aortic stenosis identified
variants at the LPA locus encoding apo(a) as the most significantly associated
genomic locus. This has led to the hypothesis that oxidized phospholipids on Lp(a)
contribute to the progression of aortic calcification and stenosis (Yeang et al. 2016).
The apo(a) peptide is encoded by the LPA gene located on chromosome 6q27
(Scanu et al. 1991). The gene is primarily expressed in liver with minor expression
in kidney. The length of each LPA allele is variable due to there being variability in
the copy number of domains that encode kringle IV type 2 (KIV-2) (Lanktree et al.
2010). The number of KIV-2 domain repeats in LPA has been estimated to range
4 Lp(a) Metabolism 77

from 2 to >40 copies (Kronenberg and Utermann 2013). Null alleles of LPA that
encode a truncated apo(a) protein that is unable to bind covalently to apoB have
been reported (Ogorelkova et al. 1999). In addition, LPA alleles with a large number
of the KIV-2 domain repeats are unable to be secreted, presumably due to being
unstable intracellularly, and are therefore also considered null alleles (White et al.
1994). In vitro studies suggest that the number of KIV-2 domain repeats are inversely
associated with circulating Lp(a) levels due to the more efficient intracellular pro-
cessing and secretion of smaller apo(a) isoforms (White et al. 1994).
A number of genome-wide association studies have been conducted that provide
insight into the genes that regulate Lp(a) levels. Quantitively, genetic variation at the
LPA locus itself is, by far, the most important factor influencing Lp(a) levels. Clarke
et al. identified two SNPs (rs10455872 and rs3798220) at the LPA locus, which
were associated with reduced KIV-2 copy number, small Lp(a) size, and increased
Lp(a) levels (Clarke et al. 2009). Mack et al. identified 30 single nucleotide poly-
morphisms (SNPs) in the LPA gene, which either increased or decreased Lp(a) lev-
els (Mack et al. 2017). They confirmed the two SNPs identified by Clarke et al. as
well as other SNPs that were associated with the number of KIV-2 domain repeats
that are inversely associated with Lp(a) levels. There was also an association with
Lp(a) levels and the apolipoprotein E (APOE) gene, specifically with the APOE2
allele being associated with decreased Lp(a) levels. Li et al. also found an associa-
tion between a SNP in the APOE gene with Lp(a) levels (Li et al. 2015). Since apoE
is an exchangeable apolipoprotein that binds to multiple lipoprotein receptors, it is
possible that apoE on Lp(a) contributes to the clearance of Lp(a) from the circula-
tion. There is also an association between the Toll-like receptor 2 (TLR2) gene and
Lp(a) levels (Mack et al. 2017) leading to speculation that TLR2 may participate in
Lp(a) clearance from plasma. TLR2 is known to bind lipopolysaccharide but, thus
far, there have been no reports regarding the interaction between Lp(a) and TLR2.

Lp(a) Metabolism

The metabolism of Lp(a) is complex and has been the subject of intensive investiga-
tion. Here we review the major aspects of what has been reported regarding Lp(a)
metabolism.

 tudies Addressing Potential for VLDL, LDL,

S
and Lp(a) Interconversion

The first study to examine the metabolism of autologous Lp(a) in humans was con-
ducted by Krempler et al. (1978) to characterize the clearance and metabolic fate of
Lp(a) in plasma. Lp(a) was isolated using a combination of ultracentrifugation, and
78 J. S. Millar and D. J. Rader

size exclusion chromatography was reductively methylated using [14C]-formaldehyde

and injected Lp(a) into four male subjects with moderate dyslipidemia. They deter-
mined an average fractional clearance rate for Lp(a) to be 0.378 pools/day that cor-
responds to a residence time of 2.6 days which they found was slightly shorter than
the residence time of LDL reported in the literature. They also found that between
3% and 8% of labeled Lp(a) appeared in the LDL density range suggesting conver-
sion of a small amount of Lp(a) to LDL, although they did not have a sufficient
radioactive signal in the LDL fraction to state this with certainty.
Krempler et al. (1980) followed up their original studies by using a radioactive
iodine tracer which gives a much higher specific activity of the Lp(a) tracer permit-
ting a longer trace period as well as higher degree of sensitivity. This allowed them
to address the question of whether there was a conversion of Lp(a) to LDL. They
isolated Lp(a) from the study participants, radioiodinated it, and injected it either in
an autologous or homologous fashion into nine subjects with a wide-range of Lp(a)
levels. They then followed the clearance of the radiolabeled Lp(a) from plasma for
up to 21 days. It was found that the radioactivity from the labeled Lp(a) injected
stayed with the Lp(a) particle indicating that there is no interconversion of the
apoB100 moiety between Lp(a) and LDL. This group also examined the potential
of VLDL and LDL apoB100 to be the precursor of apoB100 in Lp(a) (Krempler
et al. 1979). This was done by injecting radiolabeled VLDL and examining the
appearance of radioactivity in LDL and Lp(a). They found that there was a precursor-­
product between VLDL and LDL with a large proportion of the radiolabeled VLDL
appearing in LDL. However, there was essentially none of the radiolabeled VLDL
appearing in the Lp(a) fraction. They concluded that VLDL and LDL are not precur-
sors to Lp(a) and that Lp(a) is secreted as a separate lipoprotein.
Jenner et al. (2005) studied the metabolism of Lp(a) in mildly hyperlipidemic
subjects with low, medium, and high levels of Lp(a) using endogenous labeling with
a stable isotope labeled leucine tracer under continuously fed conditions. Lp(a) was
isolated, and the leucine tracer enrichments in the apo(a) and apoB100 moieties of
Lp(a) were measured. Kinetic data were analyzed using a multicompartmental
model that allowed for independent production and clearance of apo(a) and apoB100
from Lp(a). They found the apoB100 portion of Lp(a) had a faster clearance than
the apo(a) moiety suggesting that these components are metabolized differentially.
A similar study by the same group, Diffenderfer et al. (2016), found that the clear-
ance rate of apoB100 on Lp(a) was faster than that for the apo(a) component. It was
also noted that the apoB100 moiety of Lp(a) had a tracer enrichment curve that dif-
fered from that of LDL apoB100, with tracer in apoB100 from Lp(a) appearing
more rapidly than that from LDL B100. This would suggest that LDL apoB100 is
not the direct precursor of apoB100 on Lp(a).
Demant et al. (2001) examined the kinetics of VLDL, IDL, LDL apoB100, and
Lp(a) in relatively normolipidemic subjects using endogenous labeling with a leu-
cine stable isotope tracer. The subjects were fasted for the first 10 h of the 12-day
sample collection. The Lp(a) was isolated from plasma and then reduced so that the
enrichment of the leucine tracer in the apo(a) and apoB100 moieties of Lp(a) could
be measured. Kinetic data were analyzed using a multicompartmental model that
4 Lp(a) Metabolism 79

Hepatocyte Mature Lp(a)

Intracellular assembly
A and secretion of -S-S- -S-S-
mature Lp(a)

Intracellular assembly
of disulfide-linked apo(a)
B to VLDL, secretion, and -S-S- VLDL -S-S- VLDL -S-S-
extracellular conversion
to mature Lp(a)

Secretion of free apo(a)

and extracellular VLDL/
C association with VLDL/ IDL/ -S-S-
IDL/LDL and conversion LDL
to mature Lp(a)

Fig. 4.1 The biosynthesis of mature Lp(a) may occur through diverse pathways. Possible path-
ways (not mutually exclusive) include: (a) intracellular assembly and secretion of the mature Lp(a)
particle; (b) intracellular assembly of disulfide-linked apo(a) to VLDL, secretion, and extracellular
conversion to mature Lp(a); (c) secretion of free apo(a) and extracellular association with VLDL/
IDL/LDL and conversion to mature Lp(a)

allowed for formation of Lp(a) in both the liver (pre-formed) or in plasma. They
calculated that about 50% of Lp(a) was formed in plasma from LDL with the
remainder being secreted into plasma directly from liver as a preformed Lp(a) par-
ticle. They also compared the clearance rates of the apo(a) and apoB100 compo-
nents of Lp(a) and found that they were cleared from plasma at similar rates. The
potential pathways by which Lp(a) is formed from apo(a) and apoB-containing
lipoproteins are shown in Fig. 4.1.

Studies Examining the Determinants of Lp(a) Concentration

Krempler et al. (1980) addressed the question of whether circulating Lp(a) levels
were controlled primarily by production or by clearance. They measured Lp(a) pro-
duction and clearance in subjects with a wide range of Lp(a) levels and found that
there was a significant correlation between circulating Lp(a) levels and the Lp(a)
production rate. There was no relationship between Lp(a) levels and the Lp(a) frac-
tional clearance rate. These results indicated that Lp(a) levels are primarily con-
trolled by production.
While circulating Lp(a) levels were known to be associated with apo(a) isoform
size, it had been noted that there was a considerable variation in Lp(a) levels in
subjects with the same apo(a) isoform size (Utermann et al. 1987). However, the
mechanism responsible for these differences was unknown. Rader et al. (1993)
examined the metabolism of Lp(a) in humans to determine the mechanism respon-
sible for the differences in Lp(a) levels seen in individuals with the same sized
80 J. S. Millar and D. J. Rader

apo(a) isoform. Lp(a) was isolated from donors with a single apo(a) isoform size by
sequential ultracentrifugation followed by density gradient ultracentrifugation.
Isolated Lp(a) was then radiolabeled and injected into study subjects with a range of
Lp(a) levels but having a single apo(a) isoform size. The results showed that there
was no difference in the clearance of Lp(a) from plasma in subjects with the same
apo(a) isoform size. However, there were substantial differences in the production
rate of Lp(a) among individuals with the same sized apo(a) isoform, perhaps due to
variants in the LPA gene that are independent of isoform size but which affect apo(a)
production (White et al. 1994). They concluded that Lp(a) production is the most
important determinant of the plasma level of Lp(a) independent of apo(a) iso-
form size.
It is well established that plasma Lp(a) levels are inversely correlated with apo(a)
isoform size, but the mechanism behind this correlation was unknown. Rader et al.
(1994) examined the physiology responsible for differences in Lp(a) levels based on
apo(a) isoform size. The goal of the study was to determine if there were differences
in the fractional clearance rates of Lp(a) particles containing different sized apo(a)
isoforms. Healthy normolipidemic subjects were injected with either autologous or
homologous radiolabeled Lp(a) isolated from plasma by sequential followed by
density gradient ultracentrifugation. Subjects with different apo(a) isoform pheno-
types were injected with radiolabeled Lp(a) preparations containing different sized
apo(a) isoforms. They found that Lp(a) containing different sized apo(a) isoforms
had similar clearance rates consistent with what they had observed in their previous
study (Rader et al. 1993). However, there were substantial differences in the produc-
tion rate of Lp(a) containing different sized apo(a) isoforms. The production rate of
Lp(a) containing small apo(a) isoforms was considerably higher than that for Lp(a)
containing large apo(a) isoforms. They concluded that the inverse association of
plasma Lp(a) concentrations with apo(a) isoform size is not due to differences in the
fractional clearance rates of Lp(a) containing different sized isoforms but rather the
production rate. These studies provided in vivo evidence that supported earlier
in vitro observations that smaller apo(a) isoforms are more readily secreted from
hepatocytes, likely due to more efficient intracellular processing (White et al. 1994).
The relationship between apo(a) isoform size and apo(a) production is shown in
Fig. 4.2.

The Role of the LDL Receptor in Lp(a) Clearance

The receptor(s) responsible for Lp(a) clearance from plasma are currently unknown.
Since Lp(a) contains apoB100, the ligand for the LDL receptor, the potential role of
the LDL receptor in mediating Lp(a) clearance has been of great interest. One
approach to address this question has been the use of genetics. In studies with
patients with familial hypercholesterolemia (FH), Kraft et al. found a gene dosage
effect of the LDL receptor gene (LDLR) on Lp(a) levels when controlling for LPA
alleles (Kraft et al. 2000). A study conducted using the UK Biobank found that
4 Lp(a) Metabolism 81

Small K-IV2-1 K-IV2-2

* 100 % Secreted
* 0 % Unstable

Medium K-IV2-1 K-IV2-10 * Majority Secreted

* Minority Unstable

* Minority Secreted
Large K-IV2-1 K-IV2-20 * Majority Unstable

Extra-Large K-IV2-1 K-IV2-40 * 0 % Secreted

* 100 % Unstable

Fig. 4.2 The LPA gene encodes the apo(a) protein which is of highly variable length due to varia-
tion in the number of kringle IV2 domain repeats. The strong inverse association of apo(a) protein
size with plasma Lp(a) level is due to the effect of apo(a) protein size on the rate of production, not
catabolism, of Lp(a). While apo(a) peptide translation appears to occur normally, longer intracel-
lular peptides are unstable and are targeted for pre-secretory degradation. While not shown here, in
vivo evidence also indicates that variation in plasma Lp(a) levels among individuals with the same
size isoform(s) is also due to differences in Lp(a) production, not catabolism

patients carrying variants in the LDL receptor that cause FH had higher Lp(a) levels
than unaffected subjects (Trinder et al. 2020). However, it was determined that the
population of FH patients studied was enriched in the rs10455872 SNP in LPA
which is associated with higher Lp(a) levels; when the presence of the SNP was
controlled for, it was found that Lp(a) levels were similar between patients with and
without FH. Lp(a) levels are within the normal range in patients with familial defec-
tive apoB, a disorder where there is an amino acid substitution in LDL receptor
binding domain of apoB (Innerarity et al. 1987). It is also of interest to note that
genome-wide association studies have identified variants at the LDLR locus as being
strongly associated with LDL cholesterol levels (Kathiresan et al. 2008); variants at
the LDLR locus have not been found to be associated with Lp(a) levels (Clarke et al.
2009). Thus, the genetic data do not strongly support a role for the LDL receptor in
directly mediating the catabolism of Lp(a).
There have also been experimental efforts to determine the role of the LDL
receptor in Lp(a) clearance. Lp(a) can bind to the LDL receptor in vitro although the
affinity of Lp(a) for the LDL receptor has been characterized as “weak” (Reblin
et al. 1997). Knight et al. (1991) studied the role of the LDL receptor in the in vivo
clearance of Lp(a) from plasma in hyperlipidemic patients with and without hetero-
zygous FH. They radiolabeled autologous Lp(a) and LDL isolated by density gradi-
ent ultracentrifugation and examined the clearance of each from plasma. They
found that there was no difference in the clearance rate of Lp(a) from plasma
82 J. S. Millar and D. J. Rader

between individuals with and without heterozygous FH despite there being signifi-
cant differences in the clearance of autologous LDL. They also conducted in vitro
studies that examined the ability of Lp(a) to compete with LDL binding to the LDL
receptor. They found that Lp(a) was unable to compete with LDL for binding to the
LDL receptor. They did find that after injection of radiolabeled Lp(a) that there was
appearance of approximately 25% of the Lp(a) tracer in the LDL fraction which
they interpreted as resulting from loss of apo(a) from the Lp(a) particle. They
hypothesized that the resulting LDL could be cleared by LDL receptors. These
authors also found no differences in the clearance of Lp(a) containing apo(a) iso-
forms of different sizes and that Lp(a) levels were correlated with the Lp(a) produc-
tion rate.
Rader et al. studied the catabolism of Lp(a) in five patients with homozygous FH
who had little to no LDL receptor function (Rader et al. 1995). Purified radioiodin-
ated Lp(a) and LDL were simultaneously injected into homozygous FH patients and
control subjects, and the catabolism was followed over time. While the catabolism
of LDL was markedly delayed as expected, the catabolism of Lp(a) was not slower
in homozygous FH patients than in control subjects. This study provided powerful
evidence that the absence of a functional LDL receptor does not result in delayed
catabolism of Lp(a) and suggested that the LDL receptor is not a physiologically
important route of Lp(a) catabolism in humans.
In mice with marked overexpression of the LDL receptor, there was an increased
uptake of Lp(a) levels resulting in decreased levels in plasma (Hofmann et al. 1990;
Romagnuolo et al. 2017). Cain et al. addressed the question using mice deficient in
the LDL receptor (Cain et al. 2005). When the catabolism of radiolabeled Lp(a) was
studied in mice deficient in the LDL receptor compared to wild-type mice, there
was no observed difference in Lp(a) turnover. This was consistent with the human
studies and provided additional evidence that the LDL receptor is not a major con-
tributor to Lp(a) clearance.
As another type of evidence, statins reduce LDL-C levels by causing the upregu-
lation of the LDLR in hepatocytes and increased clearance of LDL and its precur-
sors. However, as reviewed below, statins do not decrease Lp(a) levels and, if
anything, cause Lp(a) levels to increase slightly. This also argues against the LDL
receptor as being an important mediator of Lp(a) clearance. Overall, the data do not
support an important role for the LDL receptor in mediating clearance of Lp(a)
from blood.

The Role of Other Receptors in Lp(a) Clearance

The role of other receptors in Lp(a) clearance has been investigated and current
information suggests that Lp(a) can be cleared from plasma through multiple recep-
tors (Fig. 4.3). Available data suggest that apo(a) is the ligand responsible for
receptor-­mediated binding of Lp(a). For example, while excess LDL was shown to
have minimal impact on Lp(a) clearance in mice, excess apo(a) significantly slowed
4 Lp(a) Metabolism 83

LDLR

LRP-1
Apo(a) ligand

LRP-8

VLDLR

SR-BI

Megalin/
Glycoprotein 330

ApoB ligand Plasminogen

Receptor

TRL-2

Other
Receptors?

Fig. 4.3 Lp(a) clearance is not a major determinant of plasma Lp(a) levels and mostly occurs by
the liver. The mechanisms of hepatic Lp(a) clearance remain unknown and likely involve multiple
pathways. This figure lists many of the cell surface receptors that have been proposed as receptors
for Lp(a)

Lp(a) clearance from plasma (Cain et al. 2005). The scavenger receptor B-I, best
known for its role in regulating HDL cholesterol uptake, has been reported to bind
Lp(a) (Yang et al. 2013). The megalin/glycoprotein 330 receptor, a member of the
LDL receptor family, has been shown to bind and take up Lp(a) into cells in vitro
(Niemeier et al. 1999). The LDL receptor-related protein-1 has been shown to bind
Lp(a) weakly in vitro (Reblin et al. 1997) but has been reported to have no effect on
Lp(a) clearance in vivo in animal models (Romagnuolo et al. 2017). The plasmino-
gen receptor PlgRKT has been shown to mediate Lp(a) uptake by HepG2 cells
(Sharma et al. 2017). It is interesting to note that following uptake by PlgRKT, the
apo(a) component of Lp(a) was trafficked to recycling endosomes and subsequently
re-secreted into the cellular media. This would help explain the results of some
in vivo human studies that had results showing a slower clearance of apo(a) as com-
pared to apoB100 on Lp(a) which could be explained by apo(a) recycling (Jenner
et al. 2005; Diffenderfer et al. 2016). Other receptors that have been shown to have
no effect on Lp(a) clearance include the VLDL receptor, LDL receptor-related pro-
tein-­8 (Romagnuolo et al. 2017), the asialoglycoprotein receptor (Cain et al. 2005),
and sortilin (Gemin et al. 2018). Variants in APOE have been identified as being
84 J. S. Millar and D. J. Rader

determinants of Lp(a) levels (Clarke et al. 2009; Mack et al. 2017; Li et al. 2015).
In addition, apoE in plasma has been shown to have a modest impact on Lp(a) clear-
ance (Li et al. 2015; Cain et al. 2005). It is of interest to note a case report of a
patient with apoE deficiency who had an Lp(a) level approximately three-fold
higher than the upper limit of normal (Mak et al. 2014) which might be expected if
apoE is involved in clearance of Lp(a) or its precursors.

Drug Effects on Lp(a) Metabolism

Statins

Statins inhibit cholesterol synthesis and result in the compensatory upregulation of

the LDL receptor, leading to increased LDL catabolism and lower LDL-C levels.
Statins were originally thought to have no effect on Lp(a) levels (Kostner et al.
1989) but a meta-analysis of several large statin trials concluded that statins
increase Lp(a) levels by ~8–20% (Tsimikas et al. 2020a). The mechanism behind
the increase in Lp(a) in response to statin treatment was examined in in vitro stud-
ies and was shown to be due to effects on LPA gene transcription (Tsimikas
et al. 2020a).

PCSK9 Inhibitors

PCSK9 inhibitors block the effect of PCSK9 in mediating LDL receptor degrada-
tion, thus leading to increased LDL receptor protein and increased clearance of
LDL. They reduce LDL-C levels by about 60%. Treatment with PCSK9 inhibitors
has been shown to modestly lower Lp(a) levels by ~20% (Ajufo and Rader 2016).
This has been shown to be due to enhanced clearance of Lp(a) from plasma (Watts
et al. 2020; Reyes-Soffer et al. 2017). While increased LDL receptor numbers
could play a role, it is also possible that PCSK9 influences other factors that affect
Lp(a) clearance. Patients heterozygous for PCSK9 gain-of-function mutations have
been reported to have Lp(a) levels that were two-fold higher than control subjects
(Tada et al. 2016), while heterozygous carriers of PCSK9 loss-of-function muta-
tions have been reported to have Lp(a) levels that are 22% lower than those found
in control subjects (Mefford et al. 2019). PCSK9 expression has been shown to
slow the uptake of Lp(a) by cultured cells expressing the LDL receptor but had no
effect on Lp(a) uptake by cells deficient in the LDL receptor (Romagnuolo et al.
2017). The modest effect of PCSK9 inhibition on reducing Lp(a) is in direct con-
trast to the effect of statins on increasing Lp(a) and this mystery has yet to be
resolved.
4 Lp(a) Metabolism 85

Niacin

Niacin can modestly reduce Lp(a) levels. The degree of Lp(a) lowering by niacin
has been shown to be greater in subjects with smaller apo(a) isoform size that have
elevated Lp(a) levels (Artemeva et al. 2015). Ooi and colleagues examined the
mechanism by with niacin reduces Lp(a) have shown that niacin reduced apo(a) and
Lp(a) associated apoB100 production with no change in the FCR of these compo-
nents in subjects treated with niacin (1–2 g/day) with background rosuvastatin treat-
ment (Ooi et al. 2015). Croyal et al. also found that niacin (2 g/day) reduced the
production rate of apo(a) in hypertriglyceridemic subjects while the also reducing
the clearance rate (FCR) to a lesser degree (Croyal et al. 2015). The mechanism by
which niacin can influence Lp(a) metabolism is not clear, although variants in the
niacin receptor (hydroxyl-carboxylic receptor 2; HCAR2) have been shown to influ-
ence the Lp(a) response to niacin (Tuteja et al. 2017) suggesting that the mechanism
may lie downstream of HCAR2 receptor signaling. HCAR2 expression is relatively
high in white adipose tissue (Jadeja et al. 2019), and activation of HCAR2 on adipo-
cytes leads to inhibition of triglyceride lipolysis within adipose resulting in reduced
fatty acid delivery to liver. However, HCAR2 is also expressed to a lesser degree in
liver (Jadeja et al. 2019) and therefore it is possible that activation of HCAR2 on
hepatocytes by niacin had direct effects that lead to reduced LPA transcription.

Inhibitors of apoB Synthesis/Secretion

The antisense oligonucleotide (ASO) to APOB, mipomersen, has been reported to

reduce Lp(a) levels by approximately 20–28% (Ajufo and Rader 2016; Santos et al.
2015). This drug, which targets the synthesis and production of cellular apoB100 in
liver, might be expected to influence Lp(a) production. However, a kinetic study
conducted in patients treated with mipomersen found that the primary effect of the
drug on Lp(a) metabolism was to increase the clearance of Lp(a) from plasma,
although the reason for this is not entirely clear (Nandakumar et al. 2018). Targeting
the hepatic production of apoB100 through use of the microsomal triglyceride
transfer protein (MTP) inhibitor lomitapide also reduced Lp(a) levels, although to a
lesser extent than mipomersen (Rader and Kastelein 2014).

Other Drugs on Lp(a) Metabolism

Another drug class that has been shown to lower circulating Lp(a) levels are inhibitors
of cholesteryl ester transfer protein (CETP) which reduce Lp(a) levels from 24% to
40% (Thomas et al. 2017; Nicholls et al. 2016; Hovingh et al. 2015). This was shown
to be due to a decrease in the production of Lp(a) (Thomas et al. 2017), although the
mechanism behind this decrease is not entirely clear and requires more study.
86 J. S. Millar and D. J. Rader

While Volanesorsen, an ASO that targets APOC3, showed no apparent effects of

lowering apoC-III on Lp(a) levels (Tardif et al. 2022), it may provide some insights
on Lp(a) biology. ApoC-III is an exchangeable apolipoprotein that has been shown
to inhibit both lipoprotein lipase mediated hydrolysis of triglyceride as well as
inhibit receptor-mediated uptake of VLDL remnant lipoproteins. Treatment with
Volanesorsen was reported to reduce the apoC-III content of Lp(a) (Yang et al.
2016). The fact that Lp(a) levels remained unchanged following treatment would
suggest that apoC-III does not influence the clearance of Lp(a) from plasma.

Targeting Apo(a) Production to Lower Lp(a)

A direct approach to lowering Lp(a) levels is to target the synthesis and production
of apo(a). The antisense oligonucleotide (ASO) pelacarsen targets the apo(a) mRNA
to promote degradation and thus reduces the synthesis of apo(a) protein. Treatment
of humans with pelacarsen resulted in a dose-dependent decrease in Lp(a) levels of
up to 80% (Tsimikas et al. 2020b). Pelacarsen is now being studied in a phase 3
cardiovascular outcome trial to assess the impact of this degree of Lp(a) reduction
on cardiovascular events.

Conclusion

Major advances have been made in our understanding of the factors that regulate
Lp(a) metabolism since its discovery 60 years ago. The synthesis of apo(a) is largely
under genetic control and ultimately determines the production rate and concentra-
tion of Lp(a) in plasma. A single receptor that regulates Lp(a) clearance has not
been identified to date; the LDL receptor does not appear to play a major physiolog-
ical role. Other receptors and apoE have been studied and may have modest effects
on Lp(a) clearance from plasma. Thus, it is possible that multiple receptors are
responsible for the catabolism of Lp(a). Future studies that examine Lp(a) metabo-
lism, particularly when conducted in response to treatment with novel drugs that
influence Lp(a) levels, should lead to further advances in our understanding of Lp(a)
biology and the factors that regulate its metabolism.

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Chapter 5
Contemporary Aspects of Lp(a)
Metabolism and Therapies Based
on Tracer Kinetic Studies in Humans

Dick C Chan, Jing Pang, and Gerald F Watts

Bullet Points
• Lipoprotein(a) [Lp(a)] is an inherited and causal risk factor for atherosclerotic
cardiovascular disease (ASCVD) and aortic valve stenosis.
• Use of stable isotope tracers and compartmental modelling has provided deeper
understanding of the physiology and pathophysiology of Lp(a) metabolism
in humans.
• Plasma Lp(a) concentration is predominantly determined by the rate of produc-
tion of Lp(a) particles, irrespective of apo(a) isoform size and background ther-
apy with statins.
• Niacin and cholesteryl ester transfer protein inhibitors lower plasma Lp(a) con-
centration by increasing the clearance or catabolism of apo(a).
• ApoB antisense oligonucleotides lower plasma Lp(a) concentration by decreas-
ing hepatic production.
• Proprotein convertase subtilisin kexin type 9 inhibitors can lower plasma Lp(a)
concentration by a dual mode of action involving both increased clearance and
decreased production of apo(a),
• Further studies should investigate nucleic acid-based inhibitors for apo(a),
angiopoietin-like 3 and apoC-III inhibitors on the metabolism of Lp(a) and other
lipoproteins.

D. C Chan · J. Pang
Medical School, University of Western Australia, Perth, WA, Australia
e-mail: [email protected]; [email protected]
G. F Watts (*)
Medical School, University of Western Australia, Perth, WA, Australia
Lipid Disorders Clinic, Department of Cardiology and Internal Medicine, Royal Perth
Hospital, Perth, WA, Australia
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 91

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_5
92 D. C Chan et al.

Introduction

Lipoprotein(a) [Lp(a)] is one of the most important genetically determined risk fac-
tors for atherosclerotic cardiovascular disease (ASCVD) and aortic valve stenosis
(Nordestgaard and Langsted 2016; Saleheen et al. 2017; Tsimikas et al. 2018; Cegla
et al. 2009; Arsenault and Kamstrup 2022; Reyes-Soffer et al. 2022). Large clinical
trials have consistently shown that patients with elevated Lp(a), even when treated
with statins, are at an increased risk of ASCVD (Khera et al. 2014; Nicholls et al.
2010). The metabolic pathways governing the metabolism of Lp(a) have been
extensively studied in cellular and animal model systems (McCormick and
Schneider 2019; Boffa and Koschinsky 2022; Chemello et al. 2022a). However,
only scare information is available on the metabolism of this lipoprotein in humans.
Use of stable isotopically labelled tracers and compartmental modelling has greatly
enhanced our understanding of Lp(a) metabolism (Chan et al. 2004; Barrett et al.
2006). In the present chapter, we review use of these techniques and its contribution
to key knowledge of the physiology and pathophysiology of Lp(a) metabolism in
humans. We focus on subjects with elevated Lp(a) and the mode of action of lipid-­
regulating agents.

Structure and Genetics of Lipoprotein(a) in Brief

Lp(a) is composed of one molecule of a highly polymorphic apolipoprotein(a)

[apo(a)] particle covalently linked to one molecule of a low-density lipoprotein
(LDL)-like particle containing apoB-100 by a single disulphide bond (Schmidt
et al. 2016). Apo(a) is composed of 10 types of kringle 4 (KIV) domains related
to plasminogen kringle 4, followed by a KV domain and an inactive protease-like
domain. KIV2 exists in variable numbers (from 3 to >30), which gives rise to
Lp(a) isoform size heterogeneity (Marcovina et al. 1996; Kronenberg and
Utermann 2013).
The gene encoding apo(a), LPA, is located on the long arm of chromosome 6 at
6q2.6–2.7, adjacent to the human plasminogen gene. While the control of LPA
expression is at present not well understood, certain factors, such as estrogen, hepa-
tocyte nuclear factor 4α, interleukin-6 and tumour necrosis factor alpha, influence
the expression of LPA (Kronenberg and Utermann 2013). Plasma Lp(a) concentra-
tion is largely controlled by the LPA gene locus. Up to 90% of its variation in Lp(a)
concentration is attributable to genetic factors (Lamon-Fava et al. 1991; Austin et al.
1992; Boerwinkle et al. 1992), with approximately 30–70% explained by a variable
number of KIV2 repeats in the LPA gene (Kronenberg and Utermann 2013). The
unexplained genetic variance in Lp(a) concentration can be contributed by other
genetic factors outside KIV2 repeat variation. Several single nucleotide polymor-
phisms (SNPs) in the LPA gene, such as rs3798220 (CT/CC) and rs10455872 (AG/
5 Contemporary Aspects of Lp(a) Metabolism and Therapies Based on Tracer Kinetic… 93

GG), are strongly associated with an elevated Lp(a) concentration (Clarke et al.
2009). Genome-wide association studies (GWAS) have also identified many com-
mon genetic variants of small effect which can aggregately influence Lp(a) concen-
tration (Coassin and Kronenberg 2022). Accordingly, a polygenic risk score for
predicting Lp(a) concentration has recently been reported, explaining approxi-
mately 60–70% of the variance in Lp(a) levels in the EPIC-Norfolk and UK Biobank
cohorts (Wu et al. 2021). APOE gene is one of the most important genetic factors
modulating Lp(a) concentrations (Li et al. 2015; Zekavat et al. 2018; Mack et al.
2017; Chemello et al. 2022b); the ε2 allele is associated with reduced Lp(a) concen-
trations, whereas the ε4 allele is associated with increased Lp(a) concentrations
compared with the ε3 allele (Moriarty et al. 2017). Several physiological states,
such as kidney, thyroid and liver disease, and ancestry, also contribute to the vari-
ability in Lp(a) concentration (Enkhmaa and Berglund 2022).

Stable Isotopic Tracer Methodologies

Plasma Lp(a) concentration in the circulation is determined by a balance between

the rates of production and catabolism of Lp(a) particles. Stable isotope tracer stud-
ies using endogenous labelling of apolipoproteins with amino acid precursor mole-
cules (isotopomers) and mathematical modelling have been employed to study
Lp(a) kinetics (Barrett et al. 2006). This approach has provided better understanding
of Lp(a) homeostasis and of the pathogenesis of elevated Lp(a), as well as the
kinetic effects of statin and newer lipid-regulating agents, such as proprotein con-
vertase subtilisin/kexin type 9 (PCSK9) inhibitors.
Briefly, stable isotopically labelled amino acids (such as D3-leucine) are admin-
istered intravenously as a bolus or primed infusion with serial blood sampling over
several days to assess the turnover of apo(a). Enrichment data (tracer/tracee ratio)
are generated by gas- or liquid-chromatography mass spectrometry (GCMS or
LCMS, respectively) analysis after separation of apo(a) from plasma (Chan et al.
2004). A novel LCMS method for quantification of apo(a) enrichment has recently
been established by employing a synthetic peptide (LFLEPTQADIALLK) that tar-
gets the proteolytic domain of apo(a) following a standardized sample trypsin diges-
tion procedure (Croyal et al. 2015, 2018). This method is more sensitive and less
labour-intensive than the traditional approach based on immunoprecipitation and
Western blotting. Enrichment data are then analysed via multicompartmental mod-
elling, from which the fractional turnover rate of apo(a) in the circulation is derived.
Fractional catabolic (or clearance) rate (FCR) refers to the fraction of trace lost from
a defined plasma pool per day. From these primary kinetic data, together with the
corresponding plasma pool size of apo(a), absolute transport rates in the circulation
are calculated. We have detailed these methods elsewhere (Chan et al. 2004; Barrett
et al. 2006). Figure 5.1 shows a multicompartmental model for the metabolism of
Lp(a)-apo(a) and Lp(a)-apoB-100.
94 D. C Chan et al.

Fig. 5.1 Compartmental model to describe Lp(a)-apo(a) and Lp(a)-apoB-100 tracer kinetics.
Plasma leucine kinetics are described by a four-compartment model, which is connected to intra-
hepatic delay compartments (compartments 5 and 6) that accounts for the synthesis and secretion
of Lp(a)-apo(a) and Lp(a)-apoB-100, with compartments 7 and 8 describing the plasma kinetics of
Lp(a)-apo(a) and Lp(a)-apoB-100, respectively

Metabolism of Lipoprotein(a)

Synthesis, Assembly and Secretion

Apo(a) is exclusively synthesized by the liver (Schmidt et al. 2016). However,

details of the assembly process have not been fully elucidated. The site of Lp(a)
assembly may occur in hepatocytes, extracellularly in the space of Disse, or in the
circulation (plasma space) (Hoover-Plow and Huang 2013; Youssef et al. 2022).
Several pathways for Lp(a) assembly and secretion have been suggested. Apo(a)
and apoB are assembled intrahepaticaly, forming an Lp(a) particle which is subse-
quently secreted into plasma. The Lp(a) particle may also be assembled in the cir-
culation (e.g. on the hepatocyte surface) from its constituent protein; these are then
independently secreted from the liver into plasma. There is also uncertainty con-
cerning whether the kinetics in plasma of the two protein components of Lp(a) are
coupled, and specifically whether apo(a) is recycled or cleared with apoB-100 as an
Lp(a) holoparticle. Using stable isotope tracers and compartmental modelling, we
demonstrated that in individuals with a wide range of plasma Lp(a) concentrations,
Lp(a)-apoB-100 and Lp(a)-apo(a) have identical isotopic enrichment curves in
plasma and similar FCRs (Watts et al. 2018). This finding was confirmed in another
kinetic study of statin-treated patients (Ma et al. 2019a) (Fig. 5.2). Hence, these
kinetic data generally support that newly synthesized Lp(a)-apoB-100 and Lp(a)-
apo(a) are secreted as a holoparticle with tightly coupled apo(a) and apoB100 resi-
dence times in the circulation. However, it remains unclear whether the covalent
binding of apo(a) to apoB-100 takes place in the liver or in the circulation.
Another outstanding issue concerning the assembly of Lp(a) particles is the
extent to which the binding of apo(a) to triglyceride-rich lipoproteins (TRLs) con-
tributes to the formation of Lp(a) particles in the circulation (Nassir et al. 1998;
5 Contemporary Aspects of Lp(a) Metabolism and Therapies Based on Tracer Kinetic… 95

Fig. 5.2 Lp(a)-apo(a) and Lp(a)-apoB tracer-tracee ratio (%) in 20 statin-treated subjects includ-
ing association of Lp(a)-apo(a) and Lp(a)-apoB-100 fractional catabolic rates (FCR)

Ramos-Cáceres et al. 2022). Earlier radiolabelled kinetic studies suggest that apo(a)
is unlikely to be adducted to a triglyceride-rich very low-density lipoprotein (VLDL)
as a precursor of Lp(a) in the LDL/HDL density range (Krempler et al. 1980). In
contrast, apo(a) can be associated with TRLs, such as chylomicrons and chylomi-
cron remnants, after oral ingestion of a fatty meal (Bersot et al. 1986). This is sup-
ported by experimental evidence that the apoB-100-apo(a) complex within Lp(a)
particles have a high affinity for TRL particles (Marcoux et al. 1997). A significant
proportion of Lp(a) particles can bind non-covalently to TRLs in the hypertriglyc-
eridemic state. Consistent with this, we and others have demonstrated a redistribu-
tion of a significant portion of apo(a) protein from Lp(a) to the TRL fraction,
particularly in the postprandial state (Cohn et al. 1991; Ying et al. 2022). In a recent
study of FH, we found that the impaired postprandial TRL-apo(a) response to a fat
load was partially corrected by fish oil supplementation (Ying et al. 2022). The
reduction in postprandial TRL-apo(a) with fish oil supplementation in response to
the fat load was significantly associated with the corresponding reduction in post-
prandial triglyceride response. Hence, interaction with TRLs may influence the
metabolism or catabolism of Lp(a). The underlying kinetic mechanism remains to
be investigated employing stable isotopes and compartmental modelling.

Clearance and Catabolism

It is well established that the liver is the main site of Lp(a) clearance and, to a much
lesser extent, the kidney and the arterial wall (McCormick and Schneider 2019).
The mechanisms of Lp(a) clearance from the circulation and the catalytic pathways
96 D. C Chan et al.

involved remain uncertain, however. Several cellular receptors have been proposed
to mediate the clearance of Lp(a) from the liver. These include LDL receptor and
other members of the LDL-receptor family such as VLDL receptor, LDL receptor-­
related protein 1 (LRP1), megalin/gp330, scavenger receptor class B type 1 (SR-BI)
and plasminogen receptor (McCormick and Schneider 2019).
The role of the LDL receptor in Lp(a) clearance remains controversial. Several
experimental studies have demonstrated that LDL receptor can facilitate Lp(a) bind-
ing and uptake (Havekes et al. 1981; Reblin et al. 1997; Romagnuolo et al. 2015),
and in mice overexpressing LDL receptor the clearance of Lp(a) particles is signifi-
cantly increased (Hofmann et al. 1990). Very few kinetic studies have specifically
investigated the metabolism of Lp(a) in patients with LDL receptor defects, such as
familial hypercholesterolemia (FH). Using exogenous radiolabelled tracers, Rader
et al. found that the clearance of Lp(a) did not differ significantly among homozy-
gous FH patients, heterozygous FH parents and non-FH control subjects (Rader
et al. 1995). Using endogenous stable isotope tracers, Croyal et al. reported that the
FCRs of Lp(a)-apo(a) were similar in patients with PCSK9 gain-of-function muta-
tions and control subjects (Croyal et al. 2020). Hence, defects in LDL receptor func-
tion do not appear to result in delayed clearance of Lp(a). In a study of healthy
normolipidemic men, there was no significant association between the FCRs of
apo(a) and LDL-apoB-100 (Chan et al. 2019). These kinetic findings suggest under
physiological conditions that the LDL receptors may not play a major role in Lp(a)
clearance. As discussed later, LDL receptor could play a role in Lp(a) clearance in
a supraphysiological condition in which the activity of LDL receptors is markedly
upregulated, such as in patients who are treated with a combination of statins and
PCSK9 inhibitors (Watts et al. 2018).

 inetic Determinants of Plasma Lipoprotein(a)

K
Concentrations

Production Rate vs. Fractional Catabolic Rate

In a kinetic study of healthy normolipidemic men with a wide range of plasma Lp(a)
concentration, Lp(a) particle concentration was significantly and positively associ-
ated with apo(a) production rate (PR) and inversely with apo(a) FCR (Chan et al.
2019). In another study of statin-treated subjects, plasma concentration of apo(a)
was significantly and positively associated with apo(a) PR in patients with both
normal and elevated Lp(a) concentrations (Ma et al. 2019b). However, there was no
significant association between plasma apo(a) concentration and FCR in either of
the groups. Hence, these observations reinforce the notion that plasma concentra-
tions of Lp(a) are primarily determined by the rates of production and not clearance,
irrespective of background statin use. Accordingly, plasma concentration and PR of
apo(a) were significantly higher in statin-treated patients with elevated Lp(a)
5 Contemporary Aspects of Lp(a) Metabolism and Therapies Based on Tracer Kinetic… 97

b c

Fig. 5.3 Kinetic parameters of apo(a) in statin-treated subjects with (a) normal (<75 nmol/L), (b)
high (75–145 nmol/L) and (c) very high apo(a) concentrations (>145 nmol/L). Data presented as
mean ± SEM. Apo apolipoprotein, FCR fraction catabolic rate, PR production rate. *P < 0.001
compared with normal apo(a) group. †P < 0.001 compared with normal and moderate-high apo(a)
group using ANOVA

compared with those with normal Lp(a) (Fig. 5.3a, b). The FCR of apo(a) did not
differ significantly between the groups (Fig. 5.3c). This finding suggests that ele-
vated plasma Lp(a) concentration is a consequence of increased hepatic production
of Lp(a) particles in patients with elevated Lp(a). In a constant-feeding study of
healthy individuals, patients with high Lp(a) had increased apo(a) PR and reduced
FCR compared with those without elevated Lp(a) concentration (Jenner et al. 2005).
Plasma concentrations of Lp(a) were correlated significantly with both apo(a) PR
and negatively with apo(a) FCR. These findings implicate a role of Lp(a) catabolism
in determining Lp(a) plasma concentrations in the fed state.

Apo(a) Isoform Size

As discussed earlier, the plasma concentrations of Lp(a) is dependent on genetic

variations in the number of KIV2 repeats (Marcovina and Koschinsky 1999;
Kronenberg and Utermann 2013). Experimental data have suggested that the size of
the apo(a) transcripts is inversely associated with hepatic LPA mRNA concentration
(Wade et al. 1991; White et al. 1994) and by implication apo(a) production. Smaller
apo(a) isoforms have been shown to have a shorter retention time in the endoplas-
mic reticulum and probably lesser intracellular apo(a) proteasome degradation,
resulting in a more efficiently secretion from hepatocytes (White et al. 1994;
Brunner et al. 1996; Lobentanz et al. 1998). On the other hand, Lp(a) with apo(a)
98 D. C Chan et al.

isoforms of different sizes may have different binding affinities for the LDL recep-
tor or other receptors (März et al. 1993). Lp(a) particles with larger isoform size
have been shown to be more effectively removed via LDL receptor independent
pathways.
In a study of healthy normolipidemic subjects, subjects with smaller apo(a) iso-
form sizes (≤22 KIV repeats) had significantly higher apo(a) concentration and PR,
and lower apo(a) FCR than those with larger sizes (Chan et al. 2019). Plasma apo(a)
concentration was significantly associated with apo(a) PR, but not with FCR in
subjects with smaller apo(a) isoform size. In contrast, both apo(a) PR and FCR
were significantly associated with plasma apo(a) concentrations in subjects with
larger isoforms. Similar observations were observed in patients who were on statin
(Ma et al. 2019c). Taken together, these findings again suggest that the plasma
Lp(a) concentration is predominantly determined by the rate of production of Lp(a)
particles, irrespective of apo(a) isoform size and background statin use. Lp(a) par-
ticle catabolism may only play a modest role in determining Lp(a) concentration in
subjects with larger apo(a) isoform size. These observations also support the clini-
cal use of agents that target the hepatic production and secretion of Lp(a)
(Tsimikas 2017).
As discussed earlier, APOE genotype can influence the concentration of Lp(a)
(Moriarty et al. 2017; Croyal et al. 2020; Chemello et al. 2022a). However, the
effect of APOE genotype, particularly the presence of apoE2 and apoE4, on Lp(a)
concentrations is known to be affected by the size of apo(a) (Klausen et al. 1996;
Blanchard et al. 2021). Accordingly, the effect of apoE genotype on the metabolism
of Lp(a) in subjects with large and small apo(a) isoform merits further
investigation.

 echanisms of Action of Lipid-Regulating Agents

M
on Lipoprotein(a) Metabolism

A major challenge in managing patients with elevated Lp(a) is a lack of effective

and specific treatment for lowering Lp(a) concentrations (Tsimikas 2017;
Tsimikas et al. 2018; Reyes-Soffer et al. 2022; Schwartz and Ballantyne 2022).
Diet and lifestyle interventions, such as weight loss or physical activity, do not
seem to influence Lp(a) concentrations. Lipoprotein apheresis is the only FDA
approved treatment for elevated Lp(a). Currently, there are no approved pharma-
cologic therapies that specifically target Lp(a) concentrations (Cegla et al. 2009;
Tsimikas 2017). The kinetic effect of several established and newer therapies,
including statins, niacin, PCSK9 inhibitors, cholesteryl ester transfer protein
(CETP) inhibitors and apoB antisense oligonucleotides (ASO), on Lp(a) metabo-
lism are discussed below and in Table 5.1, with specific reference to the mecha-
nisms of action.
5 Contemporary Aspects of Lp(a) Metabolism and Therapies Based on Tracer Kinetic… 99

Table 5.1 Mechanisms of several pharmacological interventions in regulating lipoprotein(a)

metabolism
Principal results
Author (year) Subjects Agents Concentration FCR PR
Watts et al. Healthy Atorvastatin ⟺ ⟺ ⟺
(2018) normolipidaemic men
Ooi et al. Statin-treated men with Extended-release niacin ↓↓ ⟺ ↓↓
(2015) type 2 diabetes
Croyal et al. Non-diabetic, obese Extended-release niacin ↓↓ ↓↓ ↓↓↓
(2015) men with
hypertriglyceridaemia
Reyes-Soffer Healthy Alirocumab ↓ ↑ ⟺
et al. (2017) normolipidaemic men
and women
Watts et al. Statin-treated men and Alirocumab ↓↓ ↑↑ ⟺
(2020) women with high Lp(a)
Ying et al. Statin-treated men and Alirocumab ↓↓↓ ↑↑ ↓↓
(2022) women with very high
Lp(a)
Watts et al. Healthy Evolocumab ↓↓ ⟺ ↓↓
(2018) normolipidaemic men
Watts et al. Healthy Evolocumab + Atorvastatin ↓↓ ↑↑ ⟺
(2018) normolipidaemic men
Thomas et al. Mildly Anacetrapib (CETP ↓↓ ⟺ ↓↓
(2017) hypercholesterolaemic inhibitor)
men and women
Nandakumar Healthy Mipomersen (ApoB ASO) ↓↓ ↑↑ ⟺
et al. (2018) normolipidaemic men
and women
apo apolipoprotein, ASO antisense oligonucleotide, CETP cholesteryl ester transfer protein, FCR
fractional catabolic rate, PR production rate
↑↑: mild increase; ↓↓: mild decrease; ↓↓↓: marked decrease; ⟺: no change

Statins

The value of statins in lowering LDL-cholesterol is well recognized. Statins com-

petitively inhibit HMG CoA reductase, thereby decreasing cholesterol biosynthesis,
reciprocally upregulating hepatic LDL receptors and enhancing clearance of LDL
and other apoB-100-containing particles, including TRLs (Ginsberg 2006). Given
the structural similarities between LDL and Lp(a), one would speculate that statins
could lower Lp(a) concentration by increasing the clearance of Lp(a). However, the
effect of statins on Lp(a) levels is conflicting: some studies show a neutral role
(Wang et al. 2021; de Boer et al. 2022), while others report a decrease (Takagi and
Umemoto 2012) or increase of plasma Lp(a) levels (Tsimikas et al. 2020). It appears
that the influence of stains on Lp(a) level may depend on the type of statins; atorv-
astatin and rosuvastatin increase Lp(a) levels whereas pitavastatin has no impact or
100 D. C Chan et al.

may tend to decrease plasma Lp(a) concentrations (Tsimikas et al. 2020). The
statin-induced increase in Lp(a) level is supported by experimental evidence in
HepG2 cells showing a higher LPA mRNA level in response to atorvastatin
(Tsimikas et al. 2020). In a study of healthy normolipidemic subjects, atorvastatin
(80 mg daily) did not significantly alter the FCR or PR of apo(a) (Watts et al. 2017).
This finding does not support a role of LDL receptor in the regulation of apo(a) FCR
under physiological condition. However, it remains unclear whether statin has a
potential impact on Lp(a) metabolism in subjects with high Lp(a) concentration.
There is also evidence showing that statins increase Lp(a) levels exclusively in
patients with a small size apo(a) defined as ≤22 KIV repeats (Yahya et al. 2019).
The precise mechanisms of action of this effect on Lp(a) metabolism remain to be
investigated.

Niacin

Niacin is one of few agents that can significantly lower plasma Lp(a) concentra-
tions. Experimental data suggest that niacin decreases the expression of LPA
mRNA (Chennamsetty et al. 2012). This is consistent with a kinetic study showing
that niacin lowered Lp(a) concentration by decreasing the production of apo(a) in
non-­diabetic, obese and hypertriglyceridemic men (Croyal et al. 2015). The lower-
ing of the PR of apo(a) by niacin was confirmed in another postprandial kinetic
study in statin-treated patients with type 2 diabetes (Ooi et al. 2015). In this study,
extended-­release niacin (1–2 g/day) significantly decreased plasma Lp(a) concen-
tration and the production rates of apo(a), with greater treatment effect in indi-
viduals with elevated Lp(a) concentration. This is consistent with another study
showing that extended-release niacin was more effective in lowering Lp(a) level in
subjects with small apo(a) isoform than those with large isoform (Artemeva
et al. 2015).

PCSK9 Inhibitors

Inhibition of PCSK9 in combination with statins and/or ezetimibe provides a highly

effective approach for lowering LDL-cholesterol concentrations in patients with
hypercholesterolemia (Duprez et al. 2020; Ying et al. 2021; Ferri et al. 2020).
Monoclonal antibodies (mAbs) targeting PCSK9, such as evolocumab and ali-
rocumab, have been consistently known to significantly lower plasma LDL-­
cholesterol and the incidence of ASCVD outcomes (Sabatine et al. 2017; Schwartz
et al. 2018; Deedwania et al. 2021). PCSK9mAbs can similarly lower plasma Lp(a)
concentration. The effectiveness of PCSK9 mAbs in reducing ASCVD events is
also found to be most pronounced in patients with high Lp(a) and that the reduction
5 Contemporary Aspects of Lp(a) Metabolism and Therapies Based on Tracer Kinetic… 101

in Lp(a) could also partly mediate the cardiovascular benefit of PCSK9 mAbs
(Bittner et al. 2020; Schwartz et al. 2021).
In a kinetic study of healthy normolipidemic men, evolocumab monotherapy
significantly decreased plasma Lp(a) concentration chiefly by reducing the PR of
apo(a) with no effect on the corresponding FCR (Watts et al. 2018). This effect is
consistent with a tracer study conducted in non-human primates in which ali-
rocumab decreased the PR of apo(a) (Croyal et al. 2018). The mechanistic effect of
evolocumab may involve reduced hepatic production of Lp(a) by decreasing the
assembly of Lp(a) particles through the reduction of apo(a) binding with LDL on
the surface of hepatocytes (Lambert et al. 2017). This speculation is supported by
in vitro studies showing that PCSK9 induces Lp(a) intracellular assembly and secre-
tion, whereas PCSK9 mAbs reduce the extracellular release of Lp(a) (Villard
et al. 2016).
However, as combination therapy with high-dose atorvastatin, evolocumab
reduced the plasma concentration of Lp(a) chiefly by a significant increase in the
FCR of apo(a) (Watts et al. 2018). The PR of Lp(a) was not significantly altered
with the combination. Similar results were also found in another kinetic study in
healthy individuals receiving alirocumab treatment (Reyes-Soffer et al. 2017).
However, the increase in apo(a) FCR in the latter study was not statistically signifi-
cant, probably owing to greater variability in study subject characteristics (e.g.
mixed race and gender). The mechanistic effect of evolocumab in combination with
atorvastatin may involve supraphysiological upregulation of the activity of LDL
receptors and decreased competition of Lp(a) with very low concentrations of LDLs
for clearance by these receptors. This mechanism suggests that the LDL receptor
likely plays a significant role in mediating Lp(a) clearance only when its expression
is markedly upregulated and when LDL plasma levels are substantially lowered,
allowing decreased competition between LDL and Lp(a) for receptor-mediated
uptake in the liver.
The mechanism of action of PCSK9 inhibition has recently been studied in
statin-treated patients with high Lp(a). Using stable isotopes, PCSK9 inhibition
with alirocumab-lowered plasma Lp(a) concentration by increasing apo(a) FCR in
patients with elevated Lp(a) receiving maximally tolerated statin therapy (Watts
et al. 2020). However, in patients with very high-Lp(a) concentration, alirocumab
significantly lowered plasma Lp(a) concentration by a dual mode of action involv-
ing both increased clearance and decreased production of apo(a) (Ying et al. 2022).
Taken together, the mechanistic action of PCSK9 mAbs on the PR and FCR of
apo(a) appears to be dependent on background statin use and Lp(a) concentration at
baseline.
Unlike evolocumab or alirocumab, small interfering RNA on PCSK9 mRNA
transcript (e.g. Inclisiran) is a new approach to targeting PCSK9 intracellularly
(German and Shapiro 2020; Smith and White 2022). This novel agent was shown to
inhibit hepatic synthesis of the PCSK9 protein, and lower apoB-100-containing
lipoproteins, including Lp(a) (Ray et al. 2020; Raal et al. 2020). This implies that
the effect of PCSK9 inhibition on Lp(a) is irrespective of mode of inhibition of
102 D. C Chan et al.

PCSK9 (intracellular or extracellular. However, the mechanisms of action of incli-

siran on Lp(a) metabolism remain to be elucidated.

CETP Inhibitors

CETP plays an important role in lipoprotein metabolism, primarily by its ability to

facilitate transfer of esterified cholesterol from high-density lipoproteins (HDL) to
apoB-containing lipoproteins (Tall 1993). Treatment with CETP inhibitors, either
alone or in combination with statin, can lower Lp(a) concentrations up to 30%
(Schmidt et al. 2021). In a kinetic study of patients with hypercholesterolaemia
(Thomas et al. 2017), CETP inhibition with anacetrapib lowered Lp(a) concentra-
tion by reducing the PR of apo(a) with no effect on the corresponding FCR. However,
there is no clear explanation for the reduction in apo(a) PR with anacetrapib which
merits further investigation. Despite these metabolic changes, CETP inhibitors did
overall not have clinically meaningful effects in large clinical trials. While several
CETP inhibitors, including torcetrapid, evacetrapid, dalcetrapid and anacetrapid,
have fallen after disappointing clinical trial outcomes (Berberich et al. 2017;
Schwartz et al. 2012; Lincoff et al. 2017; Schmidt et al. 2021), two clinical trials
with a newer CETP inhibitor obicetrapib (TA-8995; 10 mg) has been shown to
increase HDL-­cholesterol by 160%, and reduce LDL-cholesterol, apoB and Lp(a)
levels approximately by 50–60%, 30–50% and 25–50%, respectively, in patients
treated with atorvastatin or rosuvastatin (Hovingh et al. 2015; Ray 2022). The
mechanisms of action of this agent on Lp(a) and other lipoproteins merit
investigation.

ApoB Antisense Oligonucleotides

Mipomersen is an antisense oligonucleotide (ASO) directed to liver mRNA of apoB

that inhibits apoB synthesis (Parham and Goldberg 2019). Accordingly, mipomersen
has been shown to significantly lower plasma concentrations of apoB-containing
lipoproteins including LDL and Lp(a). In a kinetic study of healthy volunteers, treat-
ment with mipomersen caused a significant decrease of plasma Lp(a) levels that was
associated with a significant increase in the FCR of Lp(a), with no effect on corre-
sponding apo(a) PR (Nandakumar et al. 2018). These results were unexpected
because inhibition of apoB synthesis with mipomersen would reduce the availability
of apoB100 substrate for the assembly of hepatic apoB with apo(a) to form an Lp(a).
It is noteworthy that mipomersen also did not reduce VLDL apoB secretion in the
same subjects studied (Reyes-Soffer et al. 2016). These observations appear to sup-
port the presence of spare apoB pool in the liver that would be utilized for the assem-
bly of Lp(a) in order to maintain hepatic homeostasis for apoB. However, this
speculation requires further investigation. In the same study, the increase in Lp(a)
FCR observed was similar to the 30% increase in the FCR of LDL apoB100,
5 Contemporary Aspects of Lp(a) Metabolism and Therapies Based on Tracer Kinetic… 103

supporting a role for the LDL receptors or related receptors in the clearance of Lp(a)
particles.

Other Therapies

Lipoprotein apheresis effectively lowers Lp(a) and LDL levels by approximately

60–70%. Kinetic studies, using stable isotope methods, have shown inconsistent
findings when comparing Lp(a) and LDL FCRs in patients undergoing apheresis
(Parhofer et al. 1999; Armstrong et al. 1989; Kroon et al. 2000). In studying the
rebound of Lp(a) and LDL particle concentration following lipoprotein apheresis
(Ma et al. 2019c), the clearance of Lp(a) is significantly slower than that of LDL-­
apoB in patients with elevated Lp(a) and ASCVD. These findings suggest that the
clearance pathways for Lp(a) differ from those of LDL-apoB.
Selective thyroid hormone receptor (THR) agonists (such as Resmetirom) can
effectively lower plasma Lp(a) concentrations (Hovingh et al. 2022). Activation of
THR has been shown to increase LDL receptor expression, resulting in reduced
circulating LDL particles (Erion et al. 2007). Whether the lowering effect of Lp(a)
is mediated by upregulating the activity of LDL receptor remains unclear and merits
further investigation.
Administration of aspirin has been shown to lower Lp(a) levels in patients with
high-Lp(a) concentrations irrespectively of apo(a) isoform size (Akaike et al. 2002).
This observation is supported by experimental data in HepG2 cells that aspirin
reduced Lp(a) production in H2G cell via the reduction of apo(a) gene transcrip-
tional activity with suppression of apo(a) mRNA expression (Kagawa et al. 1999).
However, no kinetic studies have yet specified investigated the effect of aspirin on
Lp(a) metabolism in humans.
Lomitapide is a small molecule that inhibits lipid transfer by direct binding to
microsomal triglyceride transfer protein (MTP) in the liver and intestine (Berberich
and Hegele 2017). By inhibiting MTP in hepatocytes and enterocyte, lomitapide
reduces VLDL assembly and secretion, and lowers plasma levels of all apoB-1con-
taining lipoproteins, including VLDL, LDL and Lp(a) independent of LDL receptor
(Cuchel and Rader 2013; Harada-Shiba et al. 2017). Accordingly, lomitapide is spe-
cifically approved for lowering LDL-cholesterol in homozygous FH (Berberich
et al. 2017). Kinetic studies showed a marked reduction in the production of LDL-
apoB-100 (Cuchel et al. 2007). Whether lomitapide reduces Lp(a) concentrations
by decreasing apo(a) PR remains to be investigated.

Conclusions and Future Perspectives

Lp(a) is associated with an increased risk of ASCVD, even in patients on intensive

lipid-lowering therapy. However, elevated Lp(a) remains an under-recognized,
under-treated and under-researched condition with an extremely high risk of
104 D. C Chan et al.

ASCVD. This atherogenic disorder has received little attention due to a significant
knowledge gap in understanding Lp(a) pathophysiology. Stable isotope tracer meth-
ods provide unique information of the dynamics of Lp(a) particles in the circulation.
The interferences from these studies are important for understanding the metabo-
lism of Lp(a) and for developing new therapies. Knowledge of the mode of action
of therapeutic interventions is also important for informing shared-decision making
and improving adherences to therapies. Future research is still needed to understand
whole body metabolism of Lp(a), including the stability of the covalent bonding
between apo(a) and apoB-100, the potential recycling of apo(a) in the circulation,
the possible formation of Lp(a) complexes with TRLs, and the relative roles of
hepatic and renal receptors in the clearance of Lp(a) particles. The precise modes of
action of CETP inhibitors, apoB ASO and THR agonists on the metabolism of
Lp(a), particularly in patients with high Lp(a), also merit further clarification.
While several therapeutic interventions can lower plasma Lp(a) concentrations
(Korneva et al. 2021), it is uncertain that it would mitigate the adverse effects of
elevated Lp(a) on ASCVD. Nevertheless, some of the cardiovascular benefit of
PCSK9 mAbs in clinical outcome trials are known to be mediated by the lowering
of Lp(a) independently of the concurrent reduction in LDL cholesterol. More
aggressive treatment strategies involve use of multiple lipid-regulating agents to
treat elevated Lp(a). This approach harnesses the complementary mechanisms of
action of the different agents. Possible combinations include PCSK9 inhibitor with
niacin, CETP inhibitor or THR agonist. Inhibiting hepatic apo(a) synthesis with
nucleic acid therapeutics has emerged as a potent approach to reduce plasma Lp(a)
levels up to 90% which is not affected by LPA gene variants and isoform size
(Karwatowska-Prokopczuk et al. 2021). The effect of this novel and specific agent
on the metabolism of Lp(a) and other apoB-containing lipoproteins warrants inves-
tigation. Further studies are required to characterize the mode of action of newer
lipid-regulating agents on the metabolism TRLs and Lp(a). These include inhibitors
of angiopoietin-like protein 3 (ANGPTL3) and apoC-III (antibodies and/or nucleic
acid-based ASO therapies) (Ward et al. 2022).
Conflicts of Interest GFW has received honoraria for lectures and advisory boards
or research grants from Amgen, Arrowhead, AstraZeneca, Esperion, Kowa, Novartis,
Regeneron and Sanofi. DCC has nothing to declare.

Acknowledgement JP was supported by the National Health and Medical Research Council
(HMRC) Investigator Grant.
5 Contemporary Aspects of Lp(a) Metabolism and Therapies Based on Tracer Kinetic… 105

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Chapter 6
Role of Proprotein Convertase Subtilisin
Kexin Type 9 in Lipoprotein(a)
Metabolism

Antonio Gallo, Kévin Chemello, Romuald Techer, Ali Jaafar,

and Gilles Lambert

PCSK9, a Key Player in Lipoprotein Metabolism

PCSK9 is a serine protease mainly expressed in the liver (Seidah et al. 2014). It is
synthesized as a precursor that undergoes autocatalytic intramolecular processing to
form a mature enzyme. In 2003, Abifadel and colleagues identified missense muta-
tions in the gene encoding PCSK9 causative of familial hypercholesterolemia (FH)
(Abifadel et al. 2003). These mutations were later shown to be gain-of-function muta-
tions. In 2005, a causative association was established between loss-of-­function muta-
tions in PCSK9 and low plasma concentrations of LDL-C, which was accompanied by
an astonishing reduction in global coronary heart disease risk (Cohen et al. 2006).
Strikingly, studies of individual homozygotes for PCSK9 loss-­of-­function mutations
demonstrate that a complete or near-complete absence of PCSK9 resulting in very low
levels of LDL-C is perfectly compatible with normal human health (Zhao et al. 2006).
Evidence for a direct role for PCSK9 in LDL-C metabolism came initially from
a series of studies showing that overexpression of PCSK9 promotes the accumula-
tion of LDL-C in the plasma of control mice but not in that of LDLR-deficient ani-
mals (Maxwell and Breslow 2004; Lagace et al. 2006). Mechanistically, the LDLR
promotes the cellular uptake of LDL by endocytosis. In the absence of PCSK9, the
acidic environment of the endosome promotes the dissociation of the receptor from
the LDL particle, and the LDLR is recycled to the cell surface, while LDL is routed
to the lysosome for degradation (Nassoury et al. 2007; Qian et al. 2007). PCSK9,
which is secreted from hepatocytes, binds to the LDLR at the surface of cells.
Following endocytosis, in the presence of PCSK9, the LDLR fails to change

A. Gallo · K. Chemello · R. Techer · A. Jaafar · G. Lambert (*)

Laboratoire Inserm UMR 1188 DéTROI, Université de La Réunion,
Saint-Pierre (Ile de La Réunion), France
e-mail: [email protected]; [email protected]; [email protected];
[email protected]

© The Author(s), under exclusive license to Springer Nature 113

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_6
114 A. Gallo et al.

conformation in the endosome, which precludes normal recycling of the receptor to

the plasma membrane (Fig. 6.1). The LDLR thus traffics to the lysosome and is
degraded along with the LDL particle (McNutt et al. 2007; Li et al. 2007). The
increase in LDL-C plasma levels induced by PCSK9 directly stems from the ability
of PCSK9 to enhance LDLR lysosomal degradation (Lambert et al. 2012).

a b

c d

Fig. 6.1 Lipoprotein(a) plasma levels are reduced in the absence or upon pharmacological inhibi-
tion of PCSK9—Lp(a) plasma concentrations are primarily determined genetically at the LPA
locus that chiefly governs the level of apo(a) synthesis and the subsequent rate of Lp(a) particle
assembly and secretion by hepatocytes (Panel a). PCSK9 targets the LDLR for lysosomal degrada-
tion, reducing its abundance at the surface of hepatocytes, and thus lowering the cellular uptake of
LDL (Panel b). In the absence of PCSK9, the intracellular assembly of Lp(a) may be reduced
(Panel c), whereas the abundance of LDLR at the cell surface is maximal, allowing optimal LDL
uptake by endocytosis without significantly altering Lp(a) clearance (Panel d)
6 Role of Proprotein Convertase Subtilisin Kexin Type 9 in Lipoprotein(a) Metabolism 115

PCSK9, an Ideal LDL Cholesterol-Lowering Drug Target

Given the mode of action of PCSK9 as a circulating inhibitor of the LDLR, as well
as the healthy profile of individuals with reduced or absent PCSK9 function, PCSK9
rapidly gained status of a very clean drug target to lower LDL-C in humans. Several
drug development strategies have been tested to pharmacologically inhibit PCSK9,
the most advanced in terms of clinical development being two fully human mono-
clonal antibodies (mAbs). Large phase III programs with these mAbs have been
reported: the FOURIER program with evolocumab (Sabatine et al. 2015) and the
ODYSSEY program with alirocumab (Robinson et al. 2015) (Table 6.1). In a

Table 6.1 Lipoprotein (a) reductions by PCSK9 inhibition in Phase III trials
CV outcome
Patients’ number Pharmacological Lp(a) % % reduction
and agent and median (hazard
Trial characteristics follow-up duration reduction ratios) References
FOURIER 25,096 subjects Evolocumab, −26.9 Lp(a) upper O’Donoghue
with established 48 weeks median: et al. (2019)
CVD −23%
(HR, 0.77
[0.67–0.88])
Lp(a) lower
median −7%
(HR 0.93
[0.80–1.08])
4465 subjects Evolocumab, −25.5 – Sabatine et al.
from 5 phase 2 12 weeks (2015)
and 7 phase 3
trials
1359 subjects Evolocumab, −27.0 – Stein et al.
without CVD 12 weeks (2014)
with/without
background LLT
ODYSSEY 18,924 subjects Alirocumab, −23.6 −15% Bittner et al.
with CVD 146 weeks (HR 0.85 (2020)
[0.78–0.93])
3499 subjects Alirocumab, −25.6 (vs. −12% Ray et al.
(placebo-­ 84.6 weeks placebo) (HR 0.88 (2019)
controlled) −21.4 (vs. [0.78–0.98])
1484 ezetimibe)
(ezetimibe-­
controlled)
ORION 482 subjects with Inclisiran, −17.2 – Raal et al.
heterozygous FH 77 weeks (2020)
1561 subjects Inclisiran, −25.6 – Ray et al.
with established 77 weeks −18.6 (2020)
CVD
1617 subjects
with CVD
equivalent
116 A. Gallo et al.

nutshell, these trials have unequivocally shown that PCSK9 inhibition robustly and
safely lowers LDL-C levels regardless of background lipid-lowering therapy and
reduces cardiovascular disease (CVD). In addition to mAbs that sequester PCSK9 in
circulation and that have been approved by regulating bodies and are now prescribed
to patients in many countries, other approaches to PCSK9 inhibition are currently in
late-stage clinical development. The small interfering RNA (siRNA) inclisiran that
targets PCSK9 hepatic production was approved by the US Food and Drug
Administration in December 2021, following reports of positive results of the
ORION phase III clinical trial with this drug (Ray et al. 2020).
Statins are the most prescribed lipid-lowering drugs. They enhance LDLR gene
and protein expression and thereby markedly reduce LDL-C. As mentioned above,
PCSK9 inhibitors lower the intracellular degradation of the LDLR, thus increasing
the abundance of LDLR at the cell surface and thus reducing LDL-C levels. Whereas
statins are neutral or can even elevate the plasma concentrations of Lp(a), PCSK9
inhibitors not only reduce LDL-C (by 50–60% on average) but also concomitantly
lower Lp(a) plasma levels by 20–30% (Gaudet et al. 2014; Raal et al. 2016; Lambert
et al. 2017). This intriguing observation has led to a flurry of research aimed at
investigating the role of PCSK9 in Lp(a) metabolism.

 ipoprotein(a) Plasma Levels Are Chiefly Governed

L
by Production

As mentioned in detail in the previous chapters of this book, Lp(a) is independently

and significantly associated with CVD and calcified aortic valve stenosis
(Kronenberg and Utermann 2013). Pathophysiological, epidemiological, and
genetic studies demonstrate that elevated plasma Lp(a) levels increase the rate of
cardiovascular events at any achieved LDL-C level. The major structural difference
between Lp(a) and LDL is that Lp(a) has a second large protein, apolipoprotein(a)
[apo(a)], bound to the apolipoprotein B100 (apoB100) moiety of an LDL-sized par-
ticle by a single disulfide bond (Boffa and Koschinsky 2019). A key feature of Lp(a)
is the strong genetic determination of its concentrations, which range over 1000-­
fold distribution in the population, with the LPA gene that encodes apo(a), account-
ing for more than 90% of this variation. LPA alleles contain a variable number of
exon pairs encoding plasminogen-like kringle IV (KIV) domains, giving rise to an
important size polymorphism of apo(a) isoforms, ranging approximately from 300
to 800 kDa, which corresponds to the presence of one to more than 40 KIV type 2
(KIV2) domains (Koschinsky et al. 1990). Apo(a) is exclusively synthesized in
hepatocytes and undergoes post-translational modifications in the ER (Kraft et al.
1989). The residence time of apo(a) isoforms in the ER is proportional to their num-
ber of KIV2 domains (Brunner et al. 1996; White et al. 1999). As a result, large
apo(a) isoforms are more susceptible to proteasomal degradation, which explains to
some extent why circulating plasma Lp(a) levels are on average higher in carriers of
short apo(a) isoforms.
6 Role of Proprotein Convertase Subtilisin Kexin Type 9 in Lipoprotein(a) Metabolism 117

Several investigators have tried to elucidate the molecular, cellular, and meta-
bolic pathways governing the production of Lp(a), the contribution of Lp(a) to
lipid transport in the plasma, and the catabolic fate of Lp(a). The metabolism of
this enigmatic lipoprotein nevertheless remains incompletely understood. Unlike
LDL, Lp(a) is not the direct product of very low-density lipoprotein (VLDL)
metabolism (Krempler et al. 1979). Lp(a) assembly appears to be a two-step pro-
cess: (1) apo(a) and apoB associate noncovalently through the interactions between
weak lysine binding sites located in apo(a) KIV7 and KIV8 domains and lysine
residues located on apoB100; (2) a disulfide bond is formed between the only
“free” cysteine of apo(a) located in its KIV9 domain and a cysteine located in the
C-terminal domain of apoB100 (Gabel and Koschinsky 1998; Youssef et al. 2022).
Most in vitro and in vivo kinetic studies suggest that the noncovalent association
between apoB100 and apo(a) takes place within hepatocytes, whereas their cova-
lent attachment which is enzyme-catalyzed occurs extracellularly (Youssef et al.
2022). As referred to above, the plasma concentrations of Lp(a) have a strong heri-
table component related to genetic variations in the number of KIV2 repeats, with
epidemiological studies consistently demonstrating an inverse association between
the size of apo(a) and plasma Lp(a) concentrations (Kronenberg and Utermann
2013). Apo(a) production rate and apo(a) isoform size, but not apo(a) fractional
catabolic rate (FCR), were shown to be significant predictors of plasma Lp(a) con-
centrations (Watts et al. 2018). In addition, patients with elevated Lp(a) concentra-
tions have smaller apo(a) isoform sizes and higher apo(a) production rates than
patients with normal Lp(a) concentration, the FCR of Lp(a)-apo(a) not differing
significantly between these groups of patients. These observations clearly support
that plasma concentrations of Lp(a) are primarily determined by the rates of pro-
duction and not clearance.

The PCSK9-LDLR-Lp(a) Axis

The mechanisms of Lp(a) clearance from the blood and the catalytic pathways
involved remain highly uncertain. It is well established that the liver is the major site
of Lp(a) clearance followed to a much lower extent by the kidney (Kronenberg
2014). Multiple pathways for Lp(a) clearance have been proposed (McCormick and
Schneider 2019). For instance, the scavenger receptor BI (SR-BI) has been shown
to promote the selective uptake of Lp(a) cholesterol esters in cells and in SR-BI
transgenic mice (Yang et al. 2013). Given the strong homology between apo(a) and
plasminogen, the role of plasminogen receptors in mediating Lp(a) clearance has
been evaluated (Sharma et al. 2017). One of them, the plasminogen receptor pre-
senting a C-terminal lysine (PLGRKT), was shown to mediate the cellular uptake of
Lp(a) by human hepatoma cells and primary human fibroblasts. This study also
showed that the LDL component of Lp(a) undergoes lysosomal degradation,
whereas apo(a) traffics through recycling endosomes and is re-secreted. Several
members of the LDLR family of receptors have also been proposed to mediate
118 A. Gallo et al.

whole Lp(a) particle cellular uptake. Thus, the VLDL receptor binds apo(a) and
allows the internalization and subsequent degradation of Lp(a) in macrophages
(Argraves et al. 1997). The LDLR-related protein 1 (LPR1) and megalin/gp330
(known as LRP2) also play a role in Lp(a) binding (Reblin et al. 1997; Niemeier
et al. 1999) cellular uptake and degradation in vitro. LRP8 (formerly known as the
apoB,E receptor) is also able to bind Lp(a) at the plasma membrane (Steyrer and
Kostner 1990), but it remains to be seen whether this promotes Lp(a) particle cel-
lular uptake and degradation. The cellular uptake of Lp(a) was, however, recently
shown to be unaffected in HepG2 hepatoma cells overexpressing either the VLDLR,
LRP1, or LRP8 (Romagnuolo et al. 2017). Given the important structural similari-
ties between LDL and Lp(a), and the Lp(a) lowering effects of PCSK9 inhibitors,
the LDLR has received the most attention as a candidate receptor for Lp(a) over the
past decades.
The initial reports showed that Lp(a) can bind to the LDLR with a lower affinity
than LDL (Snyder et al. 1992). It has also been proposed that Lp(a) could associate
with LDL and undergo LDLR-mediated clearance by a hitchhiking process (Hofer
et al. 1997). In HepG2 and primary human fibroblasts, PCSK9 was shown to reduce
the binding and the cellular uptake of Lp(a) via the LDLR (Raal et al. 2016;
Romagnuolo et al. 2015). These results were confirmed in HuH7 hepatoma cells
and in primary mouse hepatocytes (Romagnuolo et al. 2017). In contrast, other
studies found no significant role for the LDLR in mediating Lp(a) cellular uptake in
primary human hepatocytes, but also in fibroblasts and HepG2 cells (Sharma et al.
2017; Villard et al. 2016). Neither did they find any significant difference in Lp(a)
cellular uptake in primary lymphocytes isolated from normolipemic individuals and
patients with homozygous FH who totally lack LDLR function (Chemello et al.
2020). Noteworthy, LDLR expression in human primary lymphocytes positively
and significantly correlates with individuals’ LDL-C, but not with Lp(a) plasma
concentrations (Thedrez et al. 2018).
Studies conducted in mice, rabbits, or nonhuman primates have also yielded
opposite conclusions regarding the role of the LDLR and the effects of PCSK9
inhibitors on Lp(a) catabolism. Thus, compared with wild-type animals, mice
overexpressing the LDLR display accelerated Lp(a) plasma clearance (Hofmann
et al. 1990), but LDLR knockout mice have similar Lp(a) clearance than wild-type
animals (Cain et al. 2005). Furthermore, the catabolism of Lp(a) in rabbits is
slower than that of LDL, suggesting that Lp(a) uptake is not fully dependent on the
LDLR and may be mediated by other mechanisms (Liu et al. 1993). In addition,
alirocumab did not alter the catabolic rate of Lp(a) but was found to enhance Lp(a)
production in nonhuman primates (Croyal et al. 2018). Likewise, alirocumab had
no effect on the hepatic capture of Lp(a) in liver-humanized mice (Chemello
et al. 2020).
Studies of FH and non-FH siblings with identical apo(a) isoforms have clearly
demonstrated that Lp(a) is approximately twice higher in FH patients than in nonaf-
fected family members (Lingenhel et al. 1998). Homozygous FH subjects with two
nonfunctional LDLR alleles also display twofold higher Lp(a) levels than their
6 Role of Proprotein Convertase Subtilisin Kexin Type 9 in Lipoprotein(a) Metabolism 119

heterozygote relatives (Kraft et al. 2000). Likewise, familial ligand-defective

apoB100 patients (FDB) have higher Lp(a) than non-FDB family members (van der
Hoek et al. 1997), and PCSK9 gain-of-function mutation carriers also similarly dis-
play higher Lp(a) than non-FH controls (Tada et al. 2016). The fact that Lp(a) is
higher in FH patients has recently been challenged by two independent studies. In
46,200 individuals from the Copenhagen General Population Study in whom Lp(a)
was measured, mean Lp(a) concentrations were 23 mg/dL in individuals unlikely to
have FH, 32 mg/dL in subjects with possible FH, and 35 mg/dL in those with prob-
able or definite FH, based on the Dutch Lipid Clinic Network diagnostic criteria
(Langsted et al. 2016). However, after adjusting LDL-C levels for Lp(a) cholesterol
to more accurately assess the FH status, those values were similar at 24, 22, and
21 mg/dL, respectively. Similar observations were made in the British Columbia FH
cohort (Trinder et al. 2020). In that cohort, elevated Lp(a) levels in FH were linked to
a twofold higher prevalence of a specific single nucleotide polymorphism
(rs10455872) on the LPA gene associated with an average of 64 mg/dL increase in
circulating Lp(a) levels (Trinder et al. 2020) compared with reference populations,
suggesting an ascertainment bias in the association between FH and elevated Lp(a).
The authors further investigated this possibility using whole-exome sequencing by
identifying 221 “true” FH patients (i.e., with pathogenic mutations on the LDLR,
APOB, or PCSK9 genes) out of 37,486 individuals in the UK Biobank, without prior
knowledge of their clinical history. As anticipated, these 221 individuals had signifi-
cantly higher LDL-C plasma levels than the 37,265 non-FH individuals, but both
groups displayed similar circulating Lp(a) concentrations (Trinder et al. 2020). These
novel insights cast a doubt on the consensus that Lp(a) is genuinely elevated in FH.
Lipoprotein kinetic studies conducted in humans also provide discrepant conclu-
sions regarding the role of the LDLR and the effects of PCSK9 inhibitors on Lp(a)
catabolism. For instance, Lp(a) FCR was similar in control individuals and in homo-
zygous FH patients totally lacking LDLR function (Rader et al. 1995). In contrast,
the PCSK9 inhibitor alirocumab was shown to increase (albeit not significantly) the
FCR of Lp(a) in one study (Reyes-Soffer et al. 2017), whereas the PCSK9 inhibitor
evolocumab in monotherapy did not alter Lp(a) FCR (Watts et al. 2018). In patients
with high Lp(a) treated with a high potency statin, alirocumab lowered Lp(a) con-
centrations by increasing the FCR of Lp(a), but also decreased the particle secretion
in those with higher pretreatment Lp(a) concentrations (Watts et al. 2020). There is
a large discordance in response to PCSK9 inhibitors in terms of LDL-C and Lp(a)
lowering (Edmiston et al. 2017; Shapiro et al. 2019; Blanchard et al. 2022), given
that at best only weak correlations between these parameters have been reported.
For instance, the FOURIER trial shows a correlation coefficient of 0.37 between
changes in Lp(a) and changes in LDL-C levels after 48 weeks of treatment with
evolocumab (Raal et al. 2016; O’Donoghue et al. 2019). Similar weak correlations
were reported for alirocumab (Mahmood et al. 2021; Gaudet et al. 2017). In a real-­
life setting, there was no significant correlation between the reduction in apo(a) and
the reduction in apoB100 specifically present on VLDL/IDL/LDL after four weeks
of PCSK9i treatment (Blanchard et al. 2022).
120 A. Gallo et al.

Conclusion

The fact that unlike statins PCSK9 inhibitors reduce Lp(a) has clearly raised interest
in deciphering the molecular mechanisms by which this may occur. Despite much
effort, there is no consensus at present indicating that the lowering of Lp(a) induced
by PCSK9 inhibitors directly results from the reduction in LDLR expression and
function, as is the case for LDL. Figure 6.1 summarizes the potential pathways by
which PCSK9 (and hence PCSK9 inhibitors) modulates Lp(a) plasma concentra-
tions. For instance, the LDLR may play some role in mediating Lp(a) clearance
when its expression is starkly upregulated (e.g., by concomitant use of statins and
PCSK9 inhibitors) and when LDL plasma levels are substantially reduced, allowing
decreased competition between LDL and Lp(a) for receptor-mediated uptake. In
addition to an effect of PCSK9 on Lp(a) plasma clearance, the latest in vitro evi-
dence points toward a direct role for PCSK9 in enhancing Lp(a) production, assem-
bly, and subsequent secretion from liver cells (Youssef et al. 2022; Villard et al.
2016) (Fig. 6.1). Further exciting research is now needed to extensively explore this
fascinating observation in vivo.

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modulation of ApoB secretion. Arterioscler Thromb Vasc Biol. 2022;42:289–304.
Zhao Z, Tuakli-Wosornu Y, Lagace TA, Kinch L, Grishin NV, Horton JD, Cohen JC, Hobbs
HH. Molecular characterization of loss-of-function mutations in PCSK9 and identification of a
compound heterozygote. Am J Hum Genet. 2006;79:514–23.
Chapter 7
The Role of Cell Surface Receptors
in Lp(a) Catabolism

Lamia Ismail, Déanna Shea, and Sally McCormick

Introduction

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein (LDL)-like molecule that is

associated with a significant risk of developing multiple forms of atherosclerotic
cardiovascular disease (Nordestgaard et al. 2010; Reyes-Soffer et al. 2022). Lp(a)
(Fig. 7.1) is distinguished from LDL by the presence of apolipoprotein(a) [apo(a)],
a variably sized plasminogen homologue (McLean et al. 1987), which confers
unique properties including the specific binding of oxidised phospholipids (OxPL)
(Leibundgut et al. 2013). Apo(a) is synthesised in the liver and requires extensive
processing for secretion before it unites with LDL. Lp(a) is cleared principally by
the liver with some involvement of the kidney. The pathway for Lp(a) catabolism is
complicated as multiple receptor types expressed in both the liver and kidney have
been shown to bind and promote Lp(a) uptake. These include members of the lipo-
protein, plasminogen, lectin, and scavenger receptor families. There is also some
interaction of Lp(a) with toll-like receptors (TLRs) in macrophages during inflam-
mation. In this chapter, we summarise the findings from biochemical and clinical
studies documenting the various cell surface receptors that promote Lp(a)
catabolism.

Lamia Ismail and Déanna Shea contributed equally with all other contributors.

L. Ismail · D. Shea · S. McCormick (*)

Department of Biochemistry, School of Biomedical Sciences, University of Otago,
Dunedin, New Zealand
e-mail: [email protected]; [email protected];
[email protected]

© The Author(s), under exclusive license to Springer Nature 125

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_7
126 L. Ismail et al.

Lipid metabolism Wound response

KIV1
proteins proteins
OxPL APOA1 A2M
APOA2 APOA2
APOA4 APOE
PL APOB C3
APOC2 C4A
KIV2 APOC3 C5
APOC4 CLU
APOE FGA
APOF FGB
apoB APOL1 FGG
apo(a) APOM HRG
CLU KNG1
Lys
Lys

S-S
P LBP LPB
3
KIV

KV
LPA ORM2
KIV 10
4
KIV

KIV9
5

PLA2G7 PLA2G7
KIV

KIV 8
KIV7
6
KIV

PON1 SAA4
SERPINA1

Fig. 7.1 Lp(a) structure. Lp(a) consists of an LDL molecule attached to apo(a) via a disulphide
link to apoB. Oxidised phospholipids (OxPL) are found bound to the apo(a) KV domain and con-
tained in the phospholipid (PL) monolayer on the LDL surface. A number of lipid metabolism
proteins (green) are associated with the LDL particle, as are many wound-healing proteins (blue),
some of which may be bound to apo(a). The proteins are designated by their gene names.
(Reproduced from McCormick and Schneider 2019 with permission)

Lp(a) Structure and Assembly

Originally identified as an LDL variant by Kåre Berg (1963), a major difference is

that Lp(a) harbours apo(a), a large glycoprotein attached to the LDL surface
(Fig. 7.1). Apo(a) is coded for by the LPA gene, a plasminogen gene (PLG) homo-
logue recently evolved from the duplication of PLG kringle IV and V (KIV and KV)
(McLean et al. 1987). Further, the LPA KIV has 10 different subtypes (KIV1–10)
(McLean et al. 1987), with the KIV2 subtype harbouring a copy number variation
from 2 to >40 (Kostner et al. 2013) resulting in many different-sized apo(a) iso-
forms. The apo(a) moiety adds some unique elements to Lp(a) structure since it
specifically binds OxPL (Leibundgut et al. 2013) and is associated with various
wound response proteins (Von Zychlinski et al. 2011) (Fig. 7.1).
Apo(a) is almost exclusively produced in the liver with small amounts expressed
in the testis and brain (Tomlinson et al. 1989). Its synthesis and secretion are regu-
lated by the effects of genetic variation on LPA expression and the processing of the
apo(a) protein. Recent studies have identified some common single nucleotide vari-
ations that are functionally associated with low levels of LPA expression (Coassin
et al. 2017; Schachtl-Riess et al. 2021). The recognition that LPA expression is an
important determinant of Lp(a) levels underlies the development of apo(a) antisense
oligonucleotides which prevent apo(a) translation in the liver and significantly
lower Lp(a) (Tsimikas et al. 2021). With respect to apo(a) processing, each kringle
domain requires extensive glycosylation and disulphide cross-linking for proper
folding to exit the endoplasmic reticulum; hence, larger isoforms are less efficiently
7 The Role of Cell Surface Receptors in Lp(a) Catabolism 127

secreted. This was first shown by studies in primary hepatocytes with different-­
sized isoforms (White et al. 1997; Brunner et al. 1996) and is supported by recent
studies of an apo(a) mutant that displayed defective glycosylation and folding
(Morgan et al. 2020). These observations underpin the well-known inverse relation-
ship between Lp(a) levels and isoform size (Kraft et al. 1996).
The location of Lp(a) assembly is not certain but there is significant evidence for
an extracellular assembly with LDL (on the surface of hepatocytes or in circulation)
after apo(a) secretion (Chiesa et al. 1992; White and Lanford 1994). The process of
assembly involves an initial noncovalent binding between apolipoprotein B (apoB)
lysine residues exposed on the LDL surface and lysine-binding sites in apo(a)
(Gabel and Koschinsky 1998; Becker et al. 2004). This is followed by the formation
of a disulphide bond between specific cysteine residues in apo(a) and apoB
(Koschinsky et al. 1993; McCormick et al. 1995).

Lp(a) Catabolism

The liver provides the principal route of Lp(a) clearance from the circulation (Cain
et al. 2005). However, the presence of apo(a) fragments in the urine of patients with
kidney disease (Kostner et al. 1996; Albers et al. 2007) and their reduction after
transplantation (Black and Wilcken 1992) also suggest a role for the kidney. In mac-
rophages, it has been documented that Lp(a) binds to and triggers the CD36/TLR2
apoptotic pathway (Seimon et al. 2010).
Unlike LDL, which has a clearly defined uptake pathway via the low-density
lipoprotein receptor (LDLR) (Brown and Goldstein 1986) that can be specifically
targeted by drugs, i.e. statins and proprotein convertase subtilisin/kexin type 9
(PCSK9) inhibitors, the clearance of Lp(a) is rather more complicated. Many recep-
tors including lipoprotein, plasminogen, lectin and scavenger receptors have been
documented as being involved in Lp(a) catabolism (Hoover-Plow and Huang 2013;
McCormick and Schneider 2019). Lp(a)’s association with multiple receptors is
likely because it has a more complex composition than LDL with more potential
ligands (Fig. 7.1); apo(a), apoB and OxPL elements of Lp(a) have been shown to be
ligands for the various Lp(a) receptors with apolipoprotein E (apoE), orosomucoid
and alpha-2-macroglobulin also reported as possible ligands (McCormick and
Schneider 2019).

Liver Receptors

There are multiple receptors that have been shown to interact with Lp(a). Figure 7.2
represents the receptors that have been reported as being involved in Lp(a) uptake
that are expressed in the liver and kidney as well as receptors expressed in macro-
phages which promote cell signalling events from Lp(a). Figure 7.3 shows the gene
128 L. Ismail et al.

Fig. 7.2 Cell surface receptors for Lp(a). Receptors expressed in the liver and kidney for which
there is evidence of a role in binding to and promoting Lp(a) uptake are shown. These include the
lipoprotein receptors, LDLR, VLDLR, LRP-1 and megalin; plasminogen receptors, annexin A2,
S100A10 and PlgRKT; lectin receptors, galectin-1 and ASGR1; and the scavenger receptor,
SR-B1. Also shown are receptors on macrophages which mediate cell signalling events via Lp(a).
These include the scavenger receptor; CD36; and toll-like receptors, TLR2 and TLR6

expression profiles of the Lp(a) receptors in both liver and kidney. Most attention
has been paid to the LDLR with early cell culture studies showing Lp(a) to bind to
fibroblasts via the LDLR (Havekes et al. 1981; Floren et al. 1981) and subsequent
hepatocytes studies showing the same (Romagnuolo et al. 2015, 2017). These find-
ings were well supported by a study on fibroblasts from familial hypercholesterol-
aemia (FH) patients with defective LDL receptors which showed a much-reduced
binding to Lp(a) (Krempler et al. 1983). In the same study, kinetic experiments also
showed a delayed clearance of Lp(a) in FH subjects (Krempler et al. 1983). However,
there are many studies countering these findings including a knockout of the LDLR
in mice which showed no difference in Lp(a) clearance compared to wild-type mice
(Cain et al. 2005) and kinetics studies indicating no difference in Lp(a) catabolism
between FH and non-FH subjects (Rader et al. 1995; Knight 1994). Most impor-
tantly, there is the conundrum that statins do not lower Lp(a) and may increase it
(Yeang et al. 2016) and that PCSK9 inhibitors, which also upregulate LDLR, have
no effect on Lp(a) catabolism (Chemello et al. 2020).
One other LDLR family member that is expressed in the liver (Fig. 7.3) and has
been shown to interact with Lp(a) is low-density lipoprotein receptor-related protein
7 The Role of Cell Surface Receptors in Lp(a) Catabolism 129

Liver Kidney
150 150
144

60 60

50 50

40 40

RPKM
RPKM

30 30

20 20

10 10

0 0

LR 1

AS S1
1
B1
P1

AS 1
1

B1
PL 10

VL R
R

P2
S1 A2

PL A10
S1 A2

P2
LR R
KT

KT

VL R

P
GR

GR
S
DL
L

DL
L
A

AR

AL
LR

AL

AR
X

LR

X
LD
GR

GR

LD
00

00
AN

AN
LG

LG
SC

SC
Plasminogen Lipoprotein Lectin Scavenger Plasminogen Lipoprotein Lectin Scavenger

Fig. 7.3 Relative gene expression of Lp(a) receptors. The gene expression data for each of the
receptors in the liver and kidney are shown based on reads per kilobase of transcript per million
reads mapped (RPKM). Gene expression data were taken from NCBI (https://www.ncbi.nlm.
nih.gov/)

1 (LRP1). LRP1 plays an important role in chylomicron remnant catabolism through

binding to apoE (Beisiegel et al. 1989). The binding able to be competed by LRP1
to bind Lp(a) (Marz et al. 1993; Reblin et al. 1997) with the binding able to be com-
pleted by LDL, plasminogen and alpha-2-macroglobulin (Marz et al. 1993) suggest-
ing multiple ligands.
Not surprisingly, apo(a) interacts with plasminogen receptors on the liver sur-
face. This was first indicated by studies showing that Lp(a) binds to liver cells with
the binding able to be blocked by both plasminogen and lysine analogues (lysine
dependence is characteristic of plasminogen receptor interactions) (Tam et al.
1996). So far, three members of the plasminogen receptor family have been con-
nected to Lp(a). Lp(a) was found to interact with annexin A2 (Hajjar and Krishnan
1999), an abundant protein associated with membranes and the actin cytoskeleton.
Annexin A2 works in conjunction with another plasminogen-binding receptor,
S100A10, in the form of a tight heterodimer (Bharadwaj et al. 2021). Interestingly,
both proteins are implicated in actin remodelling which is required for macropino-
cytosis, a clathrin-independent endocytosis pathway known to promote LDL uptake
(Kruth et al. 2005) and recently implicated in Lp(a) endocytosis (Siddiqui et al.
2022). Direct evidence for the plasminogen receptors being involved in Lp(a) uptake
comes from studies of plasminogen receptor with C-terminal lysine (PlgRKT)
(Sharma et al. 2017). Overexpression of PlgRKT in both HepG2 and fibroblast cells
130 L. Ismail et al.

enhanced Lp(a) uptake significantly in both cell lines (Sharma et al. 2017).
Furthermore, a much-diminished internalisation of Lp(a) was observed in PlgRKT
knockdown HepG2 cells and in PlgRKT−/− fibroblast cells (Sharma et al. 2017).
Whether PlgRKT is a bona fide receptor for Lp(a) or whether it enhances the sur-
face binding of apo(a) to allow for uptake by other receptors, or via macropinocyto-
sis, is not yet known.
Another receptor highly expressed by the liver (Fig. 7.3) for which evidence is
mounting for a role in Lp(a) catabolism is scavenger receptor B1 (SR-B1). Well
known for its role in high-density lipoprotein (HDL) metabolism by facilitating
selective uptake of cholesterol esters (Acton et al. 1996), it has also been shown to
mediate whole particle uptake of very-low-density lipoprotein (VLDL), LDL and
HDL (Wang et al. 1998; Zanoni et al. 2018). Evidence for SR-B1’s involvement in
Lp(a) catabolism has come from transgenic mouse models in which overexpression
of SR-B1 significantly increases Lp(a) uptake, and contrariwise, SR-B1 knockout
mice show a reduced Lp(a) clearance (Yang et al. 2013). Another study showed that
SR-B1 facilitated the uptake of OxPL from Lp(a) in liver cells (Sharma et al. 2015).
Interestingly, individuals harbouring a mutation that reduces the ability of SR-B1 to
facilitate lipid uptake display both elevated HDL and Lp(a) levels (Yang et al. 2016)
suggesting clinical relevance.
One further receptor which is highly expressed in the liver (Fig. 7.3) shown to
mediate Lp(a) uptake is the asialoglycoprotein receptor (ASGPR), a lectin receptor
that mediates endocytosis of desialylated glycoproteins (Igdoura 2017). Mice lack-
ing the ASGPR showed a much-reduced clearance and degradation of Lp(a) by the
liver compared to wild-type mice (Hrzenjak et al. 2003). As apo(a) has a significant
content of desialylated O-linked sugars, it is a likely ligand for ASGPR. Indeed,
removal of sialic acids from Lp(a) greatly enhanced the clearance rate providing
support for an apo(a)/ASGPR interaction (Hrzenjak et al. 2003). Lastly, a lectin
receptor, galectin-1, highly expressed in the liver (Fig. 7.3), has been shown to bind
to Lp(a) (Chellan et al. 2007).

Kidney Receptors

The kidney expresses many of the same receptors as the liver (Fig. 7.2) including
the plasminogen receptors and LDLR. Two other members of the LDLR family
expressed in the kidney which have been shown to bind Lp(a) are the VLDL recep-
tor and the megalin receptor (otherwise known as LRP2). The VLDLR has been
shown to promote endocytosis and degradation of Lp(a) in fibroblasts with apo(a)
mediating the binding (Argraves et al. 1997). Furthermore, mice lacking the VLDLR
showed delayed clearance of Lp(a) (Argraves et al. 1997). The megalin receptor is
highly expressed in the kidney and plays a role in nutrient uptake via many different
ligands (Christensen and Birn 2001). It has been shown to bind to Lp(a) in a yolk
sac cell line via its apoB component (Niemeier et al. 1999).
7 The Role of Cell Surface Receptors in Lp(a) Catabolism 131

Macrophage Receptors

It is well documented that Lp(a) promotes inflammation via many different signal-
ling pathways through its OxPL content (Tsimikas and Hall 2012; Van der Valk
et al. 2016). Macrophages express an array of receptors which bind OxPL and stim-
ulate inflammatory signalling pathways and immune responses (Taylor et al. 2005).
These include the TLRs, which often work as co-receptors in conjunction with the
CD36 scavenger receptor to sense ligands. The TLR2/TLR6 heterodimer along with
CD36 (Fig. 7.2) has been shown to interact with OxPLs on apo(a) to promote
inflammation and apoptosis of macrophages (Seimon et al. 2010). Another study
indicated that both TLR2 and CD36 were necessary for the ability of Lp(a) to pro-
mote IL-8 production from macrophages (Scipione et al. 2015). These studies indi-
cate that the TLRs mediate the signalling promoting properties of Lp(a).
From this array of possible Lp(a) receptors, it is difficult to speculate which
receptors might contribute the most to Lp(a) clearance in the physiological setting
of the human body. If one considers Lp(a) clearance from a tissue aspect, then evi-
dence suggests that the focus should be on receptors that are highly expressed in the
liver. If one considers Lp(a) clearance from a ligand aspect, then the focus might be
best placed on receptors that specifically interact with apo(a) since it is a ligand
unique to Lp(a).

Summary

The Lp(a) molecule has a complex structure with several of its components known
to be ligands for various receptors. Cell culture studies have shown Lp(a) to bind to
a diverse range of cell surface receptors on multiple cell types. This situation makes
it difficult to pinpoint any one receptor pathway as being important in Lp(a) catabo-
lism and currently precludes targeting Lp(a) catabolism as a route for Lp(a) lower-
ing. Future studies will require a careful teasing out of the roles of the different
Lp(a) receptors in the liver and kidney with an emphasis on in vivo studies to gauge
clinical relevance.DisclosureNothing to disclose for all authors.

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Chapter 8
Physiological Roles and Functions of
Lipoprotein(a)

Zaid N. Safiullah, Thorsten Leucker, Steven R. Jones, and Peter P. Toth

Delivery Platform of Oxidized Phospholipids

It has been proposed that Lp(a) has a role in the transport of proinflammatory oxi-
dized phospholipids (OxPL). This hypothesis was driven by observations made by
Bergmark et al. who initially developed a method to measure OxPL bound to
apoB-100 by using a monoclonal antibody. They demonstrated that plasma OxPL/
apoB levels were quantitatively predictive of the presence and extent of angiograph-
ically determined CAD, identifying the presence and progression of carotid and
femoral atherosclerosis, and predicting CVD events over a 10-year interval. The
OxPL/apoB ratio was independent of all known risk factors, except for Lp[a], and,
remarkably, in all clinical studies performed at that time, there was an unusually
strong correlation of OxPL/apoB with Lp(a) (Bergmark et al. 2008).
These clinical observations suggested that OxPL may be bound to Lp[a] and may
thereby contribute to atherosclerotic plaque. In vitro studies demonstrated that not
only did OxPL bind to Lp(a), but it was the preferential carrier of OxPL in serum
(Bergmark et al. 2008). Notably, when compared to low-density lipoprotein (LDL),
Lp(a) bound three times as much OxPL. This led to the proposition that Lp(a)’s
physiologic roles may be to preferentially bind and transport OxPLs that are derived
from apoptosis and cell death, as occurs during inflammation and oxidative stress,
or when OxPLs are mobilized from tissues during iatrogenic plaque rupture during

Z. N. Safiullah
Osler Medical House Staff, The Johns Hopkins Hospital, Baltimore, MD, USA
e-mail: [email protected]
T. Leucker · S. R. Jones · P. P. Toth (*)
Ciccarone Center for the Prevention of Cardiovascular Disease, Johns Hopkins University
School of Medicine, Baltimore, MD, USA
e-mail: [email protected]; [email protected]; [email protected]

© The Author(s), under exclusive license to Springer Nature 135

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_8
136 Z. N. Safiullah et al.

O
O O
O
O PC
O PC

Arachidonoyl-PC
HO
O O
EI-PC
H O PC O O
OV-PC
O PC
O O H
HO O PC O OH
G-PC IsoLGE2-PC
OH O O O
H
O PC O PC
O
HOOA-PC HO OH
O O
8-isoPGE2-PC
H O PC
O
OH O (CH2)15CH3
HOHA-PC O O
+
O O O P O
PAF N
HO OH
O PC
O
KOdiA-PC
O O O
O (CH2)15CH3
O PC HO O O
+
HAzPC O P O
N
OH
OH
12-HETE-PC HOO O
O
O PC
O PC

9-HPETE-PC
OH
13-HODE-PC

Fig. 8.1 Oxidized phosphatidylcholine-containing phospholipids (Ox-PL). PC1-acyl-2-lyso-sn-­­

glycero-3-phosphatidylcholine. Only sn-2 position composition is shown for all Ox-PL except
those forming an ether bond at the sn-1 position. PAF platelet-activating factor, HAz-PC hexadecyl
azelaoyl PC; 13-HODE-PC 1-palmitoyl-2-(13(S)-hydroxy-(9Z,11E)-octadeca-9,11-­dienoyl)-sn-­
glycero-­3-phosphocholine. (Figure and legend reproduced with permission from Lee S, Birukov
KG, Romanoski CE, Springstead JR, Lusis AJ, Berliner JA. Role of Phospholipid Oxidation
Products in Atherosclerosis. Circulation Research 2012;111:778–799)

PCI or during lesion regression in response to therapeutic interventions. There are

many forms of OxPL that can be transported by Lp(a) (Fig. 8.1).
If Lp(a) is the preferential binding partner of oxidized phospholipids, then what
are the specific binding domains of OxPLs on Lp(a)? Lp(a) is composed of
8 Physiological Roles and Functions of Lipoprotein(a) 137

Fig. 8.2 Comparison of LDL and Lp(a) particles. (Left) Low-density lipoprotein (LDL) particle;
(right) lipoprotein(a) [Lp(a)] particle. Apoprotein (apo) B is the scaffolding for lipidation of both
lipoprotein species. Lp(a) is an LDL particle that is modified by the covalent addition of apo(a) to
apoB. Apo(a) is comprised of a series of kringles (protein loops; kringle IV [1–10] followed by
kringle V) and a protease terminus. The number of repeats in kringle IV type 2 are highly variable
person to person, genetically determined, and correlate with serum levels of Lp(a) as well as the
magnitude of risk for cardiovascular disease exerted by this lipoprotein. LDL-C low-density lipo-
protein cholesterol. (Figure and legend reproduced with permission from Toth PP. Familial
Hypercholesterolemia and Lipoprotein(a): Unraveling the Knot That Binds Them. Journal of the
American College of Cardiology 2020;75:2694–2697)

apolipoprotein(a) [apo(a)] covalently bound to apolipoprotein B-100 (apoB) via a

single disulfide bond on kringle (K) IV type 9 (KIV9) to a site near the LDL
receptor-­binding site of apoB (Fig. 8.2). In vitro studies have shown that the amino
acid residue 17K Asp 57 of the KIV10 lysine-binding site influences the covalent
binding of OxPLs. In vivo studies in mice have also supported the importance of the
lysine-binding sites of KIV10 fir OxPL binding (Bergmark et al. 2008). In sum-
mary, oxidized phospholipids are proinflammatory and participate in atherogenesis
(Chang et al. 2004; Tsimikas et al. 2005). Lp(a) has shown to be the preferential
carrier of these molecules when compared to other lipoproteins in vitro and in vivo.

Promoter of Inflammation

Inflammation is a known contributing factor to atherosclerosis development and

progression, and to increased cardiovascular disease risk (Fig. 8.3). In this regard,
there is ample preclinical and clinical evidence confirming that inflammation plays
a pivotal role in multiple steps of atherogenesis by promoting endothelial activation,
dysfunction and loss of integrity, failure of endothelial repair capacity, intimal lipid
138 Z. N. Safiullah et al.

a b c

d e f

Fig. 8.3 Increased arterial wall inflammation in subjects with elevated lipoprotein(a) [lp(a)]. (a)
Cross-sectional 18F-fluorodeoxyglucose uptake (18F-FDG) positron emission tomographic/com-
puted tomographic (PET/CT) images demonstrating an increased 18F-FDG uptake (yellow) in the
left carotid artery (top, white arrow) and aorta (bottom) in a subject with normal Lp(a) (left) and a
subject with elevated Lp(a) (right) quantified as the maximum target-to-background ratio (TBR) in
the (b) carotid arteries and (c) ascending aorta in subjects with elevated Lp(a) (n = 30) and normal
Lp(a) (n = 30). (d) Cross-sectional single-photon emission CT (SPECT)/CT images demonstrating
increased autologous technetium-99m (99mTc)-labeled peripheral blood mononuclear cell
(PBMC) accumulation (blue; at 6 h after infusion), depicted as the arterial wall–to–blood pool
ratio (ABR) at the level of (e) the carotid arteries and (f) ascending aorta in subjects with elevated
Lp(a) (n = 15) and normal Lp(a) (n = 15). **P < 0.01. (Figure and legend reproduced with permis-
sion from Valk FMvd, Bekkering S, Kroon J et al. Oxidized Phospholipids on Lipoprotein(a) Elicit
Arterial Wall Inflammation and an Inflammatory Monocyte Response in Humans. Circulation
2016;134:611–624)

deposition, as well as plaque formation and instability. The detrimental link between
inflammation and atherosclerotic CVD risk is further supported by those studies
showing that attenuation of inflammation is paralleled by improvement of surrogate
indicators of arterial function (e.g., endothelial function, aortic stiffness, ratio of
endothelial microparticles to endothelial progenitors) and cardiovascular prognosis
(Pirro et al. 2017).
Lp(a) is a well-known acute phase reactant whose production is stimulated by
inflammation. Notably, inflammatory response elements are present on the Lp(a)
gene. In primary monkey hepatocyte cultures, interleukin-6 (IL-6), IL-4, IL-13,
transforming growth factor-β (TGF-β), and tumor necrosis factor-α (TNF-α) can
8 Physiological Roles and Functions of Lipoprotein(a) 139

modulate Lp(a) gene expression. While IL-6 significantly increases plasma Lp(a)
levels, other cytokines inhibit Lp(a) synthesis by 50% or more at 10 ng/mL, with the
most powerful effect exerted by TGF-β and TNF-α (Ramharack et al. 1998).
Although there is sufficient evidence that inflammation may increase plasma Lp(a)
levels, data have emerged suggesting that a bidirectional regulatory loop involves
Lp(a) and inflammation; thus, Lp(a) may be proinflammatory in most cases, while
exerting anti-inflammatory effects in other conditions (Pirro et al. 2017). This pro-
inflammatory relationship leads to endothelial dysfunction, fragmentation, detach-
ment, and loss of repair activity, and activates inflammatory signaling cascades.
There is a close link between lipoproteins and inflammation in the arterial wall.
Oxidized LDLs (OxLDLs) trigger both directly and indirectly a proinflammatory
cascade leading to atherosclerosis development, progression, and complications
(Orsó and Schmitz 2017). On entry and trapping by interstitial matrix molecules
within the arterial intima, triglyceride-rich lipoprotein degradation by lipoprotein
lipase liberates free fatty acids and diacylglycerols, both of which are able to pre-
cipitate local inflammation (Orsó and Schmitz 2017). Oxidatively modified lipopro-
teins can be recognized by toll-like receptors, a type of pattern-recognition receptor
that responds against invading microbes, activating early innate recognition, and
host inflammatory responses. Also, lipoprotein exposure to reactive oxygen species
(superoxide anion, hydroxyl and peroxynitrite radicals) generates diverse oxidized
phospholipids (OxPLs) which can contribute to the initiation and the amplification
of the inflammatory response. Specifically, OxPLs present on OxLDLs can elicit
strong proinflammatory cytokine and chemokine responses from murine macro-
phages and human monocytes; they can also alter intracellular redox status and
directly activate proinflammatory genes, leading to arterial wall inflammation (Pirro
et al. 2017; van der Valk et al. 2016).
Lp(a) additionally has a role in initiating and stimulating inflammation, while
inhibiting anti-inflammatory pathways (Huang et al. 2014). Lp(a) binds monocyte
chemoattractant protein-1 (MCP-1), a chemokine that promotes the binding, roll-
ing, and transmigration of monocytes into the arterial intima (Deshmane et al.
2009). OxPLs have been shown to be major determinants for MCP-1 binding in the
vascular endothelium (Wiesner et al. 2013). Lp(a) inhibits the activation of trans-
forming growth factor-beta (TGF-β), a multifunctional and pleiotropic immune
regulatory cytokine that participates in peripheral immune tolerance and a negative
regulator of inflammation (Kojima et al. 1991). Lp(a) stimulates mRNA and cell
surface expression of intercellular adhesion molecule-1 (ICAM-1) in cultured
human umbilical vein endothelial cells (HUVECs) (Takami et al. 1998). Lp(a)-
induced expression of ICAM-1 in HUVECs appears to be mediated by decreasing
active TGF-β availability. Furthermore, Lp(a) promotes monocyte adhesion and
trans-endothelial migration by stimulation of MCP-1 and chemokine I-309, which
is the principal monocyte chemotactic cytokine produced by T helper cells (Haque
et al. 2000). Lp(a) promotes the differentiation of proinflammatory M1-type macro-
phages, leading to activation of T-helper-1 lymphocytes and natural killer cells.
Thus, macrophage expression of interleukin-1 (IL-1), IL8, and TNF-α is stimulated
by Lp(a) (Klezovitch et al. 2001). Secretion of these proinflammatory cytokines can
140 Z. N. Safiullah et al.

further induce endothelial activation by promoting ICAM-1, vascular cell adhesion

molecule-1 (VCAM-1), and E-selectin cell surface expression on endothelial cells.
Other than modulating inflammatory cells’ activity, Lp(a) stimulates the release of
proinflammatory cytokines from vascular endothelial and smooth muscle cells
(Klezovitch et al. 2001; Schmitz and Orsó 2015). Finally, apo(a), the distinguishing
kringle-containing component of Lp(a), is able to directly elicit a proinflammatory
response by inducing nuclear-catenin-mediated cyclooxygenase-2 (COX-2) expres-
sion (Pirro et al. 2017; Orsó and Schmitz 2017).
Lp(a) is associated with heightened systemic inflammation (Pirro et al. 2017). In
end-stage renal disease patients on hemodialysis, increased Lp(a) levels are associ-
ated with systemic inflammation and immune dysregulation. Plasma Lp(a) levels
are significantly higher in patients with elevated C-reactive protein (CRP) levels
than in those with plasma CRP levels within the normal range. van der Valk et al.
(2016) reported that subjects with increased plasma Lp(a) levels show radiologic
evidence of arterial inflammation and increased inflammatory cell trafficking to the
arterial wall. Intriguingly, these proinflammatory effects are mediated by Lp(a)’s
OxPL content. In summary, Lp(a) may activate several pathways linked to local and
systemic inflammation (Pirro et al. 2017; Orsó and Schmitz 2017; van der Valk
et al. 2016).
There is an abundance of data that describes Lp(a)’s role in stimulating and sus-
taining inflammation. However, there is also some evidence that Lp(a) also has anti-­
inflammatory properties. Lp(a)-mediated OxPL scavenging and, in some
circumstances oxPL degradation by Lp(a)-lipoprotein-associated phospholipase A2,
may exert anti-inflammatory effects (Tsimikas and Witztum 2008; Kiechl et al.
2007). Furthermore, in two inflammatory models (i.e., thioglycollate-induced peri-
tonitis or CaCl2-induced abdominal aortic aneurysm), apo(a) inhibits neutrophil
recruitment by inhibiting cytokine release and reducing entry of neutrophils into the
vessel wall (Huang et al. 2014). However, this observation was accompanied with a
leukocytosis so a concomitant inflammatory process cannot be excluded.

Impact on Malignancies

Following from its role as a proinflammatory agent, Lp(a) has a complicated role in
cancer biology. Observational studies have elucidated varying levels of Lp(a) in dif-
ferent malignancies. Elevations in serum Lp(a) are associated with lung cancer and
metastatic breast cancer (Orsó and Schmitz 2017; Lippi et al. 2007). However,
Lp(a) levels are found to be mostly decreased in hepatocellular cancer with some
variability (Orsó and Schmitz 2017; Gao et al. 2018). Lp(a) serum levels seem to be
independent of ovarian cancer, as well as acute lymphoblastic lymphoma.
Observationally, it is unclear if there is a singular relationship between Lp(a) con-
centration and cancer.
Lp(a) staining studies have identified Lp(a) in the tumor vasculature, rather than
in the primary parenchyma of the tumor (Lippi et al. 2007; Correc et al. 1990).
8 Physiological Roles and Functions of Lipoprotein(a) 141

Angiogenesis is vital to tumor growth and proliferation. It is thought to be driven by

an imbalance in angiogenic and angiostatic factors (Ramanujan et al. 2000). A pro-
totypical example of an angiostatic molecule is angiostatin. Angiostatin is not a
novel encoded protein, but rather a degradation product of proteins with kringle
domains (Wahl et al. 2005). This domain is responsible for angiostatin’s antiangio-
genic properties. In vitro experiments have shown that disrupting the tertiary struc-
ture of the kringle domain leads to disinhibited angiogenesis (Cao et al. 1996). This
kringle domain is found in the PLG domain of Lp(a) (Lippi et al. 2007).
The specific mechanism by which kringle fragments inhibit endothelial cell pro-
liferation remains an issue of ongoing investigation. It is theorized that the antian-
giogenic potential of the different kringle domains may depend not only on
appropriate protein folding from disulfide linkages but also on the individual pri-
mary amino acid sequence. Although the amino acid sequence alignment of the
PLG kringle domains shows that kringles I, II, III, and IV display significant
sequence homology (48–50% identity) (Lippi et al. 2007), kringle I was identified
as the most potent inhibitor for endothelial cell growth. Kringle III exhibited higher
inhibitory potency than kringle II and, interestingly, kringle IV was virtually inac-
tive in the suppression of endothelial cell growth (Cao et al. 1996; Dominguez et al.
2001). Since the removal of kringle IV from angiostatin potentiates its inhibitory
activity on endothelial cells, it is conceivable that this specific domain may prevent
some of the inhibitory effect of kringles I–III. Amino acid sequence alignment
reveals that kringle V displays the highest sequence identity with kringle I (57.5%)
(Lippi et al. 2007; Cao et al. 1997). Therefore, the high degree of similarity in the
primary structure may relate to the potent inhibitory activity of these two kringles
on endothelial cell growth. In vitro studies concluded that the order of endothelial
cell inhibition may be kringle V > kringle I > kringle III > kringle II > kringle IV
(Lippi et al. 2007; Cao et al. 1996, 1997). However, it is unknown if this order of
inhibition is seen in vivo.
In addition to the angiogenesis that promotes tumor proliferation, cell adhesion
and thrombus formation are processes that also aid in tumor growth (Lippi et al.
2007; Kim et al. 2004). Fibrin deposition following local thrombin generation
increases endothelial cell motility and promotes angiogenesis (Staton and Lewis
2005). The apo(a)-dependent inhibition of fibrinolysis might play a role in promot-
ing tumor proliferation.
Evidence from animal models have supported an antineoplastic role of Lp(a)
seen in vitro. Experiments have shown that angiostatin can maintain metastases in a
dormant state and shrink primary tumors by blocking neovascularization and tumor
growth in vivo. Accordingly, several clinical investigations confirmed the potent
antineoplastic effect of a recombinant form of apo[a] in the animal model. The ret-
roviral gene transfer of murine colon carcinoma cell line CT26 with LK68 (a recom-
binant of Lp(a) kringle type IV and V) significantly suppressed tumor growth, as
well as progression of micrometastases to macroscopic tumors and peritoneal dis-
semination (Yu et al. 2004, 2005). Intramuscular administration of virus carrying
genes encoding for LK68 gave 60–84% suppression of tumor growth in mice bear-
ing subcutaneously transplanted hepatocellular carcinoma (Lee et al. 2006). Overall,
142 Z. N. Safiullah et al.

improved mortality was seen in mice expressing LK68 and bearing human colorec-
tal cancer, lung cancer, or hepatocellular carcinoma (Lippi et al. 2007).
A large prospective cohort study of over 10,000 patients showed that low serum
Lp(a) levels correlated with increased mortality from cancer, specifically hepatocel-
lular carcinoma (HCC) (Lippi et al. 2007; Katzke et al. 2017). In HCC, increased
Lp(a) has been linked with decreased recurrence after resection. Further, it has util-
ity as a prognostic marker in α-fetoprotein <400 ng/mL level and tumor size <5 cm
in subgroups from a small prospective study. Similarly, a retrospective study that
compared serum Lp(a) concentration with tumor size in papillary thyroid cancer
found a significant negative correlation and concluded that Lp(a) may have a protec-
tive effect. Finally, a German cohort study found that increased Lp(a) levels were
associated with a total reduction of cancer mortality (Lippi et al. 2007; Katzke
et al. 2017).
Alternatively, a retrospective analysis done to investigate the association between
Lp(a) levels and clinical pathologic features of prostate cancer showed that higher
Lp(a) levels correlated with a high-risk prostate cancer phenotype (Wang and Zhang
2019). These results were similar to a separate German analysis (Katzke et al. 2017).
The authors theorized that increased Lp(a) levels may be compensatory to chronic
inflammation secondary to aggressive prostate cancer. Furthermore, increased Lp(a)
may promote increased cancer cell adhesion, invasion, and metastasis through its
role as a competitive inhibitor of plasmin-induced fibrinolysis (Wang and Zhang
2019). The clinical significance of Lp(a) in malignancy requires further investiga-
tion. Presently, Lp(a) may be protective in HCC and papillary thyroid cancer (Ma
et al. 2021). Conversely, it may be deleterious in prostate cancer. More research is
needed to understand the paradoxical underpinnings of these phenotypes.
Lp(a)’s angiostatic properties may make it a novel target for cancer therapeutics.
There are preclinical studies that have shown the utility of angiostatin gene therapy
in decreasing angiogenesis. Small molecule delivery and gene therapy do not have
cytotoxic side effects seen in conventional chemotherapy and immunotherapy.
Further research is needed to investigate whether the derived angiostatic mecha-
nisms of Lp(a)’s kringle domain can be developed into cancer therapies (Lippi
et al. 2007).

Lp(a) in Thrombosis

Lp(a) has a well-characterized role in thrombogenesis. Biochemically, Lp(a)’s

apo(a) moiety has a sequence homology to plasminogen. Plasminogen is a proen-
zyme of plasmin, which participates in fibrinolysis. Functional studies have illus-
trated that apo(a) is a competitive inhibitor of pericellular plasminogen activation
(Boffa and Koschinsky 2016).
Plasminogen consists of an N-terminal tail domain, five different kringle
domains, and a latent trypsin-like protease domain (Forsgren et al. 1987). The krin-
gle domain is common to other proteases involved in hemostasis such as prothrom-
bin, Factor VIII, tissue plasminogen activator (TPA), and urokinase plasminogen
8 Physiological Roles and Functions of Lipoprotein(a) 143

activator (Patthy 1985). The apo(a) subunit consists of ten different types of kringle
domains, differing in amino acid sequence, which are most homologous to plas-
minogen kringle IV (KIV), and a single plasminogen kringle V (KV)-like domain
and a protease-like domain (McLean et al. 1987). Of the ten KIV types in apo(a),
nine are present in single copy in all apo(a) isoforms (van der Hoek et al. 1993),
while KIV type 2 (KIV2) is encoded in a variable number of tandemly repeated
copies by the apo(a) gene (LPA), generating a series of different-sized LPA alleles
and, hence, apo(a) isoforms in the human population. Known alleles encode as few
as 1 and as many as 34 KIV2 repeats, giving rise to apo(a) isoforms containing
between 10 and 43 KIV-like domains, and polypeptide molecular masses between
200 and 800 kDa (Boffa and Koschinsky 2016).
Mechanistically, kringles are thought to function in ligand interactions with
lysine-containing substrates. Several of the kringles in plasminogen contain lysine-­
binding sites (LBSs), defined structurally by a hydrophobic trough, lined by two or
three key aromatic side chains, which binds the aliphatic backbone of the lysine side
chain and that is flanked on either end by a cationic and anionic center (Hoover et al.
1993; McCance et al. 1994). Regarding LBS in plasminogen, the LBS in kringle I
has the highest affinity for lysine analogs, followed by KIV and KV (Castellino and
McCance 1997). The LBSs in plasminogen have been shown to be important for
both lysine-dependent interactions with substrates, such as fibrin and cell-surface
receptors, as well as for intramolecular interactions that maintain the closed native
conformation of plasminogen (Boffa and Koschinsky 2016; Cockell et al. 1998;
Violand et al. 1978).
The KIV types within apo(a) have varying affinities for lysine binding, with
KIV10 having a stronger affinity than KIV5–KIV8. The differential in lysine-­
binding affinity is due to conservative amino acid substitutions (Ye et al. 2001;
Rahman et al. 2002). KIV10 is the only kringle domain that interacts with lysine-­
containing substrates because the LBS in KIV5–KIV8 are masked when bound to
apoB-100 in Lp(a). In fact, KIV7–KIV8 have been explicitly shown to participate
in noncovalent interactions with specific lysine residues on apoB-100 that precede
covalent Lp(a) formation (Boffa and Koschinsky 2016).
In addition to the kringle domain, apo(a) has a protease domain. The protease-­
like domain in apo(a) is catalytically inactive, despite having an intact Ser-His-Asp
catalytic triad (Gabel and Koschinsky 1995). An Arg to Ser substitution at the loca-
tion analogous to the site on plasminogen that is cleaved by plasminogen activators
ensures that an activating cleavage of apo(a) cannot occur (Hajjar et al. 1989). In
addition, several other amino acid substitutions relative to plasminogen, as well as
a key nine amino acid deletion in apo(a), have been proposed to render the protease-­
like domain in apo(a) inactive (Boffa and Koschinsky 2016; Gabel and
Koschinsky 1995).
Lp(a) lacks protease activity while retaining the ability to bind to lysine-­
containing substrates; hence, it is possible that Lp(a) may interfere with the func-
tions of plasminogen through molecular mimicry. This has been characterized by
several in vitro studies that have shown that Lp(a) interferes with plasminogen
activity (Boffa and Koschinsky 2016; Hajjar et al. 1989; Miles et al. 1989;
Romagnuolo et al. 2014). The lysine-binding function of plasminogen is crucial to
144 Z. N. Safiullah et al.

its fibrinolytic role. Activation of plasminogen by tPA occurs slowly in the absence
of a fibrin surface. In the presence of fibrin, a ternary complex is formed that results
in efficient production of plasmin (Hoylaerts et al. 1982). Plasminogen binding to
fibrin converts the protein from a closed to an open conformation that makes it a
better substrate for tPA (Urano et al. 1988). Further, partial degradation of fibrin by
plasmin results in the formation of carboxyl terminal lysine residues that mediate
positive feedback in the fibrinolytic cascade by promoting: (1) plasminogen binding
(Suenson and Petersen 1986), (2) plasmin-mediated conversion of native Glu1-­
plasminogen to Lys77-plasminogen, which lacks the tail domain and is a better
substrate for tPA (Suenson and Thorsen 1988), and (3) binding to plasmin and thus
protecting it from consumption by antiplasmin (Boffa and Koschinsky 2016; Wiman
and Collen 1978).
The first studies that explored the functional implications of the homology
between apo(a) and plasminogen demonstrated the ability of Lp(a) and apo(a) to
inhibit binding of plasminogen to cell surface receptors on monocytes and endothe-
lial cells (Hajjar et al. 1989; Miles et al. 1989). Apo(a) was presumed to be a com-
petitive inhibitor of pericellular plasminogen activation. This was confirmed by
later experiments (Romagnuolo et al. 2014). It is also thought that this mechanism
may contribute to atherogenesis secondary to persistence of mural thrombi or extra-
cellular matrix degradation of the vascular wall (Boffa and Koschinsky 2016).
Regarding the effect of apo(a) on tPA, in vitro studies have demonstrated that
apo(a) and Lp(a) are capable of inhibiting tPA-mediated clot lysis and inhibiting
tPA-mediated plasminogen activation (Boffa and Koschinsky 2016). Furthermore,
apo(a) can inhibit the positive feedback step of plasmin-mediated Glu- to Lys-­
plasminogen conversion in the context of fibrin. However, when it comes to under-
standing the mechanistic underpinnings of this observation, the data have been
mixed. Some data support that apo(a) directly competes with plasminogen for bind-
ing to fibrin. Whereas an alternate mechanism has been postulated that apo(a) forms
a quaternary complex with plasminogen, tPA, and fibrin that has a much lower turn-
over number than the ternary complex lacking apo(a) (Boffa and Koschinsky 2016).
Thus far we have explored the antifibrinolytic mechanisms of Lp(a). Additionally,
Lp(a) has well-characterized prothrombotic mechanisms including the promotion
of platelet aggregation and tissue factor pathway inhibitor (TFPI) binding (Boffa
and Koschinsky 2016). Lp(a) has a dual role in platelet aggregation (Boffa and
Koschinsky 2016). Experiments have shown that Lp(a) enhances platelet aggrega-
tion and granule release mediated by the thrombin receptor activation peptide
SFLLRN. It has also been shown that Lp(a) potentiates arachidonic acid-induced
platelet aggregation. Mechanistically, this is mediated by binding of apo(a) to lysine
residues on platelet receptors. Alternatively, there is evidence that Lp(a) or apo(a)
decreases platelet activation induced by collagen, ADP, or platelet-activating factor.
This duality of function may exist as a counterbalance to Lp(a)’s antifibrinolytic
activity (Boffa and Koschinsky 2016).
Lp(a) also directly binds tissue factor pathway inhibitor (TFPI). TFPI inhibits the
extrinsic coagulation cascade by binding to Factor Xa and then the tissue factor/
Factor VIIa complex. Apo(a) binds to TFPI through lysine residues in the carboxyl-
terminal portion of TFPI and decreases its fluid phase and cell surface activity,
8 Physiological Roles and Functions of Lipoprotein(a) 145

thereby inhibiting coagulation. Furthermore, the binding of Lp(a) to TFPI may con-
tribute to its atherogenic potential. Immunostaining studies on coronary atherec-
tomy samples have showed TFPI and apo(a) in smooth muscle cell-rich areas of the
intima (Boffa and Koschinsky 2016; Caplice et al. 2001).
Lp(a) also interacts with the fibrin clot. The fibrin clot is the final product of
primary hemostasis. The clot structure is important in determining both the stability
and fibrinolytic capacity of the fibrin clot, which has implications for abnormal
thrombolysis. Unfortunately, as compared with the role of Lp(a) in antifibrolytic
and prothrombotic, its mechanism of interaction with fibrin is not as well understood.
Observational studies have shown that elevated Lp(a) levels have been associated
with an altered fibrin clot structure that is accompanied by reduced fibrin clot per-
meability and impaired fibrinolysis (Boffa and Koschinsky 2016). This is thought to
be one of the etiologies of how Lp(a) contributes to CAD. Furthermore, from a
genetics perspective, the LPA gene contains a SNP (rs3798220) that results in an Ile
to Met substitution at amino acid 4399 within the protease-like domain of apo(a).
The allele encoding Met has been associated with elevated plasma Lp(a) levels,
small apo(a) isoform sizes, and increased risk for congenital heart disease (Luke
et al. 2007). Caucasians heterozygous for the Ile4399Met variant exhibit elevated
Lp(a) levels, increased clot density, and increased clot lysis times, while non-­
Caucasian carriers showed increased clot permeability and shorter lysis times, with
no significant increase in Lp(a) levels. Interestingly, in the Women’s Heart Study,
individuals heterozygous for the Ile4399Met variant exhibited elevated Lp(a) levels
and an increased risk for CAD and benefited more from aspirin therapy than wild-
type subjects (Boffa and Koschinsky 2016). This could suggest a prothrombotic role
for the Ile4399Met polymorphism.
Most studies that examined the association between Lp(a) and venous thrombo-
embolic events (VTE) are cross-sectional. A meta-analysis of ten studies revealed
that among 13,541 subjects, those with a history of deep vein thrombosis were more
likely to have elevated Lp(a) (Dentali et al. 2017). Elevated Lp(a) was associated
with the presence of VTE at an odds ratio of 1.56 (95% CI 1.36–1.79). There was
also a stronger association between Lp(a) and VTE in patients with other predispos-
ing risk factors. A study of 467 patients with first VTE followed up for 1 year by
Marcucci et al. found a fivefold increased risk of recurrent VTE for Lp(a) >30 mg/
dL (OR 5.1, 95% CI 3.1–8.4), a level of risk similar to that seen in hyperhomocys-
teinemia and even higher than that for factor V Leiden or the factor II 20210GA
polymorphism (Caplice et al. 2001; Dentali et al. 2017; Crowther 2004; Nave and
von Eckardstein 2019).

Lp(a) in Diabetes

The mechanism by which Lp(a) influences the development of type 2 diabetes is not
well understood. Retrospective cohort studies have shown a negative correlation
between Lp(a) and insulin resistance (Mora et al. 2010). In a sample of 607 dyslip-
idemic patients, Lp(a) correlated inversely with serum triglycerides (TG) levels, TG/
146 Z. N. Safiullah et al.

HDL-C ratios, insulin, HOMA-IR, C-peptide, body mass index, and waist circum-
ference (Vaverková et al. 2017). Another study has also illustrated that there is a
sharp decrease in Lp(a) levels in the transition from prediabetes to type 2 diabetes
(Kaya et al. 2017). Genetic data from Chinese and Danish populations have shown
an increased risk of type 2 diabetes in individuals with genetically determined low
lipoprotein(a) plasma concentration due to large lipoprotein(a) isoform size related
to the number of kringle IV type 2 repeats. Alternatively, a Mendelian randomization
analysis showed that genetic variants associated with fasting insulin levels bore no
relation to Lp(a) concentration (Mora et al. 2010; Kamstrup and Nordestgaard 2013).
Like retrospective data, large prospective studies have also shown that there is a
negative association between Lp(a) and the risk of developing type 2 diabetes (Mora
et al. 2010; Schwartz et al. 2021). The negative association between Lp(a) and type
2 diabetes has also been observed to be dependent on the concentration of Lp(a).
Lower Lp(a) levels were associated with an increased risk of type 2 diabetes.
Similarly, it has been shown that Lp(a) levels are increased in prediabetes compared
to normoglycemic controls (Paige et al. 2017). Regarding sex differences, the
inverse relationship between Lp(a) and increased glucose levels is observed in men
prior to prediabetes. Whereas in women, the inverse relationship between Lp(a) and
glucose levels is observed only starting in prediabetes (Paige et al. 2017). The sig-
nificance of this observation is not yet clear. There was also no association between
isoform size and risk of diabetes (Kamstrup and Nordestgaard 2013). One hypoth-
esized mechanism that explains this observation is that insulin suppresses apo(a) in
hepatocytes. The biologic role of Lp(a) in insulin resistance and hyperglycemia
requires more interrogation.
In those with cardiovascular disease, low levels of Lp(a) have also been associ-
ated with a greater prevalence of type II diabetes mellitus in prospective, retrospec-
tive, and genetic studies. In an analysis of 13,480 patients in the ODESSY
OUTCOMES trial, similar findings were observed with negative correlation
between Lp(a) and the prevalence of type 2 diabetes. Furthermore, in the same
analysis, reduction of Lp(a) levels by the PCSK9 inhibitor alirocumab in those with
high baseline Lp(a) level increased the estimated risk of incident type 2 diabetes
compared with placebo hazard ratio 1.07 (95% CI 1.03–1.12; P < 0.0002) (Schwartz
et al. 2021). However, there was an interaction between treatment with alirocumab
and baseline Lp(a) on the risk of incident type 2 diabetes. The concentration of
Lp(a) at which alirocumab had a neutral effect on incident type 2 diabetes was
around 50 mg/dL. It was shown that PCSK9 inhibitor-induced reductions of Lp(a)
levels lead to an increased risk of type 2 diabetes (Schwartz et al. 2021).
Although the mechanism by which low levels of Lp(a) contribute to an increased
risk of type 2 diabetes is unknown, the risk may be modifiable as evidenced by
Lp(a) levels in the above study that were neutral. Further, it seems that those with
higher baseline Lp(a) who are treated with PCSK9 inhibitor therapy have an
increased risk to develop type 2 diabetes than those with lower baseline Lp(a) levels
(Schwartz et al. 2021). This observation, if confirmed in additional studies, is impor-
tant to consider when prescribing this therapy to mitigate cardiovascular risk.
8 Physiological Roles and Functions of Lipoprotein(a) 147

In summary, retrospective and prospective data have elucidated that Lp(a) levels
are inversely related to an increased incidence of type 2 diabetes. The mechanism
by which Lp(a) participates in diabetogenic pathophysiology is not yet understood.
However, based on the studies discussed previously, the risk is modifiable through
PCSK9 inhibitor therapy. Additional investigation is needed to understand whether
Lp(a)-directed therapies currently in development impact risk for the development
of diabetes.

Lp(a) in Wound Healing

Lp(a) is implicated in wound healing. Histologic studies have identified Lp(a) in all
four stages of wound healing from infiltration of inflammatory cells to formation of
granulation tissue. In the first stage of healing, there is an infiltration of inflamma-
tory cells followed by formation of a fibrin clot mixed with red blood cells covers
the exposed wound surface. In the second stage, the immature cell mass is replaced
by granulation tissue, which is produced by fibroblasts, endothelial cells, and vas-
cular sprouts from adjacent viable tissues, induced by growth factors released dur-
ing the first stage. In this stage, granulation tissue is often covered with loose fibrous
connective tissue with various thickness, which forms the fibrous cap. Angiogenesis
also takes place in this stage. The epithelial sheets are spread to cover the granula-
tion tissue in the third stage. In the last stage, collagen fibers replace the granulation
tissue, resulting in reduction of wound size. Finally, the healing process is com-
pleted by replacement of granulation tissue with new epithelium or by organization
(Yano et al. 1997).
The apo(a) and apoB100 subunits of Lp(a) are more strongly identified in the
fibrous cap, endothelial cells, and plasminogen and fibrinogen-rich surfaces than in
the re-epithelized tissue surface. The mechanism by which Lp(a) influences wound
healing has not been described. Following from discussions elsewhere of Lp(a)’s
role in angiogenesis and antifibrinolysis, it is possible that Lp(a) promotes the pro-
liferation of endothelium with accompanying vasculogenesis; and is also involved
in maintenance of the fibrin cap and preventing excessive fibrinolysis (Yano
et al. 1997).

Lp(a) in Autoimmune Disease

Lp(a) elevation is associated with several autoimmune diseases (Missala et al. 2012;
Toms et al. 2011). The mechanisms by which Lp(a) contributes to autoimmune
disease are through acute phase reactions, autoantibodies, and fibrinolysis (Missala
et al. 2012). The interplay of these mechanisms leads to increased inflammation
which contributes to clinical autoimmune phenotypes.
148 Z. N. Safiullah et al.

Elevated Lp(a) is associated with increased circulating levels of acute phase

reactants. In patients with rheumatoid arthritis, Lp(a) levels are associated with
elevated C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)
(Asanuma et al. 1999). Based on this association, Lp(a) is thought to have a crucial
role in the acute inflammation pathway (see discussion of Lp(a) in inflammation
previously). Observations of Lp(a) as an acute phase reactant have been identified
in other inflammatory conditions such as polymyalgia rheumatica (Missala
et al. 2012).
Autoantibodies are a prototypical mechanism of autoimmune disease.
Autoantibodies to Lp(a) have been detected in antiphospholipid syndrome (APLS)
and lupus. Specifically, for APLS, malondialdehyde (MDA)-modified lipoprotein(a)
antibodies were observed. Antibodies reacting against MDS implicate increased
oxidation in the pathogenesis of this condition (Romero et al. 1999).
Many autoimmune diseases carry an increased risk for cardiovascular disease,
namely atherosclerotic plaque formation (Missala et al. 2012). For example, in
those with rheumatoid arthritis, mortality from CVD is about 50% greater than con-
trols. The increased CVD risk conferred by autoimmune diseases is attributed to
dyslipidemia, systemic inflammation, and increased typical CVD risk factors, such
as hypertension and obesity. Lp(a) seems to be at the crossroads between autoim-
mune disease and atherosclerosis. As discussed elsewhere, oxidized and glycosyl-
ated Lp(a) contributes to atherosclerotic plaque formation. In patients with
rheumatoid arthritis and APLS, a similar glycosylated Lp(a) product is observed in
the serum, specifically beta(2) GP1-Lp(a). Beta(2) GP1-Lp(a) is known to be asso-
ciated with CAD and acute coronary syndromes. This product likely contributes to
increased atherosclerotic risk in these populations in addition to typical CVD risk
factors. There is limited data regarding the impact of Lp(a) on CVD in lupus.
However, some observational studies have shown that Lp(a) is more elevated in
those with CVD and lupus than those with lupus alone. Further research is needed
to elucidate the mechanism of how Lp(a) promotes atherosclerosis in the setting of
autoimmune disease (Wang and Zhang 2019; Missala et al. 2012).
Given its implication in the mechanism of autoimmune diseases, Lp(a) may be a
viable target of intervention. Lp(a) lowering therapy may help reduce the systemic
inflammation seen in these diseases and simultaneously mitigate the CVD risk. This
will have to be evaluated prospectively in randomized trials with agents that specifi-
cally reduce Lp(a).

Lp(a) in Calcific Aortic Stenosis

Calcific aortic valve stenosis (CAVS) is the most common valve disease in the
elderly population, affecting >1 million patients in the USA, and is associated with
significant morbidity and mortality (Guddeti et al. 2020). Elevated Lp(a) is linked
to increased risk for calcific aortic valve stenosis (CAVS). Observational studies
8 Physiological Roles and Functions of Lipoprotein(a) 149

from the early 1990s showed that increased Lp(a) levels were associated with aortic
valve calcification and stenosis. This relationship is linear, with higher Lp(a) levels
correlating with higher risk. Multivariate analyses have shown that increased Lp(a)
is an independent predictor of developing CAVS. Additionally, both prospective
and retrospective genetic studies have shown that the LPA locus carries a greater
risk of CAVS and may be causative. Interestingly, some studies have shown aortic
stenosis after the sixth decade does not correlate with Lp(a) levels (Guddeti
et al. 2020).
The relationship between Lp(a) and CAVS is driven in part by oxidized phospho-
lipids. Oxidized phospholipid apoB-100 was linked to faster progression of aortic
stenosis in those with elevated Lp(a). This observation leads to the postulation that
Lp(a) leads to aortic valve stenosis by phospholipid oxidation. Mechanistically,
OxPL are proinflammatory, can lead to endothelial dysfunction, and promote osteo-
genic differentiation which leads to calcification. Interestingly, the relationship
between aortic stenosis progression and OxPL content may be linear, based on a
subgroup analysis of the ASTRONOMER (Effects of Rosuvastatin on Aortic
Stenosis Progression) clinical trial (Vavuranakis et al. 2020).
Increased Lp(a) levels have also been associated with valve calcification in
patients with bicuspid aortic valves. Fewer KIV-2 repeats have also been linked to
more severe calcification. In the context of calcification of a bicuspid aortic valve,
Lp(a) could be a useful marker to identify those at risk to develop valve calcification
and stenosis (Guddeti et al. 2020).
CAVS is often present in the setting of CAD. Interestingly, the relationship
between Lp(a) and CAVS is independent of CAD. However, studying patients with
this comorbidity has yielded novel insights into the mechanism of how Lp(a) pro-
motes CAVS. Autotaxin (ATX), a lysophospholipase D enzyme, transforms lyso-
phosphatidylcholine into lysophosphatidic acid (LysoPA). ATX is transported in the
aortic valve via the bloodstream by Lp(a) and is also secreted by valve interstitial
cells. ATX-LysoPA has been shown to promote inflammation and leads to calcifica-
tion of the aortic valve, thus promoting CAVS. Autotaxin also indirectly promotes
the nuclear translocation of the transcription factor NF-κB, which leads to height-
ened inflammation (Nsaibia et al. 2016).
In addition to deposition of oxidized phospholipids and inflammation mediated
by autotaxin, Lp(a) has other pleiotropic mechanisms that lead to CAVS. Lp(a) is
thought to participate in cholesterol deposition on the aortic valve cusps causing
thickening. Lp(a) is also implicated in macrophage apoptosis and might contribute
to early valve lesion progression. Further, following from Lp(a)’s role in thrombo-
sis, it can cause fibrin deposition on the leaflets which can cause stenosis (Guddeti
et al. 2020; Vavuranakis et al. 2020; Nsaibia et al. 2016).
In summary, Lp(a) contributes to the pathogenesis and progression of calcific
aortic stenosis. Lp(a) has several pleiotropic mechanisms that contribute to CAVS,
including, cholesterol deposition, delivery of OxPL, fibrin deposition, and inflam-
mation mediated by autotaxin. More research is needed to determine if Lp(a) lower-
ing therapy can mitigate the development of calcific aortic stenosis.
150 Z. N. Safiullah et al.

Lp(a) in Acute Coronary Syndromes

Elevated Lp(a) leads to an increased risk of coronary artery disease (CAD) and
cardiovascular events (Tsimikas et al. 2020). Lp(a) contributes to atherosclerosis
independent of LDL-C by many of the mechanisms discussed previously. These
mechanisms include delivery of oxidized phospholipids and promotion of inflam-
mation and thrombosis (Rehberger Likozar et al. 2020). Additionally, Lp(a) is asso-
ciated with IL-8, a proinflammatory, prothrombotic, and proatherogenic cytokine,
which attracts leukocytes, triggers tissue factor production, and promotes adhesion
of monocytes to early atherosclerotic plaques (Lippi et al. 2021).
Observational data of the role of Lp(a) in CAD have shown that Lp(a) levels are
higher in the setting of stable angina compared to unstable angina.
Immunohistochemically, 90% of the Lp(a) area in coronary atheromas co-localizes
with plaque macrophages, and 30% of which correlates with plaque a-actin, which
might be related to the role of Lp(a) in plaque enlargement (Dangas et al. 1998).
Similarly, in acute myocardial infarction (MI), Lp(a) levels increase significantly
within the first 24 h and normalize within about 30 days (Rehberger Likozar
et al. 2020).
The compete mechanism of how Lp(a) contributes to atherogenesis is not yet
fully understood. In addition to the mechanisms discussed previously, it is pro-
posed that Lp(a) is deposited on the vascular wall and is readily taken up by mac-
rophage scavenger receptors. The macrophages soon become foam cells and the
canonical pathway of atherogenesis follows. Lp(a) also induces endothelial dys-
function which is proatherogenic (van der Valk et al. 2016; Rehberger Likozar
et al. 2020). As detailed previously, Lp(a) leads to coronary thrombi formation by
antifibrinolysis (i.e., competitive inhibition of tPA). In addition, Lp(a) promotes
coagulation and platelet aggregation and boosts inflammation (Boffa and
Koschinsky 2016).
Based on the increased CVD mortality conferred by increased Lp(a) levels, cur-
rent European Society of Cardiology guidelines recommend screening at least once.
The 2018 American College of Cardiology/American Heart Association guidelines
on blood cholesterol defined Lp(a) 50 mg/dL, or 125 nmol/L, as a risk-enhancing
factor; according to their guidelines, this is a relative indication for its measurement
with a family history of premature CVD. Those with elevated Lp(a) >180 mg/dL
carry a risk of atherosclerotic CVD equivalent to patients heterozygous for familial
hypercholesteremia (Rehberger Likozar et al. 2020).
Multiple studies have shown that the association of genetically predicted Lp(a)
levels with the risk of CVD is independent of changes in LDL cholesterol levels.
This is thought due to genetic variants that mimic the LDL lowering effects of
statins, PCSK9 inhibitors, and ezetimibe to the risk of CVD (Burgess et al. 2018).
This observation is more significant in younger patients. In those less than 45 years
old, in whom elevated Lp(a) levels (>120 nmol/L, 80th percentile) are associated
with a threefold increased risk of MI. The clinical benefit of lowering Lp(a) levels
is proportional to the absolute reduction in Lp(a) levels. An absolute reduction in
Lp(a) levels of approximately 100 mg/dL should result in a clinically relevant
8 Physiological Roles and Functions of Lipoprotein(a) 151

reduction in the risk of CVD. Such a decrease in Lp(a) represents the same magni-
tude of CVD risk reduction achieved by lowering LDL cholesterol levels by
38.67 mg/dL (Tsimikas et al. 2020; Rehberger Likozar et al. 2020).
Specifically, in acute coronary syndromes (ACS), Lp(a) elevation is observed for
up to 4 months after an event. Concomitantly, OxPL levels are also elevated, which
could mean that Lp(a) participates in OxPL delivery during acute plaque rupture.
Interestingly, Lp(a) and OxPL transient elevations have also been observed after
percutaneous coronary intervention for stable CAD (Tsimikas et al. 2020). Lp(a)
levels are also inversely related to the age of first presentation with ACS. This means
that younger patients presenting with ACS or observed to have higher Lp(a) levels
than older patients with a similar ACS presentation. This reflects the importance of
other, traditional atherosclerotic risk factors in older individuals, in contrast to a
more important role of Lp(a) in younger individuals (Vavuranakis et al. 2020).

Evolution of Lp(a)

The synthesis of apo(a) is confined to a certain group of primates. However, the

hedgehog produces an apo(a)-like protein composed of tandem repeats of plasmino-
gen kringle III homologous domain but without the protease domain (Lawn et al.
1997). Phylogenetic analysis has determined that the human and hedgehog genes
evolved independently from different DNA sequences. This observation signifies
convergent evolution (Lippi and Guidi 2000).
The human apo(a) is in a 400 kb gene cluster on the telomere of chromosome 6
(6q26-27) (Frank et al. 1988). Sequencing of the apo(a) gene has revealed it con-
tains ten different kringle IV subtypes. There are at least 34 different polymor-
phisms within the plasminogen kringle IV type 2 domain. This contributes to allelic
heterogeneity among the many apo(a) isoforms identified in human plasma (Lippi
and Guidi 2000).
Interestingly, there are also null alleles of the apo(a) gene that produce no circu-
lating Lp(a) (Cox et al. 1998). This is caused by an in frame 47 amino acid deletions
in the protease domain. This precludes the correct splicing of apo(a) mRNA, which
creates a nonfunctioning protein. This is thought to lead to improper post-­
translational N terminal glycosylation (Lippi and Guidi 2000). The apo(a) gene
cluster also includes the sequences encoding plasminogen. Apo(a) is homologous to
three other genes, which are together termed plasminogen-related genes. These are
located on chromosome 2 and 4 (Lippi and Guidi 2000).
Apo(a) has sequence homology with a diverse gene family (Byrne et al. 1994;
Magnaghi et al. 1994). This includes genes that encode proteins that are involved in
thrombosis and coagulation, such as, prothrombin, tissue type plasminogen activa-
tor (t-PA), and factor XII. Apo(a) also shares sequence homology with macrophage-­
stimulating factor and hepatocyte growth factor, the sequence homology of apo(a)
to these other genes informs on the structure and function relationship. This means
that the genes that apo(a) shares homology with inform on its mechanism (Lippi and
Guidi 2000).
152 Z. N. Safiullah et al.

Further genetic analysis has elucidated the boundaries of intron and exon
sequences are remarkably similar between these genes and only differ between 1%
and 5% (Lawn et al. 1997; Ichinose 1992). Also, the intron and exon junction posi-
tions are almost identical. These findings suggest that apo(a) and these other genes
may have developed during recent primate evolution from a common ancestral
component of the kringle-related serine protease, most likely plasminogen, via
duplication and exon shuffling (Lippi and Guidi 2000).
The apo(a) gene is most homologous with the proenzyme plasminogen. They
both share the Kringle V domain. However, the kringle IV domain of plasminogen
is in the apo(a) gene as multiple variable tandem repeats (McLean et al. 1987).
Furthermore, a point mutation in the domain homologous to the protease domain of
plasminogen deprives apo(a) of enzymatic activity (Lippi and Guidi 2000).
It is evident that Lp(a) has been conserved through evolution. This may be due to
its positive influence on wound healing, thrombosis in the face of injury and, when
at high concentrations, may be protective against diabetes. Its structural similarities
to other local genes inform on its diverse functions. Lp(a) has a complex mechanism
of action and although may seem deleterious in some contexts; it could also confer
an evolutionary advantage that is not yet fully understood.

Lp(a) and COVID-19

COVID-19 infection has led to increased mortality in those with significant pulmo-
nary disease, ischemic heart disease, diabetes, and human immunodeficiency virus
infection (Enkhmaa and Berglund 2022). COVID-19 infection leads to a hyperin-
flammatory state that is linked with an increased risk of venous thromboembolism
and cardiovascular complications (Enkhmaa and Berglund 2022).
It was hypothesized that Lp(a) has a synergistic effect with COVID-19 infection.
This follows from its proinflammatory and prothrombotic roles discussed else-
where. Briefly, because Lp(A) is an acute phase reactant and contributes to inflam-
mation, in part, by carrying oxidized phospholipids. Regarding thrombosis, one
major mechanism is the inhibition of endogenous fibrinolysis (Boffa and Koschinsky
2016). Furthermore, the stimulation of IL-6 from COVID-19 infection was thought
to promote the acute phase expression of Lp(a). When the relationship between
Lp(a) levels and COVID-19 infection was interrogated, there was no significant dif-
ference in the serum Lp(a) levels between those infected with COVID-19 and con-
trols (Enkhmaa and Berglund 2022).
However, Lp(a) levels increase over the course of hospitalization and increase in
concentrations that are associated with the severity of COVID-19 infection and
stage of acute kidney injury in these patients. In the COVID-19 population in a
small observational study, Lp(a) was not associated with IL-6, a well-known inflam-
matory marker. Taken together, these results suggest that a hyper Lp(a) state,
8 Physiological Roles and Functions of Lipoprotein(a) 153

independent of inflammation, may lead to severe COVID infection with kidney

injury (Lippi et al. 2021; Nurmohamed et al. 2022). Lp(a) was positively associated
with IL-8, which is a cytokine involved in coronary atherosclerosis, venous throm-
boembolism, and thrombotic microangiopathy. Further, Lp(a) was negatively asso-
ciated with ADAMTS13 (a von Willebrand factor-cleaving protease) and von
Willebrand factor. These observations suggest a thrombotic microangiopathy in the
pathogenesis of severe COVID-associated acute kidney injury (Lippi et al. 2021).
The absence of an association between Lp(a) and COVID-19 infection is con-
trary to several other studies that have observed elevated Lp(a) levels in inflamma-
tory diseases (Toms et al. 2011; Romero et al. 1999). One explanation as to why this
was not observed in COVID-19 infection is that Lp(a) elevation may occur after
chronic exposure. In addition, relative increases were observed in patients while
hospitalized (Enkhmaa and Berglund 2022).
Despite Lp(a) levels not being significantly more elevated in those with
COVID-19 infection, the SARS-CoV-2 infection enhanced the associations of ele-
vated Lp(a) concentrations with atherosclerotic events such as ischemic heart dis-
ease. The mechanism of this may be due to vascular preconditioning from chronic
Lp(a) exposure which may make the endothelium more susceptible to COVID-19-­
induced inflammation. In addition, Lp(a) is associated with IL-8 in COVID-19
infection. As discussed elsewhere, IL-8 promotes atherosclerotic plaque instability.
More research is needed to fully elucidate this mechanism. Lastly, how gender and
sex differences influence the relationship between Lp(a) and COVID-19 infection is
not understood at this time.
There was no association observed between COVID-19 infection and increased
risk for venous thromboembolism in those with elevated Lp(a) (Enkhmaa and
Berglund 2022). However, it was observed in hospitalized COVID-19 patients
who had the largest increase in Lp(a) relative to admission had an increased inci-
dence of VTE. The discordance in these observations may be explained by the fact
that Lp(a) by itself is not a prothrombotic factor; but rather an antifibrinolytic and
thus predominantly may cause clot-propagation in pre-existing thrombi as a “sec-
ond hit” agent. This “second hit” mechanism could already be activated at rela-
tively low Lp(a) levels, even below the ASCVD risk threshold of 50 mg/dL from
the 2019 ESC/EAS guidelines. In the case of COVID-19, severe endothelial injury
and ongoing active coagulation may be particularly sensitive to Lp(a) tipping the
balance to clot propagation and clinical expression of VTE (Nurmohamed
et al. 2022).
In summary, Lp(a) is associated with severity of COVID-19 infection and devel-
opment of severe AKI. Lp(a) serum concentrations have been observed to increase
over the course of infection in hospitalized patients. Patients with the greatest
increase observed to have a greater incidence of VTE. COVID-19 infection in those
with elevated Lp(a) at baseline is associated with increased atherosclerotic disease.
More research is needed to elucidate the mechanism of these observations and if
Lp(a) lowering therapies have a role in the treatment of COVID-19.
154 Z. N. Safiullah et al.

Conclusions

1. Lp(a) is highly versatile and participates in a broad range of physiological phe-

nomena that can have both good and bad biochemical and histological
consequences.
2. It will require a great deal of additional investigation to further delineate how
specific kringle domains, phospholipids, and the Lp(a) particle as a whole is
capable of driving such a wide spectrum of biochemical phenomena. Its pro-
teome and lipidome require much additional characterization. It will also be of
interest to determine if this lipoprotein, like others, can carry micro RNAs and if
its biochemical cargo varies as a function of the physiological milieu.
3. Work is ongoing with prospective randomized clinical trials using pharmaco-
logic interventions to lower Lp(a). It will clearly be of interest to determine if
Lp(a) lowering results in a reduction of risk for acute cardiovascular events.
4. Whether Lp(a) lowering can be harnessed to reduce aortic valve calcification,
risk for thromboembolic phenomena, some forms of malignancy, and attenuate
inflammation will also be of interest.
5. It will also be important to establish whether or not therapeutic Lp(a) lowering
associates with increased risk for diabetes mellitus, impaired wound healing,
and possible toxicity from reduced oxidized phospholipid transport.

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Chapter 9
The Role of Lp(a) in Atherosclerosis:
An Overview

Anastasiya Matveyenko, Marianna Pavlyha, and Gissette Reyes-Soffer

Introduction

Lipoprotein(a) [Lp(a)] is an apolipoprotein B100 (apoB100)-containing particle

that circulates in human plasma. It differs from other apoB lipoproteins by its lipid
composition and the presence of a covalently bound glycoprotein, apolipoprotein(a)
[apo(a)] (Berg 1963; Jawi et al. 2020). Recently published studies in cell models
have also described noncovalent bonding between apo(a) and apoB (Youssef et al.
2022). Plasma levels of Lp(a) are genetically regulated and variations can be traced
to the LPA gene locus (Utermann 1989). The gene has large homology with the
plasminogen gene. Similarities between them and effects on pathophysiology are
still being investigated (Zheng et al. 2020). Unlike other apoB-containing lipopro-
teins, the apo(a) within Lp(a) has a broad range of sizes from 300 to 800 kDa
(Lackner et al. 1991). This is due to the number of Kringle IV type 2 (KIV-2)
repeats, resulting in apo(a) isoforms ranging from 1 to greater than 40 KIV-2 repeats
(Kostner and Kostner 2017; Utermann 1999). Individuals can express one or two
apo(a) isoforms, which are synthesized in the liver and then bind to apoB particles.
There are particle composition similarities between Lp(a) and other apoB-contain-
ing lipoproteins, and even HDL (Scanu 1988). However, the mechanisms that regu-
late the synthesis and distribution of Lp(a) are not completely defined and can be
independent from LDL- and HDL-described functions. Lp(a) synthesis takes place
inside hepatocytes with likely association to apoB100 on the cell surface. There is

Anastasiya Matveyenko and Marianna Pavlyha contributed equally with all other contributors.

A. Matveyenko · M. Pavlyha · G. Reyes-Soffer (*)

Division of Preventive Medicine and Nutrition, Department of Medicine, Vagelos College of
Physicians and Surgeons, Columbia University, New York, NY, USA
e-mail: [email protected]; [email protected];
[email protected]

© The Author(s), under exclusive license to Springer Nature 159

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_9
160 A. Matveyenko et al.

no evidence that Lp(a) levels in plasma are related to lipoprotein lipase activity, and
it is unlikely that it is derived from catabolism of other lipoproteins. Similarly, Lp(a)
clearance may be regulated by various pathways (Reyes-Soffer and Ramakrishnan
2017; Chemello et al. 2022), depending on the particle composition. Several studies
show that Lp(a) clearance can be dependent on LDL receptors; however, studies
with PCSK9 inhibitors varied in their results (Reyes-Soffer and Ramakrishnan
2017; Chemello et al. 2022). The latter may be related to the use of statins and dif-
ferences in ethnicities of the cohorts evaluated (Reyes-Soffer and Ramakrishnan
2017; Chemello et al. 2022). Large studies have shown an inverse relationship
between Lp(a) plasma concentrations and isoform sizes, based on the KIV-2 repeats
(Sandholzer et al. 1992; Kraft et al. 1992; Stefanutti et al. 2020). This relationship
can account for 30–70% of the plasma levels. Notably, Black and Asian Indian eth-
nicities have higher levels of Lp(a) when compared to Caucasians, pointing again to
genetics as being one of the determining factors (Kronenberg and Utermann 2013;
Enkhmaa and Berglund 2016; Reyes-Soffer et al. 2021; Patel et al. 2021).
Additionally, small differences in Lp(a) levels have been found between men and
women (Markus et al. 2021; Forbang et al. 2016; Simony et al. 2022).

Lipoprotein(a) and Atherosclerotic Cardiovascular Disease

Elevated levels of Lp(a) are causal for atherosclerotic cardiovascular disease

(ASCVD) (Reyes-Soffer et al. 2021), as confirmed by epidemiological (Nordestgaard
et al. 2010; Bennet et al. 2008), Mendelian randomization (Burgess et al. 2018), and
genome-wide association studies (GWAS) (Kettunen et al. 2016; Tybjaerg-Hansen
2016; Nordestgaard and Langsted 2016). A large epidemiological study, looking at
records of over 6000 participants, found an association between Lp(a) levels and
Coronary Heart Disease (CHD) risk that remained regardless of adjusting for other
known risk factors such as diabetes, hypertension, lipids, and smoking status
(Bennet et al. 2008). This suggests that unlike triglycerides and C-reactive protein,
which are affected by these risk factors, the relationship between CHD and Lp(a) is
not. A Mendelian randomization study analyzed data from 62,240 patients with
CHD versus 127,299 controls and reported that the association of genetically pre-
dicted Lp(a) with CHD risk was linearly proportional to the absolute change in
Lp(a) levels (Burgess et al. 2018). Another group performed an extended genome-
wide association study with 24,925 individuals and found that genetic variation in
LPA appears to be associated with ischemic heart disease and provides support for
treatment of high Lp(a) levels for CHD risk reduction (Kettunen et al. 2016).
ASCVD has a complex biology and pathophysiology leading to various clinical
presentations. A therosclerosis starts with lipid build up in arterial wall, followed by
inflammatory cascase activation, and cell turnover (Libby et al. 2019). The last stage
of atherosclerotic progression - destabilization of lipid-rich plaque is what
9 The Role of Lp(a) in Atherosclerosis: An Overview 161

ultimately leads to events, such as myocardial infarction, stroke, and associated

increase in mortality (Libby 2013). The time course of disease development varies
due to the numerous interlinks of metabolic risk factors.
The role of apoB-containing lipoproteins in this process has been well-estab-
lished (Sniderman et al. 2019). Specifically, Lp(a) is known to have proatheroscle-
rotic, prothrombotic, and proinflammatory roles (Tada et al. 2019; Riches and Porter
2012) (Fig. 9.1). Some key signatures of its atherosclerotic profile include (Berg
1963) endothelium injury, (Jawi et al. 2020) development of lipid deposition, i.e.,
fatty streak within the vessel intima compartment, (Youssef et al. 2022) presence of
leukocytes and deposition of smooth muscle cells into the vascular wall, (Utermann
1989) presence of foam cell, i.e., macrophages, and (Zheng et al. 2020) degradation
of the extracellular matrix (Kobiyama and Ley 2018). Excessive uptake of Lp(a) by

Biology Pathophysiology Disease Risk

Lp(a) Particle (~ >22KVI2) Known Mechanism for Lp(a)

Atherosclerosis
Ox Lp(a)
apoB
apo(a) (1) endothelium injury
Phospholipids Oxpl 100
Macrophage
(2) development of lipid
deposition, i.e., fatty
KV1 streak within the Foam cell
Triglycerides
vessel intima
Cholesterol compartment
KV2
Associated
Proteins* (3) presence of leukocytes
and deposition of
Protease
KV3 smooth muscle cells
domain into the vascular wall
KV KV10 KV9 KV8 KV7 KV6 KV5 KV4
Stroke
(4) presence of foam cell,
i.e., macrophages, and
Lp(a) Particle (~ <22KV2)
(5) degradation of the
apoB extracellular matrix.
Phospholipids Oxpl 100

Less Established Roles Myocardial infarction

Inflammation
Triglycerides
apo(a) Thrombosis
Coagulation
Cholesterol
Associated
Proteins*
Genetic Variation
KV3
Protease
domain Thrombosis and plaque
KV KV10 KV9 KV8 KV7 KV6 KV5 KV4

Fig. 9.1 Lipoprotein(a): biology, pathophysiology, and disease development. (Panel a) The struc-
ture and function of circulating lipoprotein particles have been nicely described. Proteins (*) on the
lipoprotein(a) particle have led to further understanding of its link to disease development
(McCormick and Schneider 2018). (Panel b) Lp(a) has been linked to atherosclerosis and the roles
of genetics and the additional proteins bound to the particle are still to be fully described
(Kronenberg 2022). (Panel c) As an apoB100-containing particle, Lp(a) can lead to plaque forma-
tion, yet, further research is needed to understand its link to specific disease presentation. Genome-
wide association, epidemiological, and Mendelian randomization studies support its role as causal
in development of ASCVD
162 A. Matveyenko et al.

macrophages with subsequent transformation into foam cells sheds light onto its
integral role in atherogenesis. The role of Lp(a) in coronary artery disease (CAD)
has been studied previously (Rasouli et al. 2006), with a recent study reaffirming
that high Lp(a) levels are associated with increased progression of coronary low-
attenuation plaque (necrotic core) between baseline and 12 months of follow-up in
patients with advanced stable CAD (Kaiser et al. 2022). The role of Lp(a) in these
specific atherogenic processes has been studied by various authors (Marchini et al.
2021), but its exact pathophysiology, not related to conventional apoB injury, has
not been completely described.

Lipoprotein(a) and Links to Inflammation

Advancements in mass spectrometry have enhanced the ability to study Lp(a)

composition further. These new tools have led to identification of novel pathways
unique to Lp(a) which can explain the pathogenic nature of Lp(a) and its link to
ASCVD (Rodger et al. 2018; McCormick and Schneider 2018). Proteomic studies
of Lp(a) mouse models, which present elevated levels of both LDL and Lp(a),
showed that the apo(a) protein allows Lp(a) particle to bind more oxidized phos-
pholipid (OxPl) molecules than LDL, possibly involving antioxidant enzymes,
glutathione peroxidase 1 and peroxiredoxin 6 (Rodger et al. 2018). The binding of
OxPls to Lp(a) has been investigated as one of the possible culprits of its athero-
genicity (Stefanutti et al. 2020). Upon binding, there is an initiation effect of Lp(a)
on macrophages, leading to increased IL-8 expression. Notably, those individuals
who have higher levels of Lp(a) may have a higher potential for oxidized phospho-
lipid binding and subsequent atherogenic activation of Lp(a) (Berliner and
Watson 2005).
There is also an association between Lp(a) and several inflammatory conditions.
This is particularly interesting, as inflammation has been shown to be directly
involved in ASCVD risk (Ridker et al. 2017). For example, a positive relationship
between Lp(a) and interluekin-6 (IL-6) has been suggested in subjects with chronic
inflammatory conditions. Additionally, some studies show that Lp(a) may serve as
a chemoattractant for monocytes and affect IL-6 via this pathway as well (Syrovets
et al. 1997). It also influences the expression of vascular cell adhesion molecule
(VCAM)-1, E-selectin, and intracellular adhesion molecule (ICAM) in endothelial
cells (Schnitzler et al. 2020). All these play an integral role in the early plaque
development. Interestingly, during MI, monocyte levels increase dramatically
resulting in an inflammatory response (Nahrendorf et al. 2010). Additionally, Lp(a)
plays a major role in calcific aortic valve stenosis (CAVS). The latter highlighted by
human proteomic studies that found lifelong exposure to elevated Lp(a) contributes
to the development and progression of CAVS through multiple pathways (Bourgeois
et al. 2021).
9 The Role of Lp(a) in Atherosclerosis: An Overview 163

 ipoprotein(a) and Atherosclerosis: Brief Review

L
of Clinical Outcomes

Myocardial Infarction

Multiple studies have demonstrated the association between Lp(a) and myocardial
infarctions (MIs), although these were mostly conducted in Caucasian populations
(Afanasieva et al. 2022). Lp(a) levels greater than 50 mg/dL were linked to greater
risk of developing an MI (Kamstrup et al. 2008). A study looking at a Danish popu-
lation showed that there was a step-wise effect with Lp(a) levels and risk of MI
(Emerging Risk Factors Collaboration et al. 2009; Erqou et al. 2009; Langsted et al.
2015). Particularly those patients with Lp(a) levels in the 95th percentile had as high
as three to fourfold risk for an experiencing at event. This risk was noted to be
higher in men compared to women.

Aortic Stenosis

There is a strong association between Lp(a) and Aortic Stenosis (AS) with main
driver in the mechanism of the disease being oxidized phospholipids. There is also
an association between higher Lp(a) measurements with a more rapid progression
of stenosis and greater need for aortic valve replacement compared to the group
with lower Lp(a). Exposure of valvular interstitial cells to Lp(a) increases the
expression of the osteoblastic transcription factors, runt-related transcription factor
2 (RUNX2) and bone morphogenetic protein 2 (BMP2), suggesting that Lp(a) plays
a role in osteogenic differentiation of valvular interstitial cells with oxidizing phos-
pholipids playing an integral role in this mechanism (Zhiduleva et al. 2018).
Additionally, Lp(a) plays a major role in calcific aortic valve stenosis (CAVS)
Calcific aortic valve stenosis (CAVS). The latter highlighted by human proteomic
studies that found lifelong exposure to elevated Lp(a) contributes to the develop-
ment and progression of CAVS Calcific aortic valve stenosis (CAVS) through mul-
tiple pathways (Bourgeois et al. 2021).

Stroke

There has been conflicting data regarding the association between levels of Lp(a)
and stroke (Colantonio et al. 2022; Pan et al. 2022). It is well established that Black
populations are at a higher risk for CAD stroke, and mortality compared to other
ethnicities and tend to have higher levels of Lp(a). The REasons for Geographic
and Racial Differences in Stroke (REGARDS) study published data on Lp(a) and
164 A. Matveyenko et al.

stroke in a race and sex stratified cohort. Their findings, after adjusting for other
risk factors, showed a correlation between high Lp(a) levels and ischemic stroke
with higher hazard ration in Black populations. Women had higher on average
Lp(a) levels, but no statistically significant association with stroke. The driving
mechanisms are thought to be similar as in CAD and PAD, involving increased
cholesterol deposition in plaque, inflammation, and prothrombotic effects (Arora
et al. 2019).

Peripheral Artery Disease (PAD)

While there are numerous publications linking Lp(a) and CAD data pertaining to
PAD are not as robust (Norgren et al. 2007). Review of current publications does,
however, suggest that elevated levels of Lp(a) are associated with increased inci-
dence, progression, and post-treatment recurrence of PAD (Tmoyan et al. 2018).
The pathophysiology behind this relationship is driven by the ability of Lp(a) par-
ticles to migrate more easily into the subendothelial space when compared to LDL
particles (Kraaijenhof et al. 2021). This is facilitated by endothelial activation via
oxidized phospholipids and upregulation of chemokines and adhesion molecules.
Additionally, Lp(a) has been shown to compete for binding of plasminogen and
plasmin, generating a prothrombotic state (Boffa 2022). The latter is especially
important for those patients with limb threat due to tibial disease (below the knee)
(Tsimikas 2017). High Lp(a) levels have been shown to be associated with increased
incidence of claudication, symptom progression, re-stenosis after intervention, hos-
pitalization due to PAD, and limb amputation (Price et al. 2001). Patients with ele-
vated Lp(a) also have higher risk of combined PAD outcomes after adjusting for
other traditional risk factors (Kosmas et al. 2019). A recent prospective, observa-
tional study in symptomatic lower extremity arterial disease comparing patients
with high and low Lp(a) was performed. It showed that compared with low-­Lp(a)
group, patients with high-Lp(a) had a higher proportion of heart failure, CLTI, and
multivessel lesions as well as higher LDL cholesterol. A 5-year incidence of all-
cause mortality was significantly higher in the high Lp(a) cohort than in those with
low Lp(a) (48.1% vs. 27.3%). Additionally, the cumulative 5-year incidence of
major adverse limb occurrence was also significantly higher in patients with high
Lp(a) levels (67.9% vs. 27.2%) (Tomoi et al. 2022).

Lp(a): Cardiovascular Mortality

Lp(a) is a known independent risk factor for increased mortality. A study published
in 2019, examining a Danish population, reported high risk of both cardiovascular
and all-cause mortality with no difference in noncardiovascular-related mortality
(Langsted et al. 2019). Reported median survival was the lowest in those patients
9 The Role of Lp(a) in Atherosclerosis: An Overview 165

who had the highest measured Lp(a) (>93 mg/dL). The known causal factors driving
high mortality in patients with Lp(a) are mainly coronary heart disease, myocardial
infarction, atherosclerotic stenosis, and aortic valve stenosis (Nordestgaard and
Langsted 2016). Another cross-sectional study done in United Kingdom looking at
a large cohort of patients determined via Mendelian randomization that genetically
elevated Lp(a) levels were associated with parental life span. High Lp(a) levels were
also shown to be associated with increased all-cause mortality (Patel et al. 2021;
Arsenault et al. 2020).

Lipoprotein(a): Genetics and Atherosclerosis

Multiple genetic studies and significant observations of a link between Lp(a) and
cardiovascular disease risk have been published (Mehta et al. 2020; Arsenault and
Kamstrup 2022). These focus on examining associations between Lp(a) and cardio-
vascular risk, CHD, lifespan, using genetic make-up, including family history, to
better understand genetic role of Lp(a) in ASCVD. The role of genetic variants
outside and within the KIV2 region of the LPA gene have been recently described.
There have been numerous variants proposed for CAD (Clarke et al. 2009). In stud-
ies by Clarke et al., a number of chromosomal regions were associated with risk of
CAD, and the LPA locus region 6q26–27 has the strongest relationship between
high Lp(a) levels and risk of CAD (Clarke et al. 2009). Various single nucleotide
polymorphisms (SNPs), rs10455872 and rs3798220 within the LPA site, have been
described and are highly associated with high Lp(a) levels. These variants are more
common in those of European ancestry. In the work of Kamstrup and Nordestgaard,
the genotypes mentioned above and high Lp(a) levels were associated with an
increased risk of heart failure, consistent with causal association (Kamstrup and
Nordestgaard 2016). More recently, the same authors have highlighted the effects of
Lp(a) on morbidity and mortality (Simony et al. 2022). Beyond the effect of specific
single nucleotide polymorphisms (SNPs), possible SNP-SNP interactions and SNPs
in the KIV-2 repeat region have to be taken into account, which might not be picked
up by conventional sequencing methods (Coassin and Kronenberg 2022). Work in
the cohort from Pakistan (Saleheen et al. 2017) at risk for myocardial infarction
showed additional SNPs and that both, smaller apo(a) isoform size and high Lp(a)
levels, are independent and causal risk factors for CAD. In studies of diverse
cohorts, such as the Atherosclerosis Risk in Communities (ARIC), Lp(a) measured
at middle age of participants was significantly associated with valvular and vascular
calcification at older age, represented by aortic valve calcium, mitral valve calcifi-
cation, and other factors (Obisesan et al. 2022). In this cohort, plasma Lp(a) levels
and family history of cardiovascular disease had independent and additive joint
associations with cardiovascular risk (Mehta et al. 2020). Another study, looking at
mostly males from Southeast Asia, found that Lp(a) levels in plasma are a positive
predictor of coronary artery disease and acute myocardial infarction (Loh
et al. 2022).
166 A. Matveyenko et al.

 ffects of Available Treatments on Lp(a) and Risk

E
of Atherosclerosis

There are currently no targeted approved pharmacologic therapies that lower

Lp(a) concentrations. However, some therapies lower apoB and LDL cholesterol
(LDL-C), decrease Lp(a) modestly (niacin—20%; CETP inhibitor—24–36%;
ApoB antisense therapy—26–27%; microsomal triglyceride transfer protein
inhibitor—17%; Anti-IL6R—30–37%) (van Capelleveen et al. 2016). Proprotein
convertase subtilisin:kexin type 9 (PCSK9) inhibition showed a 25% reduction in
Lp(a), and after one year of treatment, reduced the event rate for acute cardiovas-
cular events (Kaiser et al. 2022). However, that could be due to the combined
effect of lowering LDL-C with Lp(a). Lipoprotein apheresis is approved for treat-
ment of high Lp(a) for specific patients at increased risk in the United States
(Nugent et al. 2020). Regular lipoprotein apheresis has been approved in Germany
for lipoprotein(a) hyperlipoproteinemia with progressive cardiovascular disease
since 2008 (Roeseler et al. 2016). A study conducted in Germany looked at 36,745
lipoprotein apheresis treatments of 118 patients to analyze the efficacy, safety, and
tolerability (Heigl et al. 2015). Average annual rate of major adverse coronary
events was reduced by 79.7% for all patients after beginning lipoprotein apheresis
(Heigl et al. 2015). Overall, the procedure was well-tolerated and effective for
CVD risk reduction (Heigl et al. 2015). There have been since developed aphere-
sis preferential for Lp(a) using antibodies against apo(a), targeting people with
high Lp(a) and otherwise normal lipid levels (Waldmann and Parhofer 2019).
Likewise in the US, similar results were seen with the use of lipoprotein apheresis
in patients with high Lp(a) and relatively normal LDL_C, showing an improve-
ment in cardiovascular events (Moriarty et al. 2019). Various targeted treatment
programs are in phase 2 and phase 3 of development (Reyes-Soffer et al. 2021),
mostly decreasing the synthesis of apo(a) using biologicals and RNA inteferring
agents (Tokgözoğlu and Libby 2022).
It is not clear whether decreasing apo(a) alone versus decreasing apoB and
apo(a) will be beneficial (i.e., decrease event rate and mortality) to individuals
with isolated high Lp(a) levels with these therapies. Furthermore, due to addi-
tional protein components associated with Lp(a) and links to disease mechanism
(i.e., inflammation), other targets for treatments may be considered. These promis-
ing therapies may bring an additional benefit to those patients who are already at
a high risk for CVD due to their elevated apoB and LDL-C and are on optimal
therapy. 2019 European guidelines make recommendations to access Lp(a) level
along with history of heart disease and other known risk factors when devising a
long-term patient care plan for best future outcomes (Mach et al. 2019). Further
randomized trials are needed to gain more insight whether these therapies or new
treatments targeting Lp(a) will change patient management and disease
progression.
9 The Role of Lp(a) in Atherosclerosis: An Overview 167

Conclusions

Lp(a) is an atherogenic lipoprotein present in human plasma with higher levels cor-
responding to an elevated risks for ASCVD. It has a strong genetic predisposition
and its mechanisms of action have been linked to the propagation of atherogenic
cascade via alteration of macrophage gene expression. Although there are currently
no widely prescribed Lp(a)-lowering treatments in the United States, there are avail-
able therapies, whose utility in clinical practice, needs to be further studied.

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Chapter 10
Molecular Mechanisms of Lipoprotein(a)
Pathogenicity: Tantalizing Clues
and Unanswered Questions

Michael B. Boffa and Marlys L. Koschinsky

Introduction

Although lipoprotein(a) (Lp(a)) was discovered almost 50 years ago (Berg and New
1963) and has been subsequently shown to be a causal and independent risk factor
for atherosclerotic cardiovascular disease (ASCVD) and calcific aortic valve dis-
ease (CAVD) (Arsenault and Kamstrup 2022), the mechanisms by which Lp(a)
mediates its pathogenic effects in vivo remain unclear. Lp(a) comprises an
apoB-­100-containing lipoprotein to which is attached the unique apolipoprotein(a)
(apo(a)) moiety (Fig. 10.1). Amino acid analysis followed by complete sequencing
of the human apo(a) cDNA in 1987 revealed a high level of sequence identity with
the profibrinolytic enzyme plasminogen (Eaton et al. 1987; McLean et al. 1987).
Apo(a) contains a series of tri-looped structures called kringles that are similar to
the KIV domain of plasminogen, followed by sequences similar to the plasminogen
KV and protease domains (Fig. 10.1). Due to several critical amino acid substitu-
tions and a small deletion, the apo(a) protease-like domain has been shown to be
catalytically inactive (Gabel and Koschinsky 1995).

M. B. Boffa (*)
Robarts Research Institute, Schulich School of Medicine and Dentistry, The University of
Western Ontario, London, ON, Canada
Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of
Western Ontario, London, ON, Canada
e-mail: [email protected]
M. L. Koschinsky
Robarts Research Institute, Schulich School of Medicine and Dentistry, The University of
Western Ontario, London, ON, Canada
Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of
Western Ontario, London, ON, Canada
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 173

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_10
174 M. B. Boffa and M. L. Koschinsky

Fig. 10.1 Structure and functional domains of Lp(a). Lp(a) consists of apo(a) covalently linked to
the apoB-100 moiety of an LDL-like lipoprotein particle. The lipid portion of the particle is a shell
of phospholipids (PL) and free cholesterol (FC) surrounding a neutral lipid core of cholesteryl
esters (CE) and triacylglycerols (TG). Apo(a) consists of ten types of KIV domains, a KV domain,
and an inactive protease domain. KIV2 is repeated different numbers of times in different apo(a)
isoforms. KIV5–KIV8 contain weak lysine-binding sites (wLBS), with those in KIV7 and KIV8
binding to specific lysine residues in apoB-1000 during the noncovalent step of Lp(a) assembly.
KIV9 contains a single-free cysteine that mediates disulfide bond formation with apoB-100.
KIV10 contains a strong lysine-binding site (sLBS) as well as covalently bound oxidized phospho-
lipid (OxPL). The sLBS is required for OxPL addition, and together these features promote several
pathogenic effects on vascular and immune cells. OxPL is also present noncovalently associated
with the lipid moiety of Lp(a), and accounts for up to 50% of the total OxPL on Lp(a). EC endo-
thelial cell, SMC smooth muscle cell, VIC valve interstitial cell
10 Molecular Mechanisms of Lipoprotein(a) Pathogenicity: Tantalizing Clues… 175

Apo(a) kringle IV sequences are present in ten types based on amino acid
sequence; these have been designated KIV1–KIV10 (McLean et al. 1987; van der
Hoek et al. 1993). The KIV2 sequence is present in a variable number of identically
repeated copies (from 3 to greater than 40) which is a hallmark of Lp(a) and reflects
allele size variation in LPA, the gene encoding apo(a) (Fig. 10.1) (Lackner et al.
1993; Marcovina et al. 1996). Interestingly, there is a strong inverse correlation
between apo(a) size and Lp(a) plasma levels, which likely arises due to less efficient
secretion of larger isoforms as a result of presecretory degradation of misfolded spe-
cies in the endoplasmic reticulum (Boffa and Koschinsky 2022). The KIV9 domain
houses the only unpaired cysteine in apo(a) and is involved in disulfide bond forma-
tion with a cysteine residue in the carboxyl-terminus of apoB-100 (Koschinsky
et al. 1993). The KIV5–8 domains each contain a weak lysine-binding site (wLBS);
the wLBS in KIV7 and KIV8 is required for intracellular noncovalent interaction
between apo(a) and apoB that precedes extracellular disulfide bond formation
(Fig. 10.1) (Becker et al. 2004; Youssef et al. 2022).
The apo(a) KIV10 domain contains a relatively strong lysine-binding site (sLBS)
that has been studied extensively in attempts to understand the pathophysiology of
Lp(a) in the vasculature. Lp(a) has been demonstrated to be the preferential lipopro-
tein carrier of proinflammatory oxidized phospholipids (OxPL), compared to LDL
(Bergmark et al. 2008). These species are present both on the lipid portion of Lp(a)
as well as covalently associated with apo(a) (Bergmark et al. 2008; Leibundgut
et al. 2013). Interestingly, in this regard, it has been shown that the KIV10 sLBS is
absolutely required for the covalent addition of oxidized phospholipid to this krin-
gle, likely involving addition of the OxPL adduct to a histidine side chain through
Michael reaction addition (Fig. 10.1) (Leibundgut et al. 2013; Scipione et al. 2015).
The proinflammatory effect of the OxPL on KIV10 has been demonstrated in many
studies, both in vitro and in vivo (Koschinsky and Boffa 2022; Dzobo et al. 2022).
In vitro studies have shown the role of OxPL on KIV10 in promoting proinflamma-
tory and phenotypes in a variety of vascular and inflammatory cells including valve
interstitial cells (VICs) (see below).
Many studies using a variety of vascular cell types have shown that the apo(a)
sLBS can compete with plasminogen for binding to cell surfaces, thereby inhibiting
plasminogen activation to the active enzyme plasmin (Boffa 2022). Downstream
effects on fibrin clot lysis have also been studied, with variable results, and the sig-
nificance of Lp(a) in promoting thrombosis in the arterial and venous circulation
remains controversial (Boffa 2022). Indeed, Lp(a) appears to confer risk for venous
thromboembolism only in individuals with extremely high Lp(a) levels (Kamstrup
et al. 2012). The role of Lp(a) in platelet function and coagulation, and in the lysis
of platelet-rich clots, is not clear (Boffa 2022). Importantly, it is difficult to assess
the contribution of Lp(a) to lysis of clots formed upon rupture of vulnerable athero-
sclerotic plaques (see below).
Despite the demonstration of elevated plasma Lp(a) levels as an independent and
causal risk factor for ASCVD and CAVD, the mechanism of action of Lp(a) in these
disease processes remains unclear. This reflects, in part, the complexity of the Lp(a)
structure, as well as the lack of suitable animal models for Lp(a); together these
176 M. B. Boffa and M. L. Koschinsky

present significant challenges to understanding the molecular and cellular basis of

Lp(a) pathogenicity. Indeed, LPA is only present in Old World monkeys and apes
and humans. Of note, the Old World species all lack a functional LBS in KIV10
preventing covalent OxPL addition to this kringle. Work on transgenic mice express-
ing human Lp(a) is progressing (Yeang et al. 2016), and ultimately should comple-
ment the significant insights that are being made on probing the effect of Lp(a) on
human vascular and valve interstitial cell phenotypes (Fig. 10.2).

Fig. 10.2 Overlapping pathogenic mechanisms of Lp(a) in atherosclerosis and calcific aortic
valve disease. There are several common mechanisms mediated by Lp(a) in the two disorders.
Compromised endothelial cell function leads to barrier permeability, infiltration of Lp(a) and
monocytes, expression of endothelial cell surface receptors for monocytes, and mural thromboses.
Lp(a) activates monocytes leading to cytokine secretion and enhanced potential for transendothe-
lial migration. Within the vessel wall or valve, Lp(a) promotes macrophage foam cell formation
and macrophage apoptosis, as well as stimulating the release of proinflammatory cytokines such as
interleukin-8 (IL-8) from macrophages. Lp(a) promotes smooth muscle cell (SMC) migration and
proliferation in the arterial intima. Lp(a) also promotes calcification of SMC in the atrial intima
and osteogenic differentiation and calcification of valve interstitial cells. Many functions of Lp(a),
indicated in red, are mediated by its bound oxidized phospholipid (OxPL). Lp(a) also transports
the phospholipase D enzyme autotaxin into the aortic valve leaflet, where it catalyzed generation
of the highly proinflammatory lysophosphatidic acid (lysoPA) using lysophosphatidylcholine
(lysoPC) as a substrate. The OxPL on Lp(a) thus helps to explain why elevated Lp(a) is a causal
risk factor for both atherothrombosis and aortic stenosis, despite the disease processes underlying
each of these disorders being distinct. TNF-α tumor necrosis factor-α
10 Molecular Mechanisms of Lipoprotein(a) Pathogenicity: Tantalizing Clues… 177

Effect of Lp(a) on Vascular and Immune Cell Phenotype

Effects of Lp(a) on Vascular Endothelium

The vascular endothelial cell layer is critical in maintaining a nonpermeable barrier

that protects the vessel wall from exposure to blood contents. As such, there have
been a number of studies aimed at determining the effect of Lp(a) on endothelial
function. Lp(a) deposition in the intimal layer of the arterial wall was reported nearly
30 years ago and suggested that Lp(a) can cross the endothelial cell layer and be
preferentially retained in this milieu compared to LDL (Rath et al. 1989). In 2004, it
was reported that the apo(a) component of Lp(a) elicits a dramatic rearrangement of
the actin cytoskeleton characterized by increased central actin stress fiber formation,
redistribution of focal adhesions, and VE-cadherin disruption; these effects are a
consequence of apo(a)-mediated activation of the Rho-Rho kinase signaling path-
way leading to increased myosin light chain phosphorylation (Pellegrino et al. 2004).
A subsequent study showed that these effects are the product of increased phos-
phorylation of the myosin phosphatase regulatory subunit and hence inhibition of
myosin phosphatase activity, that Lp(a) and apo(a) resulted in enhanced EC perme-
ability, and that the KIV10 sLBS was required for these effects (Cho et al. 2008).
Therefore, increasing EC permeability represents a mechanism by which Lp(a) can
elicit a program of EC dysfunction in early atherosclerosis, facilitating deposition of
Lp(a) and LDL in the vessel wall (Fig. 10.2). In a follow-up study, enhanced prosta-
glandin E2 synthesis and secretion were observed when cultured HUVECs were
treated with apo(a) as a result of stimulation of β-catenin nuclear translocation and
increased cyclooxygenase activity (Cho et al. 2013). Lp(a) and apo(a) were shown to
activate a phosphatidylinositol-3 kinase and Akt-dependent pathway that resulted in
phosphorylation and inhibition of GSK3β to promote β-catenin translocation; once
again, these effects were attributable to the KIV10 domain of apo(a) (Cho et al. 2013).
Early studies demonstrated the ability of Lp(a) to elicit a proinflammatory
response in HUVECS through enhanced expression of E-Selectin, VCAM1, and
ICAM1 (Fig. 10.2) (Takami et al. 1998; Allen et al. 1998). The apo(a) component of
Lp(a) binds to the β2-integrin Mac-1 in a lysine-dependent manner; this in turn
promotes the adhesion of THP-1 monocytes to ECs and enhances their Mac-1-­
dependent transendothelial migration (Fig. 10.2) (Sotiriou et al. 2006). Interestingly,
the Lp(a)-Mac-1 binding resulted in increased expression of tissue factor in these
cells. Furthermore, the interaction between apo(a) and Mac-1 induces activation of
the inflammatory transcription factor NFκB (Sotiriou et al. 2006). Taken together,
these studies define a novel role for apo(a)/Lp(a) in promoting inflammatory cell
recruitment, which may represent a novel mechanism for Lp(a) atherogenicity
in vivo (Fig. 10.2). In a more a recent study, the oxPL on KIV10 was shown to acti-
vate human aortic endothelial cells, resulting in transendothelial migration of mono-
cytes (Fig. 10.2) (Schnitzler et al. 2020). Transcriptome analysis of Lp(a)-stimulated
ECs showed upregulation of inflammatory pathways related to monocyte adhesion
and migration; these effects increased 6-phophofructo-2-kinase/fructose-2,6-­
biphosphatase (PFKFB)-3-mediated glycolysis and could be abolished by inhibi-
tion of PFKFB3 (Schnitzler et al. 2020).
178 M. B. Boffa and M. L. Koschinsky

Effects of Lp(a) on Vascular Smooth Muscle Cells

A number of early in vitro studies suggested that Lp(a) could contribute to cul-
tured vascular SMC migration and proliferation through the ability of apo(a) to
inhibit the plasmin-dependent activation of TGF-β (Fig. 10.2) (Grainger et al.
1993, 1994; O’Neil et al. 2004). More recently, the ability of OxPL on apo(a)
KIV10 to stimulate the expression of Klf-4, an important factor in phenotypic
switching of SMCs in atherosclerotic lesions, was attributed to the apo(a)-medi-
ated activation of the long noncoding RNA MIAT (Fig. 10.2) (Fasolo et al. 2021).
Lp(a) has also been implicated in the calcification of human aortic SMCs through
Notch1-NFκB and Notch1-BMP2-Smad1/5/9 pathways (Peng et al. 2022). The
Notch1-NFκB pathway resulted in increased osteopontin and inflammatory cyto-
kine expression, while Lp(a)-mediated Notch1-BMP2-Smad1/5/9 activation also
contributed to calcification of the cells. The ability of Lp(a) to increase VSMC
calcification is another mechanism by which Lp(a) contributes to vascular disease
(Fig. 10.2).

Lp(a) and Monocyte/Macrophage Phenotype

It is well-established that Lp(a) can contribute to macrophage foam cell formation

(Fig. 10.2) (Keesler et al. 1996). However, the role of the OxPL on apo(a) in mac-
rophage function is a relatively recent finding. The first direct evidence of the
OxPL on Lp(a)/apo(a) mediating a proinflammatory response in macrophages was
provided by Seimon and coworkers (Seimon et al. 2010); these investigators dem-
onstrated that the OxPL was capable of inducing apoptosis in endoplasmic reticu-
lum (ER)-stressed macrophages in a CD36/TLR2-dependent manner (Fig. 10.2).
Lp(a)/apo(a) has also been shown to contribute to monocyte recruitment through
enhancing secretion of chemokines I-309 and interleukin(IL)-8 from cultured
macrophages (Fig. 10.2) (Scipione et al. 2015; Haque et al. 2000; Edelstein et al.
2003). In our own studies of the apo(a)-induced IL-8 expression in macrophages,
we conclusively determined that this stimulatory effect was attributable to the
covalent OxPL modification on apo(a) (Scipione et al. 2015). The apo(a)/OxPL-
induced signaling cascade in our study also suggested a role for CD36/TLR2 and
involved the JNK- and ERK-dependent activation of NFκB—a well-known series
of molecular events in inflammatory pathways—in response to OxPL-containing
apo(a). The OxPL moiety on apo(a) has also been implicated in the proinflamma-
tory priming of human peripheral blood mononuclear cells (Fig. 10.2) (van der
Valk et al. 2016). The same study used high-resolution in vivo imaging to show
that monocytes from high Lp(a) individuals had a propensity of trafficking to the
arterial wall, a result not seen in subjects with lower Lp(a) levels (van der Valk
et al. 2016).
10 Molecular Mechanisms of Lipoprotein(a) Pathogenicity: Tantalizing Clues… 179

Lp(a) and Valve Interstitial Cell Phenotype

Recent studies have demonstrated a key role for Lp(a) in both the development and
progression of aortic valve disease (Thanassoulis et al. 2013; Capoulade et al. 2015).
This is likely mediated, in large part, by the ability of the OxPL component of
apo(a) to modify the phenotype of valve interstitial cells to resulting in proinflam-
matory and pro-osteogenic responses in these cells (Fig. 10.2). In a recent study by
Zheng and coworkers, treatment of VICs by Lp(a) or recombinant apo(a) resulted in
osteogenic differentiation in these cells through the induction of IL6, BMP2, and
RUNX2 expression (Zheng et al. 2019). The effects were attributed to the OxPL on
Lp(a) and apo(a): the anti-oxPL antibody E06 blocked the effects of Lp(a) as did
mutation of the KIV10 LBS which significantly reduced the effect of apo(a) (Zheng
et al. 2019). OxPLs transported by Lp(a) also increase the load of reactive oxygen
species in the aortic valve, loading to ROS-mediated activation of the NFκB path-
way, and induction of a program of inflammatory gene expression (Bouchareb et al.
2015; Mathieu et al. 2017). Additionally, Lp(a) binds to autotaxin and delivers it to
valves (Bouchareb et al. 2015; Mathieu et al. 2017); autotaxin promotes inflamma-
tion and osteogenic transdifferentiation of VICs through the production of LysoPA
which in turn binds and signals through the lysoPA receptor (Fig. 10.2). Taken
together, these studies suggest that Lp(a) can initiate a program of inflammation and
osteoblastic differentiation in valvular interstitial cells which is a major contributing
factor to AVS and CAVD. Lp(a) could also promote CAVD through promotion of
valve endothelial cell dysfunction, immune cell infiltration, and foam cell formation
(Fig. 10.2).

Effect of Lp(a) on Thrombosis and Thrombolysis

The homology of apo(a) and plasminogen revealed by cloning of a cDNA-encoding

apo(a) immediately invited speculation of an antifibrinolytic role for Lp(a) (McLean
et al. 1987). However, the decades of research that have ensued have yet to provide
a definitive answer on this question (Boffa 2022). There is certainly a large body of
evidence from in vitro studies pointing to the ability of Lp(a) and—especially—
apo(a) to inhibit plasminogen activation and impede fibrinolysis (Fig. 10.3). The
earliest studies showed that Lp(a) could compete with plasminogen for binding to
fibrin, endothelial cells, and monocytes (Miles et al. 1989; Hajjar et al. 1989; Rouy
et al. 1992). Subsequent studies showed that Lp(a) and/or apo(a) could inhibit lysis
of fibrin clots and inhibit plasminogen activation on the surface of fibrin, fibrin deg-
radation products, and platelets (Sangrar et al. 1995, 1997; Hancock et al. 2003;
Ezratty et al. 1993). Definitive demonstration of inhibition on plasminogen activa-
tion on the cell surface was only recently provided by our group (Romagnuolo et al.
2014, 2018a, b). Apo(a) was shown to inhibit thrombolysis in rabbit jugular vein
180 M. B. Boffa and M. L. Koschinsky

Fig. 10.3 Effects on imbalance between coagulation (formation of fibrin by thrombin cleavage of
fibrinogen) and fibrinolysis (degradation of insoluble fibrin into soluble fibrin degradation prod-
ucts) can cause thrombosis. Lp(a) and apo(a) promote this imbalance by favoring coagulation
(green mechanisms) and impeding fibrinolysis (red mechanisms). TFPI tissue factor pathway
inhibitor

and mouse pulmonary embolism models (Biemond et al. 1997; Palabrica et al.
1995); notably, however, Lp(a) itself was not tested in these studies. Indeed, the
available data from human epidemiological and genetic studies do not provide evi-
dence for a direct antifibrinolytic/prothrombotic impact of elevated Lp(a) (Boffa
2022). Moreover, in a recent study using subjects undergoing Lp(a) lowering with
antisense oligonucleotide therapy, we found that despite dramatic reductions in
plasma Lp(a) concentrations, ex vivo plasma clot lysis time was not affected (Boffa
et al. 2019). Furthermore, recombinant apo(a) had a potent antifibrinolytic effect
whereas Lp(a) purified from human plasma lacked this effect (Boffa et al. 2019).
Against this backdrop, we summarize below the clinical evidence with respect to
Lp(a) and thrombosis and thrombolysis, and we outline areas for future studies of
this issue.

Is There Direct Evidence That Lp(a) Inhibits Fibrinolysis?

Earlier observational studies provided mixed results concerning whether elevated

Lp(a) is a risk factor for the development of VTE (Boffa and Koschinsky 2016).
This is notable in the sense that similar studies of atherosclerotic cardiovascular
disease (ASCVD) quite consistently showed elevated Lp(a) to be an independent
10 Molecular Mechanisms of Lipoprotein(a) Pathogenicity: Tantalizing Clues… 181

risk factor. With the advent of genetic approaches to study the association of geneti-
cally elevated Lp(a) with disease—including Mendelian randomization studies—
the opportunity to assess a causal role for elevated Lp(a) in VTE and to eliminate
confounding variables has arisen. Several large studies using genetic approaches
have been published. All showed that genetically inherited elevated Lp(a) or geneti-
cal markers of high Lp(a) were not significant predictors of VTE (Kamstrup et al.
2012; Helgadottir et al. 2012; Danik et al. 2013; Larsson et al. 2020). Importantly,
in several of these studies, a causal role for elevated Lp(a) in the development of
atherothrombotic disorders was observed in the same population (Kamstrup et al.
2012; Helgadottir et al. 2012; Larsson et al. 2020). However, a posthoc observa-
tional study of one of these populations found that extremely high Lp(a) levels
(≥95th percentile) were significantly associated with VTE (Kamstrup et al. 2012).
A recent retrospective study of pulmonary embolism patients found no correlation
between Lp(a) levels and the severity of pulmonary embolism (Gressenberger
et al. 2022).
The general lack of association between elevated Lp(a) and risk for VTE (except
for extremely high Lp(a) concentrations) is consistent with a minimal or absent
antifibrinolytic ability of Lp(a). Venous thrombi are fibrin- and erythrocyte-rich and
form as a consequence of blood stasis, hypercoagulability, and endothelial damage.
Thrombi in the arterial tree are platelet-rich and fibrin-poor, and generally form as a
sequela of atherosclerotic plaque erosion or rupture. Thus, mechanistic implications
of associations between Lp(a) levels and ASCVD endpoints are confounded by the
possibility that Lp(a) may contribute to atherosclerosis, thrombosis, or both.
Interestingly, a consistent observation (albeit from relatively small sample sizes)
has been the association between elevated Lp(a) levels and risk of ischemic stroke
in children (Nowak-Gottl et al. 1999; Strater et al. 2002; Kenet et al. 2010;
Goldenberg et al. 2013). These events are frequently seen in patients with inherited
dispositions toward thrombophilia such as Factor V Leiden. That the events would
occur in the absence of underlying atherosclerosis are clear from the young age of
the patients, and this may speak to a prothrombotic or antifibrinolytic effect of Lp(a).
Early studies examined the proposition that elevated Lp(a) could reduce the effi-
cacy of thrombolytic therapy. Most of these occurred in the setting of acute myocar-
dial infarction (MBewu et al. 1994; Tranchesi et al. 1991; Armstrong et al. 1990;
von Hodenberg et al. 1991; Brugemann et al. 1994), although one examined isch-
emic stroke (Ribo et al. 2004). None of these studies showed that Lp(a) levels are a
significant predictor of successful recanalization, although all of them were limited
by small sample sizes (and thus few patients with high Lp(a)) and/or having sam-
pled blood for Lp(a) measurement in the postinfarct period where the acute phase
response may have increased Lp(a) levels (Boffa 2022).
Taken together, the jury is still out on whether Lp(a) inhibits fibrinolysis
in vivo. Further studies in animal models may be required to assess this question,
and further assessment of the impact of Lp(a) on thrombolytic therapy in the set-
ting of ischemic stroke, deep vein thrombosis, and pulmonary embolism may be
warranted.
182 M. B. Boffa and M. L. Koschinsky

Could Lp(a) Promote Thrombosis Indirectly?

Elevated Lp(a) is clearly and consistently associated with ASCVD events, though
less so with intermediate phenotypes such as intimal-medial thickness and coronary
calcium scores (Kivimaki et al. 2011; Raitakari et al. 1999; Razavi et al. 2021;
Mehta et al. 2022). This can be interpreted to mean that Lp(a) plays a more impor-
tant role in precipitating atherothrombotic events, rather than in promoting the
underlying atherosclerotic process. Two scenarios can be contemplated.
1. Lp(a) could be promoting thrombus formation directly through an impact on the
coagulation system or on platelet activation (Fig. 10.3). Little to no data on an
effect of Lp(a) on coagulation have been published, although early studies
showed that Lp(a) could exert a procoagulant effect by binding and inhibiting
tissue factor pathway inhibitor (Fig. 10.3) (Caplice et al. 2001). We and others
have shown that Lp(a) and apo(a) can impact fibrin clot structure, leading to a
form resistant to lysis (Scipione et al. 2017; Skuza et al. 2019; Rowland et al.
2014) (Fig. 10.3); we also demonstrated that apo(a) could accelerate the rate of
fibrin formation, which could also impact clot structure (Scipione et al. 2017).
Lp(a) does bind to platelets (Ezratty et al. 1993; Martinez et al. 2001), and Lp(a)
and apo(a) have been shown increase the proaggregant effect of certain agonists
(such as the protease-activated receptor-1 ligand peptide SFLLRN and arachi-
donic acid) in washed platelets (Martinez et al. 2001; Rand et al. 1998). However,
two recent studies in platelet-rich plasma showed that Lp(a) level did not predict
the aggregation response to several agonists including ADP, collagen, or arachi-
donic acid (Salsoso et al. 2020; Kille et al. 2021).
2. Lp(a) could be promoting a “vulnerable” plaque phenotype with a greater pro-
pensity to rupture and hence cause an atherothrombotic event (Fig. 10.3).
­Consistent with this scenario, it was reported in a carotid ultrasound study that
Lp(a) level predicted the extent of stenosis but not total plaque area (Klein et al.
2008); the extent of stenosis could be interpreted to reflect ongoing rupture and
thrombus formation. The proinflammatory effects of Lp(a) owing to its OxPL
could result in a larger necrotic core and/or a thinner fibrous cap, both hallmarks
of rupture-prone plaques. Very little direct study of this question has been
attempted. In an immunohistochemical study of human coronary atherosclerotic
plaques of varying phenotypes, apo(a) immunostaining was found in proximity
to oxidation-­specific epitopes—such as the OxPL recognized by E06—specifi-
cally in vulnerable or ruptured plaques (van Dijk et al. 2012).
It is clear from the above that, while some evidence for Lp(a)-promoting vul-
nerable plaque and/or arterial thrombosis exists, more research is necessary. This
will require both work in animal models, such as transgenic mice expressing
human Lp(a), as well as more imaging and biomarker studies in human patients.
Such research is important because it may help to stratify risk in patients with high
Lp(a) to identify those who would most benefit from emerging Lp(a)-lowering
therapies.
10 Molecular Mechanisms of Lipoprotein(a) Pathogenicity: Tantalizing Clues… 183

Concluding Remarks

These are exciting times in the Lp(a) field, with burgeoning interest in this causal
risk factor for CVD on from both basic researchers and clinicians. The quantitative
importance of elevated Lp(a) as a risk factor is now widely accepted, although at
present clinical adoption of Lp(a) measurement has lagged because of a lack, cur-
rently, of Lp(a)-lowering therapies. With Phase III cardiovascular outcomes trials in
progress on an antisense oligonucleotide-targeting LPA mRNA, we are potentially
on the cusp of having an effective treatment for lowering Lp(a) as well as definitive
proof that elevated Lp(a) is harmful. At the same time, our understanding of the
pathogenic mechanisms of Lp(a) continues to expand, with the role of Lp(a) as a
proinflammatory mediator emerging as a key factor. Further studies of these mecha-
nisms may lead to an alternative therapeutic strategy to mitigate the effect of ele-
vated Lp(a)—interference with its pathogenic effects in the vasculature.

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Chapter 11
Thrombosis, Inflammation,
and Lipoprotein(a): Clinical Implications

Maya S. Safarova and Patrick M. Moriarty

Lipoprotein(a) and Homeostasis: Multiple mechanisms mediate thrombus forma-

tion, including activation of platelets, coagulation, and fibrinolysis (Fig. 11.1). This
figure is a schematic summary of clot formation and propagation, highlighting the
complexity of interactions between lipoprotein(a) (Lp(a)), coagulation, fibrinolytic,
and inflammatory factors. Lp(a), apo(a), and its fragments can bind to the extracel-
lular matrix of arterial and venous walls. Changing conditions of blood flow and
high shear stress impact the interplay between thrombus development in the arterial
and venous vessels. Elevated levels of Lp(a) mediate thrombus formation and slow
plasmin generation through tissue- and urokinase-plasminogen activator (t-PA and
u-PA), plasminogen activator inhibitor 1 (PAI-1), alpha 2 antiplasmin, thrombin
activatable fibrinolysis inhibitor (TAFI), thrombin released from activated platelets,
elastase released from polymorphonuclear leucocytes. Lp(a) can bind to fibrin.
Apo(a) can inactivate tissue factor pathway inhibitor (TFPI), which augments factor
VII and X activation-promoting blood coagulation. Further, LPA (encodes Lp(a)/
apo(a)) and PLG (encodes plasminogen) genes are organized in a head-to-head con-
figuration and share an intergenic region (~50,000 base pairs) on the sixth
chromosome. Through an interleukin 6 (IL-6) response element -CTGGGA- of the

M. S. Safarova
Atherosclerosis and Lipid Genomics Laboratory, Mayo Clinic, Rochester, MN, USA
Department of Cardiovascular Medicine, University of Kansas Hospital and Medical Center,
Kansas City, KS, USA
e-mail: [email protected]
P. M. Moriarty (*)
Division of Endocrinology, Diabetes and Clinical Pharmacology, Department of Internal
Medicine, University of Kansas Medical Center, Kansas City, KS, USA
Atherosclerosis and Lipoprotein Apheresis Center, University of Kansas Medical Center,
Kansas City, KS, USA
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 189

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_11
190 M. S. Safarova and P. M. Moriarty

Fig. 11.1 Lp(a) mediates components of the endogenous fibrinolytic system and oxidation

LPA gene, IL-6 can induce apo(a) expression. As shown, transcription factor ELK1
mediates expression of apo(a) through the Ets domain in the LPA promoter; fibro-
blast growth factor 19 (FGF19) has inhibitory effects on apo(a) expression. The
oxidized phospholipids (oxPL)-Lp(a) complex can upregulate adhesion molecules,
increase secretion of chemo-attractants and cytokines, interact with various signal
transduction receptors on the cell surface, and modulate binding of leukocytes to
endothelial cells. Cells can recognize oxPL through scavenger receptors (SR) CD36
and SR class B type 1 (SR-B1), prostaglandin E2 receptor 2 subtype (EP2), vascular
endothelial growth factor receptor 2 (VEGFR2), and the platelet-activating factor
(PAF) receptor. Smaller Lp(a) isoforms have stronger association with
oxPL. Oxidation of Lp(a) increases clot density. Role of Lp(a)-targeted therapies in
mediating levels of pro-inflammatory, pro-thrombotic, and antifibrinolytic markers
and its effects on clinical outcomes needs further investigation. In this chapter, we
discuss clinical relevance and implications of pro-thrombotic and pro-inflammatory
states associated with Lp(a).
Clinical Vignette
A previously healthy 11-year-old boy was admitted for a 5-day history of head-
aches, dizziness, and lethargy. His exam showed visual field defects and memory
impairment. Magnetic resonance imaging of the brain demonstrated acute strokes in
the right parietal, left cerebellar, left thalamic, and bilateral frontal lobes. An
11 Thrombosis, Inflammation, and Lipoprotein(a): Clinical Implications 191

extensive hypercoagulable, cardiac, immunological, inflammatory, and oncological

work-up was unremarkable. No history of head or neck trauma was elicited. He was
found to have an elevated lipoprotein(a) (Lp(a)) of 131 mg/dL (desirable
level <30–50 mg/dL). Both his sister and mother had significantly elevated Lp(a).
Six days after his initial hospitalization, he developed a conjugate gaze palsy and
right-sided weakness due to a small left vertebral dissection associated with throm-
bus in the basilar and left posterior cerebellar artery. He was unable to ambulate but
could follow simple commands. He underwent thrombolysis with mechanical
thrombectomy allowing for near-complete arterial recanalization. Given the recur-
rent nature of thrombotic events with no clear inciting event, on the eighth day of
hospitalization he was started on lipoprotein apheresis. Over the past 6 years, his
physical capacity has improved significantly, and he returned to school and started
practicing playing baseball (Moriarty et al. 2017).
A 31-year-old woman in her second trimester was hospitalized with a new onset
change in mental status, right arm weakness, and subsequent focal seizures.
Magnetic resonance venography revealed a superior sagittal sinus thrombosis. Four
years prior, during her first pregnancy, she had another episode of cerebral venous
thrombosis. She recovered with no neurological deficits and reportedly had an
excellent exercise capacity prior to this event. There was no history of head trauma.
Her past medical and drug history was unremarkable except for prior smoking. Her
family history was unknown. Extensive work-up including autoimmune disorders
and infectious etiology was negative. Initial hypercoagulopathy work-up revealed
elevated fibrinogen 610 mg/dL and Lp(a) of 420 nmol/L (desirable level <75 nmol/L).
Repeat coagulopathy panel in 6 months was unremarkable. Except for pregnancy,
previous smoking, and elevated Lp(a), no other known risk factor for cerebral
venous thrombosis was identified. She was initially treated with low molecular
weight heparin. Following discussion of risks and benefits, she was started on
biweekly lipoprotein apheresis. Over the course of the next 5 years, no new isch-
emic or thrombotic events were observed. During this time, she completed her third
pregnancy with no complications while receiving regular apheresis.

Introduction

The point of life is to find the delicate equilibrium between dream and reality.—Lillian
Eugenia Smith

A high plasma level of Lp(a) is a causal risk factor for atherosclerotic cardiovas-
cular disease (ASCVD) through various pathways associated with increased athero-
genesis, inflammation, and thrombosis (Reyes-Soffer et al. 2022; Tsimikas 2017).
Lp(a) affects endothelial function and mediates inflammation and oxidative stress,
fibrinolysis, and plaque stability, leading to accelerated atherothrombosis. Such life-­
threatening and debilitating events as described in the above clinical vignettes moti-
vate to continue building the foundation of understanding clinical implications of
192 M. S. Safarova and P. M. Moriarty

Lp(a) in: (1) (athero)thrombosis, (2) platelet and coagulation cascade, and (3)
inflammation. According to the 2018 National Heart Lung and Blood Institute
(NHLBI) report, an estimated 1.4 billion people globally have Lp(a) levels ≥50 mg/
mL (>100–125 nmol/L) with a prevalence ranging from 10% to 30% (Tsimikas
et al. 2018). The prevalence is higher in patients with established ASCVD, calcific
aortic valve disease, and chronic kidney disease (Tsimikas et al. 2018).
Lp(a) is a lipoprotein with a density between 1.06 and 1.11 g/mL that can bind to
lysine-rich components of the coagulation system and several components of extra-
cellular and subendothelial matrix of the vascular wall via its apoB and
apolipoprotein(a) (apo(a)), including binding to fibronectin, fibrinogen, glycosami-
noglycans, and proteoglycans (Kostner and Bihari-Varga 1990; Klezovitch et al.
1998). Lp(a) can be retained in the arterial intima, localizing preferentially to ath-
erosclerotic plaques (Boffa and Koschinsky 2016). The low-density lipoprotein
(LDL) moiety of the Lp(a) particle is covalently linked to the plasminogen-like
glycoprotein apo(a) through a single disulfide link between apolipoprotein B100
Cys 3734 and apo(a) kringle IV type 9 Cys67. Lp(a) is almost completely confined
to a subset of primates. It has been proposed that the duplication of the PLG gene
evolved into the LPA gene (Lawn et al. 1995).
Similarities between the two genes (LPA and PLG) include 5′-flanking and
untranslated regions, multiple copies of kringle IV-, a single copy kringle V-like
and protease-like domains, as well as a related 3′-untranslated region (Lawn et al.
1995; McLean et al. 1987). An Lp(a)-like complex is found in hedgehogs which is
thought to have evolved independently from that of humans. While individuals
with low concentrations of plasma Lp(a) typically show no syndromic features or
pathologic conditions, the physiological role of Lp(a) in humans is not entirely
clear. An analysis of individual-level data of 112,338 UK Biobank participants
demonstrated that one standard deviation of genetically lowered Lp(a) level was
associated with reduction in risk of coronary heart disease (CHD) and peripheral
vascular disease by 30% (odds ratio, 0.71; 95% confidence interval, 0.69–0.73 and
0.69; 0.59–0.80, respectively), ischemic stroke by 13% (0.87; 0.79–0.96), aortic
valve stenosis by 37% (0.63; 0.47–0.83) (Emdin et al. 2016). These findings were
reproduced with the LPA-rs41272114 (null allele frequency, 3%) associated with
low Lp(a), providing a significant protective effect in carriers with a ~20% risk
reduction in CHD (Kyriakou et al. 2014). No association of genetically predicted
low Lp(a) levels with type 2 diabetes, malignancy, or venous thromboembolism
(VTE) was observed in this study (Emdin et al. 2016). Current hypothesis of the
evolutionary advantages of synthesizing Lp(a)-like particles includes accelerated
repair of vascular lesions, tissue injuries, healing of wounds, reduced bleeding, as
well as induction and participation in different forms of acute phase responses
(Lippi and Guidi 2000; Brown and Goldstein 1987; Kronenberg and Utermann
2013; Caplice et al. 2001; Boffa et al. 2004; von Zychlinski et al. 2011). However,
these properties can become pathogenic in the setting of increased concentrations
of the lipoprotein and homeostatic imbalance.
11 Thrombosis, Inflammation, and Lipoprotein(a): Clinical Implications 193

Lp(a) and Thrombosis

It is hard to imagine that nature is only teasing us and the structural resemblance between
apo(a) and plasminogen has no clinical consequences.—Michael S. Brown and Joseph
L. Goldstein (1987)

Apo(a) is highly homologous to plasminogen, demonstrating antifibrinolytic

activity and pro-thrombotic properties. Data suggest that the apo(a) component of
Lp(a) inhibits conversion of plasminogen to plasmin by endogenous tissue plas-
minogen activators as well as competes with plasminogen and plasmin for binding
to established fibrin clots, thus compromising clot lysis. Other potential pro-­
thrombotic effects of Lp(a) include an increase in the expression of plasminogen
activator inhibitor 1 (PAI-1) (Etingin et al. 1991), inhibiting fibrinolysis, and inacti-
vation of tissue factor pathway inhibitor (TFPI) (Caplice et al. 2001), which aug-
ments factor VII and X activation and therefore promotes blood coagulation. In a
series of experiments using human plasma, Lp(a) had a higher binding affinity for
TFPI compared to plasminogen with its apo(a) component precluding binding of
plasminogen to TFPI. Enhanced interaction between Lp(a) and TFPI and the loss of
TFPI activity was observed in the Lp(a)-rich environment within the subendothelial
space of plaques (Caplice et al. 2001). Reduced affinity of Lp(a) to the vessel wall
and decreased fatty streak formation was demonstrated in experiments with defec-
tive lysine binding sites of the apo(a) kringle IV type 10 (Hughes et al. 1997;
Boonmark et al. 1997).
It has been proposed that Lp(a) may increase clot density (Undas et al. 2006;
Scipione et al. 2017) and may skew the balance of endogenous coagulation and
fibrinolysis to propagate thrombus enlargement and provide resistance to throm-
bolysis (Angles-Cano et al. 2001). In the context of Lp(a), different weights might
need to be applied to the components of arterial and venous thrombosis driven by
platelet aggregation (white thrombus) and entrapment of erythrocytes by fibrin (red
thrombus), respectively. In patients with pre-existing chronic inflammatory condi-
tions (i.e., rheumatoid arthritis, vasculitis) and in the presence of other procoagula-
ble states (i.e., polycythemia vera, antiphospholipid syndrome, factor V Leiden
heterozygosity, protein C deficiency, etc.) (Espinosa et al. 2001; Nowak-Gottl et al.
1997), elevated Lp(a) has been shown to promote both arterial and venous throm-
botic events. In a retrospective analysis of the Hematology Clinic referrals of
females younger than 50 years with deep venous thrombosis or pulmonary embo-
lism who had negative hypercoagulable panel, Lp(a) was elevated at >75 nmol/L in
57% of cases (Nguyen et al. 2018). Most of the screened patients with high Lp(a)
did not have any other clinical risk factors for thrombosis (Nguyen et al. 2018). In
younger individuals aged <50 years, the risk for recurrent cerebral venous thrombo-
sis was fourfold (odds ratio, 3.9; 1.23–12.4) higher in patients with Lp(a) levels
above 30 mg/dL during a median follow-up of 4.4 years, especially following dis-
continuation of anticoagulation (Skuza et al. 2019). No difference in thrombophilia
risk factors among the groups was reported.
194 M. S. Safarova and P. M. Moriarty

Observational and genetic studies have demonstrated a strong association

between Lp(a) levels and such atherosclerotic traits as coronary and peripheral
artery disease, abdominal aortic aneurysm, ischemic stroke, aortic valve stenosis
(discussed in other chapters of this book). Earlier onset of coronary artery disease
was observed in the carriers of LPA-rs10455872 and LPA-rs3798220 associated
with elevated Lp(a) levels (Helgadottir et al. 2012). Increased rates of paravalvular
leaks were reported in patients with high Lp(a) treated with transcatheter aortic
valve replacement (Ma et al. 2019). Lp(a) is a significant predictor of resistance to
endogenous thrombolysis in the early phase of acute myocardial infarction (Kim
et al. 2008), mediating insufficient fibrinolysis in the infarct-related arteries early
post thrombolytic administration (Brugemann et al. 1994). Using a Mendelian
randomization-­phenome-wide association approach in individuals of European and
African ancestry with genetically predicted elevated Lp(a) levels, a 30% increase in
the risk of arterial thromboembolic disease was observed with no significant asso-
ciation with any VTE phenotypes (Satterfield et al. 2021). An increase in the risk of
atrial fibrillation (hazard ratio, 1.07; 1.04–1.10) for every one standard deviation
increase in genetically predicted Lp(a) levels was demonstrated in this cohort
(Satterfield et al. 2021). In a total of 367,586 unrelated UK Biobank participants of
the European-descent, there was no significant association with VTE (pulmonary
embolism or deep vein thrombosis) per genetically predicted 50-mg/dL increase in
Lp(a) levels (Larsson et al. 2020).
In relation to venous thrombosis and Lp(a), there are conflicting data. The like-
lihood of VTE in the general population was shown to increase with older age,
smoking, and obesity (Gregson et al. 2019; Heit et al. 2012). Positive family his-
tory has been associated with doubling of the risk of incident venous thrombosis;
risk was up to fourfold higher when more than one relative was affected, regardless
of the presence of other risk factors (Bezemer et al. 2009). In a number of observa-
tional studies, elevated Lp(a) levels were shown to be elevated in 20–36% of indi-
viduals diagnosed with VTE (Nowak-Gottl et al. 1997; von Depka et al. 2000; Sofi
et al. 2007). No significant difference in the Lp(a) levels was seen in patients with
recurrent unprovoked VTE (Rodger et al. 2010). However, a meta-analysis of
17,688 individuals showed a 2.4-fold increase in the odds of retinal vein thrombo-
sis (odds ratio, 2.4, 1.7–3.3) in patients with elevated Lp(a) levels (Paciullo et al.
2021). Lp(a) was associated with a 6.5-fold (4.46–9.55) increase in the odds of
incident arterial ischemic stroke and cerebral venous sinus thrombosis events
among other genetic factors in 4563 children and adolescents aged less than
18 years (Kenet et al. 2010). In a meta-analysis of 14 studies with 2824 VTE
patients and 11,187 healthy controls, elevated Lp(a) was associated with a 1.6-fold
(1.36–1.79) increase in the risk of VTE (a large amount of heterogeneity was
reported, I2 = 77%) (Dentali et al. 2017). In a prospective cohort study of individu-
als aged <21 years, Lp(a)-mediated impaired fibrinolysis was thought to contribute
to substantially increased risk of recurrent arterial ischemic stroke for race-specific
Lp(a) levels above the 90th percentile with an odds ratio of 14.0 (1.0–184), and
apo(a) isoform size less than 10th percentile with an odds ratio of 12.8 (1.61–101)
(Goldenberg et al. 2013).
11 Thrombosis, Inflammation, and Lipoprotein(a): Clinical Implications 195

In contrast, when applying a Mendelian randomization study design in the

Danish adult population, only extreme levels of Lp(a) (total n = 14,783) and very
small apo(a) isoforms (total n = 38,753) were associated with a 1.7-fold (1.2–2.3)
and 1.3-fold (1.0–1.7) increase in the likelihood of venous thrombosis (deep vein
thrombosis and pulmonary embolism), respectively (Kamstrup et al. 2012). In the
same population, the risk of atherothrombosis (myocardial infarction) was associ-
ated with both Lp(a) tertiles and KIV-2 repeat tertiles (Kamstrup et al. 2012).
Among 9330 white men and women from the general population during a 10-year
follow-up, a significant increase in the risk of myocardial infarction was seen start-
ing at Lp(a) levels of 30 mg/dL. The risk incrementally grew with increases in Lp(a)
levels and was estimated at 3.6-fold (1.7–7.7) for individuals with Lp(a) ≥95th per-
centile (≥120 mg/dL) (Kamstrup et al. 2008). When this population was expanded
to 53,908 individuals with a total of 2501 VTE events, there was a 30% (odds ratio,
1.33; 1.06–1.69) increase in the risk of VTE among patients with Lp(a) above
100 mg/dL (Nordestgaard et al. 2010). In a Japanese population of 10,494 individu-
als, low Lp(a) levels (<10 mg/dL) were found to be associated with a higher fre-
quency of hemorrhagic strokes with no difference in ischemic strokes (Ishikawa
et al. 2013). It is plausible that this discordance in the reported data could be attrib-
uted to: (1) the lack of power to detect significant associations as well as low enrich-
ment with extremely high Lp(a) levels, and (2) confounding with the presence of
inflammatory and pro-thrombotic conditions affecting outcomes but not registered
in the large datasets resulting in reverse causation when the exposure-disease pro-
cess is reversed.

Lp(a), Platelets, and Coagulation

Lp(a) has been shown to inhibit endogenous fibrinolysis through: (1) competitive
inhibition of pericellular plasminogen activation on vascular and blood cells medi-
ated by tissue plasminogen activator (tPA), (2) mediation of plasminogen binding to
platelets, (3) competition with plasminogen for binding to fibrin, mononuclear cells,
annexin II on endothelial cells, (4) inhibition of fibrinogen binding to platelets acti-
vated with platelet-activating factor (PAF) (Loscalzo et al. 1990; Edelberg et al.
1989; Hajjar et al. 1989; Simon et al. 1991; Ezratty et al. 1993; Edelberg and Pizzo
1995; Moliterno et al. 1993; Romagnuolo et al. 2014). In mice resistant to tPA-­
mediated thrombolysis, apo(a) was reported to reduce clot lysis in vivo (Palabrica
et al. 1995). Clot lysis was attenuated in the setting of apo(a) transgene when com-
pared to their sex-matched normal littermates. Experiments with removal of kringle
V and the lysine binding site in kringle IV type 10, respectively, negated and sub-
stantially reduced the inhibitory effect of apo(a) (Romagnuolo et al. 2014). In cul-
tured endothelial cells, Lp(a) was shown to induce rearrangements of actin filaments
(Pellegrino et al. 2004) and upregulate the expression of PAI-1, thereby reducing the
amount of tPA available for plasminogen activation (Levin et al. 1994). Lp(a)-
mediated plasmin recognition of fibrin clots was demonstrated in experiments
196 M. S. Safarova and P. M. Moriarty

showing that apo(a) upregulated α2-antiplasmin (Edelberg and Pizzo 1992), thereby
impairing fibrinolysis.
In the presence of Lp(a), washed human platelets demonstrate significant
enhancement of aggregation and release of granule contents (Rand et al. 1998). In
direct binding experiments, specific and reversible binding of Lp(a) to platelets was
observed (Ezratty et al. 1993). On the other hand, activation of platelets with ade-
nosine diphosphate (ADP) halted Lp(a) binding capacity. Further, in a dose-­
dependent manner, Lp(a) was shown to inhibit PAF-induced platelet activation as
well as primary and secondary platelet aggregation induced by ADP and calcium.
When apo(a) was completely removed from the Lp(a) particle, its inhibitory effect
on PAC-1 (a mouse monoclonal antibody indicative of platelet activation) binding
to activated platelets significantly enhanced the antiaggregatory effects in compari-
son with the “unreduced” Lp(a) (Tsironis et al. 2004).
There is controversy surrounding apo(a) isoform size dependent versus indepen-
dent inhibition of plasminogen activation by Lp(a). In the clinical setting, it has
been demonstrated that the size of apo(a) isoforms was inversely associated with an
up to eightfold increase in the risk of thromboembolic events (Espinosa et al. 2001;
Falco et al. 1998). In a case-control study, copy number variation of LPA KIV-2 was
an independent determinant of VTE (Sticchi et al. 2016). The KIV type 2 repeat
number was significantly lower in patients with VTE than in healthy controls,
including an observed higher frequency of the KIV-2 repeat number of less than 8
(Sticchi et al. 2016). Levels of Lp(a) may vary up to 200-fold for a given apo(a)
isoform (Perombelon et al. 1994). Single nucleotide polymorphisms mapped to LPA
and KIV-2 copy number have been shown to provide complementary information,
explaining the variation in plasma Lp(a) concentrations (Lanktree et al. 2010).
Thus, while some research has shown that small size apo(a) isoforms display higher
affinity to bind fibrin (Angles-Cano et al. 2001), there are data demonstrating a lack
of association between the LPA score comprising rs10455872 and rs3798220 vari-
ants (Helgadottir et al. 2012; Danik et al. 2013), number of KIV type 2 repeats, and
the level of plasminogen inhibition (Romagnuolo et al. 2014; Hancock et al. 2003),
highlighting a need for further research in this area.

Lp(a) and Inflammation

Lp(a) is the preferential lipoprotein carrier for oxidized phospholipids (OxPL), pro-
atherogenic and pro-inflammatory markers. In vitro experiments demonstrated that
products of oxidation make fibrin clot less permeable (Hoffman 2008), another
mechanism by which Lp(a) may be affecting clot lysis. In individuals with elevated
Lp(a) levels (>50 mg/dL), a local increase in the arterial wall inflammation in vivo,
enhanced peripheral blood mononuclear cells trafficking as well as transendothelial
migration and accumulation in the arterial wall with and without plaquing was
noted (van der Valk et al. 2016). Further, it was shown that patients with familial
hypercholesterolemia (average LDL-C, 236 mg/dL) and elevated Lp(a) levels
11 Thrombosis, Inflammation, and Lipoprotein(a): Clinical Implications 197

(range, 43–401 nmol/L) elicit markedly increased local arterial wall inflammation
as compared to healthy control subjects (van Wijk et al. 2014). Following lipopro-
tein apheresis, a significant reduction of the arterial wall inflammation estimated
using the target-to-background ratio of a fluorodeoxyglucose (FDG) uptake on the
PET/CT scan was demonstrated (van Wijk et al. 2014). This anti-inflammatory
effect was observed after a single session of apheresis. Milder reduction in the Lp(a)
levels with PCSK9 inhibitors did not show any significant change in target-to-­
background ratio of the index arterial vessel as compared to placebo (Stiekema
et al. 2019).
Mechanism by which cells recognize oxPL include recognition and interactions
with the cell receptors such as CD36, SRB1, EP2, VEGFR2, Toll-like receptor-4,
and the PAF receptors (Zimman et al. 2007; Berliner et al. 2009). In the middle-­
aged patients with type 2 diabetes, oxidative stress enhanced pro-thrombotic state
by unfavorably affecting the fibrin network structure and impairing fibrin clot sus-
ceptibility to lysis regardless of diabetes severity and duration (Lados-Krupa et al.
2015). Oxidized apoB100-containing lipoproteins were independently associated
with the clot lysis time in diabetes, suggesting that oxidized forms might directly
impair plasmin-mediated fibrin degradation (Lados-Krupa et al. 2015). An aggre-
gation of Lp(a) particles on the surface of the aortic smooth muscle cells was
observed in the presence lipoprotein lipase and sphingomyelinase (Tabas et al.
1993). In the same series of experiments, coincubation of Lp(a)-coated smooth
muscle cells with macrophages resulted in formation of lipid-laden macrophages
(Tabas et al. 1993). The role of macrophages in Lp(a) pathophysiology is discussed
in Chap. 10.
Several mechanisms of Lp(a)-mediated plaque instability have been described,
including increased production of interleukin-8 (IL-8). IL-8 among other effects
disinhibits matrix metalloproteinases (Moreau et al. 1999; Ezhov et al. 2019),
which increases the intensity of plaque inflammation and likelihood of plaque rup-
ture. Oxidized Lp(a) fractions induce a dose-dependent reduction of nitric oxide
synthase expression and, as a result, reduce nitric oxide production (Moeslinger
et al. 2006), thereby adversely affecting vascular homeostasis. In an assessment of
vasomotor response to acetylcholine, an association between elevated Lp(a) levels
and impaired endothelium-­dependent vasodilatation in coronary arteries with and
without angiographically detectable atherosclerotic lesions was observed (Tsurumi
et al. 1995). In a dose-dependent manner, Lp(a) promotes intercellular cell adhe-
sion molecule-1 (ICAM1) expression (Takami et al. 1998), upregulation of which
has a key role in the inflammatory response (Libby et al. 2011). Proteomic analysis
of Lp(a) revealed its association with the histidine-rich glycoprotein (HRG) known
to interact with heparin, plasminogen, fibrinogen, and complement components
(von Zychlinski et al. 2011). Further there was a strong signal for the C3 comple-
ment component in the Lp(a) position in the plasma protein fraction, highlighting
an association of C3 with the Lp(a) particle (von Zychlinski et al. 2011; Garcia-
Arguinzonis et al. 2021).
In critically ill patients with COVID-19 infection, treatment with monoclonal
antibodies against IL-6 receptor (such as tocilizumab and sarilumab) was shown to
198 M. S. Safarova and P. M. Moriarty

improve survival (Investigators et al. 2021). In patients with underlying pro-inflam-

matory conditions (rheumatoid arthritis, COVID-19 infection), tocilizumab signifi-
cantly reduced Lp(a) levels and increased LDL-C levels by affecting LDL-receptor
expression in hepatocytes (Strang et al. 2013; Pierini et al. 2021; Miller et al. 2015).
The sequence of the LPA gene contains several IL-6-responsive elements that
enhance transcription of the gene (Wade et al. 1993). In vitro studies have shown
that Lp(a) stimulates growth of endothelial and smooth muscle cells, including
through inhibition of transforming growth factor-β activation (Grainger et al. 1993).
In human hepatocytes, tocilizumab inhibited IL-6-induced LPA mRNA and protein
expression whereas monoclonal inhibition of TNF-α with adalimumab did not
affect Lp(a) levels (Miller et al. 2015).
Individuals infected with COVID-19 demonstrate a wide range of symptoms—
from mild symptoms to severe illness. Since 2020, there is ongoing research on the
role of Lp(a) in patients with COVID-19 infection. For instance, in participants of
the UK Biobank, there was no difference in the Lp(a) distribution between individu-
als who tested positive for SARS-CoV-2 (n = 13,588) and population controls (aver-
age Lp(a) level was ~20 nmol/L in both groups) (Di Maio et al. 2021). In this
predominantly white population, each 25 nmol/L increase in the Lp(a) levels was
associated with a 4% and 7% increase in the risk of ischemic heart disease in
COVID-19-negative (n = 435,104) and COVID-positive (n = 6937) individuals,
respectively. There was a significant interaction between SARS-CoV-2-positive sta-
tus and Lp(a) levels with a 2.2-fold increase in the risk in patients with Lp(a) >95th
percentile (>220 nmol/L) compared to the bottom 20% (<6 nmol/L) of the Lp(a)
distribution. This association between Lp(a) and ischemic heart disease was most
pronounced in the younger age group. The incidence of thromboembolic events was
an eight-fold higher in patients requiring an intensive care unit stay compared to
SARS-CoV-2-positive patients without ICU treatment, and a 34-fold higher when
compared to the population controls. There was no significant association between
Lp(a) levels or LPA genetic scores and the risk of thromboembolic events among the
SARS-CoV-2-positive patients and the population controls (Di Maio et al. 2021).
This heterogeneity in the findings for the role of Lp(a) in patients with COVID-19
infection can be attributed to the intrinsic differences in selected populations with
differences in disease severity, treatment impact for COVID-19-positive patients,
and changes in Lp(a) during the acute phase of the disease. Further, although human
apo(a) can bind to substrates shared by its plasminogen homologue, it does not
innately perform as an activable plasmin-like protease, potentially adding another
confounding layer to conflicting results with VTE outcomes.

Future Directions, Relevance, and Conclusions

Medicine is the art of addition and subtraction. The subtraction of all that is excessive, and
the addition of all that is missing. And he who might be the best at doing this — will be the
best doctor.—Hippocrates Asclepiades.
11 Thrombosis, Inflammation, and Lipoprotein(a): Clinical Implications 199

Antiplatelet Agents and Anticoagulation

Among carriers of the LPA-rs3798220 variant (median Lp(a) level >80 mg/dL) in
the Women’s Health Study, aspirin intake was associated with a 56% (hazard ratio,
0.44; 0.20–0.94) reduction in the risk of major cardiovascular events, including
myocardial infarction, ischemic stroke, and cardiovascular death (Chasman et al.
2009). After a median of 9.9 years, there was a twofold decrease in the absolute risk
in the placebo group: the event rate was 2.14% (0.81–3.45%) in the aspirin group
and 4.83% (2.74–6.87%) with placebo (Chasman et al. 2009). In Japanese patients,
treatment with aspirin was associated with a significant reduction in Lp(a) levels
(Akaike et al. 2002). These findings were reproduced in the South Asian population
aged 21–60 years with aspirin significantly reducing Lp(a) levels (Ranga et al.
2007). Supporting experimental data in mice suggested that aspirin can suppress
apo(a) mRNA expression (Kagawa et al. 1999). In healthy individuals, lower Lp(a)
values were measured in EDTA-treated plasma, citrated, and heparinized plasma as
compared to the serum (Lippi et al. 1996). However, there are no studies investigat-
ing effects of anticoagulation on Lp(a).

Apo-B Lowering

In patients with CHD with on-statin LDL above 130 mg/dL, elevated Lp(a) levels
>80th percentile additionally increased the risk of recurrent ischemic events by 40%
(odds ratio, 1.40; 1.15–1.71) (O’Donoghue et al. 2014). In a meta-analysis includ-
ing primary and secondary ASCVD prevention randomized clinical trials similar
odds ratios (1.43; 1.15–1.76) were observed in statin-treated patients with Lp(a)
levels >50 mg/dL (Willeit et al. 2018). In the primary prevention JUPITER trial
(Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating
Rosuvastatin) independent of the LDL-C levels, for each standard deviation change
in Lp(a) while on a statin there was a 27% (hazard ratio, 1.27; 1.01–1.59) increase
in the relative risk of incident ASCVD (Khera et al. 2014). Among 25,096 partici-
pants in the secondary prevention FOURIER trial (Further Cardiovascular Outcomes
Research with PCSK9 Inhibition in Subjects with Elevated Risk), patients with
higher baseline Lp(a) (>120 nmol/L) had a 25% relative risk reduction (0.64–0.88)
and a 2.4% absolute risk reduction with PCSK9 inhibition, translating into an esti-
mated number needed to treat of 41 to prevent one fatal or non-fatal myocardial
infarction or urgent revascularization (O’Donoghue et al. 2019). There is ample
evidence that selective lipoprotein apheresis improves clinical outcomes related to
atherothrombosis in patients with elevated Lp(a) (Jaeger et al. 2009; Leebmann
et al. 2013; Safarova et al. 2013). Lipoprotein apheresis results in greater than 60%
reduction of apoB-containing lipoproteins following a single apheresis procedure. It
has been shown to improve blood rheology, rapidly and efficiently reduce Lp(a)
levels and inflammatory markers, including IL-6 and oxPL (Moriarty 2015). In
patients with CHD, familial hypercholesterolemia, and elevated Lp(a) levels, treat-
ment with lipoprotein apheresis demonstrated a significant downregulation of
200 M. S. Safarova and P. M. Moriarty

mRNA expression for IL-1α, IL-6, and TNF-α (Stefanutti et al. 2017). Since 2020,
the US Food and Drug Administration (FDA) approved criteria for apheresis in
secondary prevention of ASCVD with LDL-C levels >100 mg/dL and Lp(a) >60 mg/
dL on maximally tolerated lipid-lowering therapy (Nugent et al. 2020).
The importance of assessing Lp(a) in patients in the primary prevention setting
as well as in those with prior thrombotic events relates to its ability to identify sub-
jects at increased risk, and the potential to modulate impaired fibrinolysis and
inflammation and as a result, improve outcomes. To date, extensive evidence exists
in support of a causal association of elevated Lp(a) levels with the risk of atheroscle-
rosis development and progression in different vascular beds. Likely, the presence
of the atherosclerotic plaque is in fact an inciting event for Lp(a) to promote throm-
bus formation in the unstable milieu. Current data suggest that except for Lp(a)
levels ≥90–95th percentile, small isoforms (KIV-2 repeats <10–6th percentile) and
in individuals with existing pro-inflammatory and pro-thrombotic conditions, Lp(a)
does not initiate venous thrombus formation, but rather contributes to its propaga-
tion and density. Lp(a) has a complex genetic architecture. For instance, studies
within or outside the LPA gene region demonstrated that in carriers of the Lp(a)-
raising genetic variants (i.e., rs10455872), the presence of a rare missense
rs41267813[A] variant was associated with substantially lower Lp(a) concentra-
tions (Said et al. 2021), potentially offering a protective effect against CHD.
Discovery of new mechanisms elucidating pathways affecting interplay between
Lp(a) and acute thrombotic events will continue to provide potential therapeutic tar-
gets addressing current gaps in residual risk. Genetic studies evaluating LPA interac-
tion using functional studies and its clinical relevance on the clinical outcomes
involving diverse cohorts are needed. Further research is warranted to assess differ-
ences in the markers of the coagulation system activation and platelet activation before
and after Lp(a)-targeted treatment to address pathophysiological relevance of pro-
thrombotic and atherogenic properties of Lp(a) in humans. Studies investigating
effects of aspirin in primary prevention and other antiplatelet agents in secondary
prevention in patients with elevated Lp(a) are needed. Polygenic risk scores can assist
in unraveling the interplay between thrombosis and inflammation in patients with
elevated Lp(a), especially when tested in racially diverse populations (Table 11.1).

• Elevated Lp(a) promotes arterial and venous thrombosis.

• Similar to plasminogen, apo(a) induces thrombotic events. Antifibrinolytic
effects are enhanced by pro-thrombotic properties in individuals with very
high Lp(a) levels, small apo(a) isoforms, and underlying inflammatory
and pro-thrombotic states.
• Similar to LDL, Lp(a) is a subject to oxidative modification, enhancing the
thrombo-inflammatory response.
• High Lp(a) levels are causal for the risk of atherothrombosis.
• Higher Lp(a) levels are seen in patients with acute VTE.
• Clinical trials focusing on Lp(a)-targeted therapies addressing residual
risk of high Lp(a) are on the way.
11 Thrombosis, Inflammation, and Lipoprotein(a): Clinical Implications 201

Table 11.1 Proposed research questions to bridge gaps in knowledge of clinical relevance of
elevated Lp(a) in atherothrombosis and venous thrombosis
Design Proposed study question
Novel experimental Mechanisms and pathways beyond plasminogen activation and tPA-­
models mediated fibrinolysis affecting interaction between high Lp(a), acute
Functional studies thrombosis, and unstable atherosclerotic lesion development.
Biomarker research Markers of the coagulation system activation, fibrinolytic potential, and
platelet activation before and after Lp(a)-targeted treatment, with and
without other conditions precipitating thrombosis.
Assessment of anti-inflammatory effects of Lp(a)-lowering therapies on
vulnerable plaques identified using novel imaging modalities
Genetic studies Clinical impact of genetic architecture of elevated Lp(a) on outcomes in
cohorts with diverse ethnic/racial background.
Clinical/pragmatic Effects of aspirin in primary prevention of ASCVD in individuals with
trials elevated Lp(a).
Effects of dual antiplatelet agents/more potent antiplatelet agents in
secondary prevention of ASCVD in patients with elevated Lp(a).
Role of elevated Lp(a) in determining duration of anticoagulation in
patients with VTE.
Role of elevated Lp(a) in addressing the risk of stroke in patients with
atrial fibrillation.
Assessing the role of Lp(a) and targeted treatment in patients undergoing
aortic valve replacement.
Clinical trials/ Lp(a)-targeted therapies and prognosis in patients with elevated Lp(a),
outcome research identifying race-specific therapeutic targets

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Chapter 12
The Kidney Is the Heart of the
Organs: Its Role in Lp(a) Physiology
and Pathophysiology

Hans Dieplinger

Introduction

Lipoprotein(a) [Lp(a)] is a complex lipoprotein particle that is independently asso-

ciated with atherosclerotic disease (Kronenberg and Utermann 2013).
Epidemiological and genetic studies demonstrate that elevated plasma Lp(a) con-
centrations are highly genetically determined and dose-dependently increase caus-
ally the risk of cardiovascular events (Arsenault and Kamstrup 2022; Coassin and
Kronenberg 2022; Kronenberg et al. 2022). The non-genetically elevated Lp(a) con-
centrations in patients with various chronic kidney diseases (CKD) suggest a role of
the human kidney in Lp(a) metabolism (Kronenberg et al. 1996). This review sum-
marizes the experimental in vitro and in vivo evidence for a role of the kidney in
Lp(a) physiology and pathophysiology and aims to combine them into a clinical
picture for various groups of CKD patients.

Definition of Lipoprotein(a)

Lp(a) consists of an LDL-sized particle covalently linked to the glycoprotein apo(a).

Plasma Lp(a) concentrations vary widely between individuals, from hardly measur-
able to >200 mg/dL, and have a highly skewed distribution in most ethnic popula-
tions. The LPA gene encodes apo(a) on chromosome 6 and accounts for more than
90% of the variation in plasma concentrations (Kronenberg and Utermann 2013;
Utermann 1989). LPA alleles contain a variable number of exons pairs encoding the

H. Dieplinger (*)
Department of Genetics, Institute of Genetic Epidemiology, Medical University of Innsbruck,
Innsbruck, Austria
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 207

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_12
208 H. Dieplinger

plasminogen-like kringle IV (KIV) domains (McLean et al. 1987). This results in

differently sized apo(a) isoforms, ranging from 300 to 800 kDa, corresponding to
the presence of 11 to >50 KIV domains (Koschinsky et al. 1990; Lackner et al.
1993; Utermann et al. 1987). For practical purposes, apo(a) isoforms with ≤22 KIV
repeats are defined as low molecular weight (LMW) isoforms, those with >22
repeats as high molecular weight (HMW) isoforms (Kronenberg and Utermann 2013).
Epidemiological and genetic studies have suggested that increased Lp(a) plasma
concentrations are causally associated with coronary heart and aortic valve calcifi-
cation (Arsenault and Kamstrup 2022; Coassin and Kronenberg 2022; Erqou et al.
2009; Nordestgaard and Langsted 2016; Sandholzer et al. 1992; Thanassoulis
2016). As a consequence, Lp(a) is a potential pharmacological target for which
treatments are currently under development.

Metabolism of Lipoprotein(a)

Since the discovery of Lp(a) 60 years ago, tremendous effort has been invested in
the elucidation of its molecular, cellular, and metabolic pathways. Despite an over-
whelming body of experimental evidence from various in vitro and in vivo systems,
the metabolism of this enigmatic lipoprotein still remains poorly understood
(Chemello et al. 2022).

Synthesis, Assembly, and Secretion

Apo(a) is exclusively synthesized in hepatocytes (Kraft et al. 1989) and undergoes

post-translational modifications, including the formation of three disulfide bonds
within each kringle motif as well as substantial N-glycosylations. The residence
time of apo(a) isoforms in the endoplasmatic reticulum is proportional to their num-
ber of KIV2 domains (Brunner et al. 1996; White et al. 1994). Large apo(a) isoforms
are more susceptible to degradation in the intracellular proteosome (White et al.
1999), explaining the (on average) higher circulating plasma Lp(a) concentrations
in carriers of small apo(a) isoforms (Utermann et al. 1987). The availability of
apoB-100 could be rate limiting for the assembly of Lp(a), as concluded by studies
in patients with abetalipoproteinemia (Menzel et al. 1990).
Several earlier studies suggested an extracellular or cell-surface-associated
assembly of Lp(a) assembly following hepatic secretion of apo(a) (Chiesa et al.
1992; McCormick et al. 1994; White and Lanford 1994; Wilkinson et al. 1994).
This has, however, been challenged by studies showing that apo(a)-apoB100 com-
plexes can be detected intracellularly (Bonen et al. 1997) and several in vivo kinetic
studies in humans attempting to address the unanswered questions regarding the
assembly and secretion of Lp(a) (Reyes-Soffer et al. 2017).
12 The Kidney Is the Heart of the Organs: Its Role in Lp(a) Physiology… 209

Kinetic studies in vivo using stable isotope tracers and compartmental modeling
demonstrated that in individuals with a wide range of plasma Lp(a) concentrations,
the isotopic tracer curves for Lp(a)-apoB-100 and Lp(a)-apo(a) were essentially
identical, with similar contour and area-under-curve suggesting intracellular assem-
bly of Lp(a) (Watts et al. 2018). This notion was confirmed in a further in vivo study
in healthy individuals (Frischmann et al. 2012) and another kinetic study of statin-­
treated patients with elevated and normal Lp(a) concentrations (Ma et al. 2019).
These kinetic data therefore generally support an intracellular assembly of Lp(a)
suggesting that newly synthesized Lp(a)-apoB-100 and Lp(a)-apo(a) are secreted as
a holoparticle with coupled apo(a) and apoB100 residence times in the circulation.
Controversial results of an extracellular Lp(a) assembly are possibly due to inap-
propriate cellular or animal models.

Clearance and Catabolism

The mechanisms of Lp(a) clearance from the circulation also remain unclear and
controversial (McCormick and Schneider 2019). Without doubt is the liver the
major site of Lp(a) clearance followed to a much lesser extent by the kidney and the
arterial wall (Cain et al. 2005; Hrzenjak et al. 2003). Renal arterio-venous differ-
ences in Lp(a) concentrations suggest that the kidney can extract substantial amounts
of Lp(a) from the circulation (Kronenberg et al. 1997). A metabolic role for the
kidney is further supported by the inverse correlation between plasma Lp(a) and
glomerular filtration rate (GFR), with a significant increase in Lp(a) in patients with
more advanced chronic kidney disease (CKD) (Kronenberg 2014a) and by in vivo
kinetic studies demonstrating diminished clearance of Lp(a) in CKD patients treated
with hemodialysis (Frischmann et al. 2007). Further support for a possibly direct
catabolic function of the kidney for Lp(a) came from the discovery of fragments of
apo(a) in human urine and the decreased urinary excretion of apo(a) in patients with
renal dysfunction (Kostner et al. 1996; Mooser et al. 1996).

Immune-Histochemical Studies

Previous immune-histochemical studies (Nakahara et al. 1999; Sato et al. 1993;

Suzuki et al. 1997; Takemura et al. 1993) demonstrated apo(a) and apoB staining in
human kidney biopsies from patients with various kidney diseases. No Lp(a) could
be detected on the other hand in normal kidney tissue. In these studies, the authors
therefore concluded a role of Lp(a) in the progression of renal disease rather than a
direct function of the kidney for catabolizing Lp(a). Unfortunately, individual Lp(a)
plasma concentrations were not provided in these studies. The negative immunos-
taining results could thus have resulted from the chance selection of probands with
no or very low Lp(a) concentrations.
210 H. Dieplinger

We therefore performed immune-histochemical studies of Lp(a) on normal

human kidney tissue in relation to Lp(a) concentration and apo(a) size polymor-
phism in order to better understand the role of the kidney in Lp(a) metabolism
(Haiman et al. unpublished).
Apo(a) was localized in glomeruli, capillaries, and blood vessels. Staining was
found in glomerular capillaries and mesangial cells, but neither in Bowman’s cap-
sule nor in podocytes. As shown in Fig. 12.1a–f, the staining intensity strongly
depended on the Lp(a) plasma concentration. Apo(a) staining in mesangial cells
appeared as granular pattern and could be seen in main cell bodies, as well as in the
faint processes of mesangial cells (Fig. 12.2). Staining of apo(a) in walls of capillar-
ies and blood vessels between tubuli was variable: In most samples, apo(a) immu-
noreactivity was present in almost each capillary and blood vessel (Fig. 12.2a–d),
but in some samples staining in these areas was seen very rarely. A positive staining
was never observed in proximal and distal convolute tubuli (Fig. 12.3), as well as in
collecting tubuli. The negative controls in the absence of primary antibodies did not
show any staining (not shown). Staining of a tissue sample from a patient with very
low plasma Lp(a) concentration which served as an additional negative control is
shown in Fig. 12.1a.
ApoB immunostaining was localized in glomeruli, capillaries, blood vessels and
faintly on erythrocyte membranes (Fig. 12.3). In glomeruli, apoB was found in glo-
merular capillaries and in mesangial cells, but never in Bowman’s capsule nor in
podocytes. The intensity of apoB-staining did not depend on the Lp(a) concentra-
tion in the plasma of the patients (not shown). Apart from a granular pattern, apoB
staining was also seen diffuse in the cytoplasm of mesangial cells. It could also be

a b c

d e f

Fig. 12.1 Immunoreactivity of apo(a) in glomeruli of different tissue sections. Lp(a) plasma con-
centrations and apo(a) isoforms are indicated. The intensity of apo(a) staining in the glomeruli
depends on Lp(a) concentrations (a–f). HMW, high molecular weight apo(a) phenotype; LMW,
low molecular weight apo(a) phenotype; bars 20 μm
12 The Kidney Is the Heart of the Organs: Its Role in Lp(a) Physiology… 211

a b

c d

Fig. 12.2 Immunoreactivity of apo(a) in the glomerulum shows apo(a) in mesangial cells (a).
Higher magnification of rectangle from picture a (b). Granular pattern of apo(a) staining in main
cell body and processes of mesangial cells, (c, d) Mesangial cells with typical staining. Bars 10 μm

found on the walls of the capillaries between tubuli and the blood vessels (Fig. 12.3e–
g) and on the membranes of erythrocytes. Similar to apo(a), no positive staining was
ever observed for apoB in proximal and distal convolute tubuli (Fig. 12.3h), as well
as in collecting tubuli. The negative controls did not show any staining (not shown),
but staining of a tissue sample from a patient with very low Lp(a) plasma concentra-
tion demonstrated apoB immunoreactivity (not shown).
Apo(a) and apoB colocalized in mesangial cells (Fig. 12.4), capillaries, and
blood vessels (data not shown). The negative controls did not show any non-specific
staining (not shown).
Mesangial cells contribute to the regulation of glomerular filtration, produce
mesangial matrix, and are continuously exposed to the plasma compartment, only
separated from the capillary lumen by a fenestrated endothelium without interven-
ing basement membrane (Latta 1992). Mesangial cells participate in a number of
glomerular diseases and it is still a matter of debate whether lipids/lipoproteins
selectively enhance mesangial matrix synthesis, proliferation of human mesangial
cells, and foam cell formation—that may induce renal damage or diseases caused
by lipid/lipoprotein deposition—induce injuries of mesangial cells (Gyebi et al.
2012; Mondorf et al. 1999; Wheeler et al. 1994). It has been postulated that the
interaction between plasma lipoproteins and mesangial cells plays a major role in
212 H. Dieplinger

a e

b f

c g

d h

Fig. 12.3 Immuno-peroxidase staining of apo(a) (a–d) and apoB (e–h) in different regions of the
human kidney tissue: (a, e) capillaries in glomeruli, (b, f) capillaries between tubuli, (c, g) blood
vessel, (d, h) proximal (p) and distal (d) convoluted tubuli (DIC image). Bars: (a, b, e, f) 10 μm;
(c, d, g, h) 20 μm
12 The Kidney Is the Heart of the Organs: Its Role in Lp(a) Physiology… 213

Fig. 12.4 Double staining a

of apo(a) and apoB in a
glomerulum (a). Apo(a) is
stained dark purple and
apoB brown. Higher
magnification of rectangle
from picture a (b). Arrows
show capillaries and
arrowheads mesangial
cells. (c) Digital image
processing of picture b.
The original dark purple
color for apo(a) was
replaced with white to
demonstrate the sites of
colocalization in the tissue
(Adobe Photoshop).
Arrowheads show b
mesangial cells. Bars: (a)
20 μm, (b, c) 10 μm

c
214 H. Dieplinger

the pathophysiology of glomerulosclerosis. Several studies have shown that, in

addition to native LDL, mesangial cells bind VLDL (Kramer-Guth et al. 1996a),
oxidized LDL (Kramer-Guth et al. 1996a; Greiber et al. 1996), native and oxidized
Lp(a) (Kramer-Guth et al. 1996b) in vivo and in vitro, in line with our immunohis-
tochemical findings, and supporting the hypothesis that lipoproteins may play a
critical role in mediating the development of glomerulosclerosis (Gröne et al. 1992).
To reveal the origin of immunologically detected apo(a) and apoB in mesangial
cells, we investigated whether mRNA for apo(a) and apoB is expressed in human
kidney. RT-PCR was performed with purified total RNA from three different kidney
tissue samples, obtained from patients with different plasma Lp(a) concentrations,
primary mesangial and proximal tubule cells, as well as from apo(a)-transfected
HepG2 cells and human liver tissue. In these studies, apo(a) was only present in
apo(a)-transfected HepG2 cells and human liver tissue. ApoB expression in human
kidney tissue and in primary mesangial cells was very small compared to HepG2
and human liver tissue (data not shown).
Our findings of apo(a) and apoB co-localization in healthy human kidney tissue
are in accordance with the previously observed intracellular accumulation of Lp(a)
in rat kidneys after intravenous injection of human Lp(a) (Reblin et al. 2001) sug-
gesting renal uptake and/or degradation of Lp(a) as a holoparticle. They are also in
line with in vivo kinetic studies in hemodialysis patients resulting in diminished
clearance of Lp(a) in these patients (see section “In Vivo Studies” (Frischmann
et al. 2007)).

Role of LDL Receptor and Other Receptors for Lp(a) Clearance

Due to the structural similarities between LDL and Lp(a), the LDL receptor (LDLR)
has been investigated and discussed extensively as a candidate receptor for Lp(a)
over the past decades. Strong arguments again a role of the LDLR for Lp(a) uptake
and degradation came from numerous reports that statins which enhance LDLR
expression and thereby markedly reduce LDL, have a neutral, or an even elevating
effect on plasma Lp(a) concentrations (Khera et al. 2014; Tsimikas et al. 2020;
Willeit et al. 2018). Unexpectedly, PCSK9 inhibitors, which also increase the cell
surface expression of the LDLR, via an inhibition of LDLR intracellular degrada-
tion, not only lower LDL-C by 50–60%, but also reduce plasma Lp(a) concentra-
tions by 20–30% (McKenney et al. 2012). This observation has renewed research
into the roles of PCSK9 and of the LDLR in mediating the clearance of Lp(a) from
the circulation.
Several earlier reports from in vitro studies suggested, however, a role of the
LDLR for Lp(a) uptake, as well. Initial reports showed that Lp(a) can bind to the
LDLR, albeit with a lower affinity than LDL (Armstrong et al. 1990; Havekes et al.
1981; Reblin et al. 1997; Snyder et al. 1992). Lp(a) was also proposed to associate
with LDL and undergo LDLR-mediated clearance via a “hitch-hiking” mechanism
(Hofer et al. 1997; Kostner 1993). In HepG2 and primary human fibroblasts, PCSK9
was shown to reduce the binding and the cellular uptake of Lp(a) via the LDLR
12 The Kidney Is the Heart of the Organs: Its Role in Lp(a) Physiology… 215

(Raal et al. 2014). In contrast, other studies found no significant role for the LDLR
in mediating the cellular uptake of Lp(a) in primary human hepatocytes and HepG2
cells (Sharma et al. 2017; Villard et al. 2016). Moreover, no significant difference
was found in the cellular uptake of Lp(a) in primary lymphocytes derived from
normolipemic individuals compared with patients with homozygous familial hyper-
cholesterolemia (FH) and complete absence of LDLR function (Chemello
et al. 2020).
Multiple alternative pathways for Lp(a) clearance using other receptors have
been proposed, as elegantly reviewed by McCormick and Schneider (2019). For
instance, in macrophages, the Toll-like receptor 2 (TLR2) acts as a receptor for
Lp(a)-bound oxidized phospholipids (oxPL). This observation is in line with a large,
although non-significant, genome-wide association study (GWAS) showing that
TLR2 is the only receptor associated with circulating Lp(a) concentrations (Mack
et al. 2017; Seimon et al. 2010). Likewise, the scavenger receptor BI (SR-BI) has
been shown to promote the selective uptake of Lp(a) cholesterol esters in cells and
in SR-BI transgenic and knockout mice (Yang et al. 2013).
Due to the high degree of glycosylation of apo(a), carbohydrate-binding proteins
(lectins), such as the asialoglycoprotein receptor (ASGPR), have also been shown
to act as Lp(a) receptors in mice (Hrzenjak et al. 2003), but not all findings are con-
sistent (Cain et al. 2005; Sharma et al. 2017). Given the strong homology between
apo(a) and plasminogen, the role of plasminogen receptors in mediating Lp(a)
clearance has been investigated (Tam et al. 1996). One of such receptors, the plas-
minogen receptor presenting a C terminal lysine (PLGRKT), was shown to mediate
the cellular uptake of Lp(a) by human hepatoma cells and primary human fibro-
blasts. This study also showed that the LDL component of Lp(a) undergoes lyso-
somal degradation whereas apo(a) traffics through recycling endosomes and is
re-secreted into the medium (Sharma et al. 2017). However, the concentration of
free apo(a) in human plasma is relatively low, which suggests minimal to no recy-
cling of apo(a) in the circulation.
Several other members of the LDLR family of receptors have also been proposed
to mediate whole Lp(a) particle cellular uptake. Thus, the VLDL receptor binds
apo(a) and allows the internalization and subsequent degradation of Lp(a) in mac-
rophages (Argraves et al. 1997). The LDLR-related protein 1 (LPR1) and megalin/
gp330 (known as LRP2) also play a role in Lp(a) binding, cellular uptake, and deg-
radation in vitro (Reblin et al. 1997; März et al. 1993; Niemeier et al. 1999). LRP8
is also able to bind Lp(a) at the plasma membrane, but it remains to be shown
whether this promotes cellular uptake and degradation of Lp(a) particles (Steyrer
and Kostner 1990). The cellular uptake of Lp(a) in HepG2 hepatoma cells was,
however, recently shown to be unaffected overexpressing either the VLDLR, LRP1,
or LRP8 (Romagnuolo et al. 2017).
Interestingly, GWAS studies could not identify a significant association between
any of the proposed receptors and Lp(a) concentrations except the LDL receptor.
This might be explained by the fact that Lp(a) also contains cholesterol and the
signal with the LDL receptor might stem from the cholesterol content of Lp(a)
(Mack et al. 2017; Hoekstra et al. 2021).
216 H. Dieplinger

In Vivo Studies

In vivo kinetic studies using stable isotopes have been performed to investigate pos-
sible mechanisms underlying elevated Lp(a) concentrations in two different groups
of kidney disease (Fig. 12.5). These studies also revealed insights into a possible
role of the kidney in Lp(a) metabolism.
Patients with nephrotic syndrome (NS) revealed increased synthesis rates of
Lp(a) without changes in the fractional catabolic rate indicating an increased pro-
duction of Lp(a)—along with many other proteins—rather than a decreased catabo-
lism (de Sain-van der Velden et al. 1998). These results have to be, however, taken
cautiously since they are based on kinetic data from only five patients and five
controls with widely varying single values. Therefore, although mean values
between patients and controls look impressively different (see Fig. 12.5), produc-
tion rates do not differ significantly between these two groups after statistical evalu-
ation of the provided single data. Therefore, human kinetic studies should be
repeated with higher numbers of included patients/controls to confirm the previ-
ously published analysis. It has been demonstrated that in NS, patients lose a signifi-
cant amount of proteins via urine, and that the increased synthesis of Lp(a) might be
a result of compensation to keep up the oncotic pressure in the circulating blood.
In contrast, CKD patients treated by hemodialysis (HD, essentially lacking renal
function) showed similar synthesis rates for both apo(a) and apoB from Lp(a) between
hemodialysis and healthy controls (Fig. 12.5). The fractional catabolic rates (FCR)
for both components of Lp(a), however, were significantly lower in HD patients com-
pared with controls. This resulted in a much longer residence time of 8.9 days for
Lp(a)-apo(a) and 12.9 days for Lp(a)-apoB in HD patients compared to controls (4.4

Fig. 12.5 In vivo kinetic studies using stable isotopes reveal different mechanisms leading to
increased Lp(a) concentrations in patients with nephrotic syndrome (NS) and chronic kidney dis-
ease (CKD) treated with hemodialysis (HD): whereas in NS patients Lp(a) production rates are
increased with no changes in catabolic rates, the situation in HD patients is opposite: Lp(a) con-
centrations are increased due to diminished catabolic rates and not synthesis. For NS patients,
kinetic parameters are given as Lp(a) total protein, for HD patients as Lp(a)-apo(a). Each bar rep-
resents mean ± standard error. Data for NS patients are taken from de Sain-van der Velden et al.
(1998), those for HD patients from Frischmann et al. (2007)
12 The Kidney Is the Heart of the Organs: Its Role in Lp(a) Physiology… 217

and 3.9 days, respectively) (Frischmann et al. 2007). These results suggest, together
with the discovery of apo(a) immunostaining in healthy human kidney tissue (see
section “Immune-Histochemical Studies”), a possible catabolic function for Lp(a) of
the human kidney. The prolonged residence time of Lp(a) in HD patients might sub-
stantially contribute to the high risk of atherosclerosis in these patients (see below).

Lp(a) in Kidney Disease

Parra et al. reported for the first time in 1987 elevated Lp(a) concentrations in hemo-
dialysis patients (Parra et al. 1987). Since then, interest in the role of the kidney in
the metabolism of Lp(a) has steadily increased as documented in the comprehensive
review articles (Kronenberg et al. 1996; Kronenberg 2014a; Hopewell et al. 2018).
The earliest report related to this topic came from Papadopoulos et al., who described
a higher frequency of a second pre-beta band in agarose gel electrophoresis in
hemodialysis patients than in controls (Papadopoulos et al. 1980). Numerous stud-
ies have since then been published related to Lp(a) in nephrotic syndrome, CKD, or
kidney transplantation (see a recent review by Enkhmaa and Berglund (2022)).

Proteinuria and Nephrotic Syndrome

Several studies reported elevated Lp(a) concentrations in patients with proteinuria

or nephrotic syndrome (NS) (Brown et al. 1995; Faucher et al. 1993; Joven et al.
1995; Karádi et al. 1989; Querfeld et al. 1993; Stenvinkel et al. 1993; Takegoshi
et al. 1991; Thomas et al. 1992; Wanner et al. 1993; Hong and Yang 1995; Vaziri
2016). In contrast to hemodialyzed CKD patients, pronounced increases in Lp(a)
concentrations were reported in all apo(a) isoform groups (Wanner et al. 1993). In
the largest study, Kronenberg et al. reported five-fold elevated Lp(a) plasma concen-
trations in non-diabetic NS patients compared to controls (Kronenberg et al. 2004).
While the increase was partly explained by different distribution of apo(a) isoforms
between NS patients and controls, both small and large isoform groups were signifi-
cantly associated with higher Lp(a) concentrations in NS patients compared to con-
trols. Elevated Lp(a) levels in NS have been demonstrated in longitudinal studies to
decrease after remission of nephrotic syndrome (Faucher et al. 1993; Joven et al.
1995; Stenvinkel et al. 1993; Takegoshi et al. 1991; Wanner et al. 1993).

Early Stages of Kidney Disease

Increased Lp(a) concentrations have been observed in patients with reduced kidney
function characterized by impaired glomerular filtration rates (GFR) (Catena et al.
2015; Kovesdy et al. 2002; Kronenberg et al. 2000; Lin et al. 2014; Milionis et al.
218 H. Dieplinger

1999; Sechi et al. 1998). In only three of these studies, apo(a) isoforms were ana-
lyzed in addition to plasma Lp(a) concentrations (Kronenberg et al. 2000; Milionis
et al. 1999; Sechi et al. 1998). Kronenberg et al. examined the association between
kidney function, Lp(a) plasma concentrations, and apo(a) isoform size in multi-
center design in 227 non-nephrotic patients with different degrees of kidney impair-
ment. Lp(a) concentrations were significantly higher in patients with kidney disease
compared with 227 age-, sex- and apo(a)-isoform-matched controls (Kronenberg
et al. 2000). Lp(a) were increased already in the earliest stages of kidney impair-
ment before GFR starts to decrease. Kidney function was inversely related with
Lp(a) concentrations, independent of the initial kidney disease. Most remarkably,
the inverse association between Lp(a) values and kidney function was only seen in
the subgroup of patients with HMW apo(a) isoforms, in line with observations in
hemodialyzed CKD patients (see section “Chronic Kidney Disease Treated by
Hemodialysis or Peritoneal Dialysis” (Dieplinger et al. 1993; Kronenberg
et al. 1995)).
Inverse correlations between Lp(a) concentrations and GFR were also found in
the Penn Diabetes Heart Study based on 1.852 patients with mild kidney impair-
ment (Lin et al. 2014) and in a population study involving 7.675 individuals from
different ethnic backgrounds, particularly in non-Hispanic blacks, eventually sug-
gesting ethnic differences (Kovesdy et al. 2002).
However, the observed association between Lp(a) and GFR in the above-­
mentioned studies could not be confirmed by others: there was no significant asso-
ciation described in 804 individuals with stage 3–4 CKD and also no suggestion of
an interaction with apo(a) isoform size (Uhlig et al. 2005). Furthermore, a study of
87 kidney donors whose average kidney function was reduced from a GFR of 112
before donation to 72 mL/min/1.73 m2 one year later showed no significant differ-
ence in Lp(a) plasma values as a result of donation (Doucet et al. 2016). The reason
for the described discrepancies remains unclear; an explanation for the findings in
kidney transplant patients may be caused by an influence of immunosuppressive
medications on Lp(a) concentrations.

 hronic Kidney Disease Treated by Hemodialysis or

C
Peritoneal Dialysis

The majority of studies of Lp(a) in CKD is devoted to hemodialysis patients and

reported significantly elevated Lp(a) plasma concentrations in these patients (Parra
et al. 1987; Dieplinger et al. 1993; Kronenberg et al. 1995; Auguet et al. 1993;
Barbagallo et al. 1992, 1993; Cressman et al. 1992; Fiorini et al. 1995; Gault et al.
1995; Haffner et al. 1992; Heimann et al. 1991; Hirata et al. 1993; Kandoussi et al.
1992; Okura et al. 1993; Parsi et al. 1988; Shoji et al. 1992; Webb et al. 1993;
Gambhir et al. 2013; Parsons et al. 2003). Few studies described Lp(a) values not
significantly different from those in controls (Buggy et al. 1993; Docci et al. 1994;
Irish et al. 1992). Similar observations of elevated Lp(a) concentrations were made
in patients treated by continuous ambulatory peritoneal dialysis (CAPD) (Querfeld
12 The Kidney Is the Heart of the Organs: Its Role in Lp(a) Physiology… 219

et al. 1993; Kronenberg et al. 1995; Barbagallo et al. 1993; Gault et al. 1995; Shoji
et al. 1992; Webb et al. 1993; Buggy et al. 1993; Irish et al. 1992; Anwar et al. 1993;
Murphy et al. 1992; Thillet et al. 1994; Wanner et al. 1995). Only one study reported
lower Lp(a) values in CAPD patients (Kandoussi et al. 1992). In these studies, the
range of differences between controls and patients was extremely broad. These
inconsistent findings can be explained by the low number of patients and controls in
several studies together with an up to 1.000-fold inter-individual variation in Lp(a)
concentrations and the otherwise strong genetic control of Lp(a) concentrations.
These circumstances require large numbers of investigated individuals to reveal
reliable results as discussed earlier (Kronenberg et al. 1996; Kronenberg 2014b).
Only few studies with an adequate number of patients have determined apo(a)
isoforms in addition to Lp(a) concentrations (Dieplinger et al. 1993; Auguet et al.
1993; Hirata et al. 1993; Wanner et al. 1995).
To overcome the limitations of small sample size, Kronenberg et al. performed a
large multicenter study that included 534 hemodialysis and 168 CAPD patients
(Kronenberg et al. 1995). Both patient groups showed significantly elevated Lp(a)
levels in comparison with the controls. Lp(a) values were significantly higher in
patients treated with CAPD than with hemodialysis. Notably, the elevations on
Lp(a) in hemodialysis and CAPD patients were less pronounced than in several
other small studies. Consideration of apo(a) phenotypes revealed that the increased
concentration of Lp(a) was not explained by different frequencies of apo(a) iso-
forms between patients and controls confirming and extending earlier findings
(Dieplinger et al. 1993). Therefore, elevated Lp(a) values in CKD are caused by the
disease and are not due a higher frequency of LMW apo(a) phenotypes in patients.
The reason for the selective elevation of Lp(a) levels in HMW isoforms in both
treatment groups is presently unclear.
Similar to nephrotic syndrome, the markedly elevated Lp(a) concentrations in
CAPD patients are probably caused by the high loss of protein, in this case through
the dialysate fluid as demonstrated by Kronenberg et al. (1995). A generally
increased hepatic synthesis and secretion of lipoproteins including Lp(a) is the most
likely reason for their higher Lp(a) values. This increased synthesis of Lp(a) might
be responsible for the trend to higher Lp(a) values in CAPD patients with LMW
apo(a) isoforms, which, however, did not reach statistical significance.
Once the final CKD stage is reached, the cause of kidney disease has no influ-
ence on Lp(a) plasma concentrations (Kronenberg et al. 1995). This observation
was confirmed by a study including hemodialysis, CAPD, and renal transplant
patients by reporting similar Lp(a) values in patients with and without insulin-­
dependent diabetes mellitus (Gault et al. 1995).
The mechanism underlying Lp(a) elevation in CKD is still not fully understood.
The rapid decrease of Lp(a) following renal transplantation, as outlined in section
“Kidney Transplantation”, argues against an elevation induced by an acute phase
reaction, as was suggested earlier (Levine and Gordon 1995). Human kinetic studies
in various groups of these patients have been performed to examine whether this
elevation is caused by synthesis or catabolism. Further mainly kinetic studies are
necessary to shed light on the apo(a)-isoform-specific elevation of Lp(a) and its pos-
sible clinical impact.
220 H. Dieplinger

Kidney Transplantation

Findings of elevated Lp(a) concentrations in various patient groups with CKD led
several researchers to study the influence of kidney transplantation on Lp(a) plasma
concentrations. Black and Wilcken were the first to observe a highly significant
decrease in Lp(a) in 20 patients following renal transplantation (Black and Wilcken
1992). The results from several subsequent studies were not consistent, probably
reflecting differences in the study design. All prospective longitudinal studies
clearly showed a decrease in Lp(a) following transplantation (Gault et al. 1995;
Azrolan et al. 1994; Kronenberg et al. 1993, 1994a; Murphy and McNamee 1992;
Murphy et al. 1995; Segarra et al. 1995; Yang et al. 1994). Lp(a) changes were inde-
pendent of the modality of immunosuppressive therapy.
Lp(a) decreased after kidney transplantation in CKD patients, previously treated
by CAPD, independently of their apo(a) isoform. In contrast, in previously hemo-
dialyzed patients, Lp(a) declined after kidney transplantation only in those with
large apo(a) isoforms (Enkhmaa et al. 2016; Kerschdorfer et al. 1999; Kronenberg
et al. 1994a, 2003; Rosas et al. 2008). These findings are in line with the previously
described increased Lp(a) concentrations depending on the apo(a) size.
These results, together with those of kinetic studies in hemodialysis patients
(Frischmann et al. 2007), are a further convincing indication of a metabolic role of
the kidney in Lp(a) catabolism and that the observed Lp(a) changes are due to loss
of functional kidney tissue.

Lipoprotein(a) and Cardiovascular Outcome in CKD

Cardiovascular disease is also a major cause of death in CKD patients (Chronic

Kidney Disease Prognosis Consortium et al. 2010). At the same time, CKD is con-
sidered as one of the “Big Five” contributing to cardiovascular disease (Kronenberg
and Schernthaner 2020). The numerous reports on Lp(a) as risk factor for athero-
sclerosis and cardiovascular disease in the general population have therefore encour-
aged researchers to investigate the predictive power of Lp(a) also in CKD. Early
studies by Cressman et al. found significantly higher L(a) concentrations in hemo-
dialysis patients with events than in those without events (78.9 mg/dL vs 35.4 mg/
dL; P < 0.001) (Cressman et al. 1994). Various inconsistent subsequent studies,
many with limited statistical power or using various control groups or reference
Lp(a) values, followed and examined whether Lp(a) concentrations contribute to
increased cardiovascular risk in CKD patients (Bajaj et al. 2017; Emanuele et al.
2004; Koch et al. 1997; Kronenberg et al. 1999; Longenecker et al. 2005; Ohashi
et al. 1999; Shlipak et al. 2005; Webb et al. 1995).
In the Cardiovascular Health Study in elderly individuals, there was no signifi-
cant association between Lp(a) and cardiovascular mortality in CKD patients
observed (Shlipak et al. 2005). However, the risk estimate was comparable to that
observed in those individuals without CKD in whom a significant association
between Lp(a) and cardiovascular mortality was reported.
12 The Kidney Is the Heart of the Organs: Its Role in Lp(a) Physiology… 221

In line with the above-mentioned apo(a) isoform-specific elevations of Lp(a) in

non-nephrotic CKD, the majority of cross-sectional as well as prospective studies
revealed that Lp(a) concentrations and, even more, the LMW apo(a) isoform are
independent risk factors for cardiovascular disease in CKD (Cressman et al. 1992;
Koch et al. 1997; Kronenberg et al. 1994b, 1999; Longenecker et al. 2002, 2005).
Similar observations have been made by Wanner et al., who investigated CHD in
62 CAPD patients (Wanner et al. 1995). Affected patients showed only a small ele-
vation of Lp(a) (51 mg/dL vs 39 mg/dL; P = 0.06), but a significantly higher fre-
quency of LMW apo(a) phenotypes compared with patients without CHD (67% v
31%; P < 0.05).
Kollerits et al. reported an association of elevated Lp(a) concentrations and
LMW apo(a) isoforms with an increased risk for all-cause mortality and death due
to infection in hemodialysis patients with type 2 diabetes mellitus in the 4D Study
(Kollerits et al. 2016). Their findings were modified by age; the association between
apo(a) isoforms and mortality was only seen in patients ≤66 years.
CKD patients with LMW apo(a) isoforms have generally higher Lp(a) plasma
concentrations during their entire life, whereas patients with HMW isoforms
develop high Lp(a) concentrations only when renal insufficiency begins to develop.
CKD is therefore the only constellation in which the apo(a) isoform has a higher
predictive power for atherosclerosis than the Lp(a) concentration. This might
explain why the apo(a) isoform, which reflects pre-disease Lp(a) values, is an excel-
lent predictor of atherosclerosis in CKD patients before a clinical atherosclerotic
complication develops (Kronenberg et al. 1996; Kronenberg 1995).
In addition to these genetic considerations, it has been postulated that prolonged
residence times of Lp(a), as shown in hemodialyzed patients by in vivo kinetic stud-
ies (Frischmann et al. 2007), may contribute to the high risk of atherosclerosis in
CKD patients.

 etabolic Relation Between Kidney and Lipoprotein(a):

M
Conclusions and Gaps in Knowledge

In contrast to the general population, the elevated Lp(a) plasma concentrations in

CKD patients are nongenetic in origin and are a consequence of the disease.
Numerous studies reporting increased Lp(a) values in CKD suggested a role of the
kidney in the catabolism of Lp(a). However, only few studies, such as human kinetic
studies (Frischmann et al. 2007), provided at least indirect evidence to support this
hypothesis.
There are two possible explanations for high Lp(a) concentrations in CKD. First,
the kidney has an indirect influence on the synthesis of Lp(a) in the liver. This might
be triggered by a factor that is secreted by the kidney and regulates hepatic Lp(a)
synthesis. This idea is supported by the findings of Azrolan et al. in five nephrecto-
mized hemodialysis patients who had Lp(a) concentrations that were not different
from those in a control group (Azrolan et al. 1994). The authors therefore proposed
that impaired or dysfunctional kidneys might play a role in elevating plasma Lp(a)
222 H. Dieplinger

concentrations. These results remain to be confirmed in longitudinal studies and

considering apo(a) isoforms.
The second explanation for high Lp(a) concentrations in CKD is that the kidney
has a direct catabolic function and degrades Lp(a). Oida et al. were the first to
describe the excretion of degraded lipid-free apo(a) fragments in urine, which
decreased with the decline in glomerular filtration rate (Oida et al. 1992). Several
subsequent studies were published confirming the generation of apo(a) fragments
(Kostner et al. 1996; Cauza et al. 2003; Trenkwalder et al. 1997). A direct role of the
human kidney in catabolizing Lp(a) was further supported by reporting clear differ-
ences in Lp(a) plasma concentrations between arteria and vena renalis in humans
(Kronenberg et al. 1997). Various renal cell types express the LDL receptor-related
protein (megalin), a member of the LDL receptor gene family (Kukida et al. 2020),
which is believed to play a role in the catabolism of Lp(a) (März et al. 1993) (see
also section “Clearance and Catabolism”).
On the other hand, Lp(a) also could play a role in the pathogenesis of kidney
disease. If elevated Lp(a) concentrations are a primary cause of kidney disease,
one would expect a higher frequency of LMW apo(a) isoforms in CKD patients.
However, this was not observed in two large studies (Dieplinger et al. 1993;
Kronenberg et al. 1995). It is conceivable, however, that high Lp(a) values accel-
erate the progression of renal disease at a later stage. Lipoprotein(a) and apoB
have been demonstrated in the glomeruli of patients with glomerular disease,
mainly in the mesangial area and occasionally along capillary loops (Sato
et al. 1993).
At least one important question regarding the metabolic interrelationship between
Lp(a) and the kidney remains unanswered: Why are Lp(a) concentrations in hemo-
dialyzed CKD patients elevated only in carriers of HMW apo(a) isoforms? Kinetic
human studies performed in larger study groups (including a sufficiently large sub-
group with HMW apo(a) isoforms) should help to clarify this issue.

Acknowledgments Work from the author described in this overview has been supported by
grants from the Austrian Science Fund (P12358-MED) and the Jubiläumsfonds of the Austrian
National Bank (6721/4).
The author highly appreciates the critical reading of this review manuscript by Florian
Kronenberg as well as fruitful and long-lasting collaborations with many scientific colleagues and
technical assistants including H Andersson, M Auinger, D Bach, U Beisiegel, C Bösmüller, JH
Bräsen, B Dieplinger, N Donarski, E Dosch, C Drechsler, A von Eckardstein, G Friedrich, ME
Frischmann, B Grabensee, H Graf, E Gröchenig, A Gruber, M Hohenegger, F Hoppichler, E Hoye,
K Huber, K Ikewaki, H Kathrein, L Kerschdorfer, B Kollerits, P König, GM Kostner, KM Kostner,
V Krane, F Kronenberg, MF Kronenberg, C Lackner, C Lamina, U Lang, I Leiter, K Lhotta, A
Lingenhel, EM Lobentanz, R Margreiter, W März, G Maurer, HJ Menzel, N Moes, U Neyer, D
Öfner, G Pinter, A Pribasnig, B Rantner, T Reblin, J Reitinger, P Riegler, E Ritz, H Salmhofer, W
Salvenmoser, C Sandholzer, JR Schäfer, K Scheiber, U Scheidle, M Schober, JP Schwaiger, H
Schweer, RA Stahl, T Stefenelli, W Sturm, F Thaiss, J Thiery, E Trenkwalder, G Utermann, C
Wanner, M Wieshofer, G Wolf.
My special thanks go to Linda Fineder for a tremendous 30-year-long collaboration.
12 The Kidney Is the Heart of the Organs: Its Role in Lp(a) Physiology… 223

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Chapter 13
Lp(a) as a Cardiovascular Risk Factor

Angela Pirillo and Alberico Luigi Catapano

Introduction

Epidemiological and genetic evidence has clearly shown that elevated levels of
lipoprotein(a) [Lp(a)] are causally linked with an increased risk of cardiovascular
disease (CVD). This has led to renewed interest in an “old” lipoprotein that, although
sharing structural similarities with LDL, is endowed with exclusive properties due
to the presence of apolipoprotein(a) [apo(a)], a protein with homology to plasmino-
gen (McLean et al. 1987). Lp(a) exerts multiple effects in CVD, as it can act simi-
larly to an LDL particle and enter the intima of the arterial wall, thus contributing to
atherosclerosis, but it can also inhibit fibrinolysis due to its homology with plas-
minogen and contribute to inflammation by mean of Lp(a)-associated oxidized
phospholipids (Koutsogianni et al. 2021).
Apo(a), encoded by the LPA gene, is a highly heterogeneous protein containing
multiple repeats of kringle 4 type 2 (KIV2). The number of repeats is genetically
determined by common copy-number variation within the LPA gene and is inversely
related to the plasma concentration of Lp(a), with isoforms containing fewer KIV2
repeats being associated with smaller Lp(a) lipoprotein size and higher circulating
levels. Two unique features of Lp(a) are its wide range of plasma level variation

A. Pirillo
Center for the Study of Atherosclerosis, E. Bassini Hospital, Milan, Italy
IRCCS MultiMedica, Milan, Italy
e-mail: [email protected]
A. L. Catapano (*)
IRCCS MultiMedica, Milan, Italy
Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano,
Milan, Italy
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 231

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_13
232 A. Pirillo and A. L. Catapano

(from <0.2 to >200 mg/dL, or <0.5 to 500 nmol/L), which for the most part reflects
genetic variations in LPA, and its profile of distribution in the population, which is
highly skewed with a long tail toward extremely high values, with ~20% of indi-
viduals showing Lp(a) levels >50 mg/dL (Nordestgaard et al. 2010). Elevated Lp(a)
levels can occur in individuals with otherwise normal lipid levels; the risk threshold
for Lp(a) is set at 50 mg/dL.
Being mostly genetically determined, circulating levels of Lp(a) are relatively
stable throughout life. Based on this observation, current European guidelines for
the management of dyslipidemias recommend measuring Lp(a) at least once in life
(Mach et al. 2020).

Lp(a) and CVD: A Causal Relationship

A large number of studies have established a causal relationship between Lp(a) and
CVD (Emerging Risk Factors Collaboration et al. 2009); above all, elevated Lp(a)
increases the risk of myocardial infarction (MI), stroke, and peripheral arterial dis-
ease, but its pathophysiological role appears to be more complex than that of
LDL. In fact, the mechanisms beyond this association likely involve both its LDL
particle-like features (promoting atherosclerosis) and plasminogen-like particle
(inhibiting fibrinolysis).
A stepwise increase in the risk of MI with increasing levels of Lp(a) was reported
in both genders in a general European population, with extreme levels of Lp(a)
(>95th percentile) predicting a threefold to fourfold increased MI risk (Kamstrup
et al. 2008). In agreement with this observation, another study reported a threefold
to fourfold higher prevalence of ASCVD and MI in adults having Lp(a) >99th per-
centile [median Lp(a) 460 nmol/L] compared to those with Lp(a) levels ≤20th per-
centile [median Lp(a) 7 nmol/L] (Nurmohamed et al. 2021). The incorporation of
Lp(a) into algorithms for CV risk assessment led to the increase of mean estimated
10-year risk and the upward reclassification of substantial percentages of patients,
both in primary and in secondary prevention (Nurmohamed et al. 2021). An inter-
esting observation reported in this study was a higher LDL-C goal attainment among
individuals with lower Lp(a) levels compared with those with high Lp(a) levels,
which may suggest that measured LDL-C in these patients mainly is the result of
high Lp(a) levels (Nurmohamed et al. 2021). It has been observed that a 15 mg/dL
(~0.39 nmol/L) increase in Lp(a) cholesterol determines a higher hazard ratio of CV
mortality compared with a corresponding increase in LDL cholesterol (1.18 vs
1.05), which may suggest that not only the cholesterol content in lipoprotein(a) is
pathogenic, but likely its unique protein apo(a) may play a relevant role as well
(Langsted et al. 2019).
Mendelian randomization studies have substantiated the causal role of Lp(a) in
CVD (Clarke et al. 2009; Kamstrup et al. 2009; Burgess et al. 2018; Lamina et al.
2019). Two common single-nucleotide polymorphisms (SNPs) have been strongly
associated with both increased levels of Lp(a) and increased risk of coronary
13 Lp(a) as a Cardiovascular Risk Factor 233

disease (Clarke et al. 2009); on the other hand, a 10 mg/dL lower genetically deter-
mined Lp(a) level was associated with a 5.8% lower risk of coronary heart disease
(CHD) (Burgess et al. 2018). The observation that a similar reduction in LDL-C will
translate into a 14.5% lower CHD risk has a relevant consequence on the magnitude
of Lp(a) level reduction required to provide a clinical benefit: a ~100 mg/dL Lp(a)
reduction concentration anticipates a CHD risk reduction similar to that achieved
with a ~39 mg/dL change in LDL-C level (Burgess et al. 2018) (Fig. 13.1). A similar
finding has been reported by another study (Lamina et al. 2019) (Fig. 13.1).
Elevated Lp(a) levels can also explain, at least in part, the residual CV risk com-
monly observed in patients with well-controlled LDL-C levels. A meta-analysis of
data from 29,069 patients included in seven placebo-controlled statin trials showed
that elevated baseline and on-statin Lp(a) levels conferred a significantly higher CV
risk, suggesting that statins do not impact the residual risk in patients with elevated
Lp(a) (Willeit et al. 2018) (Fig. 13.2). Among patients with recent ACS receiving
intensive or maximum-tolerated statin treatment, baseline Lp(a) levels predicted the
risk of MACE, nonfatal MI, and CHD and CV death, independent of baseline
LDL-C (Bittner et al. 2020). Although the reduction of MACE is mainly attributable
to the reduction in LDL-C (referred to as “corrected LDL-C”) across the range of
baseline Lp(a) levels, in patients with the highest baseline Lp(a) levels the contribu-
tion of Lp(a) reduction in reducing the risk of MACE was substantial (Bittner et al.
2020). Interestingly, moderately elevated plasma Lp(a) levels (≥15 mg/dL) appear
to confer an increased risk of all-cause mortality in patients with CAD (Liu
et al. 2021).
The role of Lp(a) in determining the residual CV risk is further supported by the
results of most recent trials evaluating therapies able to achieve considerable reduc-
tions in LDL-C levels. The FOURIER trial, which evaluated the clinical impact of

60

50
Genetic estimate
% CHD risk reduction

(lifelong exposure to low

40 LDL-C or Lp(a))

30 LDL-C Lp(a)
38,67 mg/dL 65,7-101,5 mg/dL
Trial estimate
20 (short-term clinical trials for
LDL-C)

10

0
0 20 40 60 80 100 120

Absolute lipoprotein reduction, mg/dL

Fig. 13.1 Estimates of CHD risk reduction with lowering of LDL-C or Lp(a) level
234 A. Pirillo and A. L. Catapano

1.8

Baseline
1.6 On-statin
Hazard ratio (95% CI)

1.4

1.2

0.8
15 to <30 mg/dL 30 to <50 mg/dL ≥50 mg/dL Lp(a) level

Fig. 13.2 Association of baseline and on-statin Lp(a) levels with incident CVD, age-adjusted, and
sex-adjusted according to different Lp(a) levels

evolocumab in patients with ASCVD, found that higher baseline levels of Lp(a)
were associated with an increased risk of coronary events, independent of LDL-C
levels (O'Donoghue et al. 2019). Furthermore, a post hoc analysis of the ODYSSEY
OUTCOMES trial observed that while patients having LDL-C levels ≥70 mg/dL
derive a clinical benefit from alirocumab treatment independent of Lp(a) levels, in
those having LDL-C ~70 mg/dL alirocumab treatment provides incremental clinical
benefit only when Lp(a) is at least mildly elevated (≥13.7 mg/dL) (Schwartz et al.
2021). This observation suggests that Lp(a) reduction translates into a clinical ben-
efit, or at least allows to identify patients who may benefit from PCSK9 inhibition.
It must be acknowledged that the response of Lp(a) to a PCSK9 inhibitor is highly
variable and related to the size of apo(a), since each additional kringle domain is
associated with an additional 3% reduction in Lp(a) (Blanchard et al. 2021).

 he Prevalence of Elevated Lp(a) Levels in Individuals

T
with Coronary Artery Disease

A large number of studies have shown that the prevalence of elevated Lp(a) in
patients with CVD is higher than in the general population. As an example, the fre-
quency of elevated Lp(a) among patients admitted to the coronary care unit was
27%, and it was even higher (32%) among patients with premature CAD (Ellis et al.
2018). Similarly, patients with relatively early onset CAD had a median Lp(a) of
29 mg/dL; levels ≥30 mg/dL were observed in half of the patients, 36.1% had an
13 Lp(a) as a Cardiovascular Risk Factor 235

Lp(a) 50 ≥ mg/dL, and 16.5% had Lp(a) level ≥100 mg/dL, a level that was shown
to increase CV risk by about threefold (Oo et al. 2020). Likewise, among patients
undergoing percutaneous coronary intervention, Lp(a) was elevated in 48% of indi-
viduals; it is worth remarking that elevated Lp(a) was observed among 45% of
patients with LDL-C ≤70 mg/dL, suggesting a contribution to residual CV risk
(Weiss et al. 2017).

Lp(a) in Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is a genetic condition characterized by ele-

vated levels of LDL-C since birth and increased risk of early incident acute MI
event or premature death (REF). Commonly, Lp(a) levels are higher among FH
individuals than in the general population, creating a unique condition in which a
lifelong exposure to two genetically elevated CV risk factors, namely Lp(a) and
LDL-C, further increases the risk of MI and also predispose to aortic valve calcifi-
cation (Vongpromek et al. 2015). Furthermore, a substantial proportion (~25%) of
all individuals diagnosed with clinical FH were diagnosed due to high Lp(a) levels
(Enkhmaa et al. 2019). The observation that FH patients carrying null mutations
and Lp(a) levels >50 mg/dL have a significantly increased CV risk compared with
patients carrying the same mutations and Lp(a) levels <50 mg/dL further strength-
ens the role of elevated Lp(a) levels in this specific, high CV risk, population
(Alonso et al. 2014).
Both FH and high Lp(a) are common genetic disorders; however, both are largely
undiagnosed. Direct assays for the evaluation of LDL-C, as well as LDL-C calcula-
tions, will include the cholesterol carried by Lp(a). It follows that Lp(a) concentra-
tion should be assessed in all individuals with a clinical diagnosis of FH to identify
those with the highest levels and, as a consequence, the highest risk of MI (Langsted
and Nordestgaard 2022); eventually, information on LPA variants may be useful in
improving the diagnosis of FH (Chan et al. 2019). Accurate assays quantifying
Lp(a)-cholesterol and the correct cholesterol content of LDL will be required to
define the phenotypic differences between familial hypercholesterolemia due to
elevated LDL vs. Lp(a) (Yeang et al. 2020).

 he Impact of Race and Ethnicity on Lp(a) Levels

T
and Cardiovascular Risk

The direct association between Lp(a) levels and the risk of myocardial infarction
has been clearly established in populations of European ancestry (Kamstrup et al.
2008, 2009; Clarke et al. 2009). On the other hand, the heritability of apo(a) and
Lp(a) levels varies across ethnicities, with, as an example, African Americans exhib-
iting the highest Lp(a) level despite having a lower heritability compared with
236 A. Pirillo and A. L. Catapano

% Individuals with
Lp(a) >50 mg/dL
Controls Cases
Controls Cases
2.0-53.6
7.0 13.0 Southeast Asians
2.4-74.2

2.1-61.5
9.0 18.0 South Asians 3.2-88.2

2.0-77.2
14.0 21.0 Latin Americans
2.0-100.0

2.0-89.3
14.0 18.0 Europeans
2.0-99.1

1.9-39.9
3.0 6.0 Chinese 2.3-53.4

2.0-66.8
12.0 15.0 Arabs 2.0-82.6

3.3-102.4
27.0 26.0 Africans 4.1-110.6

0 4 8 12 16 20 24 28
Median Lp(a) (mg/dL)

Fig. 13.3 Median levels (fifth and 95th percentile) of Lp(a) and prevalence of Lp(a) levels >50 mg/
dL in individuals from diverse ancestries (in healthy individuals-CONTROLS- and patients with
myocardial infarction-CASES)

Caucasians (Enkhmaa et al. 2019). Relative to Blacks, South Asians exhibit the
second highest median Lp(a) level, followed by Whites, Hispanics, and East Asians;
however, the causal relationship between Lp(a) and CVD extends to all racial and
ethnic groups (Virani et al. 2022).
An interesting analysis of data from the INTERHEART study, involving 52
countries, showed differences in Lp(a) levels in individuals from diverse ancestries,
with Africans having higher levels compared with other populations (Pare et al.
2019) (Fig. 13.3). Despite the observed differences, high Lp(a) levels were overall
associated with an increased risk of MI (OR 1.48), independently of other estab-
lished MI risk factors (Pare et al. 2019). When analyzed in single ethnic groups,
elevated Lp(a) levels (>50 mg/dL) were associated with increased MI risk in all
populations, except Arabs and Africans; in these two groups, however, the small
sample size might have limited the relevance of the observation compared with
other groups (Pare et al. 2019). In fact, a comparison of the association between
Lp(a) and incident CV events between African Americans and Caucasians in the
ARIC study showed that the hazard ratios for incident CVD and CHD were signifi-
cantly higher in the highest quintile of Lp(a) (>13.5 mg/dL) in both ethnic groups;
it must be acknowledged that Lp(a) levels in the highest quintile ranged from >24 to
81.7 mg/dL (median 32.1 mg/dL) in African Americans and from 13.5 to 80.3 mg/
dL (median 20.4 mg/dL) in Caucasians (Virani et al. 2012). These findings need to
be validated but suggest that, while Lp(a) thresholds designated on the basis of stud-
ies mainly performed in Europeans apply to different ethnic groups, they might
require an adjustment for other ethnic groups having higher mean Lp(a)
concentrations.
13 Lp(a) as a Cardiovascular Risk Factor 237

Conclusions

Although epidemiological and genetic studies have clearly established a causal role
for Lp(a) in CVD, to date the evidence that reducing Lp(a) levels translates into a
clinical benefit is still lacking. While substantial reductions in Lp(a) levels are
required to observe a clinical benefit, new agents that potently lower Lp(a) are under
clinical development. Ongoing trials will tell whether this reduction translates into
reduced CVD events. A phase III study will assess the impact of Lp(a)-lowering
with the antisense oligonucleotide Pelacarsen on major CV events in patients with
CVD and elevated Lp(a) levels (≥70 mg/dL), with a planned follow-up of 4 years
(NCT04023552).
A recent study using samples from the UK Biobank has shown that elevated
Lp(a) levels are associated with an increased risk for incident CAD in individuals
without a family history of heart disease, suggesting that Lp(a) evaluation may be
beneficial in refining CAD risk in primary prevention patients (Finneran et al. 2021).
While waiting for the results of outcome trials and looking for resolutions of major
issues in the measurement of Lp(a) (Virani et al. 2022), Lp(a) assessment should be
regarded as a plus in clinical practice and measured at least once in life (Mach et al.
2020; Reyes-Soffer et al. 2022), with the aim to improve risk stratification and iden-
tify individuals that might be at increased CV risk due to the presence of ele-
vated Lp(a).

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Chapter 14
Lp(a) and Aortic Valve Stenosis, Stroke,
and Other Noncoronary Cardiovascular
Diseases

Anne Langsted and Pia R. Kamstrup

Introduction

High lipoprotein(a) [Lp(a)] levels are now, based on more than three decades of
accumulating evidence from mechanistic, epidemiological, and genetic studies,
widely recognized as an important and likely causal risk factor for ischemic cardio-
vascular and, in particular, coronary artery disease (CAD) (Kamstrup 2021; Reyes-­
Soffer et al. 2022). High Lp(a) levels in the top 10% of the concentration distribution
(Fig. 14.1) associate with a two to threefold increase in risk of myocardial infarction
independent of conventional risk factors (Kamstrup 2021). More recently, high
Lp(a) levels, found in an estimated >1 billion individuals globally, have also been
identified as a risk factor for calcific aortic valve stenosis (AVS) with risk estimates
of at least the same magnitude as those found for CAD (Thanassoulis et al. 2013;

Anne Langsted and Pia R. Kamstrup contributed equally with all other contributors.

A. Langsted (*)
Department of Clinical Biochemistry, Copenhagen University Hospital-Herlev and Gentofte,
Herlev, Denmark
Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of
Copenhagen, Copenhagen, Denmark
e-mail: [email protected]
P. R. Kamstrup (*)
Department of Clinical Biochemistry, Copenhagen University Hospital-Herlev and Gentofte,
Herlev, Denmark
The Copenhagen General Population Study, Copenhagen University Hospital-Herlev and
Gentofte, Herlev, Denmark
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 241

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_14
242 A. Langsted and P. R. Kamstrup

Fig. 14.1 Lp(a) concentration distribution. Plasma levels of Lp(a) (total mass and particle num-
ber) in the Copenhagen General Population Study (N = 79,718 White individuals of Danish
descent, 118 measurements >200 mg/dL not displayed). All measurement values were calibrated
to fresh sample measurements by the latex-enhanced Denka Seiken (Denka Seiken, Tokyo, Japan)
immunoturbidimetric assay with traceability to an international calibrator (WHO SRM 2B).
Conversion to nmol/L was done according to the following equation based on ~13,900 individuals
with measurements in both mg/dL and nmol/L (Denka Seiken Roche distributed assay using dif-
ferent calibrations for mg/dL and nmol/L): lipoprotein(a) nmol/L = 2.18*lipoprotein(a) mg/
dL–3.83. (Adapted from Clin Chem 2021;67:154–166 with permission)

Kamstrup et al. 2014). Several clinical guidelines on cardiovascular disease (CVD)

prevention now recommend once-in-a-lifetime Lp(a) measurements in all to iden-
tify individuals at increased risk and optimize management of modifiable risk fac-
tors (Mach et al. 2020; Pearson et al. 2021).
The mounting evidence that high Lp(a) levels represent an unmet clinical need
have spurred the development of potent Lp(a)-lowering drugs opening opportunities
for future CVD prevention in individuals with levels above thresholds for increased
risk (Reyes-Soffer et al. 2022). In this chapter, we summarize findings for Lp(a) and
AVS, stroke, and other noncoronary CVD, focusing on findings from large genetic
epidemiologic studies, and provide estimates for thresholds.
14 Lp(a) and Aortic Valve Stenosis, Stroke, and Other Noncoronary Cardiovascular… 243

Aortic Valve Stenosis

Calcific AVS is a chronic, progressive disease which shares risk factors with athero-
sclerotic disease and is estimated to affect 3% of adults older than 75 years of age
and with a steeply increasing disease burden in high-income countries (Otto and
Prendergast 2014; Yadgir et al. 2020). Up to 50% of patients progress to severe
disease within 2–4 years, and valve replacement represents the only treatment
option for severe disease characterized by obstructive heart failure and increased
risk of sudden death (Coisne et al. 2021). While familial aggregation exists for both
bi- and tricuspid diseases, up until 2013, no specific genetic risk factors had been
identified (Otto and Prendergast 2014). However, in a landmark study from 2013,
and using a hypothesis-free genome-wide association study (GWAS) approach, the
LPA gene was identified as the only genome-wide significant locus for the presence
of aortic valve calcification (AVC) in a White European ancestry cohort (N = 6942)
and with replication also in African American and Hispanic American cohorts
(Thanassoulis et al. 2013). The LPA rs10455872 single nucleotide polymorphism
(SNP), previously found to be strongly associated with Lp(a) levels (Clarke et al.
2009), associated with a twofold increase per allele in risk of AVC considered an
early phenotype for AVS (Thanassoulis et al. 2013). Also, based on Lp(a) levels
available in a subgroup (N = 3670), an odds ratio of 1.62 (1.27–2.06) for AVC was
found per log-unit increase in genetically determined Lp(a) levels. Finally, in two
cohort studies, an association with incident AVS and valve replacement was found
with hazard ratios per allele of 1.7 (95% confidence interval: 1.3–2.2) and 1.5
(1.1–2.3). The combined findings of the study provided strong genetic evidence of
a causal association of Lp(a) with AVC and likely also AVS.
Prior to 2013, associations of high Lp(a) levels with increased risk of AVC or
AVS had only been sporadically reported in smaller epidemiological studies (Gotoh
et al. 1995; Stewart et al. 1997; Glader et al. 2003; Bozbas et al. 2007). However,
the 2013 GWAS study generated considerable interest in high Lp(a) levels as a pos-
sible causal risk factor for calcific AVS. Thus, in 2014, risk estimates for incident
AVS at different levels of Lp(a) were reported from large general population stud-
ies. The first was a combined analysis of the historic Copenhagen City Heart Study
and the contemporary Copenhagen General Population Study (Kamstrup et al.
2014). Risk of AVS was increased for Lp(a) levels in the top third of the concentra-
tion distribution (≥20 mg/dL, ≥40 nmol/L), and individuals with levels ≥90th per-
centile (≥65 mg/dL, ≥138 nmol/L) had a two to threefold increased risk of AVS as
compared to individuals with levels <5 mg/dL (N = 29,016, Fig. 14.2). Notably, the
risk estimates appeared independent of the presence or absence of CAD. On a con-
tinuous scale, a tenfold increase in plasma Lp(a) levels is associated with a hazard
ratio of 1.4 (1.2–1.7) comparable to the 1.6 (1.2–2.1)-fold increase in risk found for
a similar increase in genetically determined levels based on three LPA genotypes
244 A. Langsted and P. R. Kamstrup

Age and sex adjusted Multivariable adjusted

Lipoprotein(a) Participants Events

percentile mg/dL N N

<22 3(2-4) 6123 53 Trend:p<0.001 Trend:p<0.001

22-66 11(7-17) 13053 127

67-89 40(30-51) 6677 87

90-95 80(63-95) 1728 27

>95 124(104-148) 1432 30

1 2 3 4 5 1 2 3 4 5

Hazard ratio (95% CI) for aortic Hazard ratio (95% CI) for aortic
valve stenosis valve stenosis

Fig. 14.2 Risk of aortic valve stenosis by Lp(a) levels. Analyses were adjusted for age, sex, total
cholesterol, HDL (high-density lipoprotein) cholesterol, systolic blood pressure, smoking, and
diabetes. Lipoprotein(a) in mg/dL is shown as median (interquartile range). (Adapted from J Am
Coll Cardiol 2014;63:470–7 with permission)

(rs10455872, rs3798220, KIV-2) explaining 41% of the total variation in plasma

levels. The second was from the European Prospective Investigation into Cancer
(EPIC)-Norfolk study (N = 17,553), where individuals in the top third of the Lp(a)
concentration distribution had a 1.6 (1.0–2.4)-fold increased risk of AVS with con-
sistent genetic findings as hetero- and homozygous minor allele rs10455872 carriers
had a 1.8 (1.1–2.9)- and 4.8 (1.8–13.2)-fold increased risk, as compared to noncar-
riers (Arsenault et al. 2014).
In subsequent years, additional large genetic studies provided evidence that high
Lp(a) levels were at least as strong a risk factor for AVS as for CAD (Emdin et al.
2016; Gudbjartsson et al. 2019). Thus, in a study including 112,338 UK Biobank
participants and using a genetic risk score based on four LPA SNPs associated with
decreased Lp(a) plasma levels, a one standard deviation decrease in genetically deter-
mined Lp(a) levels is associated with a 37% reduced risk of AVS, compared to effect
sizes of 31% for peripheral vascular disease, 29% for CAD, 17% for heart failure, and
17% for stroke (Emdin et al. 2016). Similarly, in a large case-control study of 143,087
Icelanders from 2019, genetically imputed Lp(a) levels were associated with similar
risk estimates for CAD and AVS of 16–17% risk increase per 50 nM (~25 mg/dL)
increase in genetically determined Lp(a) levels. The association appeared entirely
mediated by increased Lp(a) levels and not by the concomitant small apolipoprotein(a)
isoform size, a previous point of contention (Gudbjartsson et al. 2019).
While genetic studies have provided strong evidence for high Lp(a) levels as a
cause of AVS, the pathophysiological mechanism is not fully understood. In vitro
studies have, however, demonstrated osteogenic differentiation of valvular intersti-
tial cells exposed to Lp(a) and associated pro-inflammatory oxidized phospholipids
(Zheng et al. 2019), and both measurements have in AVS patient cohort studies been
associated with the progression of both mild to moderate and more advanced valvu-
lar diseases (Zheng et al. 2019; Capoulade et al. 2018), and most recently also with
aortic valve microcalcification in individuals without macroscopically detectable
valve pathology (Despres et al. 2019), thus also pointing to a role in earlier-stage
14 Lp(a) and Aortic Valve Stenosis, Stroke, and Other Noncoronary Cardiovascular… 245

disease. The totality of evidence pointing to high Lp(a) levels as a potentially modi-
fiable cause of calcific AVS thus holds great promise for improved prevention of
symptomatic AVS upon future availability of effective Lp(a)-lowering drugs.

Stroke

Ischemic Stroke

Lp(a) is a firmly established risk factor for myocardial infarction and AVS by pro-
posed pathophysiological mechanisms such as atherosclerosis and thrombosis. The
association of high Lp(a) levels with risk of ischemic stroke (IS) is not as firmly
established as results from several studies are unclear and somewhat conflicting.
Most studies did find increased risk of IS with high Lp(a) levels such as in the large
prospective Atherosclerosis Risk in Communities (ARIC) study from the USA,
which included both White and Black individuals with a 79% higher risk ratio for
high versus low levels (Ohira et al. 2006). Another large prospective contemporary
study from Denmark, the Copenhagen General Population Study (N = 46,699),
found increased risk of IS per 50 mg/dL higher Lp(a) levels with a hazard ratio of
1.2 (95% CI: 1.1–1.3) in observational analyses and with genetic estimates of 1.2
(1.0–1.4) via KIV2 and 1.3 (1.1–1.5) via rs10455872, indicating a causal role
(Fig. 14.3) (Langsted et al. 2019a). In support of a genetic association, the previ-
ously mentioned large UK Biobank study found an odds ratio of 0.87 (0.79–0.96)
for risk of IS for one standard deviation genetically lower Lp(a) levels (Emdin et al.
2016). Also, meta-analyses of observational studies, for example, from the Emerging
Risk Factors Collaboration including data from 24 studies (Emerging Risk Factors
Collaboration et al. 2009) and from India including data from 41 studies (Kumar
et al. 2021) on IS, find an association of high Lp(a) levels with increased risk of
IS. In the Prospective Epidemiological Study of Myocardial Infarction (PRIME)
study, a large prospective study from Northern Ireland and France, the association
was not significant, but the point estimate indicated an increased risk of IS with high
Lp(a) levels (Canoui-Poitrine et al. 2010). On the contrary, data from the Physicians’
Health Study including White middle-aged males from the USA found no associa-
tion between high Lp(a) levels and risk of IS (Ridker et al. 1995).
Notably, the risk estimates for IS for high Lp(a) levels or corresponding genetic
variants are lower than those reported for myocardial infarction and AVS, perhaps
indicating that the pathophysiology for IS might be different.

Hemorrhagic Stroke

In most cases, the pathophysiology of hemorrhagic stroke differs greatly from the
causes of IS. High Lp(a) levels have previously been associated with a low risk of
bleeding (Langsted et al. 2017) perhaps due to the proposed antifibrinolytic
246 A. Langsted and P. R. Kamstrup

effects of Lp(a) because of its homology with plasminogen. Most studies examin-
ing the role of Lp(a) in stroke have focused on ischemic or overall stroke as
described above, and results on hemorrhagic stroke are even more mixed with
both protective and pathological effects of high Lp(a) levels reported. In a study
from Japan, it was found that high Lp(a) levels were associated with low risk of
hemorrhagic stroke, most significantly in men with a hazard ratio of 0.44
(0.21–0.96) for highest versus lowest tertile of Lp(a) levels (Ishikawa et al. 2013).
The meta-analysis from the Emerging Risk Factors Collaboration et al. (2009)
including nine studies on hemorrhagic stroke found no association of Lp(a) with
hemorrhagic stroke, in contrast to findings from two Chinese studies (Sun et al.
2003; Fu et al. 2020) which found high Lp(a) levels to be associated with increased
risk of hemorrhagic stroke.
The complex nature and fundamentally different causes of hemorrhagic stroke
compared to ischemic stroke might be one of the reasons for these highly conflicting
results. Studies including subtypes of hemorrhagic stroke based on the underlying
pathophysiology are needed to find meaningful associations.

Arterial Ischemic Stroke in the Young

In 2011, pediatric guidelines for cardiovascular disease risk reduction introduced

screening for high Lp(a) levels in children and adolescents with a previous ischemic
or hemorrhagic stroke (Expert Panel on Integrated Guidelines for Cardiovascular
Health and Risk Reduction in Children and Adolescents, National Heart, Lung, and
Blood Institute 2011). Several studies have examined the relationship of high Lp(a)

Fig. 14.3 Risk of ischemic Copenhagen General Population Study

stroke in the Copenhagen
General Population Study. Per 50 mg/dL (105 nmol/L) higher Lp(a)
Age- and sex-adjusted Number/Events
observational odds ratio
and causal genetic risk
ratios by LPA KIV2 and Observational 49,699/1666
LPA rs10455872 with 95%
confidence intervals for
ischemic stroke for 50 mg/
dL (105 nmol/L) higher Genetic
49,699/1666
Lp(a) levels. (Adapted from KIV2
J Am Coll Cardiol.
2019;74(1):54–66 with rs10455872 49,699/1666
permission from Elsevier)

Hazard ratio or causal risk ratio (95% CI)

for ischemic stroke
14 Lp(a) and Aortic Valve Stenosis, Stroke, and Other Noncoronary Cardiovascular… 247

levels and risk of arterial IS in the young. A meta-analysis including 4 studies and a
total of 90 events found that children with high Lp(a) levels have an odds ratio of 4.2
(2.9–6.1) for risk of arterial IS (Sultan et al. 2014). Another meta-analysis including
five studies (with two studies also included in the former meta-analysis) found an
odds ratio for high Lp(a) levels of 6.3 (4.5–8.7) in children with arterial IS (Kenet
et al. 2010).
Of note, as arterial IS is much less prevalent in children than in adults and is often
associated with underlying medical conditions, most of the studies on Lp(a) have
excluded children with other risk factors which may, therefore, limit the generaliz-
ability of the study findings; however, the risk estimates are substantial and should
be investigated further.

Other Noncoronary Cardiovascular Diseases

Heart Failure

The two to threefold increased risk of myocardial infarction and calcific AVS found
in individuals with Lp(a) levels in the top decile is consistent with Lp(a) also being
a possible risk factor for heart failure (HF), representing a major and increasing
health-economic burden in aging populations (Kamstrup 2021; Heidenreich et al.
2013). In 2015, a clear stepwise association of Lp(a) levels with risk of incident HF
was reported from the combined Copenhagen general population studies including
>98,000 adult individuals of Danish descent (Kamstrup and Nordestgaard 2016).
Lp(a) levels >90th percentile (>67 mg/dL) are associated with a 1.6–1.8-fold
increased risk as compared to individuals with levels in the lower third of the con-
centration distribution and with comparable genetic risk estimates in support of a
causal association. Notably, the association appeared largely driven by the likely
causal associations of Lp(a) with myocardial infarction and/or AVS, with 63% of
the Lp(a)-driven HF risk mediated by these two conditions (Kamstrup and
Nordestgaard 2016). Further, a 9% population attributable risk of HF was estimated
for high Lp(a) levels indicating that, given the development of future effective
Lp(a)-lowering treatments, a notable decrease in HF incidence may also be achieved.
The observational association of Lp(a) levels with HF was since replicated in
both the Atherosclerosis Risk in Communities (ARIC) study and in the Multi-
Ethnic Study of Atherosclerosis (MESA) (Agarwala et al. 2017; Steffen et al.
2018). Findings from the large 2019 Icelandic case-control study with informa-
tion on measured and genetically imputed Lp(a) plasma levels also provided
additional evidence of a causal association of Lp(a) with HF (Gudbjartsson et al.
2019). Thus, a 50 nM (~25 mg/dL) increase in genetically determined Lp(a) lev-
els is associated with a 5% increase in risk of HF. This is in addition to the more
pronounced risk increases reported for CAD, AVS, and peripheral arterial dis-
ease (PAD).
248 A. Langsted and P. R. Kamstrup

Peripheral Arterial Disease

The proposed pathophysiological pathway of lipoprotein(a) through interference

with fibrinolysis and thereby promoting thrombosis and the causal association with
myocardial infarction could also result in arteriosclerotic or thrombotic PAD. In a
prespecified analysis of the ODYSSEY OUTCOMES trial evaluating a PCSK9
inhibitor, it was found that the highest versus lowest quartile of lipoprotein(a) was
associated with a hazard ratio of 2.2 (1.4–3.6) for risk of PAD and further that low-
ering of high lipoprotein(a) levels reduced the risk (Schwartz et al. 2020). In a gen-
eral population study from Denmark, highest versus lowest tertile of lipoprotein(a)
was associated with an odds ratio of 1.6 (1.3–2.0) for risk of peripheral arterial
stenosis (Kamstrup et al. 2012). Further, large genetic studies examining genetically
determined high lipoprotein(a) levels have also found a solid likely causal associa-
tion with risk of PAD (Emdin et al. 2016; Laschkolnig et al. 2014).

Summary

Lp(a) has since its discovery been a lipoprotein particle of high interest in cardiovascu-
lar research due to a composition consistent with both proatherosclerotic and prothrom-
botic effects. It is now well established that high levels are associated both observationally
and genetically, and therefore likely causally, with increased risk of CAD, calcific AVS,
HF, IS, PAD, and mortality (Langsted et al. 2019b). The exact pathophysiology of high
Lp(a) levels has not yet been elucidated and may involve, in addition to proatheroscle-
rotic and prothrombotic effects, also proinflammatory and procalcific effects, and the
exact mechanisms behind different CVD manifestations may differ.
Guidelines today are transitioning from recommending measurement of Lp(a)
only in individuals at increased risk of cardiovascular disease to once-in-a-lifetime
measurement in all. Currently, promising lipoprotein(a)-lowering agents are being
tested, and studies will hopefully show that lowering of lipoprotein(a) will lower the
risk of CVD.

Disclosures PRK reports talks and consultancies sponsored by Physicians Academy of

Cardiovascular Education (PACE), Silence Therapeutics and Novartis. AL has nothing to disclose.

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Chapter 15
Lipoprotein(a) in Cardiovascular Disease:
Evidence from Large Epidemiological
Studies

Peter Engel Thomas, Signe Vedel-Krogh, and Børge G. Nordestgaard

Introduction

After the discovery of lipoprotein(a) by Kåre Berg in 1963, interest in lipoprotein(a)

was only modest until the LPA gene, coding for apolipoprotein(a)—the protein
unique to lipoprotein(a)—was sequenced in 1987 (Berg 1963; McLean et al. 1987)
(Fig. 15.1). This significantly increased scientific interest in lipoprotein(a). However,
as early general population studies failed to find a clear association between
lipoprotein(a) and cardiovascular disease, interest gradually declined (Nordestgaard
and Langsted 2016). Awareness of lipoprotein(a) was renewed in 2009 when genetic
evidence from a Mendelian randomization analysis of two large, general population
cohorts, the Copenhagen City Heart Study and the Copenhagen General Population
Study, showed that high lipoprotein(a) was causally associated with myocardial
infarction (Kamstrup et al. 2009). The interest in lipoprotein(a) was further comple-
mented the same year as evidence from the Emerging Risk Factors Collaboration
showed that lipoprotein(a) was continuously and independently associated with car-
diovascular disease (Erqou et al. 2009) and in a large study from Clarke et al. on
genetic variation in the LPA gene as the strongest genetic cardiovascular risk factor
out of 2100 candidate genes for cardiovascular disease (Clarke et al. 2009). With the
use of more accurate lipoprotein(a) assays, by accounting for regression dilution
bias, and with evidence from genetic studies which largely avoid confounding and
reverse causation, lipoprotein(a) was firmly established as a causal risk factor for
cardiovascular disease (Nordestgaard and Langsted 2016). This led to a significant
interest in developing pharmacological therapy specifically targeting lipoprotein(a),

P. E. Thomas · S. Vedel-Krogh · B. G. Nordestgaard (*)

Department of Clinical Biochemistry, Herlev and Gentofte Hospital, Copenhagen University
Hospital, University of Copenhagen, Copenhagen, Denmark
e-mail: [email protected]; [email protected];
[email protected]

© The Author(s), under exclusive license to Springer Nature 251

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_15
252 P. E. Thomas et al.

Fig. 15.1 Overview of scientific publications on lipoprotein(a) from the bibliographic database
Scopus since its discovery in 1963 and up until 2021. Lp(a) lipoprotein(a). (Illustration by Børge
G. Nordestgaard)

and HORIZON, the first phase 3 trial of specific lipoprotein(a)-lowering therapy,

was initiated in the year 2020 (Tsimikas et al. 2020). Two drugs that can reduce
plasma lipoprotein(a) by up to 90% are likewise in clinical development (Koren
et al. 2022; Nissen et al. 2022).
Herein we highlight evidence from contemporary general population studies on
lipoprotein(a) as a risk factor for cardiovascular disease in primary and secondary
prevention cohorts, including cohorts with multiple ethnicities. We especially focus
on myocardial infarction and heart failure, as evidence of lipoprotein(a) as a risk
factor for ischemic stroke, aortic valve stenosis, and peripheral arterial disease is
covered elsewhere in this book. The present chapter is not intended as a systematic
review of all published studies but rather as an overview of important contemporary
studies on elevated lipoprotein(a) as a risk factor for cardiovascular disease.

Lipoprotein(a) in Contemporary Primary Prevention Studies

Early meta-analyses of population-based prospective cohorts showed that lipopro-

tein levels were higher in individuals who developed ischemic heart disease than in
those who did not and that individuals in the top versus bottom third of the
lipoprotein(a) level distribution had a higher risk ratio of coronary heart disease
(Danesh et al. 2000). In 2008, data from the prospective Copenhagen City Heart
Study focused on risk of myocardial infarction in individuals with extremely high
lipoprotein levels and found a three to fourfold increased risk in individuals with
15 Lipoprotein(a) in Cardiovascular Disease: Evidence from Large Epidemiological… 253

extreme lipoprotein(a) levels, that is, ≥120 mg/dL (the 95th percentile) (Kamstrup
et al. 2008). The study also demonstrated that the risk of myocardial infarction
increased in a stepwise manner with increasing levels of lipoprotein(a). Further,
when combining data from two large prospective cohorts of the adult Danish popula-
tion, the Copenhagen City Heart Study and the Copenhagen General Population
Study, a stepwise increase in the risk of incident heart failure was observed with
increasing levels of lipoprotein(a) (Kamstrup and Nordestgaard 2016). For use spe-
cifically in this book, we have updated our former epidemiological studies based on
the Copenhagen General Population Study on the association between elevated
lipoprotein(a) and risk of myocardial infarction and heart failure in Fig. 15.2
(Kamstrup et al. 2008, 2009; Kamstrup and Nordestgaard 2016; Langsted et al.
2015). As can be observed, individuals with lipoprotein(a) in the 23rd–65th percen-
tile (5–17 mg/dL or 7–33 nmol/L) have an age- and sex-adjusted hazard ratio of 1.19
[95% confidence interval (CI): 1.06–1.32] for myocardial infarction when compared
with individuals with lipoprotein(a) in the 1st–22nd percentile (<5 mg/dL or
<7 nmol/L). Thus, lipoprotein(a) is a risk factor at a comparatively low level with
regard to myocardial infarction. However, the highest risk of myocardial infarction

Fig. 15.2 Age- and sex-adjusted Cox proportional hazard ratios for the lipoprotein(a)-associated
risk of myocardial infarction and heart failure. Based on ⁓69,000 individuals from the Copenhagen
General Population Study. CI confidence interval. (Data by Børge G. Nordestgaard, Signe Vedel-­
Krogh, and Peter E. Thomas)
254 P. E. Thomas et al.

is found in individuals in the top 5% of lipoprotein(a) levels (≥95 mg/dL or

≥202 nmol/L) with a hazard ratio of 2.03 (1.71–2.40) when compared with individu-
als with lipoprotein(a) in the 1st–22nd percentile. For heart failure, the risk increases
with levels of lipoprotein(a) above the 90% percentile; however, the highest risk is
again observed in individuals in the top 5% of lipoprotein(a) levels with a hazard
ratio of 1.44 (1.24–1.67) when compared with individuals with low levels of
lipoprotein(a). Compared to relative risk estimates, absolute risk considers the num-
ber of events in a fixed time period without direct comparison between groups. As
can be seen in Fig. 15.3, the highest absolute risks of myocardial infarction and heart
failure are also seen in individuals in the top 5% of lipoprotein(a) levels with abso-
lute risks of approximately 77 and 68 events per 10,000 person years, respectively.
Data from the UK Biobank support the findings from the Copenhagen cohorts.
Like the Copenhagen studies, the UK Biobank includes individuals from the adult,
general population, but the British cohort study also includes ethnicities other than
White. In total, the UK Biobank includes data on 8940 (1.9%) individuals of South
Asian origin, 7144 (1.6%) of Black origin, and 1435 (0.3%) of Chinese origin and
thus represents the largest multiethnic prospective cohort study of lipoprotein(a) to

Fig. 15.3 Absolute risk

estimates of myocardial
infarction and heart failure
in categories of
lipoprotein(a). Based on
⁓69,000 individuals from
the Copenhagen General
Population Study. (Data by
Børge G. Nordestgaard)
15 Lipoprotein(a) in Cardiovascular Disease: Evidence from Large Epidemiological… 255

date (Patel et al. 2021). As seen in Fig. 15.4, data from the UK Biobank illustrate the
markedly heterogeneous distribution of lipoprotein(a) levels in individuals of White,
South Asian, Black, and Chinese descent and a clear log-linear association between
higher lipoprotein(a) levels and increased risk of atherosclerotic cardiovascular dis-
ease in the multiethnic population. In the overall study population, a hazard ratio of
1.11 (95% CI: 1.10–1.12) per 50 nmol/L higher lipoprotein(a) levels was observed.
Crucially, despite the clear difference in the distribution of lipoprotein(a) levels
between ethnic groups, the estimated hazard ratio for atherosclerotic cardiovascular
disease per 50 nmol/L higher lipoprotein(a) was similar for Whites [hazard ratio 1.11
(1.10–1.12)], South Asian [hazard ratio 1.10 (1.04–1.16)], and Black individuals
[hazard ratio 1.07 (1.00–1.15)]; there were too few Chinese for meaningful risk esti-
mates in this group. Thus, the risk conferred by a given higher lipoprotein(a) level
was broadly similar across racial groups. This is especially important for Black indi-
viduals where the median lipoprotein(a) concentration is four times higher than the
median concentration in White individuals. These findings are consistent with previ-
ous cohort studies of multiple ethnicities such as the ARIC (Atherosclerosis Risk in
Communities) study, the MESA (Multi-Ethnic Study of Atherosclerosis) study, and
the INTERHEART study (Virani et al. 2012; Paré et al. 2019; Guan et al. 2015).
Data from the Copenhagen cohorts and the UK Biobank clearly illustrate
lipoprotein(a) as a risk factor for atherosclerotic cardiovascular disease including
myocardial infarction and heart failure in primary prevention studies; however,
many other studies have provided crucial contributions to the understanding of
lipoprotein(a) in cardiovascular disease, as reviewed previously (Nordestgaard and
Langsted 2016; Kamstrup 2021). Importantly, generally observational cohort stud-
ies cannot be used to establish causality, as they may be prone to confounding and
reverse causation. However, for lipoprotein(a) levels that are 80–90% genetically

Fig. 15.4 Lipoprotein(a) concentrations according to ethnicity (left panel) and multivariable-­
adjusted hazard ratio for atherosclerotic cardiovascular disease according to lipoprotein(a) concen-
tration using cubic natural splines (right panel). Based on 460,506 individuals from the UK
Biobank. Left panel: Median lipoprotein(a) values, dimensions of the box capture the 25th and
75th percentiles, and whiskers capture an additional one interquartile range. (Adapted with permis-
sion from Patel et al. Arterioscler Thromb Vasc Biol. 2021;41:465–474)
256 P. E. Thomas et al.

Table 15.1 Sources of evidence that elevated lipoprotein(a) causes morbidity and mortality in
adults in a primary prevention setting
Case-­ Large
control or Meta-­analyses Large Large genome-­ Randomized
cross-­ of prospective prospective Mendelian wide double-blind
sectional observational observational randomization association lipoprotein(a)
studies studies studies studies studies lowering trials
Angina Yes Not examined Yes Yes Yes Not examined
pectoris
Myocardial Yes Yes Yes Yes Yes Trial ongoing
infarction
Heart failure Yes Not examined Yes Yes Yes Not examined
Cardio­ Not Not examined Yes Yes Yes Trial ongoing
vascular examined
mortality
Table by Børge G. Nordestgaard

determined, it can be argued that even observational epidemiological studies deter-

mine causality. Nevertheless, the ascertainment of causality using direct genetic
evidence for lipoprotein(a) is covered elsewhere in this book. The current evidence
of lipoprotein(a) as a risk factor for atherosclerotic stenosis, myocardial infarction,
heart failure, and cardiovascular mortality in primary prevention and in different
types of studies is summarized in Table 15.1. To date, no randomized controlled trial
has demonstrated that lowering of lipoprotein(a) results in a reduced risk of cardio-
vascular disease in either a primary or a secondary prevention setting. Crucially,
ongoing lipoprotein(a)-lowering trials are mainly focused on secondary prevention
cohorts, and much work on unraveling the role of lipoprotein(a) as a risk factor in
the primary prevention setting therefore remains.

 ipoprotein(a) in Contemporary Secondary

L
Prevention Studies

Interestingly, the role of lipoprotein(a) as a risk factor for recurrent cardiovascular

events, that is, in a secondary prevention setting, long remained an area of contro-
versy. This controversy arose from the heterogeneity of findings in secondary pre-
vention studies, which may have been due to confounding biases such as index
event bias, and a lack of individuals with high levels of lipoprotein(a) included
(Boffa et al. 2018).
With the controversy regarding elevated lipoprotein(a) as a risk factor in second-
ary prevention in mind, Willeit et al. conducted a meta-analysis of placebo-­controlled
statin trials in an attempt to assess lipoprotein(a)-associated risk of cardiovascular
events in patients with established cardiovascular disease (Willeit et al. 2018).
Figure 15.5 shows the comparative predictive values of on-statin vs. on-placebo
lipoprotein(a) for the risk of cardiovascular disease when comparing individuals
with lipoprotein(a) levels of 50 mg/dL or higher with individuals with lipoprotein(a)
15 Lipoprotein(a) in Cardiovascular Disease: Evidence from Large Epidemiological… 257

Fig. 15.5 Age- and sex-adjusted comparative predictive value of on-statin versus on-placebo
lipoprotein(a) concentrations for incident cardiovascular disease. (Adapted with permission from
Willeit et al. Lancet 2018;392:1311–20)

Fig. 15.6 Cumulative incidence of recurrent major adverse cardiovascular events (MACE)
according to concentrations of lipoprotein(a). CI confidence interval. (Adapted with permission
from Madsen et al. Arterioscler Thromb Vasc Biol. 2020;40:255–266)

levels lower than 50 mg/dL. The data from the seven trials included shows that
higher lipoprotein(a) is positively associated with increased risk of cardiovascular
events in a linear relationship independent of other cardiovascular risk factors in
both patients on statin treatment and on placebo. The association of on-statin higher
lipoprotein(a) with cardiovascular disease risk was stronger than for on-placebo
higher lipoprotein(a), indicating that when low-density lipoprotein (LDL) choles-
terol is reduced by statins, the risk conferred by elevated lipoprotein(a) becomes
more important. Thus, individuals with previous cardiovascular disease and ele-
vated lipoprotein(a) are at substantial residual risk of cardiovascular disease even
while on statin treatment. Nevertheless, randomized trials can be affected by index
event bias, that is, bias that may occur when the occurrence of a particular event is
required for inclusion in a study (Dahabreh and Kent 2011); however, a study of
2527 individuals from the general Danish population with a history of cardiovascu-
lar disease supports the findings from the statin-trial meta-analysis (Madsen et al.
2020). Figure 15.6 shows how higher levels of lipoprotein(a) are associated with
higher risk of recurrent major adverse cardiovascular events. Compared with indi-
viduals with lipoprotein(a) <10 mg/dL (18 nmol/L), the multivariable-adjusted sub-
hazard ratios for major adverse cardiovascular events were 1.29 (95% confidence
258 P. E. Thomas et al.

interval: 1.04–1.59) for individuals with 10–49 mg/dL (18–104 nmol/L), 1.46
(1.14–1.89) for individuals with 50–99 mg/dL (105–213 nmol/L), and 2.17
(1.59–2.98) for individuals with ≥100 mg/dL (≥214 nmol/L). These findings were
independently confirmed in two other cohorts of the Danish general population.
In conclusion, evidence from both post-hoc analyses of clinical statin trials and
from general population studies of individuals with a history of cardiovascular dis-
ease suggest that elevated lipoprotein(a) is associated with an increased risk of
recurrent cardiovascular disease.

Future Perspectives

Large epidemiological studies have established an association between elevated

lipoprotein(a) levels and increased risk of cardiovascular disease in both primary
and secondary preventive settings and in multiple ethnicities. Crucially, it cannot be
deduced from these studies that lowering of lipoprotein(a) will lead to a reduction
in cardiovascular morbidity or mortality. Such an effect must be demonstrated in
randomized clinical trials, where one is ongoing as of the year 2022 (NCT04023552),
and hopefully two further studies will follow as two new drugs already show prom-
ising results with up to a 90% lowering of lipoprotein(a) (Koren et al. 2022; Nissen
et al. 2022). Results from such studies are eagerly awaited.

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Chapter 16
Lipoprotein(a) and Immunity

O. I. Afanasieva , T. I. Arefieva , M. V. Ezhov , and S. N. Pokrovsky

Introduction

Lipoprotein(a) [Lp(a)] is an atherothrombogenic lipoprotein particle that differs in

its composition and physicochemical and biological properties from other lipopro-
teins and contains a unique apolipoprotein(a) molecule [apo(a)]. The relationship
between the immune system and lipid metabolism has been evaluated for many
decades. An increased blood Lp(a) concentration is a proven risk factor for athero-
sclerotic cardiovascular disease (ASCVD). Lawn’s hypothesis about Lp(a) as a
repair factor remains relevant until today (Lawn et al. 1992). Recent studies suggest
participation of humoral and cell immunity in wound healing and regeneration and
in inflammatory diseases (Masoomikarimi and Salehi 2022; Eming et al. 2017). An
elevated Lp(a) level in long-living persons suggests possible participation of immu-
nological factors in both the physiological and pathophysiological Lp(a) pathways
(Panza et al. 2007). It is assumed that with increased life expectancy and in the pres-
ence of “inflammaging,” [Inflammaging is the long-term result of the chronic physi-
ological stimulation of the innate immune system, which can become damaging
during ageing—a period of life largely unpredicted by evolution (Franceschi et al.
2018)] Lp(a) may become a factor contributing to atherosclerosis and other inflam-
matory diseases (Franceschi et al. 2018).

O. I. Afanasieva · T. I. Arefieva · M. V. Ezhov · S. N. Pokrovsky (*)

National Medical Research Center of Cardiology named after E. I. Chazov MOH of Russian
Federation, Moscow, Russia
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 261

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_16
262 O. I. Afanasieva et al.

Lipoprotein(a) and IgG and IgM Autoantibodies (AAbs)

The production of immunoglobulins (Ig) by B cells is necessary for the recognition,

neutralization, and removal of exogenous and endogenous pathogens and for main-
taining homeostasis. The concept of “natural” antibodies synthesized by B1 cells
with specificity to alien and native proteins was first proposed in 1908 by Ehrlich
(Piro et al. 2008). Natural IgM antibodies are encoded by the germline cells, and
they are present in the umbilical cord blood of newborns. It is assumed that the level
of natural IgM antibodies is maintained constant throughout life (Holodick et al.
2017). The main biological functions of natural IgM are removal of apoptotic cells,
protection from infection, and maintenance of tissue homeostasis (Reyneveld et al.
2020; Wang et al. 2016). The protective effect of IgM AAbs to oxidized LDL (low-­
density lipoprotein) (oxLDL) produced by B1 cells has been described in several
studies and literature reviews (Tsimikas et al. 2012; van den Berg et al. 2018;
Pattarabanjird et al. 2021). The presence of circulating Lp(a)-containing immune
complexes in the plasma of patients with coronary heart disease (CHD), healthy
donors, and patients with autoimmune diseases has been reported in several studies.
Most of the immune complexes found in the plasma of healthy donors contained
IgM AAbs against Lp(a), unlike patients with CHD (Wang et al. 2003; Sabarinath
and Appukuttan 2015; Klesareva et al. 2016).
Recently, we have shown that the levels of IgM AAbs against Lp(a) were higher
in patients without atherosclerosis or non-stenosing lesions of the coronary arteries
(Afanasievа et al. 2016b). Such a protective function of these IgM AAbs was also
present in patients with severe hypercholesterolemia (Klesareva et al. 2018). In a
retrospective study of 1228 patients, the lower the IgM level of Lp(a) AAbs and the
higher the concentration of Lp(a), the more vascular beds there were with stenosing
atherosclerotic lesions (Tmoyan et al. 2021).
The autoimmune theory of atherosclerosis was formulated by Klimov more than
40 years ago. He showed that modified lipoproteins acquire autoantigenic properties
and trigger an immune response to the “altered self” (Klimov 1990). The role of
autoantigens is played by modified LDL, as well as lipoproteins containing oxi-
dized phospholipids (Virella and Lopes-Virella 2008). Elevated plasma levels of
IgG AAbs to oxLDL are associated with angiographically verified coronary athero-
sclerosis and progression of carotid lesions (Salonen et al. 1992). Previously, a
direct relationship between the level of IgG AAbs against Lp(a) and the number of
affected coronary arteries was demonstrated (Afanas’eva et al. 2014). The content
of IgG AAbs against MDA (malondialdehyde)-LDL in the upper quartile was asso-
ciated with the risk of cardiovascular events at a 10-year follow-up (Prasad et al.
2017). However, the role of Lp(a), as well as oxLDL, as possible specific autoanti-
gen for B2 cells remains controversial (Ravandi et al. 2011). Nevertheless, studies
aimed at using immunoglobulins specific to oxidized epitopes present on lipopro-
teins’ and apoptotic cells’ surfaces for the treatment of ASCVD are in progress (de
Vries et al. 2021; Pluijmert et al. 2021; Ståhle et al. 2020).
16 Lipoprotein(a) and Immunity 263

Lp(a), such as LDL-like particles, also could be affected by modification of their

protein and/or lipid compounds; such modifications activate humoral immune
responses and create AAbs formation. Lp(a) AAbs immune complexes removed by
macrophages can be transferred to foam cells.
The IgM and IgG antibody classes against Lp(a) detected in human serum appear
to have not only different origins but also different functions. Natural IgM implies
an evolutionary advantage to neutralize Lp(a) and to eliminate it. The appearance of
autoantibodies of different IgG subclasses indicates the activation of adaptive
immunity, which perceives Lp(a) as the antigen, and causes subsequent develop-
ment of inflammatory reactions.

Lipoprotein(a) and Innate Immunity Cells

Monocytes and macrophages play a critical role in innate immunity (Libby et al.
2013) and have been the subject of numerous studies in connection with Lp(a).
Lp(a) was detected in macrophage cell-rich areas of atherosclerotic plaques in
humans according to morphology and immunohistochemistry studies (Sotiriou et al.
2006). On the other hand, individuals with elevated Lp(a) level exhibit enhanced
accumulation of peripheral blood mononuclear cells in the arterial wall compared to
individuals with normal levels of Lp(a) (van der Valk et al. 2016). Apo(a) stimulates
the production of reactive oxygen species and matrix metalloproteinase-­9 by colla-
gen-adherent monocytes, and this effect was inversely associated with the molecular
weight of apo(a) (Sabbah et al. 2019). Apo(a) also caused increased secretion of
IL-8 by macrophages of the THP-1 and U-937 cell lines (Scipione et al. 2015).
Monocytes isolated from subjects with elevated Lp(a) demonstrated an enhanced
cell surface expression of chemokine receptors, adhesion molecules, and scavenger
receptors (CCR7, CD62L, CD11b, CD11c, CD29, CD36, SR-A). Apo(a) upregu-
lates the expression of the β2-integrin Mac-1 (CD11b/CD18), thereby facilitating
cell adhesion and migration capacity. Several signaling cascades leading to altered
gene expression profiles were found to contribute to Lp(a)-induced monocyte che-
motactic activity (Scipione et al. 2015; Dzobo et al. 2022).
Besides displaying an activated and proinflammatory phenotype, monocytes iso-
lated from individuals with elevated Lp(a) exhibited an increased secretion of pro-
inflammatory cytokines (IL-1β, IL-6, TNFα) and a decrease in the anti-inflammatory
cytokine IL-10 after stimulation via toll-like receptors. OxPLs associated with
apo(a) as potent danger-associated molecular patterns (DAMPs) could be respon-
sible for these effects (Koschinsky and Boffa 2022).
Apo(a) antisense treatment resulted in downregulation of proinflammatory gene
expression in monocytes, including interferon (IFN)α, IFNγ, and toll-like receptor
(TLR) pathways, and subsequent changes in monocyte phenotype and function, that
is, a reduction in chemokine receptors CCR2 and CX3CR1 and transendothelial
migratory capacity (Stiekema et al. 2020).
264 O. I. Afanasieva et al.

The number of circulating monocytes in apo(a) transgenic mice was four times
higher than in wild-type mice and remained elevated for 3 weeks after Ca2+-induced
vascular damage (Huang et al. 2014). Also, Lp(a) affects the maturation of mono-
cytes in humans (Schnitzler et al. 2020).
Monocytes are divided into three subpopulations, depending on the content of
CD14 and CD16 surface markers, classical CD14++CD16−, intermediate
CD14++CD16+, and nonclassical CD14+CD16++, while the latter two populations
have the most pronounced proinflammatory and profibrotic potential. The participa-
tion of circulating monocytes in atherogenesis has been proven (Vergallo and Crea
2020), but the contribution of various subpopulations of monocytes to chronic
inflammatory states is currently under discussion (Yang et al. 2014; Ożańska
et al. 2020).
The high content of CD16+ monocytes is associated with unstable atheroscle-
rotic plaques in the coronary arteries (Kashiwagi et al. 2010) and predicts the risk of
cardiovascular events (Rogacev et al. 2012). In CHD patients, an increased content
of intermediate monocytes CD14++CD16+ occurs with hyperlipoproteinemia(a)
(Krychtiuk et al. 2015a), atherogenic dyslipidemia (Krychtiuk et al. 2015b), and
dysfunctional high-density lipoproteins (Krychtiuk et al. 2014). The association of
elevated Lp(a) concentration with absolute and relative content of CD14+CD16++
was shown in a retrospective study (Afanasieva et al. 2021). Since the function of
this subpopulation is to remove “cellular debris,” it is assumed that it contributes to
elimination of excess Lp(a).
Neutrophil granulocytes are the largest population of circulating phagocytizing
leukocytes capable of synthesizing a wide range of substances. Neutrophils and
“neutrophil extracellular traps” (NETs) formed by them were found in atheroscle-
rotic plaques of laboratory animals and humans (Afanasieva et al. 2021). NETs
stimulate the production of IL-1 by macrophages and activate IL-17-producing
T-helpers (Th17) in apoE-deficient mice, contributing to inflammation in the vessel
wall (Döring et al. 2017). There are no data on the effect of Lp(a) or apo(a) on the
formation of neutrophil traps. The absolute number of neutrophils and the neutrophil-­
lymphocyte index, as well as the concentration of Lp(a), was significantly higher in
patients with stenosing atherosclerosis of various vascular beds (Tmoyan et al.
2021). The evaluation of the effect of Lp(a) on neutrophil activation is a promising
avenue of further research.

 he Role of Lipoprotein(a) and Proinflammatory Status

T
in ASCVD Pathogenesis

Data on the association of increased Lp(a) concentration with systemic inflamma-

tion and its markers are ambiguous (Pirro et al. 2017). The risk of cardiovascular
events associated with Lp(a) was significantly higher in the presence of “proinflam-
matory” genotype IL-1 (Naka et al. 2018) or elevated C-reactive protein level (Puri
et al. 2020).
16 Lipoprotein(a) and Immunity 265

A higher lymphocyte count is associated with a higher apoB level; Lp(a) was
inversely associated with basophil count in men but not in women according to a
population study with 417,132 participants (Tucker et al. 2021). Low molecular
weight apo(a) phenotype, reduced lymphocyte count, and increases in neutrophil
granulocytes potentiated the risk of CHD in patients with type 2 diabetes (Suzuki
et al. 2013).
The combination of a higher absolute monocyte count (>0.54 × 109 cells/mL)
with elevated Lp(a) (≥30 mg/dL) is associated with higher risk of major adverse
cardiovascular events (MACE) in patients with premature CHD manifestation
(Afanasieva et al. 2022) (Fig. 16.1). An increase of Lp(a) concentration and the
percentage of CD14++CD16+ monocytes potentiated risk of multivessel coronary
disease (Afanasieva et al. 2021; Filatova et al. 2022) (Fig. 16.2).
A lower level of IgM AAbs against Lp(a) is negatively correlated with the con-
centration of sCD25 [the soluble form of the IL-2 receptor and a surrogate marker
of T-cell activation (Brusko et al. 2009)] and associated with stenosing coronary
atherosclerosis (Afanasievа et al. 2016b). This fact may serve as a confirmation of
participation of both Lp(a) and T-cells in atherogenesis and also the immunomodu-
latory ability of IgM AAbs against Lp(a) (Wang et al. 2016).
Systemic inflammation accompanies age-related changes in lymphocyte sub-
populations (Thomas et al. 2020). In patients with ASCVD, the number of naïve
lymphocytes, including regulatory cells, decreases with age, while the level of
effector populations, that is, Th1 and Th17, remains constant (Filatova et al. 2021).
T-Lymphocytes with predominating Th1 are detected in atherosclerotic plaques
(Saigusa et al. 2020). Th17, a subpopulation of CD4+ lymphocytes producing
IL-17, also has a proatherogenic effect. Th17 cells participate in the immune
response against their own and alien antigens by attracting myeloid cells to a place
of inflammation, activating lymphocytes and secreting proinflammatory cytokines
(Gao et al. 2010; Park et al. 2005). On the contrary, regulatory T-cells have anti-­
inflammatory activity and inhibit atherogenesis (Albany et al. 2019). Thus,

Fig. 16.1 The proportion of major adverse cardiovascular events (MACE) in patients with prema-
ture coronary heart disease depending on blood monocyte count and lipoprotein(a) concentration.
Two-hundred adult patients with early coronary heart disease manifestation (before 55 years in
men and 60 years in women) were enrolled, median follow-up 12 years. MACE, nonfatal myocar-
dial infarction, ischemic stroke, coronary artery bypass grafting, and hospitalization for unstable
angina (Afanasieva et al. 2022)
266 O. I. Afanasieva et al.

Fig. 16.2 Association of lipoprotein(a), CD14++CD16+ intermediate monocyte subpopulation,

and their association with coronary atherosclerosis severity (n = 150). Odds ratio (OR) of triple
vessel disease vs no significant, and 1–2-vessel disease was calculated according to logistic
regression analysis adjusted for age, sex, type 2 diabetes, and hypertension (Afanasieva et al. 2021)
16 Lipoprotein(a) and Immunity 267

age-­related deficiency of regulatory cells and a shift of the immune balance toward
effector populations may contribute to atherosclerosis progression.
Activation and increased amounts of Th17 are related to the progression of ath-
erosclerosis and risk of coronary events (Liuzzo et al. 2013). The ratio of circulating
Treg/Th17 is reduced in patients with severe coronary atherosclerosis (Potekhina
et al. 2015). The concentration of Lp(a) is not associated with the content of various
T-cell subpopulations (Afanasieva et al. 2016a, b). However, an increased content of
circulating Th17 (% of CD4+ lymphocytes), as well as a reduced content of Treg or
IL-10 CD4+-producing cells along with Lp(a) concentrations above 12 mg/dL, is
associated with severe coronary atherosclerosis (Afanasievа et al. 2016b) and
carotid atherosclerosis progression (Afanasieva et al. 2016a). Thus, the increased
concentration of Lp(a) and proinflammatory status with some shifts in immunity
could potentiate atherosclerosis progression.

Lipoprotein(a) as a Carrier of Inflammatory Mediators

Differences in the physicochemical and immunochemical properties of LDL and

Lp(a) have been noted for a long time (Zawadzki et al. 1988). The apo(a) moiety has
a binding site for oxidized phospholipid (oxPL) that determines its proinflammatory
effects on immune cells (Koschinsky and Boffa 2022).
Proteomic analysis shows that Lp(a) may serve as a carrier of many protein mol-
ecules, and their spectrum differs in Lp(a) and LDL (Bourgeois et al. 2020a; von
Zychlinski et al. 2011, 2014). These proteins can participate in the processes of
oxidation, cell proliferation and intercellular interactions, immunomodulation and
activation of the complement system, and blood clotting (Bourgeois et al. 2021).
Such a variety of proteins can provide Lp(a) particles with the ability to partici-
pate in the response to injury or damage. Possible ways that Lp(a) participates in
activation of the immune system via its plasma components are shown in Fig. 16.3.
The complement components C3 and C4 associated with Lp(a) could determine
the interaction of Lp(a) with innate and acquired immunity. The complex of Lp(a)
with α2 macroglobulin can interact with low-density lipoprotein receptor-related
protein 1 (LRP-1) and can not only contribute to the internalization of Lp(a) with
high molecular weight isoforms of apo(a) (März et al. 1993) but also induce the
migration of myeloid cells, such as monocytes and neutrophils.
Lp(a) constitutes the main pool of lipoprotein-associated proprotein convertase
subtilisin/kexin type 9 PCSK9 (Tavori et al. 2016). There is evidence of the modu-
lating effect of PCSK9 on cell immunity (Liu and Frostegard 2018; Kim et al.
2019). Also, PCSK9 can regulate the number of CD36 and LRP-1 receptors (Shapiro
et al. 2018), which are expressed by hematopoietic cells, participating in the pro-
cesses of hemostasis, inflammation, and tissue regeneration. The binding of PCSK9
to CD36 (Qi et al. 2021) can be recognized as a “danger signal” of innate immunity
(Silverstein 2021).
268 O. I. Afanasieva et al.

Fig. 16.3 Possible mechanisms of lipoprotein(a) contribution to immune cell activation. lysoPC
lysophosphatidylcholines, LPARs LPA receptors or G-protein-coupled receptors, IL-1β interleukin
1β, IL-6 interleukin 6, TNF tumor necrosis factor, CXCL2 chemokine (C–X–C motif) ligand 2,
MCP-1 monocyte chemoattractant protein 1, mRNA messenger RNA, LRP-1 low-density lipopro-
tein receptor-related protein 1, TGFβ transforming growth factor beta, IFNγ interferon γ, PRRs
pattern recognition receptors, oxPL oxidized phospholipids, PLA2 phospholipase A2, α2M
alpha-­2-macroglobulin, PCSK9 proprotein convertase subtilisin/kexin type 9, C3 and C4 comple-
ment components 3 and 4, and apo(a) apolipoprotein(a)

Lp(a) as a possible carrier of autotaxin and a source of lysophosphatidic acid is

associated with calcification and aortic valve stenosis (Bouchareb et al. 2015;
Bourgeois et al. 2020b). The lysophosphatidic acid participates in the differentia-
tion and homing of T-lymphocytes (Zhang et al. 2012; Knowlden and Georas
2014). Both facts suggest another possible mechanism of Lp(a) action on the
immune system.
An association of MCP-1 with Lp(a) via oxidized phospholipids of Lp(a) has
been described (Wiesner et al. 2013). The attachment of Lp(a) containing MCP-1 at
the site of injury can lead to increased recruitment of monocytes. Thus, proteins
associated with the Lp(a) particle as well as oxPL may explain its proinflammatory
properties.
Many properties of Lp(a), as well as its biological roles, remain a mystery despite
more than 50 years of research. Lp(a) is able to carry affected areas not only the
cholesterol necessary for the synthesis of new cells but also biologically active com-
ponents that attract phagocytes of the innate immune system. It can be assumed that
the original role of Lp(a) as a factor in damage repair and transport systems has
largely been lost at the present time. An increased concentration of Lp(a) set against
the background of genetic, epigenetic, and environmental variables has become a
powerful risk factor for atherosclerotic cardiovascular diseases. We designed an
immunosorbent for specific Lp(a) apheresis and proved that specific Lp(a), but not
16 Lipoprotein(a) and Immunity 269

LDL, removal by extracorporeal treatment can lead to stabilization and even regres-
sion of atherosclerotic lesions in coronary and carotid arteries (Pokrovsky et al.
2016, 2020). This study was the first direct clinical observation and confirmation of
Lp(a) atherogenicity in humans (Pokrovsky et al. 2017). The elucidation of molecu-
lar and cellular mechanisms of Lp(a) involvement in inflammatory remodeling of
the arterial wall engaging the Lp(a) immunity axis is a promising direction for the
development of new therapeutic approaches.
Lp(a) is an extremely interesting polymolecular complex, and as we learn more
about it, it is clear the less we understand about its enormous functional range and
its capacity to interact with and influence important pathways, such as immunity,
inflammation, thrombosis, and oxidation.

Acknowledgements The authors are grateful to Professor Gilbert Thompson for his help in
proofreading text of the manuscript.

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Chapter 17
When Should We Measure Lipoprotein(a)?

Karam Kostner

Introduction

Lipoprotein(a) was originally described by K. Berg in 1963 as a genetic variant of

β-lipoprotein (Kostner and Kostner 2017). Evidence from large observational and
epidemiological studies support causality of Lp(a) as one of the strongest risk fac-
tors for atherosclerotic cardiovascular disease (ASCVD) and calcific aortic valve
disease (CAVD). These are further supported by genome-wide association and
Mendelian randomisation studies (Kostner et al. 2018). Levels above 50 mg/dL are
considered elevated and seen in up to 20% of the population. Over the last 10 years,
there has been much discussion about when to measure Lp(a) and how to treat it
(Kostner et al. 2013). It is generally accepted to measure Lp(a) in individuals with
premature CV disease when traditional risk factors do not account for this. Lp(a) is
also often measured to reclassify risk in intermediate-risk individuals, where ele-
vated levels lead to more aggressive treatment of other risk factors. Imaging modali-
ties such as coronary calcium scores are often used in conjunction with traditional
and emerging plasma markers to estimate risk. Several phase II and III trials with
antisense and Si RNA-targeted therapies are currently under way and will help us
understand whether Lp(a) lowering in and of itself reduces CV risk. With the avail-
ability of effective therapies, it will be possible to define groups who benefit from
these therapeutic interventions. The cost-effectiveness of routine screening and test-
ing for Lp(a) also remains to be shown.

K. Kostner (*)
Department of Cardiology, Mater Hospital and University of Queensland, Brisbane, Australia
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 275

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_17
276 K. Kostner

Lp(a) and ASCVD Risk Assessment

Lp(a) levels are relatively stable over a lifespan as they are mainly genetically deter-
mined which is why a single measurement of serum Lp(a) is sufficient for most
patients unless a secondary cause is suspected or a specific treatment is started to
reduce its concentration. Availability and reimbursement of cost-effective methods
to measure Lp(a) as well as standardisation of assays are important and are dis-
cussed in different chapters of this book. It is generally more practical and cost-­
effective to measure Lp(a) concentrations instead of its genetic determinants. Lp(a)
measurement may be considered in both primary and secondary preventions. In
children with familial hypercholesterolemia (FH), for example, Lp(a) is a better
predictor of CV disease in family members than LDL (Zawacki et al. 2018).
In primary prevention, focus should be directed towards absolute CV risk assess-
ment, where patients with elevated Lp(a) are treated more aggressively for traditional
risk factors such as LDL, especially if they are in an intermediate-risk group (Verbeek
et al. 2018). The availability of imaging methods such as calcium scoring by CT and
plaque assessment by CT, MRI and ultrasound has improved CV risk assessment and
is often used in conjunction with lipid risk factors such as Lp(a). In secondary pre-
vention, elevated Lp(a) is a driver of residual CV risk. The Justification for the Use
of Statins in the JUPITER study (Khera et al. 2014) supported the premise that Lp(a)
is a significant independent contributor to residual risk. This is further supported by
data from the Atherothrombosis Intervention in Metabolic syndrome with low HDL/
high triglycerides: Impact on Global Health outcomes (AIM-HIGH) study (Albers
et al. 2013) and a recent meta-analysis (Willeit et al. 2018).
Recent outcome studies with proprotein convertase subtilisin/kexin type 9
(PCSK9) inhibitors have underlined the importance of Lp(a) measurement. In the
Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects
With Elevated Risk (FOURIER) trial, reduction in risk of major acute coronary
events (MACE) with evolocumab was associated with the baseline and change in
Lp(a) levels (O’Donoghue et al. 2019). In the Evaluation of Cardiovascular
Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab
(ODYSSEY OUTCOMES) trial, reduction in risk of total cardiovascular events
with alirocumab was also associated with baseline and change in Lp(a) levels
(Szarek et al. 2020). Reduction in risk of major adverse limb events (MALE) with
alirocumab was also associated with baseline and change in Lp(a) levels (Schwartz
et al. 2020). These trials support the conclusion that elevated Lp(a) is a major driver
of residual risk. Although large cardiovascular outcome trial data does not currently
exist to guide Lp(a) therapeutic intervention, indicators of significant increased risk,
including multivessel disease, PAD (peripheral artery disease), premature disease
onset, familial hypercholesterolaemia (FH), diabetes, renal disease and recurrent
presentations with acute coronary syndrome (ACS), will likely be considered as
clinical indicators for consideration of agents specifically targeting Lp(a) and
17 When Should We Measure Lipoprotein(a)? 277

Table 17.1 Indications for measurement of Lp(a)

Measurement of Lp(a)
(1) Should be considered in adults to assess or stratify ASCVD risk in those with the following
clinical features: a personal history of premature ASCVD (<60 years), family history of
premature ASCVD, family history of high Lp(a) (>200 nmol/L), familial
hypercholesterolaemia, significant renal impairment and early-onset calcific aortic stenosis
(<60 years)
(2) Should be considered in those with intermediate 10-year ASCVD risk
(5–15%) when classical risk algorithms are used such as the Framingham risk score, the
PROCAM risk score, the ESC Heart Score or the Australian and New Zealand risk
calculator, if it allows patients to be re-stratified into a higher-risk category if Lp(a) is
elevated above >200 nmol/L, which in turn leads to more intensive management of treatable
risk factors, especially LDL cholesterol
(3) Should be considered in those with suboptimal lowering of low-density lipoprotein
cholesterol (LDL-C) despite adherence to guideline-recommended treatment
(4) Should be considered in those with recurrent or progressive ASCVD despite of optimally
treated plasma LDL-C concentrations
(5) Should be considered in children and adolescents with familial hypercholesterolaemia,
premature ASCVD, a first-degree relative with significantly elevated Lp(a) (>200 nmol/L)
and a family history of premature ASCVD

already lead many clinicians to try to achieve very low LDL targets with statins,
ezetimibe and PCSK9 inhibitors. Measurement of Lp(a) is discussed as follows and
is summarised in Table 17.1:
1. Measurement of Lp(a) should be considered in adults to assess or stratify
ASCVD risk in those with the following clinical features: a personal history of
premature ASCVD (<60 years), family history of premature ASCVD, family
history of high Lp(a) (>200 nmol/L), familial hypercholesterolaemia, significant
renal impairment and early-onset calcific aortic stenosis (<60 years).
2. Measurement of Lp(a) should be considered in those with an intermediate
10-year ASCVD risk (5–15%) when classical risk algorithms are used such as
the Framingham risk score, the PROCAM risk score, the ESC Heart Score or the
Australian and New Zealand risk calculator, if it allows patients to be re-­stratified
into a higher-risk category or if Lp(a) is elevated above >200 nmol/L, which in
turn should ultimately lead to more intensive management of treatable risk fac-
tors, especially low-density lipoprotein cholesterol (LDL-C).
3. Measurement of Lp(a) should be considered in those with suboptimal lowering
of LDL-C despite adherence to guideline-recommended therapy.
4. Measurement of Lp(a) should be considered in those with recurrent or progres-
sive ASCVD despite optimally treated plasma LDL-C concentrations.
5. Measurement of Lp(a) should be considered in children and adolescents with
familial hypercholesterolaemia, premature ASCVD, a first-degree relative with
significantly elevated Lp(a) (>200 nmol/L) or a family history of prema-
ture ASCVD.
278 K. Kostner

Recommendations from International Guidelines

Even though most major international guidelines recognise that Lp(a) is a risk enhanc-
ing factor, there is still no unanimous agreement as to when to measure Lp(a) and how
to deal with increased Lp(a) values. The reasons for this are that few commonly
accepted assays and reference standards exist, there is a lack of effective medications
available to lower Lp(a) and apart from LDL apheresis, therapeutic interventions to
lower Lp(a) have not yet shown a reduction in MACE. Traditionally, levels >30 mg/
dL were considered elevated, with thresholds for inclusion into outcome trials gener-
ally higher (>50 mg/dL). Table 17.1 shows risk thresholds for different Lp(a) levels.
The European Atherosclerosis Society and European Cardiology Societies, how-
ever, likely underestimate the importance of elevated Lp(a) as they focus only on
people with extremely elevated levels (>180 mg/dL or >430 nmol/L) who they sug-
gest may have a lifetime risk of ASCVD equivalent to that of heterozygous FH (Mach
et al. 2019). They do recommend that Lp(a) measurement be considered at least once
in each adult person’s lifetime to assist with risk stratification, particularly in those
considered at moderate or higher risk (Mach et al. 2019). The HEART-UK consensus
statement on Lp(a) also supports the measurement of Lp(a) levels in patients with a
personal or family history of premature ASCVD, those with FH or other genetic dys-
lipidaemias (such as familial combined hypercholesterolaemia) or early-onset
ASCVD and patients with first-degree relatives who have significantly elevated Lp(a)
(>200 nmol/L) levels. The statement suggests that the cardiovascular risk conferred
by Lp(a) is determined by its serum concentration, with 32–90 nmol/L equivalent to
minor risk, 90–200 nmol/L to moderate risk and 200–400 nmol/L to high risk, with
concentrations >400 nmol/L equivalent to very high risk (Kostner et al. 2018),
Table 17.2.
The National Lipid Association (NLA) suggests that the 80th percentile in pre-
dominantly Caucasian US populations is ~100 nmol/L and ~150 nmol/L in African
Americans, although it is unclear whether different risk thresholds should be applied
(Wilson et al. 2019). The American Heart Association (AHA) and American College
of Cardiology (ACC) recognise elevated Lp(a) as a ‘risk-enhancing factor’ in the
development of ASCVD, with levels ≥125 nmol/L (≥50 mg/dL) considered high
risk (Grundy et al. 2018). Other groups, including the Canadian Cardiovascular
Society and the Mighty Medic Group, suggest that Lp(a) might aid risk assessment
in patients at high risk or with premature CVD/CAD, with Lp(a) levels <30 mg/dL
considered normal (Anderson et al. 2016). Two International Classification of

Table 17.2 Risk thresholds for Lp(a) concentration (adapted from Heart UK consensus statement)
ASCVD risk Lp(a) level, (nmol/L) Lp(a) level, (mg/dL) Percentile of population
Moderate 100–200 40–90 80–95th
High 200–400 90–180 95–99th
Very high >400 >180 99th
Source: Cegla et al. (2019)
17 When Should We Measure Lipoprotein(a)? 279

Diseases (ICD)-10 codes have been added to justify Lp(a) testing, E78.41 = ele-
vated Lp(a) and Z83.430 = Family History of elevated Lp(a).

Synopsis (Authors’ Recommendations)

Knowledge of Lp(a) levels is particularly valuable in reclassification of patients at

intermediate risk of ASCVD, as assessed by established risk algorithms, especially
if it leads to more aggressive therapy of other risk factors such as LDL. Lp(a) should
also be measured in individuals with a personal or family history of premature
ASCVD (or calcific aortic valve stenosis) and familial hypercholesterolaemia (FH)
and in those with recurrent vascular events despite optimal LDL lowering.
Information on Lp(a) levels may guide more aggressive treatment of conventional
risk factors or lead to assessment of subclinical atherosclerosis with newer imaging
methods such as CT.
The value of cascade testing first-degree relatives of an index case with very high
Lp(a) has not been demonstrated. However, it could help define and consolidate the
family history of ASCVD and improve adherence to existing therapies in secondary
prevention, as well as adherence to healthy lifestyle and behaviour in primary pre-
vention in family members. Elevated Lp(a) with a co-existent polygenic hypercho-
lesterolaemia or familial combined hyperlipidaemia may mimic FH and should
always be considered in patients who return a negative genetic test for FH. Finally,
results from large clinical trials with Lp(a)-lowering agents that are currently under-
way will likely have an impact on Lp(a) measurement and likely provide us with
effective therapies for this atherogenic lipoprotein.

References

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SM. Relationship of apolipoproteins A-1 and B, and lipoprotein(a) to cardiovascular outcomes:
the AIM-HIGH trial (atherothrombosis intervention in metabolic syndrome with low HDL/
high triglyceride and impact on Global Health outcomes). J Am Coll Cardiol. 2013;62:1575–9.
Anderson TJ, Gregoire J, Pearson GJ, et al. 2016 Canadian Cardiovascular Society Guidelines for
the management of dyslipidemia for the prevention of cardiovascular disease in the adult. Can
J Cardiol. 2016;32:1263–82.
Cegla J, Neely RDG, France M, Ferns G, Byrne CD, Halcox J, Datta D, Capps N, Shoulders C,
Qureshi N, Rees A, Main L, Cramb R, Viljoen A, Payne J, Soran H, HEART UK Medical,
Scientific and Research Committee. HEART UK consensus statement on lipoprotein(a): a call
to action. Atherosclerosis. 2019;291:62–70.
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SC Jr, Sperling L, Virani SS, Yeboah J. AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/
APhA/ASPC/NLA/PCNA guideline on the management of blood cholesterol: a report of the
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guidelines. Circulation. 2018;39(25):e1082–143.
Khera AV, Everett BM, Caulfield MP, Hantash FM, Wohlgemuth J, Ridker PM, Mora
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from the JUPITER trial (justification for the use of statins in prevention: an intervention trial
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Kostner KM, Kostner GM. Lipoprotein(a): a historical appraisal. J Lipid Res. 2017;58:1–14.
Kostner KM, Marz W, Kostner GM. When should we measure lipoprotein(a)? Eur Heart
J. 2013;34:3268–76.
Kostner KM, Kostner GM, Wierzbicki AS. Is Lp(a) ready for prime time use in the clinic? A pros-­
and-­cons debate. Atherosclerosis. 2018;274:16–22.
Mach F, Baigent C, Catapano AL, et al. ESC/EAS Guidelines for the management of dyslipidae-
mias: lipid modification to reduce cardiovascular risk. Eur Heart J. 2019;2019:1–78.
O’Donoghue M, Fazio S, Stroes E, et al. Lipoprotein(a), PCSK9 inhibition and cardiovascular risk.
Insights from the Fourier trial. Circulation. 2019;39:1483–92.
Schwartz GG, Steg GG, Szarek M, et al. Peripheral artery disease and venous thromboembolic
events after acute coronary syndrome. Role of lipoprotein(a) and modification by alirocumab.
Prespecified analysis of the Odyssey Outcomes Randomized Clinical Trial. Circulation.
2020;141:1608–17.
Szarek M, Bittner V, Aylward P, et al. Lipoprotein(a) lowering by alirocumab reduces the total
burden of cardiovascular events independent of LDL lowering: ODYSSEY OUTCOMES Trial.
Eur Heart J. 2020;75:133–44.
Verbeek R, Hoogeveen RM, Langsted A, Stiekema LCA, Verweij SL, Hovingh GK, Wareham NJ,
Khaw KT, Boekholdt SM, Nordestgaard BG, Stroes ESG. Cardiovascular disease risk associ-
ated with elevated lipoprotein(a) attenuates at low low-density lipoprotein cholesterol levels in
a primary prevention setting. Eur Heart J. 2018;39:2589–96.
Willeit P, Ridker PM, Nestel PJ, Simes J, Tonkin AM, Pedersen TR, Schwartz GG, Olsson AG,
Colhoun HM, Kronenberg F, Drechsler C, Wanner C, Mora S, Lesogor A, Tsimikas S. Baseline
and on-statin treatment lipoprotein(a) levels for prediction of cardiovascular events: individual
patient-data meta-analysis of statin outcome trials. Lancet. 2018;392:1311–20.
Wilson DP, Jacobson TA, Jones PH, et al. Use of lipoprotein(a) in clinical practice: a biomarker
whose time has come. A scientific statement from the National Lipid Association. J Clin
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Zawacki AW, Dodge A, Woo KM, Ralphe JC, Peterson AL. In pediatric familial hypercholesterol-
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Chapter 18
Measurement of Lipoprotein(a)
in the Clinical Laboratory

David Sullivan, Catherine Woolnough, Nimalie Perera, Jay Ramanathan,

and Tony Badrick

 ackground to Lipoprotein(a) Measurement

B
for Clinical Purposes

Lipoprotein(a) [Lp(a)] represents one of the most promising, causal independent risk
factors for a chronic disease like atherosclerotic cardiovascular disease [ASCVD]
that has emerged this century. Its appropriate use hinges on well-targeted implemen-
tation. This requires understanding of the whole analytical cycle (pre-­analytical, ana-
lytical and post-analytical), as well as the involvement of many stakeholders. Patients
who require testing need to be aware of the importance of undertaking the test.
Ordering physicians need to be cognisant of target populations and the ways in
which results should be applied. Laboratory scientists should consider the subtleties
and intricacies of Lp(a) measurement. Implementation requires dialogue with the
diagnostic industry which carries much of the responsibility for the provision of
robust, validated products with traceable standardization processes which fulfil the
governmental approval and monitoring processes, thereby guaranteeing minimum
standards. Preventive health experts can optimize the manner in which Lp(a) results

D. Sullivan (*) · C. Woolnough · N. Perera

Department of Chemical Pathology, Royal Prince Alfred Hospital, Sydney Local Health
District, NSW Health Pathology, University of Sydney, Sydney, NSW, Australia
e-mail: [email protected]
J. Ramanathan
Department of Chemical Pathology, Royal Prince Alfred Hospital, Sydney Local Health
District, NSW Health Pathology, University of Sydney, Sydney, NSW, Australia
Department of Medicine, Liverpool Hospital, Sydney South West Local Health District,
Liverpool, NSW, Australia
T. Badrick
Royal College of Pathologists of Australasia Quality Assurance Program,
St Leonards, NSW, Australia

© The Author(s), under exclusive license to Springer Nature 281

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_18
282 D. Sullivan et al.

are applied, whilst healthcare system administrators need to be able to appreciate the
health and economic benefits associated with testing, as well as the limits beyond
which this may become counterproductive. All stakeholders must be involved in the
establishment of these objectives for the optimum widespread clinical testing of
Lp(a). Ongoing attention will need to be directed towards utilization of Lp(a) testing
in the future because it is likely to evolve over time, especially during the introduc-
tion of specific forms of treatment which target Lp(a) directly.
The transition from a research biomarker to a clinically relevant laboratory risk
factor requires appreciation of the expectations that apply to the clinical environ-
ment in which Lp(a) will be measured. Like research laboratories, clinical service
laboratories need to maintain the highest standards of process control throughout
the analytical cycle. This extends from the pre-analytical sample collection and
preparation to the reporting and management of Lp(a) levels. It represents a setting
in which fastidious attention to correct patient identification and curation of the
sample is paramount. One of the advantages of laboratory automation is the reduc-
tion in the opportunity for sources of human error such as transcription errors.
Optimization and monitoring of accuracy and precision, which are components of
total laboratory error, are implicit in the objectives of any analytical laboratory.
Whilst research laboratories appropriately exploit a degree of independence in their
approach to analytical problems, clinical service laboratories need to function
within the context of laboratory networks and the wider healthcare system. This
requires a high level of collaboration and collegiate activity. Such collaboration
forms the basis for national and international standardization programmes, harmo-
nization initiatives, reference range and laboratory report consensus as well as pub-
lic health recommendations. More subtle considerations such as equity, accessibility
and intrinsic value within the healthcare system also require careful consideration.
The clinical setting in which Lp(a) is tested also affects interpretation. Lp(a) is
an acute phase reactant, so sample collection should be postponed until patients
have recovered from acute inflammatory episodes or concurrent illness. One excep-
tion is in the setting of acute coronary syndrome because the opportunity to identify
high-risk patients outweighs the risk of false-positive results. On the other hand,
requirements for urgent laboratory turnaround time are less applicable to Lp(a) for
the time being. In comparison to other analytes, technical aspects of Lp(a) measure-
ment have posed substantially greater challenges to the implementation of Lp(a)
testing than is usually the case. Nevertheless, clinicians can be assured that Lp(a)
testing is fit for purpose. Furthermore, current developments are rapidly overcoming
the remaining challenges, as will be discussed in more detail.

Method Selection

Before discussing Lp(a) method selection in detail, it is worth acknowledging the

limited distribution of its hallmark apolipoprotein, apolipoprotein(a) [apo(a)] in
nature. The presence of apo(a) in hedgehogs is thought to represent convergent
18 Measurement of Lipoprotein(a) in the Clinical Laboratory 283

evolution (Utermann 1999), whilst its absence from other species except humans
and higher apes remains difficult to explain (Utermann 1999). The absence of apo(a)
in research models such as mice and rabbits has created logistic limitations because
studies in such models require gene expression that is limited to one or two specific
isoforms in each model. Furthermore, the high degree of polymorphism of the LPA
gene locus creates an unusual degree of inter-individual genotypic and phenotypic
variability. This poses important demands on clinical laboratory measurement
which impact the commercial production and validation of diagnostic reagents such
as monoclonal antibodies.
Another noteworthy aspect of Lp(a) is its highly skewed distribution (Kronenberg
and Utermann 2013), which deviates markedly from normal distribution. Statistical
analysis requires transformation, such as logarithmic transformation, or the use of
non-parametric statistics. Another consequence of the skewed distribution is Lp(a)’s
wide analytical range. Methods need to be able to quantify levels which may be
nearly tenfold higher than the upper limit of ideal methods (Stefanutti et al. 2020).
Lp(a) is a polymorphic particle that consists of a low-density lipoprotein (LDL)
particle covalently linked via a di-sulphide bond to apo(a). There is one apo(a) and
one apolipoprotein B [apoB] molecule per particle (Albers et al. 1996). Apo(a) is
the protein product of the highly polymorphic LPA gene locus which codes for this
large protein in which a variable number of plasminogen-like kringle repeats are
present (Cegla et al. 2021). In plasma, Lp(a) is the major transporter of oxidized
phospholipid. This may contribute towards Lp(a)’s pro-atherogenic, pro-­
inflammatory and pro-thrombotic properties (Scipione et al. 2015). In theory, the
separate components of Lp(a) offer alternative options for quantification.
The cholesterol and other lipid components of lipoproteins are strongly impli-
cated in the pathophysiology of ASCVD. The measurement of Lp(a)’s cholesterol
component would provide a consistent frame of reference for the pathogenicity of
different lipoprotein classes, including Lp(a). On the other hand, the confounding
effect of triglyceride via modification by cholesterol ester transfer protein can alter
the size and density of atherogenic lipoproteins. This confuses the relationship
between lipoprotein cholesterol and lipoprotein number (Carr et al. 2019). There is
clear evidence that the atherogenic effect of most Apo B100-containing particles is
proportional to their number rather than their cholesterol content. Efforts have been
made to quantify Lp(a) in terms of cholesterol content, but the methods are not
robust and evidence of specific advantages over other methods is lacking.
Furthermore, the concordance of Lp(a) cholesterol measurement with Lp(a) molar
results has been called into question (Konerman et al. 2012).
The measurement of Lp(a)’s oxidized phospholipid content has been deduced
from immunoassay quantification via antibody E06 (Tsimikas et al. 2009). This
suggests that most oxidized phospholipid is transported by Lp(a). Whilst excellent
correlation between oxidized phospholipid immunoassay results and measured
Lp(a) levels has been demonstrated, other techniques such as lipidomic measure-
ment by mass spectroscopy suggest that the transport of oxidized phospholipid on
Lp(a) is potentially more complex (Leibundgut et al. 2013). The main advantage of
quantification of Lp(a)-associated oxidized phospholipid is the quantification of a
284 D. Sullivan et al.

particular potentially toxic component, but as has already been explained, other
components such as cholesterol are likely to have a modifying effect. The current
clinical laboratory approach to Lp(a) is that it is best measured via its unique apo(a)
component and that measurement of the cholesterol or oxidized phospholipid com-
ponents of Lp(a) is not warranted because they are not consistent with the need to
measure Lp(a) particle number.
Lp(a) levels have been assessed in terms of mass or molar units. LPA genotype
has been assessed mainly in terms of kringle IV type 2 [KIVT2] repeats. The pres-
ence of a greater number of KIVT2 repeats is associated with a relative reduction in
plasma Lp(a) molar concentrations. Metabolic turnover studies suggest that the
effect is mediated via Lp(a) synthesis (Chan et al. 2019). Other genetic variations
further modify the relationship (Coassin et al. 2017, 2019), but overall the relation-
ship between mass and molar assessments of Lp(a) concentration is confounded
because larger molecular weight isoforms are associated with a smaller number of
particles. For example, the protein composition of Lp(a) has been shown to vary
between 30 and 46% by weight (Ruhaak and Cobbaert 2020). Given that mass (mg/
dL) measurements are affected by all components of the Lp(a) particle, they are
inherently more variable than measures of particle concentration. This has estab-
lished the need to quantify Lp(a) in molar units (nmol/L) (Ruhaak and Cobbaert
2020). Unfortunately, many of the historically seminal clinical studies were con-
ducted at a time when this relationship was less evident. As a result, quantification
in terms of mass units (g/L, mg/dl) lingers as a legacy.
Based on these principles, Lp(a) should be measured by a method (e.g. immuno-
assay or mass spectroscopy) that is apo(a) isoform independent. This has required the
introduction of appropriate standards, calibrators and calibration protocols designed
to permit estimation and reporting in molar rather than mass units. The necessary
processes have been pursued throughout the past two to three decades and have
involved phase 1 and phase 2 standardization programmes conducted by IFCC
(International Federation of Clinical Chemistry) (Tate et al. 1998, 1999), leading to a
WHO/IFCC reference reagent for immunoassay (SRM 2B) (Dati et al. 2004). This
has occurred in parallel with the development of mass spectroscopy methods
(Cobbaert et al. 2021) including a proposed candidate reference method (Marcovina
et al. 2021). Sustained efforts by dedicated clinical scientists have put in place the
associated safety and quality measures which are required to maintain laboratory
performance (Marcovina and Albers 2016). This will be discussed in the next section.
Table 18.1 demonstrates that these initiatives continue to penetrate the market for
diagnostic Lp(a) immunoassays. As a result, isoform-specific assays which report in
molar units have started to predominate. Diagnostic companies will continue to
drive this process provided such a prerequisite continues to be demanded by clini-
cians (Wyness and Genzen 2021). The transition from mass units (mg/dL) to molar
units (nmol/L) is necessary because the inverse relationship between genetically
determined apo(a) KIVT2 repeats and Lp(a) particle number confounds the concept
of a single standard conversion factor (Tsimikas et al. 2018). Conversion factors
also vary with the assay, Lp(a) concentration and storage. Although equivalent mass
levels could be identified for the molar levels designated as medical decision-­
making cut-offs, mass units should be phased out as soon as possible. Lp(a)-
lowering treatment may require serial measurements in individual patients,
18 Measurement of Lipoprotein(a) in the Clinical Laboratory 285

Table 18.1 Lipoprotein(a) assay methods available

Analytical
Number principle (IT,
of labs immunoturbi­­ Reference
enrolled dimetric; IN, interval
Measurement in immunonephe­­ Units of from reagent Information
system RCPAa Reagent lometric) reporting IFU sources
Thermofisher 1 Randox IT nmol/Lb <30 mg/dL Laboratory
Konelab <75 nmol/Lc
20XTi
Beckman 1 Randox IT nmol/Lb <90 nmol/Ld Laboratory
Coulter
AU5800
Roche Cobas 9 Rochee IT nmol/Lb <75 nmol/Lc RCPA,
c501/c502/ Roche, kit
c503 IFU,
laboratory
Binding site 1h Binding site IT nmol/Lb <75 nmol/L Binding site
Optilite
Abbott Alinity 1 Abbott IT mg/dL <30 mg/dL RCPA,
c Abbott, kit
insert,
laboratory
Abbott 2 Abbott IT mg/dL <30 mg/dL RCPA,
Architect Abbott, kit
c4000 c8000 insert,
laboratory
Beckman 3f Beckman IN mg/dL Caucasian RCPA,
Coulter Coulter males Beckman
Immage 800 5.6– coulter, kit
33.8 mg/dL, insert
females
5.7–
31.2 mg/dL
Beckman 1g Beckman IT mg/dL <30 mg/dL Beckman
Coulter Coulter Coulter
AU480
Siemens 1 Siemens IN g/L <0.3 g/Li Siemens, kit
Nephelometer insert
II
Thermofisher 1 Thermofisher IT g/L 0.3 g/L Thermofisher
Konelab 30i kit insert,
laboratory
Data from the 2021 RCPA Special Lipids Survey
a
Numbers are from the 2021 RCPA Special Lipids Survey (RCPAQAP Special Lipids QAP 2021)
b
Calibrators are standardized to the WHO/IFCC international reference reagent SRM2B
c
Reference interval quotes Framingham data
d
RI from https://www.austinpathology.org.au/test-­directory/1247
e
Data from the RCPA Special Lipids Program end-of-cycle report 2019
f
Two of the three labs are outside of Australia
g
The lab using this method is in New Zealand
h
The lab enrolled is the manufacturer, not a pathology lab
i
Additional gender- and ethnicity-specific RIs are given in the reagent package insert
286 D. Sullivan et al.

preferably using the same assay, which makes the sole use of molar unit measure-
ments more logical and more urgent.
Immunoassay is usually the preferred method for high-throughput laboratories
due to logistic requirements. On the other hand, immunoassays may struggle in
comparison to the sensitive and specific results that can be achieved with dedicated
mass spectroscopy methods. Sophisticated and highly informative mass spectros-
copy methods have been described for the dedicated measurement of Lp(a)
(Lassman et al. 2014). Whilst such methods are generally robust, they may be more
difficult to align with external quality assurance programme method groups. This is
an important consideration because inter laboratory bias would lead to inconsis-
tency in the application of the cut-offs for medical decision-making.
Clinical laboratories are also able to analyse or refer samples for LPA genetic
analysis. LPA genetic polymorphisms exert most of their effects via the quantitative
phenotype of the associated Lp(a) level. Currently, LPA genotyping offers little in
the way of additional clinical benefit beyond quantitative plasma Lp(a) levels, so
there is little incentive to study the genotype separately for clinical purposes. On the
other hand, LPA genotype is one of the major contributors to “polygenic” risk scores
for cardiovascular disease [CVD] (Trinder et al. 2021). The separate contribution of
the two LPA gene alleles is usually managed by summation which reinforces the
concept of co-dominant inheritance of the plasma Lp(a) trait. Another potential
application of LPA genotyping is the possibility that pharmacogenomic assessment
of LPA may identify subjects who are likely to benefit from aspirin therapy for the
prevention of ASCVD (Shiffman et al. 2012). If required, routine genotyping meth-
ods such as massively parallel sequencing should suffice, even though this is not
ideal for detection of nucleotide repeats.

Safety and Quality

One of the main distinguishing features of clinical laboratories is the obligation to

meet the highest standards for safety as well as quality. This role is often played out
“behind the scenes”. Clinicians may be unaware that changes in patient results for
tests they order are monitored for unexplained discrepancies (so-called delta check-
ing) and potential medical emergencies (“critical results”). Tests are established in
a manner which tries to guarantee that results are available within the timeframe
required for medical decision-making. Lp(a) results are unlikely to be acutely life-­
threatening, so “turnaround time” is consistent with the average for non-urgent
immunoassays. This contrasts with urgent assays like troponin T or I, which may
warrant the provision of “point-of-care” testing options. Whilst this is not necessary
for Lp(a), a case can be made for the benefits associated with rapid notification of
results to patients whilst in the medical care setting because this could enhance the
management of complex problems such as ASCVD risk reduction. A point-of-care
test for Lp(a) has been developed, but it may not fulfil all current expectations. For
example, when last reviewed, it was reported in mass units.
18 Measurement of Lipoprotein(a) in the Clinical Laboratory 287

The quality aspects of clinical laboratory service need to be seen in a “whole of

health system” context. A test like Lp(a) must be performed consistently and return
the same results across all laboratories. There must be harmonization of all aspects
of the analytical process, and there must be a consistent interpretive framework in
which the results are applied. These requirements are usually overseen by regula-
tory processes which provide accreditation of diagnostic services. Sample collec-
tion for Lp(a) testing resembles the routine approach for collection and processing
of blood samples. Issues concerning recent illness have been discussed already. The
question of fasting status arises with lipoprotein analysis, particularly in the case of
triglyceride-rich lipoproteins [TRL]. Fasting is not necessary for Lp(a) measure-
ment because it does not affect the total plasma Lp(a) level. On the other hand,
reversible redistribution from its usual density fraction to d < 1.006 due to reversible
non-covalent association with TRL has been described (Cohn et al. 1991). The pro-
cess is influenced by isoform size and may need to be considered in mechanistic
studies.
Standardization of pre-analytical components such as patient preparation and
sample collection is underpinned by analytical protocols which apply standard
operating procedures for quantification using standard calibrators, calibration pro-
tocols and internal quality control processes. The process of establishing the frame-
work for these essential materials has been long and rigorous (Kostner et al. 1999;
Kostner and Steinmetz 1997). They allow the identification of different sources of
error to ensure that tests remain within analytical and clinical performance specifi-
cations. Laboratory errors are usually conceptualized as inaccuracy (“trueness” or
“bias”) and imprecision (variation around the “true” value). Clinical laboratories
place particular emphasis on precision because this provides a narrower range of
uncertainties, thereby aiding the detection of changes which cannot be attributed to
laboratory error. This needs to be considered in the context of the biological varia-
tion or fluctuations reflected by intra-individual variability. Intra-individual vari-
ability in Lp(a) levels is thought to be minor; however, levels may not be quite as
static (Marcovina et al. 2018a) as some clinicians imagine. One of the reasons for
clinical laboratories’ slight preference for precision over accuracy is that adjustment
can be made to correct accuracy via calibrators or correction factors provided preci-
sion is yielding reproducible results. Accuracy also requires a reference method and
a reference standard which give the highest level of trueness and precision possible.
There are currently no reference methods for Lp(a), though they are being devel-
oped. There is a reference material, but not all methods currently available are trace-
able to this material. There is current work on an improved reference material.
Nevertheless, Lp(a) results need to be interpreted in the context of designated quan-
titative cut-points, so accuracy of Lp(a) measurements cannot be compromised either.
The “whole of health system” integrity of the quality of laboratory results is
underpinned by systems of external quality assurance (EQA). Whereas research
tests may require sample exchanges with other labs to objectively monitor accuracy
and other aspects, widely used tests are scrutinized by a structured process in which
unknown samples are regularly circulated for analysis and the results are aggregated
to reflect the performance of individual laboratories according to their peers. Results
288 D. Sullivan et al.

are compared to all participants, and they are also grouped according to method,
reagents, equipment, etc. (Fig. 18.1). Target values are based on participant medians
and acceptable variation about the target determined using biological variation.
Duplication of some samples allows assessment of precision as well as accuracy.
Figure 18.1 shows an EQA result to illustrate several considerations that apply to
Lp(a). Firstly, the results were reported in mass units. The programme has responded
to requests to switch to molar units. Secondly, the samples must cater to several
analytes. In the case of the special lipid programme, the samples are created by
spiking with a lipoprotein concentrate to create a linear escalation of concentration
in duplicate samples. In this case, the concentrate lacked sufficient Lp(a) to approach
the lowest medical decision point. It will be logistically difficult to adjust for the
complexities of Lp(a) for several reasons. Firstly, the lipoprotein concentrate may
be derived from pooled samples, in which case the samples will comprise a mixture
of isoforms rather than the homozygous or heterozygous pattern expected in indi-
vidual patient sera. Secondly, whilst it may be possible to increase levels towards
the lower medical decision points, it may be difficult to encompass higher levels.
This may become important if change in Lp(a) levels becomes a treatment target. It
may be commercially difficult for EQA programmes to deal with Lp(a) separately
from other lipoproteins. The fact that some EQAs add TRL and high-density lipo-
protein in parallel rather than in reciprocal amounts illustrates that certain lipopro-
tein EQA results need to be interpreted with caution (Perera et al. 2010). The
standardization and harmonization initiatives which were mentioned earlier have
been particularly active in this area. EQA programmes for Lp(a) have been estab-
lished and analysed (Cegla et al. 2021; Cobbaert et al. 2012), but ongoing efforts
will be required (Scharnagl et al. 2019).

RCPAQAP Chemical Pathology

Lipoprotein (a) (g/L) Participant No. 241

0.17 IMPRECISION: Standard Deviation
Slope = 0.89 0.003 0.005 0.007 0.012 Your SD
i’cept = 0.030 Best 20% 50% 90%
0.010
0.13 Last Cycle
0.003 0.005 0.007 0.009 0.011
0.009
IMPRECISION: Coefficient of Variation
Your Results

X 3.5 15.0 18.3 25.9 Your CV

0.08 X X X
Best 30% 50% 90%
X X X
15.9
X X 5.0 10.0 15.0 20.0 25.0 Last Cycle
X X 16.0
0.04 X Low High INACCURANCY : Average Bias
0.004 0.011 0.033 Your Bias
Median 0.02 0.06 Best 50% 90%
Your Data 0.05 0.08 0.025
0.00 0.005 0.010 0.015 0.020 0.025 0.030 0.035 Last Cycle
0.00 0.04 0.08 0.13 0.17 0.022
Median Values
Analytical Performance Specifications
=0.06 up to 0.30: =20% >0.30 g/L

Fig. 18.1 Representative report (RCPAQAP Special Lipids QAP 2021) of EQA results for Lp(a)
prior to transition to molar units
18 Measurement of Lipoprotein(a) in the Clinical Laboratory 289

Clinical Application

Clinical laboratories need to be mindful of the clinical circumstances in which the

Lp(a) test has been performed. The intra-individual variability of Lp(a) measurements
before adulthood is moderate (Gidding et al. 1998). Intercurrent medical conditions
and therapy may influence the result. The presence of apo(a) fragments in urine
implies that Lp(a) levels may be affected by renal impairment. There is evidence to
suggest a complex relationship with renal function (Kostner et al. 2000, 2001;
Kronenberg et al. 1997; Cauza et al. 2003; Frank et al. 2001; Uhlig et al. 2005). Lp(a)
does increase in renal impairment, but it is uncertain whether this reflects decreased
catabolism. Hepatic synthesis is thought to be pivotal (Dieplinger and Utermann
1999), but hepatic function has not received a great deal of attention as a determinant
of Lp(a) levels. Bile acid metabolism has a strong influence which should not be over-
looked (Chennamsetty et al. 2011). Changes in endocrine status such as hypothyroid-
ism and acromegaly can increase Lp(a) levels whilst exogenous steroid hormones can
reduce Lp(a) levels. Other clinical factors affecting Lp(a) level have been summarized
comprehensively (Kostner and Kostner 2004), but perhaps the most important per-
spective is the role of Lp(a) as an exemplar of the use of Mendelian randomization to
identify a biomarker in the era of Precision Medicine (Hopewell et al. 2021).
Many patients undergoing testing for Lp(a) will be receiving therapy for dyslipi-
daemia, in which case those taking statins may experience an increase in Lp(a)
levels (de Boer et al. 2022; Ma et al. 2019) whilst those taking evolocumab or ali-
rocumab (Bittner et al. 2020) may experience a decrease in Lp(a) levels (O’Donoghue
et al. 2019; Gencer et al. 2021). Lp(a) levels are important risk determinants in
patients with familial hypercholesterolaemia due to reduced activity of LDL recep-
tors, but the receptor pathway for Lp(a) degradation is uncertain and any increase in
Lp(a) in FH remains to be fully explained (Kraft et al. 2000; Scholtz et al. 2000).
Several receptors have been implicated (McCormick and Schneider 2019) in Lp(a)
clearance. The functions of these receptors relate to other atherothrombotic phe-
nomena such as inflammation and thrombosis. There is some evidence that Lp(a)
may be prothrombotic (Koschinsky and Marcovina 2004) and hence a risk factor for
venous thrombosis (Sofi et al. 2007) and pulmonary emboli (Ignatescu et al. 1998).
This evidence is inconsistent, so Lp(a) is yet to take a place amongst the laboratory
markers of thrombophilia. The remaining clinical aspects of Lp(a) have been exten-
sively reviewed (Jawi et al. 2020; Nordestgaard and Langsted 2016; Kostner and
Kostner 2017; Marcovina et al. 2018b).

Interpretation

The epidemiological and Mendelian randomization studies (Nordestgaard and

Langsted 2016; Reyes-Soffer et al. 2022) which demonstrate that Lp(a) is an inde-
pendent and causative risk factor for ASCVD, myocardial infarction [MI], stroke
290 D. Sullivan et al.

and peripheral artery disease [PAD], as well as calcific aortic valve disease [CAVD],
are based on phenotypic and genotypic techniques. Due to its skewed distribution,
Lp(a) is not suitable for traditional definitions of a “normal” reference interval.
Plasma Lp(a) levels are positively skewed, with the median for Caucasian popula-
tions ~20 nmol/L (<10 mg/dL). However, like many other risk factors for chronic
disease, the upper limit of normal is less relevant than the threshold level at which
increased risk necessitates a particular medical decision. Notions of sensitivity and
specificity of Lp(a) testing will depend on the level at which such a cut-point is set.
Conversely, there is the reassurance that low levels of Lp(a) are associated with
reduced CVD risk (Coassin et al. 2017) and do not seem to be associated with any
pathological outcomes (Langsted et al. 2021).
One perplexing aspect of Lp(a) is its variation in association with racial differ-
ences. This seems to reflect multiple genetic variations including some in the region
of kringles KIV T6–T10 (Utermann 1999). Evidence suggests that this confounds
the quantitative relationship between Lp(a) level and CVD risk in some racial
groups (Geethanjali et al. 2003). This implies that medical decision points may need
to be adjusted to take account of the widely reported effects of race on Lp(a) level
(Stefanutti et al. 2020; Reyes-Soffer 2021; Ogorelkova et al. 2001). Studies are
lacking in the many Indigenous groups in whom socio-economic determinants of
health have created an excessive burden of CVD.
The perceived utility of Lp(a) testing depends on its ability to reclassify CVD
risk, particularly amongst those who are deemed to be at “intermediate risk” by
traditional methods. Lp(a) has demonstrated excellent capability in this regard in
the Bruneck Study (Willeit et al. 2014). The added benefit of Lp(a) in CVD risk
assessment may be presented as its contribution to the “C” statistic, but the author
of studies in which this estimate has been modest or gender-specific (Cook et al.
2018; Khera et al. 2014) has cautioned against the exclusion of biomarkers on this
basis (Cook 2007). Lp(a) levels may also modify the management (Burgess et al.
2018) of individuals who are not identified by routine risk factor assessments. This
includes young MI patients (Berman et al. 2021) and the relatives of those with
increased Lp(a). In due course, measurement of Lp(a) concentration could be con-
sidered in adults on at least one occasion to assess risk of ASCVD, but this amounts
to population screening, which will require convincing cost-benefit evidence. Lp(a)
testing and interpretation of results will be governed by policies determined by the
local healthcare system.

Healthcare Systems

The implementation of Lp(a) testing will depend on the policies of the relevant
healthcare systems. Their expectations will reflect the safety, quality and value per-
spectives outlined above. Clinical laboratories may consider the use of alerts and
interpretive comments on laboratory reports. These may emphasize the potential
need for assessment of ASCVD risk and cascade testing. Digital health technologies
18 Measurement of Lipoprotein(a) in the Clinical Laboratory 291

and decision support systems could be employed to enhance the management of

patients with elevated Lp(a), whilst telehealth services could be utilized for patients
in remote areas. Calculators for ASCVD risk stratification in both primary and sec-
ondary preventions could be modified to include Lp(a) as a predictor variable, and
clinical quality registries could be used to monitor the effectiveness of intervention.
Healthcare system policies will be guided by expert opinion in the form of guide-
lines and position statements such as those published by the Heart UK (Cegla et al.
2019), National Lipid Association (Wilson et al. 2019), Canadian Cardiovascular
Society (Pearson et al. 2021), EFLM (Langlois et al. 2020), NHLBI (Marcovina
et al. 2003), AHA (Grundy et al. 2019; Reyes-Soffer et al. 2022), ESC and EAS
(Mach et al. 2020). An Australian perspective was recently published (Ward et al.
2019), but modification is anticipated due to the emergence of targeted therapy for
Lp(a) (Swerdlow et al. 2021), the use of which will depend on its effect on CVD
outcomes. A global perspective will be required, as reflected by the size of the popu-
lation at risk, which exceeds one billion people (Kamstrup 2020). The previously
mentioned effects of race on Lp(a) levels will necessitate policies which have been
adapted to local circumstances. This includes the avoidance of financial barriers to
testing, especially for those who may be at increased risk of ASCVD due to socio-­
economic deprivation.

Conclusion

Cumulative research and international guidelines provide a foundation for the

imminent need to manage Lp(a) in the context of diverse international healthcare
systems. Evidence concerning the accuracy and application of Lp(a) measurement,
the safety and efficacy of therapy and the selection and monitoring of patients for
primary and secondary prevention is progressing rapidly, but it is still in its relative
infancy. Laboratory measurement of Lp(a) is fit for purpose, but it requires action-
able recommendations and supporting rationales along with recommendations for
implementation. Furthermore, the incorporation of Lp(a) measurement into clinical
practice for the prevention of ASCVD is likely to evolve via an iterative process.

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Chapter 19
Standardization of Analytical Methods
for the Measurement of Lipoprotein(a):
Bridging Past and Future Initiatives

Noemie Clouet-Foraison, Tomas Vaisar, and Santica M. Marcovina

Abbreviations

Apo(a) Apolipoprotein(a)
ApoA-I Apolipoprotein A-I
ApoB-100 Apolipoprotein B100
CV Coefficient of variation
CVD Cardiovascular diseases
ID Isotope dilution
IFCC International Federation of Clinical Chemistry
ISO International Organization for Standardization
JCGM Joint Committee for Guides in Metrology
KIV Kringle IV
KIV2 Kringle IV type 2
KIV9 Kringle IV type 9
LC-MS/MS Liquid chromatography-tandem mass spectrometry
LDL Low-density lipoproteins
Lp(a) Lipoprotein(a)
NIST National Institute of Standards and Technology
NWRL Northwest Lipid Research Laboratories
QC Quality controls
SD Standard deviation

N. Clouet-Foraison · T. Vaisar
Division of Metabolism, Endocrinology, and Nutrition, University of Washington,
Seattle, WA, USA
e-mail: [email protected]; [email protected]
S. M. Marcovina (*)
Medpace Reference Laboratories, Cincinnati, OH, USA
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 297

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_19
298 N. Clouet-Foraison et al.

SI International System of Units

SIL Stable isotope labelled
WHO World Health Organization

Standardization: What, Why, and How?

Importance of Standardization in Clinical Laboratory Medicine

Clinical laboratory measurements represent the foundation of medical care for

many pathologies. Diagnostics based on laboratory test results play a major role for
both clinical decision-making, treatment, and patient follow-up. It is therefore of
utmost importance that clinical laboratory measurements be reliable, precise, and
accurate.
From a clinical standpoint, the lack of reliability of medical test results can have
significant consequences on patient care. A study by the Mayo Clinic in the United
States demonstrated that a 3% error on the measurement of total cholesterol in a
clinical laboratory resulted in a 10% increase of erroneous diagnostics (National
Institute of Standards and Technology 2000). In the case of a false positive, patients
are needlessly treated, therefore increasing the risk of iatrogenic diseases and the
associated burden for medical care teams. On the other hand, in the case of a false
negative, patients are not treated, which can have dramatic consequences for their
life expectancy and quality of life. The lack of reliability of medical test results
additionally hinders the development of new therapies and understanding of pathol-
ogies, especially in the context of clinical trials (Plebani 2006).
From a financial standpoint, erroneous diagnostics lead to multiple additional
expenses for healthcare systems. The most important extra costs arise from provi-
sion of unnecessary treatments but also from the medical care of patients suffering
from the consequences of a late or absent treatment. Another major source of addi-
tional cost is the repetition of medical analyses (Miller et al. 2014a). Therefore,
ensuring reliability of laboratory test results represents a potentially significant
financial savings. A study performed in 2000 by the National Institute of Standards
and Technology (NIST) evidenced that the return on investment of the standardiza-
tion of total cholesterol measurements, calculated as the ratio benefit/associated
costs, was 4.5 with a social rate of return of approximately 154% (National Institute
of Standards and Technology 2000). The Cholesterol Reference Method Laboratory
Network also published in 2011 a study on the socioeconomic benefits associated
with the standardization program for lipid measurements in clinical laboratories.
The authors asked a panel of experts to estimate the share of the lipid standardiza-
tion program on the reduction of death from cardiovascular disease between 1980
and 2000, assuming 24% of this reduction was due to reduction of cholesterol levels
in patients. They then considered that every life-year saved represented either
$50,000, $115,000, or $300,000 based on different sources (Hoerger et al. 2011).
Socioeconomic benefits of implementing standardization of cholesterol in the
United States resulted in estimates of $338 million dollars per year for the most
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 299

conservative model and 7.6 billion dollars per year considering medians (Hoerger
et al. 2011).
Finally, in a globalized society, the availability of internationally recognized
clinical decision thresholds and reference intervals for treatment is of major impor-
tance. However, this requires that clinical test performed worldwide provides com-
parable results, which can only be achieved through the implementation of the
concept of metrological traceability.

Metrological Traceability, Standardization, and Harmonization

Implementing metrological traceability is the first step to standardization. The Joint

Committee for Guides in Metrology (JCGM) defines metrological traceability as
“the property of a measurement result whereby the result can be related to a refer-
ence through a documented unbroken chain of calibrations, each contributing to the
measurement uncertainty” (Joint Committee for Guides in Metrology (JCGM) 2012).
The first step in establishing a metrological traceability chain is to develop a
primary reference measurement procedure directly connected to the units of the
international system of quantities (SI). The reference method is then used to assign
a value to an ultrapure material, with certified purity and associated uncertainty,
called a primary reference material. This primary reference material is then used to
calibrate a secondary reference measurement procedure, which is in turn used to
value-assign a secondary, matrix-based reference material. In vitro diagnostic man-
ufacturers produce their own working calibrators that are commercialized with the
respective assays with value assigned by the secondary reference material. Following
this chain, measurements performed in a clinical laboratory for a given biomarker
are traceable directly to a unique reference point, usually called the anchor. All
along the traceability chain, uncertainties associated with the measurement increase.
Therefore, to obtain uncertainties that are fit-for-clinical-purpose at the bottom of
the chain, that is, in clinical laboratories, it is necessary that uncertainties associated
with the higher-order reference measurement procedure be minimized and well
controlled. It is commonly considered that uncertainties associated with the refer-
ence method should be at least two-fold smaller, if not three-fold, than that expected
in a clinical setting.
Establishing metrological traceability is the prime way to ensure comparability
between results obtained by different methods and laboratories, independent of time
and location in the world. However, there are two different possible scenarios: (1)
the case of a well-known and characterized analyte for which traceability to the SI
is achievable and (2) the case of a complex, heterogenous analyte for which knowl-
edge is incomplete. In the first case, the “simplicity” of the analyte and the technical
mastery of the measurement procedure make it possible to produce a high-purity
primary reference material traceable to the SI, making standardization possible.
However, in the second case where the analyte is of high complexity or heteroge-
neous, establishing standardization is hindered by methodological or technical
300 N. Clouet-Foraison et al.

issues (Stoppacher et al. 2015; Josephs et al. 2019). Then, instead of proceeding
with standardization, the alternative strategy is to establish harmonization of the
methods by producing a matrix-based secondary reference material with value
assigned by an arbitrarily designated reference method. The use of this material as
common calibrator for all the other procedures usually improves comparability of
the methods to a certain degree. However, since it is not anchored to the SI, harmo-
nization of the methods does not ensure accuracy of the measurements nor stability
of the values over time.
Furthermore, it is important to highlight that even though standardization and
harmonization may both improve between-method comparability, they do not guar-
antee it (Miller et al. 2014b). Indeed, there are multiple additional pre-analytical,
analytical, or post-analytical factors that can negatively impact method comparabil-
ity. In particular, the use of different methodologies to isolate, target, and measure
the analyte such as different antigen epitopes, different isolation techniques or
detection systems and methodologies, varying interferences, and different measure-
ment units can drastically impact method comparability. In this situation, even
though standardization is achieved, method comparability will remain poor until the
assays are improved, properly validated and common measurement units used.
Therefore, a prerequisite for a successful standardization is that the methods dem-
onstrate the necessary analytical performances to be deemed “standardizable.”

What Does “Establishing Standardization” Mean?

International organizations and expert groups, like the International Federation of

Clinical Chemistry and Laboratory Medicine (IFCC), the World Health Organization
(WHO) or the International Organization for Standardization (ISO), have made
major efforts in standardization and published norms, guidelines, and recommenda-
tions for the establishment of metrological traceability in clinical laboratories like
the ISO 15189 for in vitro diagnostic manufacturers and the ISO 17511:2020
(International Organization for Standardization 2020). Ideally, every single bio-
marker or clinically relevant analyte should have its full traceability chain. In prac-
tice, this is an arduous and challenging task. The practical aspects and associated
challenges will be covered in more depth in the following sections, but the first steps
to establishing a metrological traceability chain for a defined analyte can be sum-
marized as follows:
1. Production of a high-purity primary reference material. Its value should be
assigned by a primary reference measurement procedure, directly traceable to
the SI, and its purity should be certified.
2. Establishment of a higher-order secondary reference measurement procedure for
the measurement of the concentration of the analyte in the targeted matrix. This
procedure is calibrated with the pure primary reference material and should
demonstrate high levels of precision and accuracy.
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 301

3. Production of a secondary reference material to disseminate traceability along

the metrological chain. This matrix-based material should demonstrate a high
level of commutability, that is, propensity to behave similarly to a native
­individual patient sample, healthy or diseased, when measured by different ana-
lytical methods.

The Specific Case of Lp(a)

Measuring Lp(a): A Major Challenge

Lp(a) is a highly complex lipoprotein formed by a particle very similar in lipid and
protein composition to low-density lipoproteins (LDL) but characterized by the
presence of a single molecule of a unique protein, apolipoprotein(a) [apo(a)], bound
to the ApoB100 of LDL by a single disulfide bond (Schmidt et al. 2016). Circulating
serum levels of Lp(a) are predominantly genetically determined by the LPA gene
and do not substantially vary over time (Kronenberg 2016), although physiological,
dietary, hormonal, and environmental factors do contribute to its biological varia-
tion (Enkhmaa et al. 2016; Garnotel et al. 1998).
Apo(a) is a heavily glycosylated protein and its presence imparts distinct proper-
ties to Lp(a) distinguishing it from LDL (Nordestgaard et al. 2010; Tsimikas 2017;
Van Der Valk et al. 2016). Apo(a) shares a high amino acid sequence homology with
several regions of plasminogen, including the protease domain, and the kringles IV
(KIV) and V domains (Koschinsky and Marcovina 2004) and exhibits a high degree
of size polymorphism. The KIV domain of apo(a) is formed by ten distinct KIV
types numbered from 1 to 10. All KIV types, except KIV type 2 (KIV2), are present
in apo(a) as a single copy, while the KIV2 varies from <3 to >40 identical repeats,
resulting in the large number of apo(a) isoform sizes circulating in human plasma
(Marcovina et al. 1993). Being mostly determined by its hepatic production rate, the
concentration of apo(a) is largely inversely correlated to its size, smaller isoforms
being produced faster (Karwatowska-Prokopczuk et al. 2021). The distribution of
apo(a) serum levels and isoforms varies widely between individuals and popula-
tions of different ancestry, and because most individuals express two different
alleles of the LPA gene, a majority of individuals presents two different size iso-
forms circulating in plasma (Marcovina et al. 1993; Karwatowska-Prokopczuk et al.
2021; Stefanutti et al. 2020; Kamstrup 2021; Marcovina and Albers 2016). In addi-
tion, apo(a) is heterogeneous in its glycosylation pattern, which occurs both within
the core of the KIV motifs and within the linker sequences connecting the different
kringles, resulting in an extremely heterogeneous population of Lp(a) particles in
circulation (Marcovina and Albers 2016).
Because there is one molecule of apo(a) in Lp(a), the measurement of apo(a) is
used as a surrogate measure of Lp(a) in plasma. At present, Lp(a) concentrations are
reported either in nmol/L of Lp(a) protein or in mg/dL of total Lp(a) mass including
302 N. Clouet-Foraison et al.

the protein, lipid, and carbohydrate components. However, because mass units rely
on doubtful hypotheses regarding Lp(a) particle composition, and because the mass
of apo(a) is highly variable (Marcovina and Albers 2016), guidelines now recom-
mend the use of molar units for Lp(a) reporting in clinical laboratories (Marcovina
and Albers 2016; Wilson et al. 2019; Mach et al. 2019; McCormick 2004). A variety
of immunochemical methods is available to measure Lp(a) in plasma or serum such
as ELISA (enzyme-linked immunosorbent assay), nephelometry, immunoturbidim-
etry, and fluorescent immunoassays. All of them are based on the measurement of
the signal generated by the formation of a complex between apo(a) and specific
monoclonal or polyclonal antibodies. By measuring the signal generated by the
antigen-antibody complex in a calibration material containing a known amount of
the analyte, the signal in the sample can be calculated back to a concentration of the
analyte in the sample. For an assay to be accurate, (1) the antibody needs to be spe-
cific to the analyte measured, (2) the analyte should have the same structural char-
acteristics in the sample and in the assay calibrator to ensure a similar degree of
immunoreactivity per particle, (3) an appropriate reference material should be used
to value-assign the assay calibrator to guarantee reproducibility and comparability
of the results, and (4) harmonized protocols should be available to accurately trans-
fer the value from the reference material to the assay calibrator and to verify that
results obtained on test samples are accurate (Marcovina and Albers 2016).
Although specificity of antibodies to apo(a) is not a major issue because possible
immunoreactivity with apoB-100 or plasminogen can be easily eliminated, the high
degree of intra- and interindividual variation in apo(a) size, due to the variable num-
ber of KIV2 repeats, makes it practically impossible to select assay calibrators with
identical structural characteristics as individual samples. So far, only two monoclo-
nal antibodies have been reported that bind to epitopes that are not present in KIV2
(Marcovina and Albers 2016; Gonen et al. 2020), while all polyclonal antibodies
used in various immunoassays also recognize epitopes located in the variably
repeated KIV2 domain (Marcovina and Albers 2016; Kronenberg and Tsimikas
2019). In this situation, the number of antigen-antibody complexes formed during
analyses reflects the number of KIV2 repeats and thus the isoform distribution of
apo(a) in the individual rather than the apo(a) concentration. If the isoforms are
smaller in the sample than in the calibrator, less immunocomplexes will be formed
in the sample, resulting in underestimation of the concentration of Lp(a), and vice
versa will occur in samples with larger isoforms. It has been estimated that the effect
of apo(a) size variability may result in over- or underestimation of Lp(a) concentra-
tion of up to 25–30% with consequent possible misclassification of the individual’s
cardiovascular risk (Kamstrup 2021; Marcovina and Albers 2016; Marcovina
et al. 1995).
In addition, because the concentration of apo(a) is directly correlated to its pro-
duction rate which in turn is correlated to the size of apo(a), concentrations span
more than 1000-fold range from <1 nmol/L in individuals with large isoforms to
>1000 nmol/L in individuals with small isoforms (Kamstrup 2021; Marcovina and
Albers 2016). The immunoassays must therefore have appropriate calibration
dynamic ranges, meaning that most calibrators will be formed by sample pools with
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 303

high Lp(a) concentration and therefore predominantly small isoforms. Raising

polyclonal antibodies that do not target the variably repeated domains of apo(a) is
more challenging than it appears because of the preponderance of epitopes available
in the repeated domains compared to others. Unless monoclonal antibodies are
demonstrated to not cross-react with KIV2, their use does not guarantee an isoform-­
size independent assay either (Kronenberg and Tsimikas 2019). At present, only
one commercially available latex-enhanced turbidimetric method appears to be able
to measure Lp(a) with a reduced impact from apo(a) size polymorphism (McCormick
2004). The polyclonal antibodies used in this assay are bound to latex particles, and
therefore the formation of very large immunocomplexes helps reduce the impact of
the size variation of apo(a). However, the unique feature of this assay is the use of
five independent sample pools with Lp(a) concentrations ranging from low to high
levels and pools of apo(a) isoforms ranging from predominantly large to predomi-
nantly small. This approach significantly decreases the inaccuracy of the assay asso-
ciated with apo(a) size polymorphism (Marcovina and Albers 2016). However, the
inverse relationship between apo(a) size and apo(a) concentration is not always con-
sistent, and therefore the impact of apo(a) size cannot be equally minimized in all
samples (Marcovina and Albers 2016; Kronenberg and Tsimikas 2019).
Overall, the high degree of size heterogeneity of apo(a), its covalent association
with apoB-100, and the high sequence homology with plasminogen, parameters that
all impact the analytical performances and robustness of the assays, constitute a
significant challenge to the development of immunoassays to measure Lp(a) in clin-
ical laboratories. As a consequence, there is a significant lack of comparability
between methods measuring Lp(a) (Scharnagl et al. 2019; Ruhaak and
Cobbaert 2020).

A History of Standardization Initiatives for Lp(a)

Following standardization efforts of apoA-I and apoB-100 (Marcovina et al. 1991),

standardization initiatives for Lp(a) started in the early 1990s (Labeur et al. 1994;
Albers and Marcovina 1994). Labeur and colleagues, in collaboration with the
Center for Disease Control (CDC), showed for the first time the poor comparability
of Lp(a) measurements performed by different clinical laboratories through two
worldwide surveys organized in 1989 and 1990 (Labeur et al. 1994). Sixteen labo-
ratories (ten in Europe and six in the USA) participated in the first survey and thirty-­
nine in the second (twenty-nine in Europe, seven in the USA, and three in Japan)
using primarily ELISA and turbidimetric assays. For each survey, participants ana-
lyzed a set of either lyophilized or frozen samples with different Lp(a) concentra-
tions. Depending on the methods, the interlaboratory coefficients of variation (CV)
ranged from 33 to 70% highlighting that standardization efforts were urgently
needed (Labeur et al. 1994).
To evaluate to what extent a common calibration material could improve compa-
rability of Lp(a) measured by different immunoassays, Albers and Marcovina
304 N. Clouet-Foraison et al.

organized a study providing collaborating laboratories with a common calibrator

formed by a fresh-frozen sample with a high Lp(a) concentration and an average
apo(a) isoform size along with a set of 15 samples with different Lp(a) concentra-
tions and isoform size. Their results showed that the use of a common calibrator
improved between-method comparability. However, high sample-specific variabil-
ity was still observed, and results highlighted remaining issues with Lp(a) routine
measurements, even across similar methods performed in different laboratories
(Albers and Marcovina 1994).
Aware of the potential impact of the repeated KIV2 on the measurement of Lp(a)
by immunochemical methods, Marcovina and colleagues from the Northwest Lipid
Research Laboratories (NWRL), University of Washington, produced a large num-
ber of monoclonal antibodies to specifically target apo(a). Among them, they identi-
fied and characterized one monoclonal antibody (a-40) that interacted with a unique
epitope of KIV9 and showed no interaction with KIV2 and used it as detecting anti-
body to develop a sandwich ELISA assay (Marcovina et al. 1995). This method was
extensively optimized and validated, and the results showed that using this ELISA
assay, Lp(a) could be measured in equimolar basis, independent of the size of apo(a)
in the samples. To reflect that the method accurately measures the number of apo(a)
molecules and not its variable mass, Lp(a) values were reported in nmol/L
(Marcovina et al. 1995).
Following these first initiatives, the IFCC created a working group for the stan-
dardization of Lp(a) assays with the aim to produce a secondary reference mate-
rial to improve between-method and between-laboratory comparability (Tate
et al. 1998). In a first phase, the IFCC working group organized a worldwide
comparison study involving 33 diagnostic manufacturers and clinical chemistry
laboratories in 12 countries performing a total of 40 different Lp(a) assays based
on widely different approaches to target and detect Lp(a). The analytical perfor-
mances of the 40 assay systems were evaluated by testing serum samples and
manufactured Lp(a) calibrator materials for precision, linearity, and parallelism.
Twenty systems were not optimized based on the use of a serum pool which tested
nonlinear in sixteen systems and highly imprecise in four. When excluding the
assays that did not meet the minimum acceptable analytical performances, the
between-method CV was reduced down to 16% with some of the manufactured
calibration materials, suggesting their potential as candidate reference materials
(Tate et al. 1998).
Using results from Phase 1 regarding performances of the materials and assays,
four manufactured Lp(a) materials, two lyophilized (PRM1B and PRM2B) and
two liquid stabilized (PRM3B and PRM5B), were evaluated in Phase 2 in collabo-
ration with the NWRL (Tate et al. 1999). The 4 materials were compared for ana-
lytical performance and commutability in 27 different test systems. Linearity and
precision were comparable for all materials, however, depending on the material
used as common calibrator; among-assay CV ranged from 11 to 22%. The mate-
rial that resulted in overall best comparability between systems achieved a
CV below 8% across 18 of the 27 test systems. On the basis of its analytical per-
formances, best potential for harmonization, and documented stability, the
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 305

lyophilized serum PRM-2B was selected as a proposed secondary reference mate-

rial (Tate et al. 1999).
In Phase 3 of the standardization project, the NWRL prepared and distributed 30
individual frozen plasma samples spanning a large range of Lp(a) concentrations
and isoform sizes; 3 fresh-frozen quality controls with low, medium, and high Lp(a)
concentrations; and the proposed reference material 2B to be tested by 22 different
test systems (Marcovina et al. 2000). To be used as pure calibrator, two research
laboratories isolated Lp(a) from fresh plasma from a donor with a single apo(a)
isoform using two different isolation procedures. The total protein concentration of
the isolated Lp(a) was determined by amino acid analysis after acid hydrolysis, and
the molar concentration of apo(a) was calculated (Marcovina et al. 2000). To value-­
assign the proposed secondary reference material 2B, each isolate was diluted to
prepare a set of standards to calibrate the apo(a)-size insensitive, double monoclo-
nal antibody-based ELISA (Marcovina et al. 1995) designated as the reference
method. Preparation 2B was analyzed six times in duplicate on three separate plates
for each calibration material. The same protocol was carried out for 4 consecutive
days, yielding a total of 144 values. The final value assigned to secondary material
2B was 107.1 ± 8.6 nmol/L (Marcovina et al. 2000). Using this material as common
calibrator, inter-assay CV was below 10% for 18 out of the 22 measurement sys-
tems, while the others still obtained CVs above 10%. Based on the results of these
international joint efforts, the proposed serum reference material SRM-2B was
endorsed in 2004 by the WHO as the WHO/IFCC “first international reference
reagent for Lp(a) for immunoassays” (Dati et al. 2004).
Overall, the results of these studies showed that the use of a suitable reference
material reduced the variability related to the calibration component of the different
analytical systems. However, as expected, the use of a common secondary material
did not reduce the impact of the apo(a) size polymorphism, most prominent in some
system than in others, resulting in strong systematic errors that impacted the overall
comparability of the methods (Marcovina et al. 2000). Taken together, the results of
these IFCC studies clearly highlight that not all the methods available to measure
Lp(a) meet the prerequisites to be considered “standardizable.”

The Current Harmonization System

Following the collaboration with the IFCC standardization working group,

Marcovina and colleagues from the University of Washington developed a multistep
approach to evaluate the suitability of different assays to produce comparable Lp(a)
results. The first step consisted in value-assigning the assay working calibrators
using the WHO/IFCC SRM-2B reference material. Six QC samples with Lp(a) con-
centrations ranging from low to high, and predominant apo(a) isoforms ranging
from large to small, were used to validate this first calibration step. In a second step,
the accuracy of Lp(a) results was verified using 80 fresh-frozen samples from indi-
vidual donors selected to encompass a suitable range of Lp(a) values and apo(a)
306 N. Clouet-Foraison et al.

isoforms. The QCs and the 80 donor samples were value-assigned by the ELISA-­
designated reference method (Marcovina et al. 1995) performing repeated measure-
ments over several weeks and the apo(a) isoforms determined by agarose gel
electrophoresis (Marcovina et al. 1993). Criteria were established to determine the
acceptability of the bias obtained between the observed and the target values for
these samples and the contribution of apo(a) isoform variability on the results. All
requesting manufacturers received the multistep validation protocol, the WHO/
IFCC reference material SRM-2B, the six QC materials, and the set of 80 samples.
Among the analytical systems evaluated during the IFCC standardization pro-
gram, only one latex-enhanced turbidimetric method produced by Denka Seiken,
Japan, demonstrated good agreement with Lp(a) values measured by the ELISA-­
designated comparison method, most of the inaccuracy being due to overestimation
of Lp(a) levels in samples with large apo(a) isoform size. An extensive evaluation
of the Denka Seiken method was thus performed at the NWRL and showed that
careful optimization of the assay, coupled with value assignment of the five-point
calibrators with the WHO/IFCC SRM-2B reference material, resulted in improved
agreement of Lp(a) values with those obtained by the ELISA-designated compari-
son method (Marcovina and Albers 2016). Following this work, a fairly large num-
ber of manufacturers implemented the use of the Denka Seiken method on their
instruments or distributed the Denka kits to be used by clinical chemistry laborato-
ries on different analytical systems. Between 2012 and 2015, calibration and per-
formance of 42 analytical systems based on the Denka Seiken kit using different
lots of calibrators and reagents, and 6 methods from different manufacturers based
on single diluted calibrators, were evaluated. The 42 analytical systems based on
the use of the Denka kits meet the established performance criteria while the 6
methods using single calibrators did not, due to high apo(a) isoform-size-depen-
dent biases. After uniform calibration with the WHO/IFCC SRM-2B reference
material, the among-­method CV across the 42 measurement systems on the 80
samples was 5.5% and ranged from 2.1% in samples with high Lp(a) concentra-
tions to 10.5% in samples with low Lp(a) concentrations. These findings demon-
strate that harmonization of results obtained by a variety of different instruments
and different calibrator lots can be achieved in optimized test systems (Marcovina
and Albers 2016).
In the period 2015–2020, the analytical performances of 29 test systems using
the Denka Seiken reagents were evaluated by the NWRL. All systems were trace-
able to the WHO/IFCC SRM-2B through the NWRL harmonization process. As
presented in Fig. 19.1a, results show that the average CV between the 29 systems
was 5.0% and ranged from 2.3 to 10.5% with only one sample slightly exceeding
the 10.4% desirable allowed imprecision recommended for clinical measurements
by the Westgard biological variation database (Fraser 2022). Comparison of the
average Lp(a) concentration calculated across the 29 systems versus the concentra-
tion measured by the Lp(a) designated comparison method shows a near perfect
correlation with a 1.01 slope (Fig. 19.1b). As evidenced in Fig. 19.1c, the average
relative difference from the Lp(a) assigned values was consistently below the 6.9%
recommended allowable bias from the Westgard biological variation database
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 307

b c d

Fig. 19.1 Performance assessment of 29 systems measuring Lp(a) concentration using Denka
Seiken kits harmonized to the WHO/IFCC SRM-2B. Performance was evaluated on a set of 80
individual patient samples. (a) Average coefficient of variation (CV) of Lp(a) values calculated
across the 29 systems for the 80 samples sorted by increasing Lp(a) concentrations (nmol/L). (b)
Average Lp(a) concentration measured by the 29 systems as a function of the Lp(a) concentrations
determined by the Lp(a) designated comparison ELISA method. Black line is the unity line; slope
and 95% confidence interval are indicated on the graph. (c) Average relative difference to the des-
ignated comparison ELISA across the 29 systems for each sample as a function of the assigned
Lp(a) value measured by the designated comparison ELISA. Black dotted line is the 6.9% desir-
able bias recommended by the Westgard biological variation database. (d) Average relative differ-
ence to the designated comparison ELISA as a function of the predominant Lp(a) isoform size.
Slope is not statistically different from zero (green full line)

(Fraser 2022), with the exception of 8 samples with low Lp(a) concentration.
Finally, evaluation of the mean relative bias as a function of apo(a) isoforms con-
firmed that the Denka Seiken-based assays were minimally affected by systematic
accuracy errors caused by different Lp(a) isoform sizes present in the test samples
(Fig. 19.1d).
The availability of optimized assays for measuring Lp(a) with calibrators trace-
able to the WHO/IFCC SRM-2B reference material and with analytical perfor-
mances monitored by the NWRL had a very beneficial effect on Lp(a) research:
wide use and acceptance of common expression of Lp(a) values in nmol/L, compa-
rability of data obtained in different laboratories and studies, and establishment of
reliable risk thresholds for Lp(a) as a clinical biomarker of increased CVD risk
(Cegla et al. 2019; Patel et al. 2021). Establishing traceability of Lp(a) measure-
ments to the WHO/IFCC SRM-2B through the NWRL validation process provided
consistent harmonization of a good number of Lp(a) assays, thus ensuring a suitable
degree of comparability across traceable methods. Unfortunately, since the closure
in 2020 of the NWRL by the University of Washington, the ELISA-designated com-
parison method and the Lp(a) harmonization protocol are no longer available, effec-
tively ending decades of harmonization efforts.
308 N. Clouet-Foraison et al.

However, in 2018, the IFCC formed a new working group for the standardization
of Lp(a) assays with the intent to develop a higher-order reference measurement
procedure to establish full standardization of clinical methods for measuring seven
apolipoproteins including apo(a) (Ruhaak and Cobbaert 2020; Cobbaert et al. 2020).
In parallel, a new method, based on the quantification of apo(a)-specific peptides by
liquid chromatography-tandem mass spectrometry, has been developed and pro-
posed as a candidate reference method for Lp(a) standardization (Marcovina
et al. 2021).

 evelopment of a New Approach to Measure Lp(a):

D
LC-MS/MS

Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an

increasingly used alternative to antibody-based immunoassays for the quantifica-
tion of proteins in clinical practice (Hoofnagle et al. 2020). Although intact protein
quantification has been achieved for few proteins, most methods rely on the so-­
called bottom-up approach where peptides liberated by proteolysis (typically with
trypsin) of the target protein (proteotypic peptides) are quantified as surrogate mea-
sures of the protein by LC-MS/MS. The most common method used is Selected
Reaction Monitoring, where a combination of a specific peptide precursor and asso-
ciated fragment ion (a “transition”) is monitored, typically in a triple quadrupole
MS to provide high selectivity and sensitivity (Kulyyassov et al. 2021). Alternatively,
Parallel Reaction Monitoring performed on high-resolution instruments provides
a similar degree of selectivity and sensitivity with high mass accuracy MS/MS spec-
tra (Kulyyassov et al. 2021; Villanueva et al. 2014).
Absolute quantification of proteins by LC-MS/MS is achieved by the use of
heavy stable isotope-labelled (SIL) peptides or proteins as internal standards
(Villanueva et al. 2014; Shuford et al. 2017) that may be combined with external
calibration. With SIL peptides, the absolute concentration of the endogenous pep-
tides, and thereby of the target protein, is usually determined based on the absolute
concentration of each individual SIL peptide. While this strategy has the advantage
that peptides are easily synthesized and quantified in an SI-traceable manner, this
approach however relies on the assumption that the measured endogenous protein is
quantitatively (with 100% efficiency) and reproducibly digested into its proteolytic
peptides. This assumption is very difficult to prove and rarely achieved because the
proteolysis kinetics is highly dependent on protein structure and local environment
around each proteolytic cleavage site. It also depends on stability of the formed
peptides (i.e., resistance to further hydrolysis, rearrangements, and modifications).
These issues are avoided with the use of recombinant SIL full-length protein.
However, producing full-length SIL proteins that are fully post-translationally mod-
ified (i.e. glycosylated) to match the endogenous proteins is generally challenging,
and purification and value assignment can be highly complex. As an alternative,
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 309

multiple approaches have been investigated that entail various forms of recombinant
protein fragments that include proteolytic digestion sites which produce the SIL
internal peptide standards such as protein fragments or cleavable labelled peptides
(Kulyyassov et al. 2021; Villanueva et al. 2014; Oeckl et al. 2018). While these are
generally short and easy to characterize to the required level, these strategies are
based on the assumption that digestion is comparable to that of the endogenous
protein, a prerequisite for accurate absolute quantification. However, the local envi-
ronment of each proteolytic cleavage site, the higher-order structure of the con-
structs, and their post-translational modifications differ from that of the endogenous
protein which can significantly affect the proteolytic kinetics.
To achieve accurate and reproducible absolute quantification, the strategy called
double isotope dilution (ID) is considered as the gold standard (Hoofnagle et al.
2020). In this approach, the SIL peptides (or protein) are spiked into the analyzed
samples and into an external calibration curve constructed of the pure recombinant
protein or pure proteotypic peptide (Villanueva et al. 2014; Shuford et al. 2017;
Bunk and Lowenthal 2012). It was used in several higher-order reference measure-
ment procedures for small molecules or large peptides like hemoglobin A1c and
C-peptide (Hoofnagle et al. 2020) and has also been used for the absolute quantifi-
cation of larger proteins (Cobbaert et al. 2020; Huynh et al. 2021; Neubert et al.
2020; Jin et al. 2019; Dittrich et al. 2018; Sabbagh et al. 2016).

 otential for Standardization Using a Higher-Order LC-MS/

P
MS Method

Based on the characteristics described above, LC-MS/MS is also the method of

choice for higher-order reference measurement procedures for the standardization
of clinically relevant proteins. The LC-MS/MS method presented by Marcovina and
colleagues for Lp(a) quantification laid the groundwork to this process and pro-
posed a candidate reference measurement procedure for the quantification of apo(a)
by LC-MS/MS that demonstrated high levels of accuracy and direct traceability to
the SI units (Marcovina et al. 2021). This method was developed to meet the strin-
gent requirements of a reference method for standardization and therefore focused
on the absolute quantification of apo(a) rather than on multiplexing or high
throughput.
To calibrate this method, the authors used a pure recombinant apo(a) that was
expressed in human HEK 293 cells transfected with a 14K-pRK5 expression vector
and purified by Lys-Sepharose affinity chromatography (Koschinsky et al. 1991).
As previously reported, this expression protocol ensures the proper folding and gly-
cosylation of the recombinant apo(a), which retains the same structural and func-
tional characteristics as the endogenous protein (Koschinsky and Marcovina 2004;
Koschinsky et al. 1991; Gabel and Koschinsky 1995). To provide traceability to the
SI for this recombinant apo(a) calibrator, its concentration was assigned by a
310 N. Clouet-Foraison et al.

higher-­order reference method for amino acid quantification certified by the NIST
(Lowenthal et al. 2010). The size of the recombinant apo(a) (14 kringles) was con-
firmed by agarose gel electrophoresis, and purity of the preparation was verified by
SDS-PAGE electrophoresis (sodium dodecyl-sulfate polyacrylamide gel electro-
phoresis), Electrospray Differential Ion Mobility Analysis, and anion exchange Fast
Protein Liquid Chromatography (Marcovina et al. 2021). This first intention assess-
ment did not indicate the presence of significant impurities in the preparation.
However, for a material to be considered as a primary reference material, its purity
should be thoroughly assessed and certified, which is a significant challenge for a
full-length protein (Stoppacher et al. 2015; Josephs et al. 2019). Indeed, the sheer
complexity of a full-length protein in terms of post-translational modifications and
glycosylation patterns, particularly for apo(a), and the considerable size of the pro-
tein (200–>800 kDa) present major obstacle to the use of most reference methods to
assess purity (Stoppacher et al. 2015; Josephs et al. 2019). Consequently, even
though the purity of the material reported by Marcovina and colleagues appears
satisfactory, assigning a certified purity, and therefore a certified concentration, is
still needed to propose this material as candidate primary reference material for
apo(a) standardization.
In this published method, the authors used a double isotope dilution strategy
involving both SIL peptides and the pure recombinant apo(a) for calibration
(Marcovina et al. 2021) with an external six-point calibration curve constructed in a
blank human serum. Each calibrator level and all samples were spiked with a mix-
ture of the pure SIL peptides corresponding to the proteotypic peptides of interest.
No sample clean-up or pre-concentration were included in this protocol, allowing to
limit losses that could impact assay accuracy. The authors investigated six candidate
quantification peptides of which three were validated for quantification of apo(a).
Because the method was proposed as a candidate reference method, it underwent
a thorough validation. Linearity, limits of quantification, intermediate precision,
reproducibility, and digestion kinetics were assessed for all measured peptides
(Marcovina et al. 2021). To confirm that the recombinant apo(a) calibrator behaved
similarly to endogenous apo(a), parallelism was verified, and no significant differ-
ences were found between the endogenous protein and the recombinant apo(a) cali-
brator. As first intent, the method was also transferred to a high-throughput LC-MS/
MS in a clinical laboratory, and high degree of agreement was achieved (Marcovina
et al. 2021). The authors additionally evaluated accuracy of the method on a set of
64 individual samples with a wide Lp(a) concentration range and varying isoform
sizes. Comparison of Lp(a) values with those obtained by the designated ELISA
method comparison (Marcovina et al. 1995) showed a Pearson correlation r2 = 0.958.
The Bland–Altman difference plot indicated minimal differences of LC-MS/MS
values compared to ELISA with a 1.7 nmol/L mean difference (2.5%) {1.96 × SD
limits of agreement −29.8 to 33.2 nmol/L} (Marcovina et al. 2021). The measure-
ment of the WHO/IFCC SRM-2B secondary reference material produced a value of
104.7 ± 8.4 nmol/L, in close agreement with its assigned reference value of
107.1 ± 8.6 nmol/L (Dati et al. 2004). Even though this does not represent a metro-
logically sound assessment of the method’s accuracy, it is a first indication that the
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 311

LC-MS/MS method proposed is a viable option for standardization. It further pro-

vides confidence that implementing such a method on a new preparation of a sec-
ondary reference material to replace the WHO/IFCC SRM-2B may not result in a
significant change in values and risk thresholds for clinical practice.
Currently, no other LC-MS/MS method aiming at high accuracy quantification
of Lp(a) has been reported. In 2018, the IFCC and the Joint Committee for Guides
in Metrology (JCTLM) appointed a working group for the standardization of major
apolipoproteins, including apo(a) (Cobbaert et al. 2020). It is still unclear which
methodology the working group has chosen for the development and validation of
the secondary reference measurement procedure for Lp(a). However, in their article
from 2020, Cobbaert and colleagues laid the background and detailed the approach
envisioned to finally establish traceability to the SI for Lp(a) using LC-MS/MS. The
working group intends to use a peptide-based quantification approach for which the
primary reference material would be a pure non-labelled proteotypic peptide of
apo(a) (Cobbaert et al. 2020). As discussed earlier, this approach has the advantage
that peptide purity can be more easily assessed, even though it still represents a
significant challenge (Josephs et al. 2019). Synthesis and purification of peptides
are well established, and reproducibility from batch to batch is more reliable than
for pure recombinant proteins. However, the major downside of this approach is that
peptide-based quantification is highly prone to bias, especially since, as discussed
earlier, its accuracy relies on the rarely met assumption of a complete digestion of
the protein, and several studies have shown that this approach can result in signifi-
cant quantification errors (Shuford et al. 2017; Hoofnagle et al. 2016; Clouet-­
Foraison et al. 2017).
As demonstrated by Marcovina and colleagues, when a full-length recombinant
apo(a) calibrator is used, it is possible to demonstrate equivalence between the cali-
brator and the endogenous apo(a) even when the protein digestion may not be com-
plete. However, endorsing a pure recombinant apo(a) as a primary reference material
would represent a significant challenge. Indeed, its concentration was assigned by
amino acid analysis by only a single laboratory and would thus have to be confirmed
by additional accredited reference laboratories to ensure its reliability. The purity of
the recombinant protein will have to be certified, a process that will represent a
considerable challenge (Stoppacher et al. 2015; Josephs et al. 2019). Furthermore,
the stability of the protein over time will have to be established. Finally, a world-
wide standardization would require that this material be made available in substan-
tial amounts to distribute to metrology institutes for regular international comparisons
and certification.
Both methodological approaches will require that the suitability of the calibra-
tion system chosen be thoroughly evaluated and validated by international compari-
sons between several metrology institutes and reference laboratories implementing
the method in order to ensure the reliable value assignment of the primary reference
material finally produced (Cobbaert et al. 2020). Its stability, homogeneity, and
commutability will further have to be assessed in order to prevent accuracy errors
that would later impact the entirety of the traceability chain and ultimately the
patients.
312 N. Clouet-Foraison et al.

LC-MS/MS for the Measurement of Lp(a) in Clinical Laboratories

LC-MS/MS measurement of Lp(a) based on quantification of apo(a) was first

reported by Lassman et al. (2012, 2014). Since then, several methods to measure
Lp(a) by LC-MS/MS have been reported, and they are summarized in Table 19.1
(Stefanutti et al. 2020; Ruhaak and Cobbaert 2020; Marcovina et al. 2021; Lassman
et al. 2014; Blanchard et al. 2020; Van Den Broek et al. 2016). These methods
include the method by Marcovina et al. (2021), proposed as a reference method, but
that could also be used for routine quantification of Lp(a) after optimization for such
use. Overall, all methods available, except that developed by Marcovina et al., target
the same quantification peptide LFLEPTQADIALLK used by Lassman and col-
leagues and located in the C-terminal protease domain of apo(a). Interestingly,
Marcovina et al. reported in their paper that the LFLEPTQADIALLK peptide was
not selected for quantification because a larger variability of the digestion and a
poorer performance in terms of repeatability and reproducibility were observed for
this peptide compared to the other evaluated peptides (Marcovina et al. 2021).
Lassman and Blanchard additionally included the peptide GTYSTTVTGR from the
repeatable KIV-2 domain to measure the average isoform size of apo(a) (Lassman
et al. 2014; Blanchard et al. 2020).
The five LC-MS/MS methods use very different calibration strategies, and only
two out of the five are traceable to the WHO/IFCC SRM-2B (Stefanutti et al. 2020;
Lassman et al. 2014). Even though the method developed by Marcovina and col-
leagues and calibrated with the primary recombinant apo(a) is not traceable to the
WHO/IFCC SRM-2B, it showed a close agreement between the results obtained on
the WHO/IFCC SRM-2B material and its assigned reference value (Marcovina
et al. 2021). Blanchard and colleagues chose to use a double isotope dilution strat-
egy for quantification using a stable isotope-labelled peptide and pure non-labelled
analog. However, they do not report how the peptide concentrations were obtained,
making it impossible to determine what reference the results are traceable to.
Finally, Ruhaak and colleagues show results from the absolute quantification of
apo(a) by LC-MS/MS included in a set of apolipoproteins quantified by a multiplex
LC-MS/MS quantification method (Ruhaak and Cobbaert 2020; Van Den Broek
et al. 2016), but neither the calibration strategy nor method validation has been
reported. Precision and robustness were assessed for most methods, and intra- and
inter-assay coefficients of variation were generally below 15% for all methods.
In the absence of a reference method, accuracy can be estimated by comparison
of the results to a designated comparison method such as the NWRL ELISA assay
in the case of Lp(a). Out of the five LC-MS/MS methods published, two have evalu-
ated accuracy and showed excellent agreement with ELISA with minimum biases
(Marcovina et al. 2021; Lassman et al. 2014). On the contrary, the other three meth-
ods only report comparison with routine immunoassays. Ruhaak and colleagues
compared their method to a Roche clinical assay calibrated against the WHO/IFCC
SRM-2B and showed a −16% bias with the comparison method (Ruhaak et al.
2018). Similarly, Stefanutti and colleagues reported a comparison to a routine clini-
cal immunoassay and showed substantial deviation of the values at higher
Table 19.1 Comparison of published methods for the quantification of apo(a) by LC-MS/MS
19

Van den Broek et al.

Lassman et al. (2014) (2016) Blanchard et al. (2020) Stefanutti et al. (2020) Marcovina et al. (2021)
Peptide(s) Quantification: Quantification: Quantification: Quantification: Quantification:
LFLEPTQADIALLK LFLEPTQADIALLK LFLEPTQADIALLK LFLEPTQADIALLK TPAYYPNAGLIK,
confirmation: TPENYPNAGLTR, and
GISSTTVTGR GISSTTVTGR
confirmation:
LFLEPTQADIALLK
Calibration External calibration with Not published Double isotope dilution External calibration using Double isotope dilution
strategy six calibrators value strategy with synthetic the WHO/IFCC SRM-2B with a pure recombinant
assigned by the NWRL-­ non-labelled and stable apo(a) protein and stable
designated comparison isotope-labelled peptides isotope-labelled proteotypic
method for Lp(a) peptides
Throughput Medium High High Medium Low
Units nmol/L nmol/L nmol/L mg/dL nmol/L
Traceability WHO/IFCC SRM-2B Not published Not published WHO/IFCC SRM-2B SI-traceable
Accuracy – Mixing experiments did – Comparison to a – Comparison to an – Comparison with a – Accuracy was estimated
not evidence dilution bias routine Lp(a) clinical ELISA assay but results routine immunoassay by comparison with the
– Accuracy was estimated assay from Roche not detailed in shows a clear deviation at NWRL-designated
by comparison with the validated against the publication higher concentrations. comparison method for
NWRL-designated NWRL and calibrated Spearman Lp(a) on a set of 64
comparison method for using the WHO/IFCC correlation = 0.8315 samples. Deming
Lp(a) on a set of 80 SRM 2B slope = 0.98 (0.94–1.02)
samples. Deming
slope = 0.98 (R2 = 0.96)
(continued)
Standardization of Analytical Methods for the Measurement of Lipoprotein(a…
313
Table 19.1 (continued)
314

Van den Broek et al.

Lassman et al. (2014) (2016) Blanchard et al. (2020) Stefanutti et al. (2020) Marcovina et al. (2021)
Advantages – Targeted quantification – Multiplex – Multiplex – Targeted quantification – High accuracy targeted
of Apo(a) quantification of nine quantification of 18 of apo(a) method developed to meet
– Intra- and inter-assay apolipoproteins different apolipoproteins the stringent requirements
CV below 10% – Small sample volume – Rapid sample of a higher-order reference
– Excellent comparison – Short and automated preparation procedure measurement procedure
with the designated sample preparation – Intra- and inter-assay – Full validation
comparison method procedure CV below 5% and 15%, procedure published
– Robust reproducibility respectively – Intermediate precision
assessed over a 2-year – Transfer to a below 10% and inter-­
period different LC-MS/MS laboratory CV below 10%
system showed good
Bland–Altman
comparison
Limitations – Long sample – Validation data not – Use of an expensive – No data published on – Purity of the
preparation procedure published for apo(a) commercial RapidGest validation. The authors recombinant protein
kit that can be subject to state that they used the calibrator was not formally
lot-to-lot variations method by Lassman and assessed
colleagues with minor
modifications
N. Clouet-Foraison et al.
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 315

concentrations, suggesting an apo(a)-size dependence of the assay (Stefanutti et al.

2020). Moreover, even though they claimed traceability to the WHO/IFCC SRM-2B
material, the authors reported Lp(a) concentrations in mg/dL without specifying
how these values were obtained considering that the value of SRM-2B is in
nmol/L. Finally, Blanchard and colleagues did not publish comparison data but did
assess transferability of the method by comparison with another LC-MS/MS proce-
dure (Blanchard et al. 2020).
Overall, the LC-MS/MS-based quantification of apo(a) as a surrogate measure of
Lp(a) concentration is a promising technique that can be used in clinical chemistry
laboratories even though its overall analytical performance needs to be validated
and the throughput increased for routine use. Contrary to immunoassays, LC-MS/
MS can be made independent from apo(a) isoform size through the choice of a pep-
tide outside the repeatable KIV2 domain, and its routine implementation would
allow to address the issue of isoform dependence of current clinical assays. Although
LC-MS/MS requires significant initial investment in terms of instrumentation and
method development, once the method is established and validated, LC-MS/MS has
the potential to be a robust and powerful technique in clinical laboratories. It pro-
vides exceptional selectivity and specificity, has a potential for multiplexing, and is
generally less sensitive to matrix interferences than antibody-based assays
(Hoofnagle and Wener 2009; van den Broek et al. 2017).
The further development of routine LC-MS/MS assays for the measurement of
Lp(a), which would meet the prerequisites to be “standardizable,” could thus be
envisioned as the way to achieve full standardization of Lp(a) assays. However, all
the methods currently available were developed with different approaches to quan-
tification, and different goals, and therefore, have very different performance targets
and throughputs. It would nevertheless be interesting to evaluate their comparability
because even though LC-MS/MS could be more easily standardizable than immu-
noassays, the major differences observed could influence comparability of the
results.

What Is the Clinical Relevance of Standardizing Lp(a)?

So far in this book chapter, we presented the general benefits of standardization;

discussed the specific issues impacting the standardization of methods to measure
Lp(a); detailed the successful harmonization of Lp(a) results through the use of the
WHO/IFCC secondary reference material SRM-2B that greatly improved between-­
method comparability, even though only for a specific group of assays; and pre-
sented the latest efforts of the IFCC to implement full traceability and standardization
of Lp(a) measurements through the development of a new LC-MS/MS-based refer-
ence system. In light of all the previously discussed issues, we here propose to take
a step back and ask what we consider a critical question: is Lp(a) standardization
feasible and what is the clinical relevance of standardizing Lp(a) assays?
316 N. Clouet-Foraison et al.

First of all, considering a metrology-oriented approach to this question, estab-

lishing full traceability and standardization is of prime importance to obtain reliable
measurements. As explained earlier, standardization differs from harmonization in
that it is anchored to SI units while harmonization is not (Joint Committee for
Guides in Metrology (JCGM) 2012; Armbruster and Miller 2007). While harmoni-
zation allows the same degree of confidence in comparing data across methods and
laboratories (Miller et al. 2014a, b), it does not prevent issues related to batch
changes of the secondary reference material, nor storage or stability issues of the
materials that would result in a slowly decreasing accuracy of the whole traceability
chain over time. In the case of a standardized assay, the secondary reference mate-
rial is traceable to a pure primary material which value is directly traceable to the SI
units. This primary reference material is regularly tested within a network of higher-­
order primary reference measurement procedures; its purity is assessed regularly,
and its value is readjusted if needed, ensuring proper anchoring of the whole trace-
ability chain and maintained accuracy. When standardization is achieved, whenever
a batch of the secondary reference material is close to depletion, a new one can be
produced by value-assigning it using the same primary reference material, thus
ensuring continuity from batch to batch. Obviously, this does not exclude stringent
validation processes regarding the stability and commutability of the new batch
(Miller et al. 2018). Nevertheless, this process is done on a regular basis by metrol-
ogy institutes worldwide and can be achieved with reasonable efforts and financial
input. On the contrary, when a secondary reference material is depleted without a
primary reference material available to anchor the chain, like in the case of SRM-2B,
producing a new batch is equivalent to starting the whole process from the begin-
ning. For Lp(a), this means preparing the pure isolated Lp(a) fractions again, pre-
paring several batches of candidate secondary reference materials, and repeating all
the phases of the standardization efforts initiated by the IFCC working group in the
1990s, with the risk of seeing a significant change in Lp(a) values and associated
clinical thresholds. This would represent tremendous work and would be a very
lengthy, complex, and undesirable process. Therefore, establishing full traceability
to the SI and standardization should always be the aim.
However, Lp(a) is a peculiar case. Even though the implementation of the WHO/
IFCC SRM-2B efficiently improved comparability between a specific group of
methods (Marcovina and Albers 2016; Kostner et al. 2018; Watts and Boffa 2018;
Kronenberg 2019), clinical assays to measure Lp(a) still suffer from the exact same
issues evidenced during the IFCC’s first standardization process. As clearly pointed
out in 2000 by Marcovina and colleagues (Marcovina et al. 2000), while the avail-
ability of a secondary reference material plays an important role in the standardiza-
tion process, no reference material can eliminate the substantial differences in Lp(a)
values measured by different analytical methods affected by apo(a) isoform-size
heterogeneity. Similarly, recent studies reported yet again a lack of comparability
across routine clinical assays harmonized to the WHO/IFCC SRM-2B (Scharnagl
et al. 2019; Ruhaak and Cobbaert 2020; Wyness and Genzen 2021) with the most
common issue being the variable number of epitopes on the repeatable KIV2 domain
(Ruhaak and Cobbaert 2020). As long as these assays are in use in clinical practice,
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 317

the inaccuracy of Lp(a) measurement will persist, regardless of the standardiza-

tion status.
In addition, the assays most affected by apo(a) isoform-size polymorphism still
measure the mass of Lp(a) and report results in mg/dL. However, determining Lp(a)
mass with accuracy is not possible because of its extreme heterogeneity both within
and between individuals; apo(a) is present in two alleles in more than 80% of the
population, and Lp(a) composition in lipids, sphingolipids, and carbohydrate is
highly variable (Marcovina and Albers 2016). Nevertheless, a large number of clini-
cal chemistry laboratories still use these assays, and it is quite unclear how the new
IFCC standardization group will deal with assays that clearly do not have the pos-
sibility to express the Lp(a) values in SI units. To complicate the issue further, while
the methods based on a specific five-point calibration system appear to produce
comparable results and the impact of apo(a)-size variation is greatly reduced, none
of these methods are able to measure Lp(a) without any impact from apo(a) isoform
size. Strictly speaking, it can be argued that these methods do not accurately mea-
sure the number of Lp(a) particles, and doubts have been cast on their suitability to
express Lp(a) values in nmol/L. If this is the case, then it is reasonable to ask again
what assays are considered standardizable by the new IFCC group and what criteria
have been established for their selection. Finally, even after almost 20 years of har-
monization efforts, studies show that some commercially available assays still
exhibit suboptimal performances in terms of precision and robustness (Scharnagl
et al. 2019; Ruhaak and Cobbaert 2020; Wyness and Genzen 2021). Based on these
overall issues, it appears evident that even though the implementation of the elegant
LC-MS/MS reference measurement procedure proposed by the IFCC group to stan-
dardize the major apolipoproteins is highly desirable, its applicability to apo(a) will
prove challenging for the existing methods, and it will most certainly not solve all
the issues associated with Lp(a) measurements.
Following along this line of thought, the question as to why try to standardize
Lp(a) methods rises again. In a context of primary prevention, most individuals, due
to the lifelong stability of Lp(a) levels in individuals, do not even need to be repeat-
edly tested for Lp(a) concentration (Trinder et al. 2022). For clinicians who need to
measure Lp(a) in patients to estimate their risk of developing CVD, or to establish
a diagnostic and treatment strategy, knowing whether Lp(a) is low or high is mostly
enough because Lp(a) is a biomarker that should be used as a risk range more than
as a defined cutoff point (Kronenberg and Tsimikas 2019). Moreover, Lp(a) concen-
trations are genetically determined (Lamon-Fava et al. 1991) and do not signifi-
cantly vary over time, and behavioral and environmental factors have limited impact
(Garnotel et al. 1998; Clouet-Foraison et al. 2020; Reyes-Soffer et al. 2021).
Therefore, most individuals will need only one measure of Lp(a) in their lifetime
(Trinder et al. 2022; Marcovina and Shapiro 2022; Deconinck et al. 2022). Moreover,
the only situation where accuracy is needed is around the risk threshold where an
error in the measurement will have the most impact on the patient’s diagnostic and
potential follow-up. Devil’s advocate could thus argue that accuracy is less relevant
in the case of Lp(a) and that what is really needed is precision and comparability of
Lp(a) assays (Kronenberg and Tsimikas 2019; Kostner et al. 2018).
318 N. Clouet-Foraison et al.

Establishing a primary reference measurement procedure, a suitable primary ref-

erence material, a traceable secondary reference material, and a SI traceability is a
complex and laborious task that will require several years to be completed. Still,
completing the standardization project will only be the first step to a far more gar-
gantuan task: implementing standardization in clinical practice. Following the suc-
cessful model established by the Center for Disease Control for the standardization
of lipids, an international network of centrally monitored reference laboratories
should be created with the task to ensure accuracy of the calibration system set in
place for standardization and to certify that the accuracy of the calibration does
result in accurate results in patient samples. Because ensuring accuracy does not
guarantee method comparability, performances of methods and laboratories mea-
suring Lp(a) should be regularly assessed. External quality assessment schemes and
regular proficiency testing of clinical laboratories and manufacturers should be
established to allow implementation of corrective strategies to ensure results com-
parability across methods and laboratories (Cobbaert et al. 2020).
Overall, even though standardization of Lp(a) assays is a hot and open topic, the
discussion on accuracy and traceability of Lp(a) measurements should not over-
shadow the practical aspects of everyday measurements of Lp(a). Even though the
presently available analytical methods may not possess the desired spectrum of
attributes required to establish full metrological traceability, priority should be
given to timely achieving uniformity of Lp(a) values and its unit of expression.

Conclusions

In this chapter, we first presented and discussed the rationale for method standard-
ization and its necessity for clinical biomarkers. We then described the challenges
associated with the measurement of Lp(a), the past standardization efforts, and the
subsequent implementation of a calibration protocol spearheaded by the NWRL to
verify that a common calibration traceable to the WHO/IFCC reference material
SRM-2B results in harmonized Lp(a) results in patient samples in selected methods.
We then presented and discussed the implementation of a new higher-order refer-
ence measurement procedure for Lp(a) using LC-MS/MS and the different strate-
gies envisioned for this new approach. Finally, we provided a critical discussion on
the feasibility of Lp(a) standardization and its practical implementation in routine
clinical laboratories in contrast to what we believe is more urgently needed, which
is among-method comparability and reliability of Lp(a) measurements.
There are still many obstacles to overcome and several technical challenges to
solve such as developing and implementing an accurate reference measurement pro-
cedure, producing in-matrix commutable serum reference materials, and imple-
menting the whole scheme in clinical practice. The experience of the IFCC working
group on the initial standardization efforts of Lp(a) highlighted the complexity of
this process for Lp(a) and clearly defined the limitations of the immunochemical
methods available for its measurement indicating the need to compromise between
19 Standardization of Analytical Methods for the Measurement of Lipoprotein(a… 319

what are the goals of standardization and what is possible to achieve with the exist-
ing methods.
Implementing a new traceability chain is a long and arduous process that will
require significant international collaboration and efforts from metrology institutes
and regulatory institutions, scientific and clinical communities, assay manufactur-
ers, and routine clinical laboratories.
Meanwhile, while the new standardization procedure is being developed, a paral-
lel activity should be implemented to continue the verification of the manufacturers’
calibration process and the comparability of Lp(a) results previously performed by
the NWRL, with the ultimate goal to transition from harmonization to
standardization.

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Chapter 20
On the Way to a Next-Generation Lp(a)
Reference Measurement System Based
on Quantitative Protein Mass
Spectrometry and Molar Units

Christa Cobbaert, Liesbet Deprez, and Renee Ruhaak

Abbreviations

ALTM All-lab total mean

Apo(a) Apolipoprotein(a)
ApoB Apolipoprotein B
CDC Centers for Disease Control and Prevention
CV Coefficient of variation
cRMP Candidate reference measurement procedure
CSP Clinical Standardization Programs
CVD Cardiovascular disease
DELFIA Dissociation-enhanced lanthanide fluorescence
immunoassay
ELISA Enzyme-linked immunosorbent assay
EQA External Quality Assessment
IFCC International Federation of Clinical Chemistry and
Laboratory Medicine
IFCC WG APO-MS IFCC working group on apolipoproteins by mass
spectrometry
IVD In vitro diagnostic test
K-IV2 Repetitive kringle subunit 4, type 2 in the apo(a) molecule
LDL Low-density lipoprotein particle
Lp(a) Lipoprotein(a) particle

C. Cobbaert (*) · R. Ruhaak

Leiden University Medical Center, Leiden, The Netherlands
e-mail: [email protected]; [email protected]
L. Deprez
European Commission Joint Research Center (JRC), Geel, Belgium
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 325

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_20
326 C. Cobbaert et al.

RMP Reference measurement procedure

SI International System of Units
WHO World Health Organization

Introduction

Metrological Traceability of Lp(a) Test Results

In order for Lp(a) test results to be comparable in time and space across the globe,
we need traceability of test results. Traceability is the ability to trace, for example,
the origin of a product, the ancestors of an individual, or the absolute value of a test
result. The word “traceability” comes from the Latin verb trahere: to draw.
Traceability can refer to documentation such as sampling procedures, laboratory
methods, lab processes, etc., but as in ISO/IEC 17025, we are dealing with trace-
ability of medical test results. It is key that test results are traceable to endorsed
metrological references. Metrology refers to the science of measurement. In the
case of medical test results, we use the wording metrological traceability. The
current VIM definition of metrological traceability is property of a measurement
result whereby the result can be related to a reference through a documented unbro-
ken chain of calibrations each contributing to the measurement uncertainty (JCGM
2012). Ideally, the references are international standards that mimic the measurand
of interest and have assigned values expressed in the International System of Units
(SI units). For temperature and many other physical quantities, for example, mass
and time traceability is relatively easily established. Also, in forensic toxicology or
chemistry, the working standards are substances with defined purity and solutions of
pure substances. Yet, in laboratory medicine, metrological traceability of protein
test results measured in body fluids within total allowable error (Westgard QC 2014;
Fraser 2001; EFLM 2019) is an enormous challenge due to the huge interindividual
variability of endogenous measurands and the complexity of the human body fluid
matrix in which the measurands are dissolved.

Evolution in Science and Metrology

Proteins are the primary effectors in human biology systems. Hence, complete
knowledge of their structure and biological function is fundamental for understand-
ing their potential role as promising biomarkers and/or future medical tests. Proteins
from a single gene can vary widely in their amino acid sequence, and posttransla-
tional modifications also give rise to a variety of proteoforms. As it is now recog-
nized that the variation at the protein level is functionally relevant and much larger
than the variation at the genetic level, the Human Proteoform Project has recently
been launched. This ambitious initiative aims to define the human proteome by
generating a reference set of proteoforms produced from the ~20,000 genes encoded
20 On the Way to a Next-Generation Lp(a) Reference Measurement System Based… 327

in the human genome. The human proteome is the set of all proteoforms expressed
by humans. The underlying rationale is that proteoform-level knowledge is essential
to understand biological function of proteins. Unraveling the human proteome will
lead to a more refined, molecular definition of human health and disease.
Conventional immunoassays that measure human proteins are generally blinded to
the underlying proteoforms of the protein of interest and hence do not recognize or
differentiate dysfunctional from functional proteoforms. This may of course nega-
tively impact patient management and patient outcome. Enabling technology is
needed to detect proteoforms at the molecular level. The Consortium for Top-Down
Proteomics that runs the Human Proteome Initiative uses such technology (Smith
et al. 2021).
Clinicians and laboratory professionals should be aware that improved technol-
ogy is instrumental to advance science and the science of measurement. In order to
enable accurate apolipoprotein quantitation, protein methods should specifically
measure the measurand intended to be measured rather than an ill-defined mixture
of proteoforms with different biological functions. This implies that in this era of
precision medicine, we should introduce higher-order measurement procedures that
can recognize molecular forms, that is, proteoforms, of interest. To that end, triple-
quadrupole mass spectrometry-based technology has built up a reputation as a
higher-­order reference measurement procedure for direct, immunoassay-indepen-
dent protein measurement in complex mixtures.
In this chapter, the authors clarify the current state-of-the-science around Lp(a)
measurement, with special attention to the unique structural characteristics of the
highly polymorphic apolipoprotein(a) [apo(a)] in Lp(a). The apo(a) features should
be considered during Lp(a) test standardization, in order to guarantee that commer-
cial Lp(a) tests are fit for clinical purpose and produce accurate results within allow-
able limits of uncertainty. To accomplish this, a reference measurement system that
produces test results traceable to the SI units is needed. The rationale for the devel-
opment of a higher-order reference measurement system and for transitioning from
the former WHO-IFCC immunoassay-based reference measurement system for
Lp(a) into a more robust immunoassay-independent mass spectrometry-based refer-
ence measurement system and molar units is pointed out.

Current Lp(a) Measurement Procedures

 tructural Properties of Lp(a) in the Continuum

S
of apoB-­Containing Lipoprotein Classes

The Lp(a) particle is the most complex and polymorphic of all serum lipoproteins.
Lp(a) was discovered by Kare Berg in 1963 and is only present in humans and the
hedgehog and not in other mammals. Lp(a) is an apoB100-containing LDL-like
particle that is rich in cholesterol and is associated with a unique hydrophilic, highly
328 C. Cobbaert et al.

glycosylated second major protein, that is, the apo(a). Apo(a) is covalently attached
to apoB in Lp(a), shows ~80% amino acid homology with plasminogen, and con-
tains multiple copies (3–> 40) of plasminogen-like kringle IV type 2 (K-IV2). This
peculiarity of apo(a) is known as apo(a)-size polymorphism. Beyond apo(a)-size
polymorphism, other prominent determinants of Lp(a) levels are genetic variants
such as K-IV2 4925G>A and K-IV2 4733G>A (Schachtl-Riess et al. 2021). Although
apo(a) has structural homology to plasminogen, it lacks fibrinolytic activity. As a
consequence of its composite structure, Lp(a) can trigger prothrombotic and antifi-
brinolytic actions favoring clot stability as well as atherosclerosis progression via its
tendency for retention in the arterial intima, with deposition of its cholesterol load
at sites of plaque formation. In addition, Lp(a) can induce inflammation and calcifi-
cation in the aortic leaflet valve interstitium, leading to calcific aortic valve stenosis.
Recent epidemiological and genetic evidence support the proposition that elevated
concentrations of Lp(a) are causally related to atherothrombotic risk and calcific
aortic valve stenosis.
The concentration of Lp(a) in blood is largely determined by genetic factors and
hardly influenced by diet or lifestyle conditions. After reaching adulthood, the con-
centration remains constant over the lifetime of an individual. Remarkably, the
interindividual variation is large as blood Lp(a) levels among individuals vary up to
1000-fold. Its frequency distribution differs across populations, being skewed to the
right in Caucasians and displaying a more Gaussian distribution in Blacks. Its meta-
bolic role is after 60 years of intense research still not understood. As Lp(a) is an
independent genetic risk factor which accelerates cardiovascular disease (CVD)
through pro-atherogenic, pro-thrombogenic, and proinflammatory mechanisms, the
downstream consequences for patients are as devastating as in the case of untreated
patients with heterozygous familial hypercholesterolemia (Reyes-Soffer et al. 2022;
Kronenberg and Tsimikas 2019). Consequently, the pharmaceutical industry has
invested in the development of novel therapies for Lp(a) lowering that target hepatic
synthesis of apo(a). These therapies are in various phases of clinical trials, and the
completion of these studies will provide critical insight into the cardiovascular ben-
efits of Lp(a) lowering.

Characteristics and Design of Conventional Lp(a) Tests

In medical laboratories, serum/plasma Lp(a) is measured by a variety of immuno-

chemical, CE-marked, or FDA-approved methods, based on immunoturbidimetry
or immunonephelometry, dissociation-enhanced lanthanide fluorescence immuno-
assay (DELFIA), enzyme-linked immunosorbent assay (ELISA), or other readouts.
Several requirements have to be fulfilled for an Lp(a) immunoassay to produce
accurate results. Firstly, the antibodies need to be specific for the analyte intended
20 On the Way to a Next-Generation Lp(a) Reference Measurement System Based… 329

to be measured. Secondly, the analyte being measured in the patient sample should
have the same structural characteristics as the analyte in the assay calibrator(s) to
achieve the same degree of immunoreactivity per Lp(a) particle. Thirdly, an
accuracy-­based target value should be assigned to the assay calibrator(s) using an
internationally recognized reference measurement system to guarantee consistency
and comparability of results. Fourthly, common protocols should be used for trans-
ferring an accurate value from the reference material to the assay calibrators. Most
commercial Lp(a) tests express Lp(a) in mass units [in mg/L or mg/dL Lp(a) mass]
and make assumptions and oversimplifications to that end, which all confound
Lp(a) levels (Ruhaak and Cobbaert 2020). Few routine tests express Lp(a) levels in
molar units (nmol/L).
Most routine Lp(a) tests with mass units use multiple calibrators that are inter-
nally prepared by the IVD-manufacturer and produce results that are not traceable
to a common internationally accepted standard. The few routine Lp(a) tests with
molar units were—until recently—calibrated by the WHO-IFCC Lp(a) Reference
Measurement System developed at the Northwest Lipid Metabolism and Diabetes
Research Laboratories (NLMDRL), Washington DC, USA (Tsimikas et al. 2018).
That reference measurement procedure (RMP) is a reference ELISA that uses
monoclonal K-IV2-independent antibodies both for capturing and detecting intact
Lp(a). The RMP was calibrated with a matrix-based WHO-IFCC Reference Material
named SRM2B. This reference material was value-assigned based on its apo(a)
protein content and expressed in nmol/L, thus reflecting the number of Lp(a)
particles.
The results of a correlation study performed in 2020 (Dikaios et al. 2023) have
shown that expressing Lp(a) concentrations in molar units traceable to the SRM2B
largely improves the intermethod variability (Fig. 20.1). Twenty-one human serum
samples were analyzed with six methods. Three of the six methods provided the
results in both molar and mass concentration units (Roche, Diasys and Sentinel).
For the mass units, the average coefficient of variation (CV) between the methods
was 12.8% (range: 8.4–20.5%) for all six methods and 9.7% (range: 3.4–21.8%) for
the methods from Roche, Diasys, and Sentinel. For the results in molar units, the
intermethod CV of these same three methods was only 3.1% (range: 1.3–5.1%)
(Tables 20.1 and 20.2). These results also clearly indicate that commercial assays
which are based on one serially diluted calibrator will underestimate the Lp(a) con-
centration in serum samples with small apo(a) isoforms.
Notwithstanding the availability of SRM2B since 2003, most commercial
Lp(a) assays still use mass units. Conservatism and ignorance together with the
limitations of the WHO-IFCC Lp(a) Reference Measurement System have pre-
vented worldwide implementation of Lp(a) results in molar units. The important
limitation was the fact that SRM2B could not be used directly by the test manufac-
turer as part of their internal traceability procedures to value-assign their product
calibrators.
330 C. Cobbaert et al.

Fig. 20.1 Intermethod variability among six commercial immunoassay-based assays for the quan-
tification Lp(a) concentration in mass (a) and molar units (b). This study was performed in 2020
(Dikaios et al. 2023). Twenty-one human serum samples were analyzed, and the average results
from three replicate measurements in one run are shown in the graphs. The apo(a) isoforms present
in the serum samples were determined by Western blot at the Institute of Genetic Epidemiology,
Medical university of Innsbruck, Austria. All assays used a multipoint calibration curve, and for
five of the six assays, the curve consisted of independent calibrator solutions. Only for the Siemens
Healthineers N latex method one serially diluted calibrator was used. According to the information
provided by the assay manufacturers, the values expressed in mass units were traceable to internal
standards, while the values in molar units were traceable to the WHO-IFCC Reference Material
SRM2B. The inserts demonstrate that at an Lp(a) mass level of 40–50 mg/dL, the relative differ-
ence between the highest and the lowest method result is 22% for the sample with average molecu-
lar weight apo(a) isoforms (28 and 31 K-IV) rising to 37% in a sample with small apo(a) isoforms
(homozygous for 20 K-IV). In the same specimens, Lp(a) levels expressed in molar units showed
an relative difference between the highest and the lowest method result ranging between 5.7 and
8.7%, that is, three to fourfold lower. The larger intermethod variability between the methods in
mass units can be explained by the fact that these test results are traceable to different internal
manufacturer’s standards and by the fact that different calibration approaches are used, that is, five
independent calibrators with specific apo(a) isoforms per calibrator level versus serial dilutions
from one master calibrator solution. The variable protein/lipid content of Lp(a) particles also
brings along confounded Lp(a) measurements
Table 20.1 The measurement results obtained in 2020 by measuring 21 human serum samples with 6 commercial immunoassay-based assays for the
20

quantification Lp(a) concentration in mass units (Dikaios et al. 2023)

Lp(a) phenotype Lp(a) mass concentration (mg/dL)
Roche Roche
Siemens Tina-­ Tina-­
Percentage Abbott Sentinel Healthineers Siemens Diasys quant quant
Serum First Second first isoform Alinity c Lp(a) Atellica CH Healthineers N Lp(a) LPA LPA Intermethod
sample isoform isoform (%) Lp(a) ultra Lp(a) latex Lp(a) 21FS (lab1) (lab2) CV (%)
1 19 38 5 15.1 14.1 16.1 10.9 15.8 10.2 10.1 20.5
2 32 17.9 17.1 19.3 16.7 19.4 13.3 12.7 16.1
3 32 19.9 19.6 21.4 16.6 21.8 14.7 13.9 17.5
4 22 26 15 21.9 19.6 23.3 15.0 24.2 15.6 15.4 20.5
5 27 39 90 23.2 22.1 24.3 18.2 24.9 17.9 17.1 15.6
6 26 41 95 25.6 25.7 27.2 22.1 27.1 20.3 19.4 13.6
7 33 29.5 29.7 31.3 26.9 31.1 24.8 23.9 10.6
8 29 40 70 31.0 31.2 32.9 27.3 32.3 26.3 25.1 10.6
9 30 34 60 38.2 38.4 40.9 31.4 40.1 34.8 32.6 10.2
10 25 39 85 37.4 36.4 40.3 30.3 39.3 33.5 32.7 10.2
11 28 31 60 45.2 42.2 46.8 37.6 46.0 40.7 37.7 9.1
12 20 100 46.3 42.0 49.1 32.9 48.4 43.6 41.2 12.7
13 26 47.1 45.7 50.0 35.4 48.0 44.6 43.0 10.6
14 27 34 80 59.1 51.9 58.2 39.8 57.8 52.8 50.4 12.7
15 23 35 90 53.8 52.4 57.8 44.1 55.4 52.1 50.0 8.4
16 19 59.2 55.4 62.4 43.0 59.2 55.5 54.7 11.2
17 26 69.7 60.6 67.6 48.4 67.0 62.8 60.7 11.4
On the Way to a Next-Generation Lp(a) Reference Measurement System Based…

18 21 78.0 70.8 78.1 59.2 75.3 72.0 68.9 9.2

19 17 125.7 110.8 123.4 77.7 120.6 109.7 113.6 14.5
20 24 127.2 114.5 125.4 93.2 123.3 115.1 115.6 9.9
331

(continued)
Table 20.1 (continued)
332

Lp(a) phenotype Lp(a) mass concentration (mg/dL)

Roche Roche
Siemens Tina-­ Tina-­
Percentage Abbott Sentinel Healthineers Siemens Diasys quant quant
Serum First Second first isoform Alinity c Lp(a) Atellica CH Healthineers N Lp(a) LPA LPA Intermethod
sample isoform isoform (%) Lp(a) ultra Lp(a) latex Lp(a) 21FS (lab1) (lab2) CV (%)
21 17 30 70 170.6 147.2 166.5 109.4 162.7 148.7 148.7 13.6
Each sample was measured in three replicate measurements in one run and the average result is shown here. The apo(a) isoforms present in the serum samples
were determined by Western blot at the Institute of Genetic Epidemiology, Medical university of Innsbruck, Austria. All assays used a multipoint calibration
curve, and for five of the six assays, the curve consisted of independent calibrator solutions. Only for the Siemens Healthineers N latex method one serially
diluted calibrator was used. According the information provided by the assay manufacturers, the values expressed in mass units were traceable to internal
standards. The average intermethod CV over the 21 samples was 12.8%
C. Cobbaert et al.
Table 20.2 The measurement results obtained in 2020 by measuring 21 human serum samples with 3 commercial immunoassay-based assays for the
quantification of Lp(a) concentration in molar units and the cRMP developed by the IFCC WG APO-MS (Dikaios et al. 2023)
20

Lp(a) phenotype Lp(a) molar concentration (nmol/L)

Percentage of Diasys Roche Roche Intermethod CV of Intermethod CV
Serum First Second first isoform Lp(a) Tina-quant Tina-quant Sentinel immunoassay-based cRMP of all methods
sample isoform isoform (%) 21FS LPA (lab1) LPA (lab2) Lp(a) ultra methods (%) (LFLEPT) (%)
1 19 38 5 24.6 24.5 24.1 25.8 2.9 19.9 9.4
2 32 29.0 32.0 30.5 32.5 5.1 30.9 4.4
3 32 34.7 35.2 33.3 35.6 2.9 35.6 2.8
4 22 26 15 38.3 37.4 37.0 39.2 2.6 29.5 10.7
5 27 39 90 38.5 42.9 41.0 41.9 4.6 44.7 5.5
6 26 41 95 46.4 48.8 46.5 49.4 3.2 52.5 5.1
7 33 54.9 59.4 57.4 58.0 3.3 56.2 3.0
8 29 40 70 59.1 63.0 60.3 61.3 2.7 56.5 4.1
9 30 34 60 78.8 83.6 78.3 78.2 3.2 66.3 8.3
10 25 39 85 79.5 80.4 78.6 77.5 1.6 62.8 9.6
11 28 31 60 89.5 97.6 90.6 94.8 4.0 75.7 9.4
12 20 100 97.1 104.6 99.0 98.7 3.3 105.9 3.9
13 26 101.0 107.0 103.1 102.8 2.4 94.8 4.4
14 27 34 80 114.0 126.8 120.9 118.7 4.4 93.4 11.2
15 23 35 90 111.1 125.0 119.9 118.3 4.9 109.0 5.6
16 19 128.9 133.1 131.2 130.9 1.3 112.0 6.8
17 26 139.5 150.7 145.6 144.5 3.2 139.1 3.3
18 21 167.7 172.7 165.5 172.7 2.2 161.4 2.9
19 17 268.9 263.3 272.6 268.0 1.4 272.5 1.4
20 24 256.8 276.3 277.4 265.6 3.6 218.1 9.4
On the Way to a Next-Generation Lp(a) Reference Measurement System Based…

21 17 30 70 367.6 356.8 357.0 374.5 2.4 325.1 5.3

Each sample was measured in three replicate measurements in one run and the average result is shown here. The apo(a) isoforms present in the serum samples
were determined by Western blot at the Institute of Genetic Epidemiology, Medical university of Innsbruck, Austria. All assays used a five-point calibration
333

curve with independent calibrator solutions. According to the information provided by the assay manufacturers, the values in molar units were traceable to the
WHO-IFCC Reference Material SRM2B. The average intermethod CV over the 21 samples was 6.0%
334 C. Cobbaert et al.

Effect of Lp(a) Test Inaccuracy on Clinical Utility

In the 1980s and 1990s of the twentieth century, many research groups were inter-
ested in Lp(a) studies and its implementation in patient care. Unfortunately, a turn-­
point came after the publication of negative findings from the Physician Health
Study in JAMA (Journal of the American Medical Association) which demonstrated
no association between Lp(a) levels and the risk of future MI (myocardial infarc-
tion), stroke, or peripheral vascular disease (Ridker 1995; Ridker et al. 1993, 2001).
Fortunately, Rifai et al. reanalyzed the specimens from the Physician Health Study
with the gold standard reference ELISA and found that median Lp(a) in cases were
significantly higher than in controls with reference ELISA, whereas no significant
difference was found between cases and controls when using a commercial immu-
nonephelometric assay (Rifai et al. 2004). It was revealed that Lp(a) test inaccuracy
in the immunonephelometric assay had obscured the true relationship between
Lp(a) levels and CVD in the former JAMA papers. It is very essential to have accu-
rate Lp(a) tests that are not confounded by apo(a)-size polymorphism. In Fig. 20.2,
the masking impact of apo(a)-size polymorphism on Lp(a) levels is visualized in
case of isoform-dependent Lp(a) tests using a one-point calibration strategy
(Tsimikas et al. 2018). The attenuating effect of apo(a)-size polymorphism occurs
irrespective of the use of mass or molar units.
In the last years, the situation has improved as current commercially available
Lp(a) tests make use of multiple independent calibrators, well spread across the
Lp(a) concentration range and representing different apo(a) isoforms. This calibra-
tion strategy enables IVD manufacturers to measure Lp(a) with reduced impact of
apo(a)-size polymorphism (Tsimikas et al. 2018).

250
isoform independent assay Higher Lp(a)
values are
200 underestimated
by isoform
dependent
Lower Lp(a) assays
Lp(a) Value

150 values are

overestimated
by isoform
dependent
100 assays Isoform dependent assay

50

0
0 40 35 30 25 20 15
Lp(a) Isoform Size

Fig. 20.2 Theoretical relationship of Lp(a) values according to isoform size in isoform-­
independent and isoform-dependent assays in case of a one-point calibration strategy. Lp(a) values
are inversely related to isoform size, with large isoforms being associated with lower levels and
vice versa. Irrespective of the expression of Lp(a) values (nmol/L or mg/dL), isoform-dependent
assays will tend to overestimate low values and underestimate high values. Lp(a) lipoprotein(a).
[Reprinted with permission from (Tsimikas et al. 2018)]
20 On the Way to a Next-Generation Lp(a) Reference Measurement System Based… 335

 p(a): The Most Misunderstood Metric and the No(n)-sense

L
of Lp(a) Mass Results

The determinants of variability in Lp(a) particle composition are multiple and were
theoretically modeled based on existing literature (Ruhaak and Cobbaert 2020)
(Fig. 20.3). To that end and beyond the effect of apo(a)-size polymorphism, post-
translational modifications such as N- and O-glycosylations in apo(a) and apoB and
the lipid/protein ratio were considered. Depending on the number of K-IV2 repeats,
the theoretical protein content of the Lp(a) particle varies between 30 and 46%
(w/w), which inescapably confounds Lp(a) mass measurements. Based on variation
in number of K-IV2 repeats alone, the composition of lipid/protein in Lp(a) ranges
from 31% (w/w) in case of apo(a) with 6 K-IV2repeats to 42% (w/w) in case of
apo(a) with 35 K-IV2 repeats. This brings along a difference in Lp(a) mass of 19%:
2821 kDa Lp(a) mass in case of 6 K-IV2 repeats compared to 3344 kDa Lp(a) mass
in case of 35 K-IV2 repeats. This model also clarifies why using fixed factors for
converting Lp(a) particle mass into molar units that represent Lp(a) particle num-
ber—or vice versa—is metrologically not sound (Reyes-Soffer et al. 2022;
Guadagno et al. 2015). Therefore, it is of utmost importance to measure Lp(a) in
terms of its apo(a) component and no longer in terms of Lp(a) mass because varia-
tion in mass content can occur in each of its constituents, leading to large heteroge-
neity among individuals and confounded Lp(a) mass values. Because each Lp(a)
particle carries one molecule of apo(a), the molar apo(a) concentration reflects
unequivocally the number of Lp(a) particles.

 he Degree of Lp(a) Test Harmonization and Its Impact

T
on Cardiovascular Risk Management

In the Netherlands, the Dutch External Quality Assessment (EQA) organizer, named
SKML, performs national Lp(a) surveys using frozen, native human sera. In
Fig. 20.4, the national EQA data from 2018 are displayed. In total, 17 accredited
laboratories participated with nearly complete data sets, which comprised 11 rounds
of 2 blinded samples each. EQA samples were analyzed with two weekly intervals.
Each of the blinded samples was included twice in the EQA survey, that is, in the
first, respectively, and in the second half of the calendar year, to evaluate indepen-
dent duplicates within a year. A scatterplot of Lp(a) mass results (in mg/L) produced
by individual labs and stratified by IVD manufacturer is presented. Different IVD
manufacturers are marked with specific colored symbols, whereas the all-lab total
mean (ALTM) is presented with a black horizontal stripe. The overall interlabora-
tory variation of Lp(a) mass tests ranges from 16.4 to 32.1% at Lp(a) mass levels of
~150–450 mg/L. Siemens Healthineers demonstrates a negative bias whereas
Abbott and Beckman reveal a positive bias compared to the ALTM. The Roche
Lp(a) test is closest to the ALTM.
336 C. Cobbaert et al.

Particle size
6 KIV-2
repeats

Lipid composition # of KIV-2 repeats

35 KIV-2
repeats

Protein glycosylation Protein composition

PL FC CE TG apoB apo(a)

Kringles Lp(a) mass PL FC CE TG apoB apo(a) apo(a) lipids protein

IV-2 (kDa) (%) (%) (%) (%) (%) (kDa) (%) (%) (%)
6 2821 14.9 6.5 37.8 9.4 20.1 319 11.3 68.6 31.4
10 2893 14.6 6.4 36.8 9.1 19.6 391 13.5 66.9 33.1
20 3074 13.7 6.0 34.7 8.6 18.5 572 18.6 62.9 37.1
30 3254 12.9 5.7 32.7 8.1 17.5 752 23.1 59.4 40.6
35 3344 12.6 5.5 31.8 7.9 17.0 842 25.2 57.8 42.2

Fig. 20.3 Theoretical model of Lp(a) mass and compositional variation depending on apo(a)
K-IV2-size polymorphism based on literature. Lp(a) particle mass is dependent on the lipid/protein
composition and amount, the apo(a)-size polymorphism, and the N- and O-glycosylation of apo(a)
and apoB (upper left). Based on the variation in number of K-IV2 repeats in apo(a) alone, the dis-
tribution of lipid/protein in Lp(a) varies from 31% (w/w) protein with 6 K-IV2 repeats to 42%
(w/w) protein with 35 K-IV2repeats (upper right and bottom), leading to 19% difference in Lp(a)
mass. PL phospholipids, FC free cholesterol, CE cholesteryl esters, and TG triglycerides.
[Reprinted with permission from (Ruhaak and Cobbaert 2020)]

Taking into consideration published clinical thresholds of 150 and 300 mg/L for
Lp(a) particle mass, the EQA findings suggest serious impact of Lp(a) recovery on
CVD risk classification of patients and on patient management depending on the
IVD manufacturers’ reagents used. Yet, the systematic differences between IVD
manufacturers suggest that use of a common calibrator traceable to a generally rec-
ognized point of reference (i.e., SI unit through mass spectrometry) can signifi-
cantly improve the method variability. A more accurate assessment of each
individual’s CVD risk is also anticipated if molar Lp(a) assays rather than mass
assays become the norm, because of improved reflection of low risk in patients with
a small number of very large Lp(a) particles and high molecular weight apo(a) iso-
forms and high risk in patients with a high number of small Lp(a) particles and low
molecular weight apo(a) isoforms.
20 On the Way to a Next-Generation Lp(a) Reference Measurement System Based… 337

Abbott Siemens Roche Beckman Randox LDT ALTM (N=17 labs)

500

450

400

350
mg/L Lp(a) mass

300

250

200

150

100

50

0
1A 3B 2F 4A 2B 4C 2C 3D 1C 4E 1F 3C 2A 3F 1B 4B 1E 4F 2D 3E 2E 4D
Paired EQA-samples (NL, 2018)

Fig. 20.4 Dutch External Quality Assessment data of Lp(a) mass as measured in 11 paired,
blinded human serum samples analyzed at two weekly intervals in 2018 in 17 accredited medical
laboratories. Each of the blinded samples was included twice in the EQA survey to evaluate inde-
pendent duplicates within a year. For example, samples 1A and 3B are identical, but sample 1A
was analyzed in the first half of 2018 and sample 3B in the second half. Absolute Lp(a) levels vary
from half to double, with interlaboratory coefficients of variation ranging between 16 and 32%.
[Reprinted with permission from (Ruhaak and Cobbaert 2020)]

 evelopment of a Next-Generation SI-Traceable apo(a)/Lp(a)

D
Reference Measurement System

A comprehensive reference measurement system is key to achieving equivalent

apo(a)/Lp(a) test results that are traceable to the SI units among different measure-
ment procedures. Essential elements of this system are an unequivocally defined
analyte (including the unit) and a clearly described calibration hierarchy, which
integrates the reference measurement procedure and the suitable reference materials
[ISO17511:2020, (International Organization for Standardization 2020)].
Figure 20.5 shows the highest-order calibration hierarchy for Lp(a) in the envi-
sioned Reference Measurement System.
338 C. Cobbaert et al.

Calibrators Measurement procedures

SI units

Primary reference material

Primary reference method

Measurement uncertainty
Secondary reference material
SRM-2B Secondary reference
measurement procedure
(NWLRL, Seattle, WA, USA)

Manufacturer’s master
calibrators
Manufacturer’s Standing
Measurement procedure
Commercial product
calibrators from different lots

End user’s measurement

procedure
Patient sample with results in
nmol/L

Adapted from ISO 17511

Fig. 20.5 Metrological traceability chain as outlined in ISO 17511:2021. Currently, apo(a) test
results are at best traceable to WHO-IFCC secondary reference material SRM2B (blue), through
an ELISA-based K-IV2-independent method (green). However, the top of the traceability chain is
not in place (grey) and under development (Cobbaert et al. 2021), as described in this chapter. To
ensure SI traceability, primary reference materials (i.e., peptide-based calibrators) and a higher-­
order, K-IV2-independent reference measurement procedure (MS-based cRMP) are needed.
[Reprinted with permission from (Ruhaak and Cobbaert 2020)]

cRMP in a Network of Calibration Labs

A reference measurement procedure is required to transfer concentration values

from primary reference materials to the secondary reference materials or to clinical
specimens. Such a procedure should have very high accuracy, be specific and robust,
and importantly also sustainable. Quantitative mass spectrometry proteomics, in
which proteins are enzymatically digested into their representing peptides, which
are then quantified, may fulfill these requirements for protein measurands. Within
the IFCC WG APO-MS, a candidate reference measurement procedure (cRMP) is
being developed for the quantitation of apo(a) from human serum in nmol/L, inde-
pendent of the number of K-IV2 repeats (Cobbaert et al. 2021). Notably, the measur-
and was specifically defined to address the number of apo(a) molecules rather than
addressing the Lp(a) mass content. See Fig. 20.6.
20 On the Way to a Next-Generation Lp(a) Reference Measurement System Based… 339

Fig. 20.6 The infographic illustrates how a high-order mass spectrometry-based measurement
procedure allows for K-IV2-independent measurement of apo(a) at the molecular level, in contrast
to immunoassays which are by design confounded by the apo(a)-size polymorphism. Heterogeneous
apo(a) with variable sizes (3, 10, 20, and 30 K-IV2 repeats) are measured by immunoassay with
polyclonal antibodies, resulting in a poor definition of the apo(a) measurand and K-IV2-dependent
results, while LC-MS-based quantitation of apo(a) at the peptide level is K-IV2 independent with
high specificity of the measurand and in molar concentration

A group of three laboratories allowing global coverage developed a common

cRMP comprising the direct measurement of apo(a) in congruence with the six
other apolipoproteins A-I, B, C-I, C-II, C-III, and E. Briefly, serum is diluted, inter-
nal standard peptides are added, and the proteins are reduced, alkylated, and digested
using a combination of LysC and trypsin enzymes. Proteotypic peptides are then
quantified relative to the internal standard. Importantly, MS allows not only quanti-
tation but confirmation of identification at the same time, thus allowing definition of
the measurand at the molecular level with high specificity. The developed method
has an LoQ of 3.8 nmol/L, a linear range of 3.8–450 nmol/L, and a total imprecision
of 9.8% (Ruhaak et al. 2023). The method inherently allows apo(a) quantitation
independent of the K-IV2 polymorphism through the selection of unique quantify-
ing peptides.
340 C. Cobbaert et al.

Development of Primary and Secondary Reference Materials

A major requirement for establishing, implementing, and maintaining a reference

system relying on an MS-based reference measurement procedure is the availability
of fit-for-purpose primary and secondary reference materials. The primary refer-
ence materials will be used for the calibration of the MS-based reference measure-
ment procedure, and they consist of peptide solutions with a well-characterized
purity. A very precise determination (i.e., with very low uncertainty) of the molar
concentration of these calibration solutions is essential as they will form the basis of
the metrological traceability of the final results to the SI units.
The peptide-calibrated reference measurement procedure will then be used to
assign the Lp(a) concentration in molar units to the secondary reference materials.
These secondary reference materials should resemble the real clinical specimens
measured with the immunoassay-based Lp(a) tests. The term commutability is used
to express the closeness of agreement between results for a reference material and
results for clinical specimens when measured with various measurement procedures
(Miller et al. 2018). The complex and polymorphic nature of the Lp(a) requires the
production of multiple secondary reference materials with concentrations spread
across the Lp(a) concentration range and well-selected apo(a) isoforms. The IVD
manufacturer will be able to use these commutable secondary reference materials
directly in their internal traceability procedures to value-assign their product
calibrators.

 ow Do Current Immunoassay-Based Lp(a) Tests Compare

H
to the Next-Generation Reference Measurement Procedure?

The Department of Clinical Chemistry and Laboratory Medicine at the Leiden

University Medical Center performs clinical Lp(a) testing using the higher-order
MS-based method which recognizes each Lp(a) particle only once and allows mea-
surement of Lp(a) in molar terms. Three-hundred sixty-five subsequent clinical
samples were measured in 2020 with both the MS-based higher-order method and a
Roche immunoassay (Fig. 20.7). The comparison of the immunoassay-based
method (Y) and the MS-based method (X) revealed for the majority of clinical speci-
mens a good agreement (R > 0.975), although in individual specimens, significant
scatter was noted and sometimes extraordinary high biases occurred. The scatterplot
shows a Deming regression line with a slope that does not significantly deviate from
1.0, an intercept that is not different to 0.0 and a correlation coefficient R = 0.984.
From the method comparison data, it can be concluded that anno 2020, the com-
mercial Roche immunoassay test, produces on average equivalent results compared
to the higher-order MS-based test. Both are WHO-IFCC standardized. Yet, two
 20 On the Way to a Next-Generation Lp(a) Reference Measurement System Based… 341

Scatter Plot Bias Percent Bias

150 300
Deming Regr Mean Bias
1:1 Line
600
100 200
LP(a)(ITA) (nmol/L)

50 100

Bias (nmol/L)

Percent Bias
400
0 0

200 –50 –100

–100 –200

0 –150 –300
0 200 400 600 0 200 400 600 0 200 400 600
apo(a) (LFLEPT) (nmol/L) apo(a) (LFLEPT) (nmol/L) apo(a) (LFLEPT) (nmol/L)

Fig. 20.7 Method comparison in molar units between Roche immunoassay and an in-house-­
developed immunoassay-independent higher-order MS-based method. Three-hundred sixty-five
serum leftover serum specimens from the Leiden lipid clinic were compared. Deming regression
line: Y = 0.985 X + 0.52 in nmol/L, Xmean = 75.2 nmol/L, and Ymean = 74.6 nmol/L; R = 0.984.
Outliers were confirmed in independent runs. The red arrow points to a clinical specimen that
demonstrates a pronounced discordance between the two assays: 227 nmol/L with the MS-based
test and 87 nmol/L with the Roche immunoassay. It was demonstrated with Western blotting that
apo(a) in this discordant specimen had only nine K-IV repeats (unpublished data)

observations are remarkable: substantial scatter is noted in the absolute bias plot (up
to +/− 50 nmol/L) and marked discordances may occur in rare samples, poten-
tially leading to discrepant classifications with effect on patient management.
Apo(a) isoforms have been determined in all clinical specimens, and apo(a) isoform
data corroborate the average equivalence of Lp(a) test results between MS test and
IA test as well as individual scatter in clinical specimens across all apo(a) isoform
groups (see Fig. 20.1). For personalized medicine, accurate Lp(a) test results are
expected that are not confounded by apo(a)-size polymorphism and/or by molecular
diversity arising from apo(a) variants or truncated proteins (Coassin et al. 2017).
From the above head-to-head comparison, we deduce that as immunoassays are
routinely used in clinical practice, immunoassay test results (Roche) are fit for clini-
cal purpose and equivalent within total allowable error for >99% of the specimens
but can be inaccurate in rare individual cases due to genetic variants/mutants who
go undetected.

 ransitioning from the WHO-IFCC RMS to an SI-Traceable

T
Reference Measurement System

Background and Current WHO Reference Material for Lp(a)

The Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA,
is the custodian laboratory for the WHO Biological Reference Material for
lipoprotein(a) (i.e., IFCC SRM2B). As part of this function, it holds and distributes
342 C. Cobbaert et al.

this material. The current WHO/IFCC International Reference Material for

lipoprotein(a) [Lp(a)] (SRM2B) was generated in 2003. Several measurement pro-
cedures for Lp(a) are traceable to this material, which was previously value-assigned
through an ELISA-based K-IV2-independent method (Tate et al. 1999). Currently,
the IFCC SRM2B material is almost depleted, and also the ELISA-based K-IV2-­
independent method is no longer available.

New JRC-IFCC/LNE Reference Materials

The IFCC Working Group for Apolipoproteins by Mass Spectrometry (IFCC WG

APO-MS) is developing an MS-based reference measurement procedure for apoli-
poproteins, as well as primary and secondary reference materials for apolipopro-
teins including apo(a) (https://www.ifcc.org/ifcc-­scientific-­division/
sdworking-­groups/wg-­apo-­ms/). The MS-based reference measurement procedure
will be used to assign reference values to commutable, serum-based reference mate-
rials. A reference measurement procedure and primary and secondary reference
materials for Lp(a) are planned to be available in the nearby future (Cobbaert et al.
2021). The new, value-assigned, serum-based reference materials will be made
available by the European Commission’s Joint Research Centre (EC-JRC) (https://
crm.jrc.ec.europa.eu/). The new primary peptide-based reference materials for cali-
bration of the reference measurement procedure in IFCC-endorsed calibration labs
will be stored at the Laboratoire National de Métrologie et d’Essais (LNE)
­([email protected]).

Interim Solutions

The IFCC WG APO-MS members and CDC as envisioned future network coordi-
nator of Lp(a) calibration laboratories can help IVD manufacturers, clinical trial
laboratories, and EQA providers transitioning to the new IFCC reference system
by making available individual donor and pooled serum materials with Lp(a) val-
ues assigned by the IFCC’s LC-MS/MS method (molar units, not K-IV2 depen-
dent). Although this cRMP is still under development, data obtained with these
materials can provide information about the agreement of a laboratory method
with the MS-based method. Also, the CDC Clinical Standardization Program
(CSP) will prepare a formal standardization program for guiding manufacturers
on their path to molar (first step), respectively, SI-traceable (second step) Lp(a)
test results and certification. In preparation of this activity, CDC-CSP will be
conducting an interlaboratory comparison study for Lp(a) with routine clinical
laboratories as participants. An infographic on the transition is shown at the IFCC
website: https://www.ifcc.org/media/479001/210514_ifcc-­apo-­traceability_info-
graphic_def.pdf.
20 On the Way to a Next-Generation Lp(a) Reference Measurement System Based… 343

 ransitioning from Old to New Lp(a) Reference Measurement

T
System: Recommendations to Cardiologists
and Other Stakeholders

Relevance of Accurate Test Results Within Allowable Total Error

Accurate medical test results are key for safe and effective management of patients.
Moreover, exchangeability of test results, reference intervals, and decision limits
among healthcare institutions is essential for efficiency reasons along the patients’
journey in primary, secondary, and tertiary care settings. In case of inaccuracy
exceeding the total allowable error, Lp(a) test results can be misleading and harm
patients due to misclassification and undetected or untreated cardiovascular risk.
The (in)accuracy and analytical performance of a test affects its clinical perfor-
mance as both key elements of test evaluation are interdependent. On top, flawed
test results may mask the clinical utility and medical value of a test, preventing its
adoption and implementation in clinical practice. These general principles also
apply for Lp(a) testing. Nevertheless, a multitude of Lp(a) test design flaws are
causally related to about 15 years of unjustified silence around Lp(a) testing.
Currently, a revival of Lp(a) testing is taking place and its clinical relevance has
been reinvented (Ellis et al. 2017).

 hat Were the Determinants of Inaccuracy in Case of Lp(a)

W
Testing in the Twentieth Century?

Firstly, the definition of the measurand intended to be measured should have been
unequivocal, but that was not the case. In the past decades, Lp(a) particle mass,
Lp(a) particle concentration, and Lp(a) cholesterol have all been considered and
used mixed. As most routine Lp(a) tests were based on an immunoassay-based read-
out, the apo(a)-size polymorphism confounded the Lp(a) recovery, especially in
case of polyclonal antibodies. But also the type and number of calibrators are rele-
vant: multiple calibrator levels, composed of well-selected apo(a) isoforms across
the Lp(a) concentration range that mimic clinical specimens, are preferred. However,
if IVD manufacturers make serial dilutions from a single calibrator, the isoform
composition of the diluted calibrators is not aligned with that in the clinical speci-
mens, and Lp(a) test results become inaccurate (Fig. 20.1). Finally, also the post-­
analytical phase and unit choice (mass or molar) have further aggravated the
inaccuracy of Lp(a) test results due to assumptions and oversimplified models for
estimating the Lp(a) particle mass (Ruhaak and Cobbaert 2020) and Figs. 20.1 and
20.4). The cumulative errors of former Lp(a) kit designs often exceeded the allow-
able total error, making those Lp(a) tests not fit for clinical purpose.
344 C. Cobbaert et al.

 oncordance and Discordance of Contemporary

C
Immunoassay-­Based Methods Compared to a Higher-Order
Mass Spectrometry-Based Method, Being a Predecessor
of the SI-Traceable cRMP

Currently, immunoassay-based Lp(a) tests are still the methods of choice in medical
labs. Test design and analytical performance of immunoassay-based tests have been
substantially improved, especially those that are standardized against the former
IFCC-WHO reference measurement system and express Lp(a) test results in molar
units. In these tests, on average, equivalent results and correlation coefficients
>0.975 are found between immunoassays and higher-order MS-based measurement
procedures (Dikaios et al. 2023). A representative method comparison is presented
in Fig. 20.7. IVD manufacturers took lessons from the past and currently use inde-
pendent calibrators with well-selected apo(a) isoforms which are good mimics of
clinical specimens. Notable is the fact that unexplained Lp(a) scatter—within the
total allowable error zone—can be observed in the absolute/relative bias plots, also
within all predominant apo(a) isoform classes when comparing routine and higher-
order MS-based measurement procedures (data not shown). In addition, less than
1% discordances—exceeding the total allowable error—were found between immu-
noassay and higher-order MS-based measurement procedure results, pointing to
further molecular variation/truncation on top of apo(a)-size polymorphism and
affecting immunoassay-based measurements. As >99% of the results from current
immunoassays are concordant with higher-order mass spectrometry-­derived results,
we can state that contemporary Lp(a) immunoassay tests are in general fit for clini-
cal purpose with a manageable risk for patient harm.

 ransitioning from Old to New Lp(a)/apo(a) Reference

T
Measurement System: Recommendations for Stakeholders
and End-Users

Awaiting the establishment of the complete SI-traceable Reference Measurement

System for Lp(a) standardization (Cobbaert et al. 2021), it becomes obvious from
Fig. 20.1 that a lot can already be gained if one starts with the movement toward
introducing molar standardization and molar units. Therefore, a two-step
approach is recommended as it provides IVD manufacturers, clinicians, and
researchers insight in the degree of change and harmonization that can be accom-
plished in the transition phase. After all, average intermethod CVs from immunoas-
say kits expressing Lp(a) in mass units are excessive (ranging up to 21.8%) and can
be reduced three to fourfold (ranging up to 5.1%) in case Lp(a) immunoassay kits
are standardized in molar units and results are expressed in nmol/L apo(a). By doing
so, Lp(a) tests can easily meet the total allowable error requirements, which makes
20 On the Way to a Next-Generation Lp(a) Reference Measurement System Based… 345

the molar Lp(a) immunoassay kits already better fit for clinical purpose while still
being standardized to the former WHO-IFCC RMS. A second step can be made by
IVD manufacturers once the SI-traceable MS-based RMS is in place and interna-
tionally recognized. To guide IVD manufacturers toward sustainable standardiza-
tion of Lp(a) tests with SI traceability of Lp(a) test results, commutable,
value-assigned secondary RMs will be made available by JRC which IVD manufac-
turers can purchase for internal standardization.
The calibration laboratories running the SI-traceable cRMP together with JRC,
LNE, CDC, and the IFCC WG APO-MS will prepare the transition from old to new
Lp(a) RMS along these lines, in a two-phased process.

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Chapter 21
Therapy of Elevated Lipoprotein(a)

S. Ibrahim and Erik S. G. Stroes

Expected Benefit of Lp(a) Lowering

The association between elevated lipoprotein(a) (Lp[a]) and cardiovascular risk as

well as calcific aortic valve disease has been established in a large number of stud-
ies. Given the consistent genetic, mechanistic, and observational evidence support-
ing a (causal) role of Lp(a) in the development of different types of cardiovascular
disease (CVD), the European Society of Cardiology (ESC)/European Atherosclerosis
Society (EAS) guidelines recommend measuring Lp(a) levels at least once in each
person’s lifetime to identify individuals who have inherited an extremely elevated
level (Mach et al. 2020). Consequently, this leaves clinicians facing the challenge to
act on an Lp(a)-associated atherosclerotic CVD (ASCVD) risk.
Genetic Mendelian randomization studies have demonstrated that the clinical
benefit of lowering Lp(a) is likely to be proportional to the absolute reduction in
Lp(a) concentration (Madsen et al. 2020; Burgess et al. 2019). The first Mendelian
randomization analysis, by Burgess et al. involving 80,000 patients and more than
150,000 controls, estimated that an Lp(a) reduction of 100 mg/dL (210 nmol/L)
resulted in an equal ASCVD risk reduction as achieved by 1 mmol/L (38.7 mg/dL)
low-density lipoprotein cholesterol (LDL-C) lowering (Burgess et al. 2019). A more
recent observational study, including 58,527 participants from the Copenhagen
General Population Study (CGPS), estimated that this Lp(a) equivalent is lower,
approximately 55 mg/dL (116 nmol/L) (Madsen et al. 2020). Nevertheless, both
studies suggest that particularly individuals with very high Lp(a) concentrations
will benefit from therapies that reduce Lp(a) concentrations, which has important

S. Ibrahim · E. S. G. Stroes (*)

Department of Vascular Medicine, Amsterdam UMC, University of Amsterdam,
Amsterdam, The Netherlands
e-mail: [email protected]; [email protected]

© The Author(s), under exclusive license to Springer Nature 347

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_21
348 S. Ibrahim and E. S. G. Stroes

implications for clinical practice guidelines on the use of future Lp(a) lowering
therapies. These results also imply that randomized controlled trials evaluating
Lp(a) lowering therapies should enroll individuals with very high baseline Lp(a)
levels of 70–100 mg/dL or more, to demonstrate clinically meaningful reductions in
the risk of cardiovascular events.
Since the largest ASCVD risk reduction can be achieved in patients with the
highest baseline cardiovascular risk with concomitant highest Lp(a) levels, second-
ary prevention patients with Lp(a) elevation are the first to qualify for therapies
specifically and potently lowering Lp(a). In anticipation of the new drugs, this
patient group could benefit from more intensive LDL-C lowering [using either
statins or proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors].
Lifestyle and blood pressure lowering are also effective in partly attenuating the
Lp(a)-induced CV risk increase. Importantly, not only secondary prevention patients
are likely to benefit from Lp(a) lowering. An analysis from the Justification for the
Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin
(JUPITER) trial demonstrated that Lp(a) was also a significant determinant of
residual CVD risk in a cohort of primary prevention participants with low LDL-C
(Khera et al. 2014). In this group of primary prevention patients with very high
Lp(a), earlier and more intensive health behavior modification counselling and
management of other ASCVD risk factors are therefore often recommended
(Pearson et al. 2021).
As specific Lp(a) lowering therapies are still in development; for now, Lp(a) can
be incorporated into established ASCVD risk algorithms (Nurmohamed et al.
2021a). This holds true for both primary and secondary preventions. Nurmohamed
and colleagues demonstrated that in individuals with very high Lp(a) (>99th percen-
tile), the addition of Lp(a) into ASCVD risk algorithms resulted in 31% reclassifica-
tion in primary prevention and 63% reclassification in secondary prevention
(Nurmohamed et al. 2021a).

Effect of Current Therapies on Lp(a)

As circulating Lp(a) levels are, for more than 85%, mediated by genetic variation at
the LPA gene locus with only modest influence of environmental factors, pharmaco-
logical strategies to lower Lp(a) levels hold a major promise (Table 21.1) (Tsimikas
and Hall 2012). New therapies potently reducing apolipoprotein(a) (apo[a]) produc-
tion by the liver have shown the potential to lower Lp(a) levels by 80–98%.
Nonetheless, the clinical benefit of such substantial reductions in Lp(a) remains to
be established in phase 3 cardiovascular outcome trials. This leaves clinicians with
existing approaches to reduce ASCVD risk, which have varying effects on
Lp(a) levels.
21 Therapy of Elevated Lipoprotein(a) 349

Table 21.1 Therapeutic approaches in cardiovascular risk management that lower lipoprotein(a)
concentration
FDA/
Lp(a) lowering Lp(a) Cardiovascular EMA
strategies reduction risk reduction approval Reference(s)
Monoclonal PCSK9 23.0– Yes Yes Sabatine et al. (2017),
antibodies 27.0% Schwartz et al. (2018a),
O’Donoghue et al. (2019) and
Szarek et al. (2020)
PCSK9 siRNA 18.6– Under Yes Ray et al. (2020)
25.6% investigation
Niacin 23.0% No Yes Sahebkar et al. (2016)
CETP inhibitors 10.0– No or minimal Not Arsenault et al. (2018),
56.5% currently Schwartz et al. (2018b),
Bowman et al. (2017),
Thomas et al. (2017) and
Nicholls et al. (2022)
MTP inhibitors 17.0% Not investigated Yes Samaha et al. (2008)
Apheresis 30.0– Not investigated Yes Moriarty et al. (2019)
45.0%
IL-6 inhibitors 30.0– Under Yes Schultz et al. (2010), McInnes
40.0% investigation et al. (2015), García-Gómez
et al. (2017), Gabay et al.
(2016) and Ridker et al.
(2021)
Antisense 80.0% Under Not Viney et al. (2016)
oligonucleotides investigation currently
targeting Apo(a)
mrna
Small interfering >90.0% Under Not Koren et al. (2020),
RNA targeting investigation currently NCT04270760,
Apo(a) mrna NCT04606602

Statins

As compelling evidence has shown that statins are highly effective in reducing both
LDL-C levels and cardiovascular events, this drug class now represents the corner-
stone for treating patients with dyslipidemia and for attenuating cardiovascular risk
in both primary and secondary preventions (Baigent et al. 2010). Clinical trials
exploring the effect of statin therapy on Lp(a) levels, however, have shown mixed
results. The JUPITER trial, for example, showed no median change in Lp(a) with
rosuvastatin and placebo (Khera et al. 2014). Nonetheless, rosuvastatin did result in
a small but statistically significant positive shift in the overall Lp(a) distribution
(Khera et al. 2014). Furthermore, cell culture studies have found a time- and dose-­
dependent, statin-mediated increase in the expression of LPA mRNA and
apolipoprotein(a) protein production when incubating HepG2 hepatocytes with
atorvastatin (Tsimikas et al. 2020). A meta-analysis, conducted by Tsimikas et al.
including 5256 patients randomized to receive rosuvastatin, atorvastatin,
350 S. Ibrahim and E. S. G. Stroes

pravastatin, pitavastatin, or placebo, demonstrated that Lp(a) levels increased sig-

nificantly in patients starting statin therapy compared to placebo (Tsimikas et al.
2020). The mean percent change in Lp(a) level from baseline ranged from 8.5 to
19.6% in the statin groups and—0.4–2.3% in the placebo groups. A more recent
meta-analysis, including 24,448 patients from 39 placebo-controlled trials with dif-
ferent statins, however, found no significant increases in Lp(a) levels in different
types of statins, as well as different intensities of statin therapy, compared with
placebo groups (de Boer et al. 2021). Although some studies suggest that statins
mildly increase Lp(a) levels, in clinical practice, these effects are considered of no
clinical importance since the cardiovascular benefits from statins by far outweigh
the risk associated with potential mild increases in Lp(a).

Ezetimibe

Based on the available data that has been published, ezetimibe has a neutral
effect on circulating Lp(a) levels. A meta-analysis including 5188 individuals
from 10 randomized placebo-controlled trials demonstrated that ezetimibe ther-
apy had no effect on plasma Lp(a) concentrations (Sahebkar et al. 2018). In addi-
tion, a subgroup analysis indicated no significant alteration in plasma Lp(a) in
trials assessing the impact of ezetimibe monotherapy versus placebo nor in trials
evaluating the impact of adding ezetimibe to a statin versus statin therapy alone
(Sahebkar et al. 2018). Another meta-analysis, including 2337 patients with pri-
mary hypercholesterolemia from 7 randomized controlled trials, demonstrated
that ezetimibe 10 mg/day significantly reduced Lp(a) by 7.1% (Awad et al.
2018). However, investigators concluded that this small reduction was not clini-
cally significant.

PCSK9 Inhibitors

To date, PCSK9 inhibitors are the only class of LDL-C lowering drugs that has been
shown to lower Lp(a) as well as reducing CVD risk. The monoclonal PCSK9 anti-
bodies, alirocumab and evolocumab, have been evaluated in two large placebo-­
controlled outcome trials, together comprising more than 100,000 patient years of
observation (Sabatine et al. 2017; Schwartz et al. 2018a). Both outcome trials
reported a significant reduction in major adverse cardiovascular events (MACE)
during treatment with PCSK9 antibodies for a median 2.3–2.7 years. Interestingly,
in both trials, the absolute cardiovascular risk reduction with a PCSK9 inhibitor was
higher in patients with higher baseline Lp(a) levels. In the FOURIER (Further
Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with
Elevated Risk) study, the absolute reduction in MACE with evolocumab was 2.41%
for Lp(a) >50 mg/dL versus 1.41% at lower levels (O’Donoghue et al. 2019). In the
21 Therapy of Elevated Lipoprotein(a) 351

ODYSSEY OUTCOMES (Evaluation of Cardiovascular Outcomes After an Acute

Coronary Syndrome During Treatment With Alirocumab) trial, the absolute reduc-
tion in MACE with alirocumab was 3.7% for Lp(a) >60 mg/dL versus 0.5% in the
lowest quartile for Lp(a) (Szarek et al. 2020). Since evolocumab and alirocumab
also lower median baseline Lp(a) levels with 27% and 23%, respectively (Sabatine
et al. 2017; Schwartz et al. 2018a), it has been a matter of debate whether and to
what extent the Lp(a) lowering may contribute to the reduction in MACE, indepen-
dent from the profound LDL-C reduction hallmarking these drugs. For alirocumab,
it was estimated that at the 25th percentile of baseline Lp(a) (6.7 mg/dL), reduction
in Lp(a) levels was small (1.6 mg/dL) and accounted for only 4% of reduction in
risk of MACE, while 96% of risk reduction was attributable to lowering of corrected
LDL-C (Schwartz et al. 2018a). At the 75th percentile of baseline Lp(a) (59.6 mg/
dL), however, Lp(a) reduction was 13.4 mg/dL and accounted for 25% of reduction
in risk of MACE.
Trials evaluating inclisiran, a small interfering RNA that suppresses PCSK9
mRNA translation and therefore reduces PCSK9 protein synthesis, have shown
similar reductions in plasma levels of Lp(a) as the monoclonal antibodies (Ray et al.
2020). The ORION-10 and ORION-11 trials demonstrated Lp(a) reductions of
25.6% and 18.6%, respectively, with LDL-C reductions of approximately 50% (Ray
et al. 2020). The potential benefit of inclisiran on MACE is being evaluated in the
ongoing ORION-4 trial. Although PCSK9 inhibitors have been shown to reduce
levels of Lp(a), with the monoclonal antibodies simultaneously reducing the risk of
MACE, the extent to which Lp(a) lowering with PCSK9 inhibition contributes to
the overall clinical benefit of this drug class remains, however, debatable.

Other Lipid-Modifying Agents

There are several lipid-modifying agents that have been shown to reduce Lp(a) lev-
els in studies, however, without evidence substantiating that the Lp(a) lowering abil-
ity leads to improved clinical outcomes. Niacin, for example, which is an essential
micronutrient that raises the concentration of high-density lipoprotein cholesterol
(HDL-C) and reduces triglycerides (TG) as well as LDL-C concentrations, has also
been shown to reduce Lp(a) levels (Sahebkar et al. 2016). A meta-analysis of 14
randomized placebo-controlled trials demonstrated a mean Lp(a) reduction of 23%
in patients treated with extended-release niacin (Sahebkar et al. 2016). However,
two placebo-controlled trials evaluating the cardiovascular efficacy of extended-­
release niacin demonstrated no cardiovascular efficacy of the drug and even showed
an increased risk of serious adverse events (Boden et al. 2011; Landray et al. 2014).
However, it should be noted that the median Lp(a) levels in these trials were low and
the number of patients with very high Lp(a) levels was very limited, which may
reduce the validity of these findings in the group with markedly elevated Lp(a) lev-
els. Nevertheless, due to adverse effects and the lack of evidence that Lp(a) reduc-
tion with niacin is associated with a cardiovascular benefit, niacin is not recommended
352 S. Ibrahim and E. S. G. Stroes

in patients with Lp(a) elevation. In Europe, niacin is no longer on the market. A

similar picture emerges for cholesteryl ester transfer protein (CETP) inhibitors.
CETP is a liver-synthesized glycoprotein that facilitates bidirectional transfer of
cholesteryl esters and triglycerides between cholesterol-rich lipoproteins (LDL,
HDL) on the one hand and triglyceride-rich lipoprotein particles (VLDL) on the
other hand. Inhibition of CETP has been shown to reduce levels of non-HDL cho-
lesterol and to increase the concentration of HDL-C. Studies evaluating different
CETP inhibitors have demonstrated reductions in Lp(a) ranging from 10 to 56.5%
(Arsenault et al. 2018; Schwartz et al. 2018b; Bowman et al. 2017; Thomas et al.
2017; Nicholls et al. 2022). However, to date, there is no data that has related Lp(a)
reductions with CETP inhibitors to clinical outcomes. In fact, three inhibitors
(torcetrapib, dalcetrapib, evacetrapib) failed to show any cardiovascular benefit, and
only anacetrapib showed a small cardiovascular benefit directly related to the level
of non-HDLc lowering independent from Lp(a) change. The remaining CETP
inhibitor in clinical trials is the obicetrapib, of which phase II trials suggest an Lp(a)
reduction up to 56.5% (Nicholls et al. 2022). Awaiting the clinical outcomes trial of
obicetrapib (PREVAIL; NCT03260517), none of the CETP inhibitors has been
approved for therapeutic use (Nurmohamed et al. 2021b).
Plasma Lp(a) reductions can also be achieved by microsomal TG transfer protein
(MTP) inhibitors. MTP is an intracellular endoplasmatic reticulum transfer protein
responsible for the assembly and secretion of apolipoprotein B (apoB)-containing
lipoproteins in hepatocytes and enterocytes. A study evaluating monotherapy with
the MTP inhibitor lomitapide in patients with moderate hypercholesterolemia dem-
onstrated a mean reduction in Lp(a) levels of 17% on top of an approximate 30%
mean reduction in LDL-C levels (Samaha et al. 2008). However, widespread use of
this drug is hampered by adverse effects, including gastrointestinal complaints,
hepatic fat accumulation, and elevated liver enzymes, which occur in a significant
proportion of the treated patients. Moreover, since this compound has only been
approved for use in very rare patients with homozygous FH, the high price pre-
cludes its use in larger patient groups.
Other lipid-modifying agents, like bempedoic acid, fibrates, and bile acid seques-
trants, have not shown any significant effect on Lp(a) levels (Rubino et al. 2021;
Eraikhuemen et al. 2021).

Apheresis

Lipoprotein apheresis provides significant reductions in apolipoprotein B-containing

lipoproteins including Lp(a) with median Lp(a) reductions ranging between 30 and
45% during biweekly and weekly apheresis, respectively. It is, besides PCSK9
inhibitors, the only therapeutic approach that has been shown to reduce cardiovas-
cular events in individuals treated for elevated Lp(a), albeit no randomized trials
have evaluated this therapeutic intervention (Moriarty et al. 2019). The plasmapher-
etic methods for reducing Lp(a) levels will be discussed extensively in another
chapter.
21 Therapy of Elevated Lipoprotein(a) 353

Non-apoB-Directed Therapies

Various chronic inflammatory conditions, such as rheumatoid arthritis (RA) and

Crohn’s disease, have been associated with elevated Lp(a) levels (Missala et al.
2012), which is comprehensible since an IL6-responsive element is present in the
promotor region of the LPA gene. In that regard, it is of interest to investigate
whether anti-inflammatory drugs are effective in reducing Lp(a) levels and CVD
risk in patients with elevated Lp(a) levels. Several clinical studies evaluating tocili-
zumab, a specific monoclonal antibody against interleukin 6 (IL-6), have shown
reductions in plasma Lp(a) levels of approximately 30–40% in RA patients (Schultz
et al. 2010; McInnes et al. 2015; García-Gómez et al. 2017; Gabay et al. 2016).
Moreover, recent studies have demonstrated that IL-6 inhibition with ziltivekimab
also decreases Lp(a) dose-dependently (Ridker et al. 2021). The ability of these
IL-6 inhibitors to reduce CVD events is currently being evaluated in cardiovascular
outcomes trials (ZEUS; NCT05021835).

Future Perspectives: Experimental Drugs

As discussed, current lipid-modifying agents only modestly reduce elevated Lp(a)

levels, whereas Mendelian and epidemiological studies suggest that large reduc-
tions in Lp(a) are required in order to achieve a clinically meaningful benefit in
cardiovascular outcomes. Currently, therapies targeting RNA for apo(a) have been
developed in order to specifically and potently inhibit the synthesis of Lp(a). These
investigational agents are currently being tested in clinical trials to determine their
Lp(a) lowering ability, their safety and tolerability, and their potential to reduce
CVD risk.

Antisense Oligonucleotides

Pelacarsen [formerly known as IONIS-APO(a)-LRX and AKCEA-APO(a)-LRX] is

a gal-nac-conjugated, single-stranded antisense oligonucleotide that targets hepatic
apo(a) mRNA. The gal-nac moiety increase selective hepatic uptake of the antisense
via the asiologlycoprotein receptor 1; after hepatic uptake the antisense leads to
degradation of the apo(a)-messenger RNA within the nucleus of the hepatocyte. The
drug has shown median Lp(a) reductions of 80% with good tolerability of monthly
injections of the GalNac antisense (Viney et al. 2016). With 8324 patients enrolled,
the HORIZON cardiovascular outcomes trial is expected to report in 2025
(NCT04023552). Antisense therapy targeting Lp(a) will be further discussed in the
next chapter.
354 S. Ibrahim and E. S. G. Stroes

Small Interfering RNA

There are currently two small interfering RNA (siRNA) agents targeting LPA that
are being tested in different phases in clinical trials. SiRNAs are mostly 21–25
nucleotides long, double-stranded RNA that have sequence-homology-driven gene-
knockdown capability (Tromp et al. 2020). Following incorporation into the RNA-
induced silencing complex (RISC) in the plasma, the modified RISC allows for
gene blocking by binding and then degrading the mRNA produced by the gene by
the protein Argonaut-1. Two major differences with the antisense mechanism is it’s
mode of action in the cytoplasm rather than the nucleus; and second the long half-
life of the inhibitory effect of the siRNA due to the stability of the siRNA in the
RISC complex. Olpasiran (formerly known as AMG-890 and ARO-LPA) is one
example of an siRNA targeting apo(a) mRNA, which has shown reductions in Lp(a)
levels of more than 90% with no safety concerns identified in healthy volunteers in
a phase 1 study (Koren et al. 2020). A phase 2 dose finding study was recently com-
pleted in 290 patients with established ASCVD and Lp(a) levels ≥200 nmol/L,
showing a 97.4% reduction in placebo-adjusted changes of Lp(a) with the 75-mg
dose administered once every 3 months sucutaneously, (O’Donoghue et al. 2022). A
cardiovascular outcomes trial using Olpasiran has recently started (Ocean(a);
NCT05581303). Another GalNAc-conjugated siRNA targeting the mRNA tran-
script of LPA is SLN360 has reported a >95% Lp(a) reduction in the APOLLO trial
(Nissen et al. 2022). A larger phase II trial using SLN360 in patient with atheroscle-
rotic cardiovascular disease and Lp(a) elevation is expected to start in the beginning
of 2023 (NCT05537571).

Conclusion

In summary, current available therapeutic agents have only mild to moderate Lp(a)
lowering effects with PCSK9 inhibitors and apheresis being the only existing thera-
peutic approaches that have been shown to lower Lp(a) levels and reduce CVD risk.
The magnitude of treatment benefit for PCSK9 inhibitors is associated with baseline
Lp(a) levels and seems to be associated with the degree of Lp(a) reduction. Specific
and potent RNA-targeted interventions have the potential to greatly reduce Lp(a) con-
centrations. Cardiovascular outcomes trials will have to show whether such substan-
tial Lp(a) reductions are associated with meaningful clinical benefit, the outcomes of
which are expected in 2025 (HORIZON) and 2026/7 (Ocean(a)), respectively.

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Chapter 22
Antisense Oligonucleotide Therapy
to Treat Elevated Lipoprotein(a)

Sotirios Tsimikas

Since the initial report of the discovery of lipoprotein(a) [Lp(a)] in a single-author

paper by Kare Berg in 1963 (Berg, 1963) and the subsequent association to cardio-
vascular disease (Berg et al. 1974), it has been anticipated that a specific therapy
could be developed to lower Lp(a) levels and reduce cardiovascular risk. However,
despite several nonspecific approaches, until now, it has been very difficult to
potently and specifically lower plasma Lp(a) levels. The main impediments have
been the fact that Lp(a) has no enzyme activity or receptor function, and therefore
the only viable approach is to reduce synthesis and/or increase clearance of the
actual particle. Clearance pathways are ill-defined and are mediated by multiple
pathways and receptors whose individual quantitative contributions are not known.
Additionally, they do not materially affect plasma levels, which are primarily influ-
enced by the genetically driven synthetic capacity of the hepatocyte. The emergence
of a novel therapeutic modality, interfering with messenger ribonucleic acid transla-
tion to make protein, has allowed potent and specific Lp(a) lowering therapy to
become a reality (Crooke et al. 2018). This work will review the preclinical and
clinical development of antisense oligonucleotides (ASOs) in the quest to develop a
specific method to lower plasma Lp(a) and to test the “Lp(a) hypothesis,” namely,
that potently lowering Lp(a) will reduce risk of cardiovascular disease and aortic
stenosis.

S. Tsimikas (*)
Division of Cardiology, Sulpizio Cardiovascular Center, University of California San Diego,
La Jolla, CA, USA
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 359

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_22
360 S. Tsimikas

Preclinical Proof of Concept in Lowering Plasma Lp(a) Levels

One of the limitations in developing modalities to lower Lp(a) in plasma has been
the narrow species distribution of the LPA gene, limited to hedgehogs, Old World
monkeys, apes, and humans (McLean et al. 1987; Tomlinson et al. 1989). Thus,
appropriate animal models that can encompass the entire Lp(a) pathophysiology,
including regulatory elements, have generally been lacking (Yeang et al. 2016).
One of the earliest reported attempts to interfere with apolipoprotein(a) biosyn-
thesis was by Frank et al. (2001). They doubly transfected mice with an adenovirus-­
mediated N-terminal truncated apolipoprotein(a) construct comprising the
5′-untranslated region, the signal sequence, and the first three kringles of native
apolipoprotein(a) along with a concomitant antisense molecule directed to
apolipoprotein(a) mRNA. Evidence of transient but efficient reductions of
apolipoprotein(a) synthesis was shown. However, this approach could not be easily
translated to human applications due to the limitations of the animal models and the
rapid degradation and presumed inefficiency of the antisense constructs used.
Several approaches have been shown to nonselectively and modestly lower
Lp(a), including lipid apheresis, niacin, CETP inhibitors, and PCSK9 inhibitors
(Tsimikas et al. 2021). The first proof-of-concept description of effective and long-­
lasting in vivo lowering of Lp(a) was reported by Merki et al. (2008) in 2008 in a
collaborative effort of the Ionis Pharmaceuticals (then named Isis Pharmaceuticals)
led by Mark Graham and Rosanne Crooke and our laboratory at UCSD (University
of California, San Diego). In this study, Lp(a) lowering was achieved by the anti-
sense oligonucleotide mipomersen, directed to human apoB-100, which signifi-
cantly reduced human apoB-100 levels in Lp(a) transgenic mice expressing both
human apoB-100 and apolipoprotein(a) needed to generate authentic Lp(a) parti-
cles. Over the 11-week treatment period, compared with baseline, mipomersen
reduced Lp(a) levels by up to 75% (p < 0.0001) in a time-dependent fashion
(Fig. 22.1a). This was primarily due to limiting the availability of apoB-100 to bind
to apolipoprotein(a), as LPA mRNA expression and plasma apolipoprotein(a) levels
were not affected by mipomersen. Furthermore, mipomersen significantly reduced
plasma levels of oxidized phospholipids on apoB (OxPL-apoB) and apolipoprotein(a)
[OxPL-apo(a)] particles (Fig. 22.1b). This study provided proof of concept that
reducing the availability of apoB-100 is a limiting factor in Lp(a) particle assembly
in this Lp(a) transgenic mouse model. These preclinical findings were later con-
firmed in clinical studies of mipomersen in a variety of settings, including in patients
with homozygous and heterozygous familial and multifactorial hypercholesterol-
emia, showing approximately 25% reduction in Lp(a) that was independent of
LDL-C lowering (Santos et al. 2015). The study also taught us that targeting apoB
is not an ideal mechanism to lower Lp(a), as it does not affect the pathognomonic
protein of Lp(a), apolipoprotein(a). Furthermore, the pathophysiological effect of
free apolipoprotein(a), and whether it is more or less atherothrombotic and proin-
flammatory than Lp(a), is not known.
22 Antisense Oligonucleotide Therapy to Treat Elevated Lipoprotein(a) 361

Fig. 22.1 Temporal changes in Lp(a) levels (A) and in OxPL-apoB levels in Lp(a)-transgenic
mice treated with control ASO or mipomersen directed to human apoB. Temporal changes in
apo(a) levels (A) and in OxPL-apo(a) levels in apo(a)-transgenic mice treated with saline, control
ASO or ASO 144367. *P<001, **P<0.01 vs baseline values. Merki et al. 2008, 2011)

Due to the lack of apolipoprotein(a) reduction with mipomersen, a new approach

was reported in 2011 using a specific antisense oligonucleotide, ISIS 144367, to
target LPA mRNA (Merki et al. 2011). Three transgenic mouse models were uti-
lized: 8K-apo(a) mice expressing eight kringle IV (KIV) repeats (KIV1, with a sin-
gle copy of KIV2, a fusion of KV3–5 and KIV6–10), 8K-Lp(a) mice expressing both
the 8K apo(a) and human apolipoprotein B-100, and 12K-apo(a) mice expressing a
12K apo(a) with three KIV2 repeats. The mice were treated intraperitoneally with
saline, a control ASO, or ASO 144367 directed to KIV2 for 4–6 weeks. ASO 144367
significantly reduced Lp(a) by 24.8% in 8K-Lp(a) mice and reduced apolipoprotein(a)
levels by 19.2% in 8K Lp(a) mice, 30.0% in 8K-apo(a) mice, and 86% in 12K-apo(a)
mice (Fig. 22.1c). ASO 144367 also significantly reduced OxPL-apo(a) by 92.5%
362 S. Tsimikas

in 8K-apo(a) mice (Fig. 22.1d). These studies provided proof that targeting liver
expression of apolipoprotein(a) with ASOs directed to KIV2 repeats may provide an
effective approach to lower elevated Lp(a) levels in humans.

Evolution of Various Generations of Antisense Technology

Natural DNA and RNA are not suitable as effective drugs due to insufficient stabil-
ity mediated by their rapid plasma degradation by nucleases and limited tissue dis-
tribution in animals. Antisense oligonucleotides (ASOs) are single-stranded
modified DNA molecules comprised of 15–20 nucleic acids that display a comple-
mentary sequence to a target messenger ribonucleic acid (mRNA). To overcome the
limitations of naked DNA/RNA as pharmaceutical agents, ASOs contain certain
modifications of the phosphate backbone and the 2′ ribose position. ASOs have a
specific sequence to the target of interest that is not repeated throughout genome,
reducing the potential for off-target binding.
Using medicinal chemistry approaches, first-generation ASOs led to the substitu-
tion of phosphodiester (PO) bonds with phosphorothioate (PS) at one of the two
available PO bonds at each phosphate group in the backbone to improve stability of
the DNA:RNA complex and improve distribution to tissues. The PS bonds provide
stability and protection against nucleases. Second-generation ASOs are called
MOE-gapmers, in that the middle ten nucleic acids are unmodified DNA, which is
required for RNAse H1-mediated cleavage, whereas the five nucleic acids on each
wing are modified at the 2′ position by MOE. The 2′-MOE moiety increases stabil-
ity in biological systems, increases potency due to improved binding affinity to its
target mRNA, and improves the safety profile by decreasing proinflammatory
effects and class toxicities. The nucleic acid bases are in their native chemical con-
figuration and generally not modified in ASOs. Examples of 2′-MOEs are inotersen
(Benson et al. 2018) that is approved clinically for hereditary amyloid polyneuropa-
thy and volanesorsen (Witztum et al. 2019) that is approved clinically for familial
chylomicronemia syndrome in the European Union.
A further advance was made in generation 2+ molecules by improving the
screening process for proinflammatory and other undesirable side effects, as well as
by removing some of the PS groups and replacing them with their native PO groups
in the backbone. This class of drugs are represented by IONIS-APO(a)LRx, ole-
zarsen (Tardif et al. 2022), targeting ApoCIII to treat hypertriglyceridemia and
AGT-LRx (Morgan et al. 2021) targeting angiotensinogen for the proposed treatment
of resistant hypertension and heart failure.
Additional improvements in antisense oligonucleotides include generation 2.5
molecules, where the 2′-MOE has been replaced by a constrained ethyl moiety, as
exemplified by ION409/AZD8233 targeting PCSK9 (Gennemark et al. 2021). The
changes in chemistry, from first generation to second generation and to generation
2.5, each improved potency by approximately tenfold and cumulatively by approxi-
mately 1000-fold, along with additional improvements in safety and tolerability
(Crooke et al. 2018).
22 Antisense Oligonucleotide Therapy to Treat Elevated Lipoprotein(a) 363

Finally, each of these modifications can be coupled with a triantennary

N-acetylgalactosamine (GalNAc3) complex. GalNAc3 is a modified galactose moi-
ety that is generated on proteins and aging cells and is a ligand for the asialoglyco-
protein surface receptor (ASGPR) on hepatocytes. The clearance of GalNAc3 is an
evolutionarily conserved mechanism for their removal from plasma by hepatocytes.
This is a very high-capacity system in that each hepatocyte has up to one million
asialoglycoprotein surface receptors (ASGPR) that allow rapid and specific uptake.
This additional modification allows a further tenfold increase in potency.

 evelopment of a Human Candidate to Lower Lp(a)

D
Plasma Levels

There are two unique challenges in developing RNA-targeted therapeutics specifi-

cally for LPA mRNA. The first is that the LPA gene is very large (~10–15 kb depend-
ing on the number of KIV2 repeats) and has 1–>40 KIV2 repeats at the DNA/RNA
level. The second is that the homology to plasminogen is 75–94% in the coding
region. Both of these properties limit available sites for targeting ASOs without
adversely affecting the coagulation system.
The screening process for identifying a human candidate is shown in Fig. 22.2.
An in silico analysis is first performed for targeting sites, and ASOs are then
designed with optimal predicted selectivity and tolerability. Next, multiple rounds
of in vitro activity assessments are performed. For LPA screening, this involved
both primary human hepatocytes and mouse hepatocytes obtained from transgenic
mice expressing human LPA. Initial activity assessments involve evaluation of

Fig. 22.2 Screening process to identify human antisense oligonucleotides to lower plasma Lp(a)
364 S. Tsimikas

ASOs that span broad regions of the gene of interest. Top leads are then evaluated
in dose-response comparisons with leads demonstrating the greatest potency
selected for in vivo evaluation. Additionally, lead targeting sites are interrogated
with “microwalks” to identify the most potent ASOs with an optimal safety and
tolerability profile. In the case of identifying pelacarsen, this process required the
synthesis of over 2800 unique ASOs prior to the identification of the clinical candi-
date. The most promising candidates, in this case 38 ASOs, were further interro-
gated for efficacy in Lp(a) or apo(a) transgenic mice and in rodent toxicology and
pharmacokinetic studies. This process narrowed the choice to six potential candi-
dates that were then evaluated in cynomolgus monkey tolerability studies, and the
best candidate was identified to enter IND (Investigational New Drug) enabling
toxicology and pharmacokinetic studies.
ISIS-APO(a)Rx (later named IONIS-APO(a)Rx) is a second-generation
2′-O-(2-­methoxyethyl) (2′-MOE)-modified ASO drug with the sequence
5′-TGCTCCGTTGGTGCTTGTTC-3′ (Fig. 22.3) (Graham et al. 2016). ISIS-­
APO(a)Rx/IONIS-APO(a)Rx contains five 2′-MOE-modified ribonucleosides at the 5′
and 3′ ends and ten 2-O-deoxyribonucleosides within the central portion of the mol-
ecule. A modified version of IONIS-APO(a)Rx containing GalNAc3 was generated,
initially named IONIS-APO(a)-LRx, and then AKCEA-APO(a)LRx and ultimately
received the generic name pelacarsen. Pelacarsen contains the same 20-nucleotide
sequence as IONIS-APO(a)Rx and five 2′-MOE-modified ribonucleosides at the 5′

Position on NM_005577.2 apo(a)

ISIS-APO(a)Rx Binding Site mRNA transcript Binding Site on First Exon Binding Site on Second Exon

kringle IV2 repeat 2

Exon 4-5 505-524 bp CTTGTTC TGCTCAGTCGGTG
kringle IV2 repeat 3
Exon 6-7 847-866 bp CTTGTTC TGCTCAGTCGGTG
kringle IV2 repeat 4
Exon 8-9 1189-1208 bp CTTGTTC TGCTCAGTCGGTG
kringle IV2 repeat 5
Exon 10-11 1531-1550 bp CTTGTTC TGCTCAGTCGGTG
kringle IV2 repeat 6
Exon 12-13 1873-1892 bp CTTGTTC TGCTCAGTCGGTG
kringle IV2 repeat 7
Exon 14-15 22I5-2234 bp CTTGTTC TGCTCAGTCGGTG
kringle IV2 repeat 8
Exon 16-17 2557-2576 bp CTTGTTC TGCTCAGTCGGTG
kringle IV2 repeat 9
Exon l8-19 2899-2918 bp CTTGTTC TGCTCAGTTGGTG
kringle IV2 repeat 11
Exon 22-23 3583-3602 bp CTTCTTC TGCTCCGTTGGTG
kringle IV2 repeat 12
Exon 24-25 3901-3920 bp CTTGTIC TGCTCCGTTGGTG
kringle IV2 repeat 14
Exon 28-29 4584-4604 bp CTTGTIC TTCTCAGGTGGTG
kringle IV2 repeat 15
Exon 30-31 4927-4946 bp CTGCTTC TGCTCAGTGGTG

Fig. 22.3 ISIS-APO(a)Rx complementary binding sites within the human LPA transcript (GenBank
accession NM_005577.2) at position 3901–3920 bp. ISIS-APO(a)Rx was designed to perfectly
match only the exon 24–25 splice sites (indicated with bold type) but may also bind at 11 other
apolipoprotein(a) exon splice sites containing 1–4 mismatched nucleotides (indicated by under-
lined letters) (Graham et al. 2016)
22 Antisense Oligonucleotide Therapy to Treat Elevated Lipoprotein(a) 365

and 3′ ends while retaining ten 2-odeoxyribonucleosides within the central portion
of the molecule. However, 6 of the 19 PS linkages were replaced with PO linkages
at positions 2, 3, 4, 5, 16, and 17. The GalNAc3 complex was covalently attached
with a proprietary linker to the 5′ end (Viney et al. 2016) (Fig. 22.4).

Fig. 22.4 Structure and sequence of ISIS-APO(a)Rx/IONIS-APO(a)Rx (a) and IONIS-APO(a)-

LRx/AKCEA-APO(a)LRX/TQJ230/pelacarsen (b). The figure depicts space-filling models with the
nucleic acid sequence in capital letters below. IONIS-APO(a)Rx contains 20 nucleic acids with ten
2-O-deoxyribonucleosides within the central portion of the molecule and incorporates five
2′-methoxyethyl (MOE)-modified ribonucleosides at the 5′ and 3′ ends. IONIS-APO(a)-LRx
(pelacarsen) contains the same nucleic acid sequence but has only 6 of the 19 sulfur groups in the
backbone and additionally contains the triantennary N-acetylgalactosamine (GalNAc3) complex.
The phosphodiester (PO) linkages are indicated by an oxygen (O) for native linkage and by a sulfur
(S) for phosphorothioate (PS) substitution. The GalNAc3 complex is attached to the 5′ end via a
proprietary THA linker for rapid and specific uptake within hepatocytes via the asialoglycoprotein
receptor (Viney et al. 2016)
366 S. Tsimikas

 echanisms of Antisense Oligonucleotide

M
Pharmaceutical Activity

The mechanism of efficacy of ASOs utilizes the ubiquitous intracellular ribonuclease

RNase H1 that recognizes the RNA:DNA duplex formed when ASOs bind to the com-
plementary mRNA sequence. RNAse H1 binds to this duplex, irrespective of the

Fig. 22.5 Mechanism by which LPA-directed antisense oligonucleotides suppress apolipoprotein(a)

protein synthesis. Following formation of the LPA mRNA:pelacarsen duplex, the ubiquitous intra-
cellular ribonuclease RNase H1 recognizes the duplex and cleaves the target LPA mRNA sense
strand, thereby preventing translation of apolipoprotein(a) protein. The hepatocytes continue to
generate apoB-100 particles, but the relative absence of apolipoprotein(a) does not allow the
assembly of Lp(a) particles (Viney et al. 2016)
22 Antisense Oligonucleotide Therapy to Treat Elevated Lipoprotein(a) 367

specific sequence of the ASO or the target, and cleaves the target mRNA, thereby
disrupting protein translation (Fig. 22.5). The ASO is relatively resistant to RNAse
H1-mediated cleavage and becomes available to bind to additional mRNA LPA mol-
ecules. This is part of reason the intra-hepatocyte half-life is relatively long (2–4 weeks).

 ompleted Clinical Trials with ISIS-APO(a)Rx/

C
IONIS-APO(a)Rx

A total of four clinical phase 1 or phase 2 trials have been performed with ISIS-­
APO(a)Rx/IONIS-APO(a)Rx/and pelacarsen (Table 22.1). For historical purposes
and to be consistent with the literature, the names of the drugs will be given
­according to those used when the trials were published.

Table 22.1 Competed clinical trials with antisense oligonucleotides

Mean Absolute
Number baseline Mean Lp(a)
Year of Dose/dose Lp(a), Lp(a) reduction,
Study published Drug subjects regimen (nmol/L) reduction (nmol/L)
Tsimikas 2015 ISIS-­ 16 Single doses 8–66 No N/A
et al. APO(a)Rx of 50, 100, significant
(2015) 200, and change
400 mg
ISIS-­ 31 100, 200, and 82–152 40–78% 34–95
APO(a)Rx 300 mg/week,
six doses over
4 weeks
Viney 2016 IONIS-­ Cohort 100–300 mg/ 252–254 67% 183
et al. APO(a)Rx A—50 week for
(2016) 13 weeks
IONIS-­ Cohort 100–300 mg/ 445–488 72% 305
APO(a)Rx B—11 week for
13 weeks
Viney 2016 Pelacarsen 28 Single doses 111–219 26–85% 59–107
et al. of 10, 20, 40,
(2016) 80, and
120 mg
Pelacarsen 30 Multiple doses 143–165 66–92% 86–141
10, 20, and
40 mg/week
for 4 weeks
Tsimikas 2020 Pelacarsen 286 20, 40, or 205–247 35–80% 96–188
et al. 60 mg every 4
(2020) weeks, 20 mg
every 2 weeks,
or 20 mg every
week for
6–12 months
Lp(a) molar concentration in nmol/L cannot be scientifically converted to mass units in mg/
dL. However, a rough estimate is to divide nmol/L by 2.5 to approximate values in mg/dL, with the
realization that significant error may occur depending on isoform size (Tsimikas et al. 2018)
368 S. Tsimikas

The first clinical demonstration that Lp(a) levels can be potently reduced in
patients was documented in a randomized, double-blind, placebo-controlled, phase
1 study of healthy adults with Lp(a) concentration of ≥25 nmol/L assigned to
receive ISIS-APO(a)Rx or placebo. Multiple doses of ISIS-APO(a)Rx (100–300 mg)
resulted in dose-dependent, mean percentage decreases in plasma Lp(a) concentra-
tion of 39.6% from baseline in the 100 mg group (p = 0.005), 59.0% in the 200 mg
group (p = 0.001), and 77.8% in the 300 mg group (p = 0.001) (Fig. 22.6). Similar
reductions were observed in OxPL-apoB and OxPL-apo(a) (Fig. 22.6). Mild injec-
tion site reactions were the most common adverse events. No serious or severe
adverse events were recorded. Two of the 37 participants treated with ISIS-APO(a)Rx
discontinued the study drug for tolerability reasons, which was an improved experi-
ence compared to previous earlier drugs in this class of chemicals.
ISIS-APO(a)Rx was renamed IONIS-APO(a)Rx in concert with the change in the
company’s name. The phase 2 study that followed was performed in participants
with elevated Lp(a) concentrations (125–437 nmol/L in cohort A with 51 partici-
pants, ≥438 nmol/L in cohort B with 13 participants) who were randomly assigned
to escalating-dose subcutaneous IONIS-APO(a)Rx (100 mg, 200 mg, and then
300 mg, once a week for 4 weeks each) or saline placebo, once a week, for 12 weeks.
At day 85/99, participants assigned to IONIS-APO(a)Rx had mean Lp(a) reductions
of 66.8% in cohort A and 71.6% in cohort B (both p < 0.0001 vs pooled placebo)

Fig. 22.6 Plasma trough concentrations of ISIS-APO(a)Rx and mean percent change in Lp(a),
OxPL-apoB, and OxPL-apo(a) with time by treatment group in the multidose cohorts measured 7
days after the last dose in the 300 mg dose cohort in relation to change in concentration of plasma
Lp(a), OxPL-apoB, and OxPL-apo(a). The shaded area represents the dosing window, and arrows
indicate dosing at days 1, 3, 5, 8, 15, and 22 (Tsimikas et al. 2015)
22 Antisense Oligonucleotide Therapy to Treat Elevated Lipoprotein(a) 369

(Fig. 22.7). Mean concentrations were also reduced in OxPL-apoB (35.2% for
cohort A and 42.5% for cohort B), OxPL-apo(a) (26.6% for cohort A and 36.7% for
cohort B), LDL-C (13.0% for cohort A and 23.9% for cohort B), and apoB-100
(11.3% for cohort A and 18.5% for cohort B) (Fig. 22.7).
Baseline hsCRP concentrations were 2.39 mg/L for the placebo group, 1.78 mg/L
for cohort A, and 3.46 mg/L for cohort B. At day 85/99, mean absolute change in
hsCRP was −0.64 mg/L (SD 4.38, p = 0.44 vs baseline) for the pooled placebo
group, −0.23 mg/L for cohort A (SD 1.54, p = 0.92 vs baseline and p = 0.63 vs
change for placebo), and − 1.5 mg/L for cohort B (SD 3.27, p = 0.37 vs baseline
and p = 0.20 vs change in placebo). Overall, IONIS-APO(a)Rx was generally well

Lp(a) OxPL-apoB
Placebo
Mean change in fasting ApoB from baseline (%) Mean change in fasting OxPL-apo(a) from baseline (%) Mean change in fasting Lp(a) from baseline (%)

Mean change in fasting Lp(a) from baseline (%)

25 25
Cohort A
Cohort B

0 0

–25 –25

–50 –50

–75
–75

OxPL-apo(a) LDL-C
Mean change in fasting Lp(a) from baseline (%)

25 25

0 0

–25 –25

–50 –50

–75 –75

1 15 29 43 57 78 85/99 120 162 190

ApoB-100 Study day
25

–25

–50

–75

1 15 29 43 57 78 85/99 120 162 190

Study day

Fig. 22.7 Mean percent changes in plasma concentrations of Lp(a), OxPL-apoB, OxPL-apo(a),
LDL-C, and apoB in the IONIS-APO(a)Rx trial. Error bars are SEM (standard error of the mean).
The shaded area represents the dosing window and arrows indicate dosing every week. p values
show differences between treatment and pooled placebo at day 85/99. *p ≤ 0.0001, †p = 0.0002,
‡
p = 0.0005, §p = 0.0007, ¶p = 0.0003 (Viney et al. 2016)
370 S. Tsimikas

tolerated with 10% of injections in cohort A and 19% of injections in cohort B

associated with injection site reactions (overall 12%). Approximately 10% of
patients had individual components that could be consistent with influenza-like
symptoms.

 ompleted Clinical Trials with IONIS-APO(a)LRx/

C
AKCEA-­APO(a)LRx,/TQJ230/Pelacarsen

The rapid development of hepatocyte-targeting technology, with the promise of

lower dosing and improved safety and tolerability, led to the decision to switch
clinical development to IONIS-AP0(a)LRx/AKCEA-APO(a)LRx,/TQJ230/pelac-
arsen, all of which are the same molecule (the generic name pelacarsen will be used
subsequently). A new phase 1/phase 2a study was initiated in 58 healthy partici-
pants with doses of 10, 20, 40, 80, and 120 mg in the single-ascending-dose phase
and 30 participants of 10, 20, and 40 mg weekly in the multiple-ascending-dose
phase. Significant dose-dependent reductions in mean Lp(a) concentrations were
noted in all single-dose pelacarsen groups at day 30 with mean reductions at day 30
of 26.2% in the 10 mg group, 33.2% in the 20 mg group, 43.5% in the 40 mg group,
78.6% in the 80 mg group, and 85.3% in the 120 mg group versus a 2.8% mean
increase in the placebo group (Fig. 22.8a). In the multidose groups, pelacarsen
resulted in mean reductions in Lp(a) of 66% in the 10 mg group, 80% in the 20 mg
group, and 92% in the 40 mg group (p = 0.0007 for all vs placebo) at day 36
(Fig. 22.8b). Pelacarsen was associated with no injection site reactions. Compared
to IONIS-APO(a)Rx, pelacarsen was documented to be approximately 30-fold more
potent (Fig. 22.8c).
This trial provided the proof of concept that GalNAc-modified ASOs could be
targeted to the hepatocyte in a safe and tolerable manner and that potency could be
substantially increased. A phase 2 dose-ranging trial with dosing regimens ranging
from weekly to monthly was then performed in 286 patients with established car-
diovascular disease and screening Lp(a) levels of ≥60 mg/dL (≥150 nmol/L)
(Tsimikas et al. 2020). The median baseline Lp(a) levels in the six groups ranged
from 204.5 to 246.6 nmol/L. Administration of pelacarsen resulted in dose-­
dependent decreases in Lp(a) levels, with mean percent decreases of 35% at a dose
of 20 mg every 4 weeks, 56% at 40 mg every 4 weeks, 58% at 20 mg every 2 weeks,
72% at 60 mg every 4 weeks, and 80% at 20 mg every week, as compared with 6%
with placebo (p values for the comparison with placebo ranged from 0.003 to
<0.001, Fig. 22.9a). There were no significant differences between any pelacarsen
dose and placebo with respect to platelet counts, liver and renal measures, or
influenza-­like symptoms. The most common adverse events were injection site
reactions.
The temporal changes in Lp(a) levels reveal significant declines as early as 4
weeks post first injection, reaching a steady state at approximately 16 weeks
22 Antisense Oligonucleotide Therapy to Treat Elevated Lipoprotein(a) 371

Fig. 22.8 Mean

percentage change in Lp(a)
concentration in the
IONIS-APO(a)-LRx trial
and comparison of
dose-response curves of
IONIS-APO(a)Rx and
IONIS-APO(a)-LRx. (a)
Single-ascending-dose and
(b) multiple-ascending-­
dose phases. The shaded
area represents the dosing
window and arrows
indicate dosing days. p
values are only shown at
day 30 for the single-­
ascending-­dose phase and
day 36 for multiple-­
ascending-­dose phase as
determined by the exact
Wilcoxon rank-sum test
comparing IONIS-APO(a)-
LRx versus placebo.
*p = 0.0333, †p = 0.0167,
‡
p = 0.0012, §p = 0.0007.
(c) Comparison of
dose-response curves of
IONIS-APO(a)Rx and
IONIS-APO(a)-LRx after 4
weeks of subcutaneous
administration. Error bars
are SEM. The upper left
side of the curve was
extrapolated based on the
curve fit of the data due to
the fact that lower doses
were not tested (Viney
et al. 2016)
372 S. Tsimikas

Fig. 22.9 Effect of

pelacarsen (AKCEA-
APO(a)-LRx) on Lp(a)
levels. Panel (a) shows the
least squares mean percent
changes from baseline to
the primary analysis time
point (PAT) (i.e., 6 months
of exposure [at week 25 in
the groups who received
monthly doses and at week
27 in the groups who
received more frequent
doses]). I bars denote 95%
confidence intervals. Panel
(b) shows the least squares
mean percent changes from
baseline in Lp(a) over
time. Error bars denote
95% confidence intervals.
Panel (c) shows the percent
of patients with Lp(a)
levels of <50 mg/dL
(<125 nmol/L) in each
group at the PAT (Santos
et al. 2015)
22 Antisense Oligonucleotide Therapy to Treat Elevated Lipoprotein(a) 373

(Fig. 22.9b). The percent of patients with an Lp(a) level of ≤50 mg/dL (125 nmol/L)
or lower at 6 months of exposure was 23% in the group who received 20 mg of pelac-
arsen every 4 weeks, 63% in the group who received 40 mg every 4 weeks, 65% in
the group who received 20 mg every 2 weeks, 81% in the group who received 60 mg
every 4 weeks, and 98% in the group who received 20 mg every week (Fig. 22.9c).
The mean percent reductions in OxPL-apoB were 37% at a dose of 20 mg every
4 weeks, 57% at 40 mg every 4 weeks, 64% at 20 mg every 2 weeks, 79% at 60 mg
every 4 weeks, and 88% at 20 mg every week, as compared with a 14% increase in
the placebo group. The corresponding mean percent reductions in OxPL-apo(a)
were 28%, 49%, 45%, 63%, and 70%, respectively, compared with a 20% decrease
in the placebo group. Corresponding absolute reductions in LDL-C were −5.6,
−13.5, −13.2, −8.2, and −16.4 mg/dL, respectively, compared to −1.2 mg/dL in
placebo. Corresponding absolute reductions in apoB were −2.2, −8.3, −6.3, −3.9,
and −10.9 mg/dL, respectively, compared to 0.6 mg/dL increase in placebo.
Corresponding absolute changes (nonsignificant) in hsCRP were −0.9, −0.7, −0.3,
−0.5, and −0.1 mg/L, respectively, compared to −0.8 mg/L in placebo.
Individual responses revealed that all patients in the pelacarsen 20 mg weekly
dose had declines in Lp(a) (−42.6 to −99.5%), OxPL-apoB (−37.0 to 99.7%), and
OxPL-apo(a) (−12.6 to −99.5%), compared to +16.1 to −40.6%, −28.7 to +150.0%,
and −66.6 to +18.1 for the three variables in the placebo groups, respectively.
There were no significant differences between any pelacarsen dose and placebo
with respect to platelet counts, liver and renal measures, or influenza-like symp-
toms. The most common adverse events were injection site reactions.

 p(a) Horizon Cardiovascular Outcomes Trial and Testing

L
of the “Lp(a) Hypothesis”

Based on the totality of epidemiologic, genetic, and clinical evidence, as well as the
proof of concept in potently lowering Lp(a) these four trials provided, further devel-
opment of pelacarsen was undertaken. The Lp(a) HORIZON trial (NCT04023552,
Assessing the Impact of Lipoprotein(a) Lowering With Pelacarsen on Major
Cardiovascular Events in Patients With CVD [Lp(a) HORIZON] is a pivotal phase
3 study designed to support an indication for the reduction of cardiovascular risk in
patients with established CVD and elevated Lp(a). It is a global, international mul-
ticenter, randomized, double-blind, placebo-controlled study in >8000 patients with
elevated Lp(a) levels (≥70 mg/dL, ≥175 nmol/L) and history of CVD (myocardial
infarction, ischemic stroke, peripheral artery disease). Key inclusion criteria include
1-Lp(a) ≥70 mg/dL, 2-myocardial infarction ≥3 months from screening and ran-
domization to ≤10 years prior to the screening visit, 3-ischemic stroke ≥3 months
from screening and randomization to ≤10 years prior to the screening visit, and
clinically significant symptomatic peripheral artery disease.
Subjects are required to have risk factors, particularly LDL-C, optimized accord-
ing to local guidelines. They are then randomized to pelacarsen 80 mg
374 S. Tsimikas

Fig. 22.10 Design of the Lp(a) HORIZON trial

subcutaneously monthly versus matching placebo. The primary outcome measure is

the time to first occurrence of expanded major adverse cardiovascular events
(MACE), consisting of cardiovascular death, nonfatal MI, nonfatal stroke, and
urgent coronary revascularization requiring hospitalization in the overall study pop-
ulation with established CVD and Lp(a) ≥70 mg/dL. A co-primary outcome mea-
sure is the time to first occurrence of expanded MACE, in the overall study
population with established CVD in the overall study population and Lp(a) ≥90 mg/
dL (Fig. 22.10). Secondary outcome measures include (1) the time to the first occur-
rence of MACE (CV death, nonfatal MI, and nonfatal stroke), (2) the time to first
occurrence of the composite endpoint of coronary heart disease (coronary heart
disease death, nonfatal MI, urgent coronary revascularization requiring hospitaliza-
tion), and (3) the number of participants with confirmed all-cause death.
In conclusion, targeting LPA mRNA with antisense oligonucleotides is a funda-
mentally new approach to potently reducing circulating Lp(a) levels. The Lp(a)
HORIZON trial is testing the “Lp(a) hypothesis,” namely, that lowering plasma
Lp(a) levels will lead to a reduced rate of recurrent cardiovascular events. It is
expected to have primary results in 2025.

Acknowledgements We thank Rosanne Crooke, Mark Graham, Richard Geary, Nick Viney, and
other members of the Ionis Pharmaceuticals and Joseph L. Witztum for their contributions in dis-
covering and developing pelacarsen and Trace Reigle of the Ionis Pharmaceuticals for the prepara-
tion of some of the artwork.

Disclosures ST is a coinventor and receives royalties from patents on oxidation-­specific antibod-

ies and of biomarkers related to oxidized lipoproteins held by UCSD. ST is a co-founder and has
equity interest in Oxitope, Inc., and its affiliates (“Oxitope”) as well as in Kleanthi Diagnostics,
LLC (“Kleanthi”). The terms of this arrangement have been reviewed and approved by the
University of California, San Diego, in accordance with its conflict-of-interest policies. ST has a
dual appointment at the UCSD and Ionis Pharmaceuticals.

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nary heart disease. Clin Genet. 1974;6(3):230–5.
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2018;27(4):714–39.
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Gennemark P, Walter K, Clemmensen N, Rekić D, Nilsson CAM, Knöchel J, et al. An oral anti-
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Graham MJ, Viney N, Crooke RM, Tsimikas S. Antisense inhibition of apolipoprotein(a) to lower
plasma lipoprotein(a) levels in humans. J Lipid Res. 2016;57(3):340–51.
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human apolipoprotein(a) is homologous to plasminogen. Nature. 1987;330(6144):132–7.
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cleotide directed to human apolipoprotein B-100 reduces lipoprotein(a) levels and oxidized
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Transl Sci. 2021;6(6):485–96.
Santos RD, Raal FJ, Catapano AL, Witztum JL, Steinhagen-Thiessen E, Tsimikas S. Mipomersen,
an antisense oligonucleotide to apolipoprotein B-100, reduces lipoprotein(a) in various popula-
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2015;35(3):689–99.
Tardif JC, Karwatowska-Prokopczuk E, Amour ES, Ballantyne CM, Shapiro MD, Moriarty PM,
et al. Apolipoprotein C-III reduction in subjects with moderate hypertriglyceridaemia and at
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Tomlinson JE, McLean JW, Lawn RM. Rhesus monkey apolipoprotein(a). Sequence, evolution,
and sites of synthesis. J Biol Chem. 1989;264(10):5957–65.
Tsimikas S, Viney NJ, Hughes SG, Singleton W, Graham MJ, Baker BF, et al. Antisense therapy
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Chapter 23
Lipoprotein Apheresis for Reduction
of Lipoprotein(a)

Ulrich Julius and Sergey Tselmin

Abbreviations

A Atorvastatin
AIC Arteria iliaca communis
Aorta-mes bypass Mesenteric artery bypass
A. mes sup Arteria mesenterica superior
ApoE Apolipoprotein E-polymorphism
BMS Bare metal stent
CAD Coronary artery disease
CX Left circumflex coronary artery
DEB Drug-eluting balloon
DES Drug-eluting stent
LAD Left anterior descending artery
LCA Left coronary artery
LMS Left main stem
nk Not known
MI Myocardial infarction
PLA Posterolateral artery
PTA Percutaneous transluminal angioplasty
PTCA Percutaneous transluminal coronary angioplasty
RCA Right coronary artery
RDI Ramus diagonalis, diagonal branch 1
R Rosuvastatin

U. Julius (*) · S. Tselmin

Department of Internal Medicine III, Lipidology and Center for Extracorporeal Treatment,
University Hospital Carl Gustav Carus at the Technische Universität Dresden,
Dresden, Germany
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature 377

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_23
378 U. Julius and S. Tselmin

S Simvastatin
TEA Thromboendarteriectomy
Tr. coel Truncus coeliacus

Introduction

An elevation of lipoprotein(a) [Lp(a)] is an internationally recognized risk factor for

cardiovascular events (CVEs) such as myocardial infarction, stroke, or peripheral
arterial disease (Nordestgaard et al. 2010; Tsimikas 2017; Reyes-Soffer et al. 2022;
Kronenberg 2022). Statins usually do not have an impact on Lp(a) levels or even
may increase them (Reyes-Soffer et al. 2022; Korneva et al. 2021). Niacin is no
longer available; ezetimibe and bempedoic acid do not exert any effect on Lp(a). On
the other hand, monoclonal antibodies directed against PCSK9 (alirocumab, evo-
locumab) decrease Lp(a) concentrations by up to 30% (Julius et al. 2019). In two
prospectively conducted placebo-controlled intervention studies (Fourier, ODYSSEY
OUTCOMES), it could be shown that the lower Lp(a) levels in the verum groups led
to a reduction in CVEs, independent of the effect on LDL cholesterol (LDL-C)
(Szarek et al. 2020; O'Donoghue et al. 2019). In studies with inclisiran, a similar
decrease of Lp(a) was seen (up to 26%); outcome data are not yet available (Wright
et al. 2021). But PCSK9 inhibitors are not officially accepted for the indication of an
elevation of Lp(a), and they are not effective in this direction in all patients.
Thus, lipoprotein apheresis (LA) is at present the only therapeutic approach
which has been shown to effectively reduce Lp(a) concentrations and CVEs. This
article describes the current knowledge about the impact of LA on lipid concentra-
tions and cardiovascular outcomes. One major advantage of LA in comparison with
drug treatment is that the extracorporeal therapy is rather well tolerated. Besides
heparin and/or citrate, isotonic saline solution, and in some cases calcium, no other
foreign substances are applied in the daily routine of an apheresis center. In the
1960s, a plasma exchange was performed to treat patients with homozygous famil-
ial hypercholesterolemia (Thompson and Parhofer 2019)—albumin or fresh frozen
plasma coming from donors substituted the plasma of the patients. Modern LA
methods usually do not induce a loss of blood, and the plasma components which
are removed do not have to be replaced.

 istory of Lipoprotein Apheresis (LA) Regarding

H
the Indication of High Lipoprotein(a) [Lp(a)]

Between 1975 and 2004, several LA systems were developed to treat patients with
familial hypercholesterolemia (Julius 2017; Thompson 2022). Lp(a) appeared as a
target molecule only after 2000.
The largest number of patients treated with LA worldwide exists in Germany. In
2008, the Joint Federal Committee (Gemeinsamer Bundesausschuss), a paramount
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 379

decision-making body of the German Healthcare System, accepted an elevation of

Lp(a) as an indication for LA (Bundesministerium für Gesundheit 2008). In the
anteceding years, several patients were treated after courts had supported this indi-
cation in high-risk patients.
An Lp(a) level exceeding 60 mg/dL was set as the threshold value at which LA
is indicated. Comparative measurements in large apheresis centers established
120 nmol/L as equivalent (Schettler et al. 2015).
Moreover, the following two additional conditions had to be fulfilled
1. The LDL-C concentration should be optimized (by drugs when tolerated; in
some patients, this requirement is difficult to be met).
2. A progress of atherosclerosis needed to be documented either clinically (several
CVEs) or by imaging techniques (in the clinical practice, the first myocardial
infarction in a young patient with a positive family history for CVEs is accepted
as an indication; some specialists regard a CVE already as progress per se).
In the United States, the Food and Drug Administration’s approval specifically
for Lp(a) lowering also requires an Lp(a) >60 mg/dL (about 150 nmol/L) (Reyes-­
Soffer et al. 2022).
The HEART UK Lipoprotein apheresis guidelines recommend that apheresis
should be considered for those patients with progressive coronary disease and Lp(a)
greater than ~150 nmol/L (>60 mg/dL) whose LDL-C remains 3.3 mmol/L or
higher despite maximal lipid-lowering therapy (Cegla et al. 2019). The attitude
toward LDL-C differs from that in Germany.
In Russia, LA is recommended for patients with homozygous or heterozygous
familial hypercholesterolemia in combination with Lp(a) >60 mg/dL and early
onset of atherosclerotic cardiovascular disease (Pokrovsky et al. 2020).
As far as the authors know, other countries where this indication is recognized
are Italy (Stefanutti et al. 2013, 2020) and Poland (Mickiewicz et al. 2021).
It has to be emphasized that an LA treatment is only justified in patients within a
secondary prevention strategy, meaning that CVEs had occurred or severe progres-
sive atherosclerosis is documented. There is no indication to treat patients extracor-
poreally just to reduce elevated Lp(a) concentrations in a primary prevention
concept.
In the last years, the number of patients who were treated with LA has continu-
ously increased in Germany. The approval for the reimbursement of costs by the
health insurance companies is based on an individual application to be renewed
annually to regionally appointed committees of the Associations of Statutory
Health Insurance Physicians. This major hurdle represents an important step of
quality control for strict selection of patients. The rate of refusals to accept the
application is rather high, especially among patients with the diagnosis “isolated
elevation of Lp(a).” These numbers are also published for the federal states of
Germany (in Table 23.1, only basic data are given). The number of physicians
who got the permission to perform an LA therapy amounted to 1286 in the end
of 2020.
380 U. Julius and S. Tselmin

Table 23.1 Numbers of patients treated with LA at the end of the given years in Germany

Year 2013 2014 2015 2016 2017 2018 2019 2020

hoFH 187 120 122 103 89 97 93 95
Severe HCH 1221 1472 1597 1700 1538 1477 1575 1663
Isolated 753 954 1303 1468 1895 2163 2396 2448
Lp(a)
(35 %) (37 %) (43 %) (45 %) (54 %) (57 %) (59 %) (58 %)
elevation
Total 2161 2546 3022 3271 3522 3737 4064 4206

Data are officially published by the National Association of Statutory Health Insurance Physicians
(Kassenärztliche Bundesvereinigung) (Kassenärztliche Bundesvereinigung 2014, 2015, 2016,
2017, 2018, 2019, 2020, 2021)
hoFH homozygous familial hypercholesterolemia, HCH hypercholesterolemia

The percentage of patients who were included into LA treatment with the indica-
tion of an isolated Lp(a) elevation was increasing from 35% in 2013 to 59% in 2019.
At the Dresden Center for Extracorporeal Therapy, in the last years, the vast major-
ity of new patients had elevations of Lp(a). Unfortunately, our 160 patients are not
included into the data shown in Table 23.1 because working in a university hospital,
we do not belong to the National Association of Statutory Health Insurance
Physicians.

 fficiency of LA with Respect to Lowering

E
of Lp(a) Concentrations

An LA session acutely reduces lipid concentrations which rise again in the follow-
ing days. Thus a sawtooth picture is seen. In our experience, after 7 days, the Lp(a)
level is again at its maximum.
In order to describe the efficiency of LA on Lp(a) levels, the following
criteria are used
1. Acute reductions—comparing the Lp(a) level after LA sessions with those
before the sessions.
2. Interval mean values—taking into account that Lp(a) increases following an LA
session. This parameter defines the burden imposed by Lp(a) on arteries. We
have published a formula to calculate interval mean values (Tselmin et al. 2017).
3. Comparison of the presession and post-session and interval mean values with the
initial Lp(a) concentrations measured before the first LA session (reflecting the
steady-state condition till the start of the extracorporeal therapy).
The following data have been measured at our center in 2019; the first LA ses-
sion usually took place years ago (Julius et al. 2020). The vast majority of our
patients are treated with LA weekly.
In our patients, the acute reductions amounted to 128 nmol/L (−77%) (see
Table 23.2). When comparing with the initial Lp(a) level before the first LA session,
the interval mean values were decreased by 134 nmol/L (−55%). Waldmann and
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 381

Table 23.2 Lp(a) before the first LA session, before and after an LA session, and interval mean
values after years of extracorporeal therapy—percent reductions comparing with the initial levels
are marked in red (n = 97; data have been recalculated on the basis of a publication in 2020) (Julius
et al. 2020)

Timepoint of Before 1st LA Before LA After LA Interval mean

blood session after session after values after
sampling years years years

Median 242 / baseline 167 / - 31 % 39 / - 84 % 108 / - 55 %

(nmol/l) /
Percent
reduction
comparing
with Lp(a)
before 1st LA

IQR (nmol/l) 192 / 308 127 / 212 29 / 53 84 / 152

Min / Max 70 / 820 50 / 391 10 / 122 27 / 288

(nmol/l)

IQR interquartile range

Fig. 23.1 Interval mean values of Lp(a) (n = 97; x-axis: upper bounds of intervals, nmol/L)

Parhofer reported a decrease of this parameter between 25 and 40% (Waldmann and
Parhofer 2016). Evidently, the results obtained in Dresden are superior to these data.
But it has to be taken into account that an additional Lp(a)-lowering effect may be
due to the application of PCSK9 antibodies in some patients at our center.
The following histogram (Fig. 23.1) shows the interval mean values of Lp(a).
Only in 20 (21%) patients the interval mean value was below 75 nmol/L.
382 U. Julius and S. Tselmin

Almost half of the patients showed interval mean values above 120 nmol/L.
The corresponding LDL-C interval mean value [calculated according to Kroon
et al. (2000)] was 1.75 mmol/L (IQR 1.32/2.22 mmol/L), which is −15% lower than
the initial level [2.07 mmol/L (IQR 1.77/3.04 mmol/L)].
There are three lipoprotein classes carrying apolipoprotein B (ApoB): VLDL,
LDL, and Lp(a). All these lipoproteins are removed by LA.
We measured ApoB in our patients in January 2021 and obtained the results
shown in Fig. 23.2.
The median of ApoB is effectively acutely reduced by −70%. The mean ApoB
concentration (between pre- and post-values) is 55 mg/dL.
In a recently published American statement on Lp(a), it was recommended to
calculate the percentage of ApoB transported with the Lp(a) particles (Reyes-Soffer
et al. 2022). Interestingly, in the HEART UK consensus statement, an expression of
Lp(a) in molar units in order to appreciate its concentration relative to ApoB
expressed in molar units is discussed (Cegla et al. 2019).
Our data before and after an LA session are depicted in Fig. 23.3.
The median is a little bit decreased. In Fig. 23.3b, it is shown that the percentage
of ApoB contained in Lp(a) exceeding 20% is increased after the LA session com-
paring with the initial data.
The rebound of LDL and Lp(a) particle concentrations following LA allows the
determination of fractional catabolic rate (FCR) and hence production rate (PR)
during nonsteady-state conditions (Ma et al. 2019). The FCR of Lp(a) was signifi-
cantly lower than that of LDL-ApoB, implying that different metabolic pathways
are involved in the catabolism of these lipoproteins, with no significant differences
in the corresponding PR.
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 383

a median 80 mg/dl (IQR 68 / 98 mg/dl)

40

30
Frequency

20

10

0
0 50 100 150 200 250

b median 24 mg/dl (IQR 10 / 30 mg/dl)

40

30
Frequency

20

10

0
20 40 60 80

Fig. 23.2 ApoB concentrations (mg/dL) before (a) and after (b) one LA session. (a) Median
80 mg/dL (IQR 68/98 mg/dL). (b) Median 24 mg/dL (IQR 10/30 mg/dL)
384 U. Julius and S. Tselmin

a median 11.3 % (IQR 6.9 / 14.6 %)

25

20

15
Frequency

10

0
.00 10.00 20.00 30.00 40.00 50.00

b median 9.8 % (IQR 5.0 / 18.0 %)

30

20
Frequency

10

0
.00 10.00 20.00 30.00 40.00 50.00

Fig. 23.3 Percentage of ApoB transported with Lp(a) particles before (a) and after (b) an LA ses-
sion. (a) Median 11.3% (IQR 6.9/14.6%). (b) Median 9.8% (IQR 5.0/18.0%)
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 385

Oxidized Phospholipids

Oxidized phospholipids (OxPL) are mainly transported by Lp(a) particles and play
an important role in atherogenesis (Yeang et al. 2016; Tsimikas 2019).
A small study in 18 patients with familial hypercholesterolemia and with low
(≈10 mg/dL; range 10–11 mg/dL), intermediate (≈50 mg/dL; range 30–61 mg/dL),
or high (>100 mg/dL; range 78–128 mg/dL) Lp(a) levels was performed to check
the effect of LA on OxPL-ApoB and OxPL-apolipoprotein(a) [Apo(a)] concentra-
tions (Arai et al. 2012). Plasma OxPL-ApoB was not reduced in the low Lp(a)
group, but the levels were very low and near the level of detection of this assay.
There was a strong trend for acute reduction (48%) in OxPL-ApoB in the intermedi-
ate Lp(a) group, and there was a significant decline (62%) in the high Lp(a) group.
OxPL-Apo(a) was significantly reduced in all groups.

Adverse Effects and Contraindications of LA

Blood is handled outside the body. Different LA methods are characterized by a

differing extracorporeal blood volume (Julius 2016). Moreover, an anticoagulation
with heparin and/or citrate is needed to prevent blood clotting in the system.
Major adverse effects are hypotension, bleeding, and hypocalcemia (citrate
effect). These occur rarely and can easily be treated (Dittrich-Riediger et al. 2015;
Heigl et al. 2015). Some patients may show an iron deficiency (blood is taken to
check lipid concentrations before and after LA sessions, other parameters are mea-
sured for security reasons) which often is relieved by intravenous iron application.
A venous access on both arms is needed. Usually, experienced medical staff handles
puncturing very well. Some patients need fistulas or shunts (less than 10% at our
center, up to 30% in centers where nephrologists are in charge). These procedures
may cause additional problems (e.g., thrombotic occlusions).
An extreme fear of patients with regard to this may be a contraindication
against LA.
The list of contraindications (Table 23.3) against LA is rather limited (Julius 2016).

Table 23.3 Contraindications against LA [according to (Julius 2016)]

• No accessible veins (no possibility to establish a fistula)

• Severe heart failure, malignant cardiac arrhythmias
• Therapy-resistant hypotension
• Lack of compliance
• Foreseeable very short life expectancy
• Severe physical or intellectual inability of a given patient
• Presence of a malignant tumor with poor prognosis
• Severe psychiatric disorder
386 U. Julius and S. Tselmin

The intake of oral anticoagulants, especially new oral anticoagulants, is no con-

traindication for LA. When the HELP method is used, warfarin therapy should be
avoided, due to danger of bleeding, because this method has a high impact on coag-
ulation factors.

 fficiency of LA on Cardiovascular Events (CVEs) in Patients

E
with High Lp(a) Concentrations

Observational Studies

Up to now, only observational studies on the effects of LA on outcome data in

patients with elevated Lp(a) concentrations were published. German authorities
asked for a prospective randomized controlled study to be performed in 2008. Ethics
committees did not approve this type of study.

Observational Studies

In 2009, a longitudinal, multicenter, cohort study was published by Jaeger et al. In

120 patients [with an initial Lp(a) level of about 118 mg/dL], who were treated
extracorporeally for about 5 years, the mean annual rate of major adverse coronary
events per patient was reduced from 1.056 to 0.144 (−87%) (Jaeger et al. 2009). The
rate of myocardial infarction was decreased by −97%. In the Pro(a) Life study, a
prospectively conducted multicenter study, 170 patients were included (Leebmann
et al. 2013; Roeseler et al. 2016; Klingel et al. 2019). The initial Lp(a) concentration
was 108 mg/dL; LA sessions acutely reduced Lp(a) by −68%. When comparing
with the situation before the start of LA therapy, major coronary adverse events
declined by −78%; this finding remained stable up to the end of the 5-year follow-up.

Our Own (Dresden) Experience

The Dresden Center for Extracorporeal Therapy was involved in both studies.
Moreover, we were the first to report that LA is more efficient with respect to the
reduction of CVEs in patients whose Lp(a) levels are elevated when comparing with
patients with normal or non-detectable Lp(a) (von Dryander et al. 2013; Schatz
et al. 2017). This difference had been confirmed by another group (Heigl et al. 2015)
and also in the German Lipoprotein Apheresis Registry (see below).
Moreover, we compared patients who developed CVEs while being treated with
LA (n = 48) to those who did not suffer from CVEs (n = 60) (Julius et al. 2020).
Both groups were on extracorporeal therapy for years already, for a mean period of
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 387

5–6 years. Interestingly, no difference with respect to lipid concentrations, includ-

ing Lp(a), was observed. But two factors had a significant impact on the occurrence
of new CVEs during LA: (1) older age at the start of the extracorporeal therapy
(Patients with events were about 5 years older, in the mean 60 years.) and (2) a
higher number of CVEs before the initiation of LA—a positive correlation between
this number and the number of CVEs during LA was calculated.

I talian Retrospective Multicenter LA Study in Patients

with Elevated Lp(a) and Coronary Artery Disease

Twenty-three patients with Lp(a) levels above 60 mg/dL and a pre-apheresis LDL-C
<100 mg/dL on maximally tolerated lipid-lowering therapy were included (Bigazzi
et al. 2018). They were treated with LA for several years (median 7, interquartile
range 3–9 years) by heparin-induced LDL precipitation apheresis (16/23), dextran-­
sulfate (4/23), cascade filtration (2/23), and immunoadsorption (1/23). The time lapse
between first cardiovascular event and beginning of apheresis was 6 years (interquar-
tile range 1–12 years). The rates of adverse cardiovascular events were reduced by
74% when comparing the situation before and after the LA treatment inception.

 tudy in Patients with Elevated Lp(a) and Peripheral Artery

S
Disease (PAD)

Ten patients with severe PAD and isolated Lp(a)-HLP who recently underwent revas-
cularization (index procedure) were included (Poller et al. 2017). When comparing
the situation before LA with the results after 12 months, the pain level, ankle-­brachial
index (ABI), transcutaneous oxygen pressure (tcpO2), and walking distance all
improved. Importantly, the frequency of revascularization procedures was strongly
decreased under LA. All patients combined underwent 35 revascularizations within
the 12 months prior to the index procedure (mean interval between two revascular-
izations: 104.3 days). Since the index procedure, only one revascularization was nec-
essary within 79 patient-months under LA (mean interval: 2404.5 days, p < 0.001).

 merican Single-Center, Retrospective Cohort Study in Patients

A
with High Lp(a) Levels

Fourteen patients with cardiovascular disease with elevated Lp(a) and near-normal
LDL-C were treated with LA over a mean treatment period of 48 months (range
8–105 months) (Moriarty et al. 2019). The authors describe a 94% reduction in
major adverse cardiovascular events.
388 U. Julius and S. Tselmin

German Lipoprotein Apheresis Registry

This registry has existed since 2011. In the annual report for 2020, data on 1111
patients (from 44 LA centers, 6791 LA sessions) have been documented (Schettler
et al. 2020). Following the suggestion from Dresden, three hyperlipoproteinemia
(HLP) groups have been defined (based on the initial lipid values): (a) with isolated
elevation of LDL-C [Lp(a) level <60 mg/dL or <120 nmol/L or not detectable,
n = 180], (b) with isolated elevation of Lp(a) [Lp(a) ≥60 mg/dL or ≥120 nmol/L
and LDL-C <2.6 mmol/L, n = 500), and (c) with combined elevation of both LDL-C
and Lp(a) (using the abovementioned criteria, n = 228). The latter group is totally
neglected in the officially published data.
The following mean acute reduction rates were reported for the LA sessions:
LDL-C—69%
Lp(a)—73%
The mean Lp(a) concentrations (the mean levels are a surrogate parameter for the
interval mean values; formula: mean = ½ × (pre value + post value); levels have
been separately reported according to the dimension provided by the lab): Group
B—54.20 mg/dL and 98.60 nmol/L, respectively; Group C—61.50 mg/dL and
104.75 nmol/L, respectively.
Mean LDL-C levels ranged between 2.00 mmol/L (Group A) and 1.59 mmol/L
(Group C). LDL-C levels for Group B are not given in detail.
CVEs have been subdivided into MACE [major adverse cardiac events: acute
coronary syndrome (unstable angina pectoris, NSTEMI, STEMI), coronary inter-
vention/surgery (PTCA, stent, CABG)] and MANCE [major adverse noncardiac
events: arterial occlusive disease (AOD) at noncoronary arteries with occlusion or
necessity for intervention/operation (PTA, stent, bypass, amputation), AOD of brain
arteries with TIA/stroke (CAOD), AOD of aorta thoracalis or aorta abdominalis
including visceral vessels and renal arteries, peripheral arterial occlusive disease
(PAOD)] (Table 23.4).
Follow-up data are contained in the annual report of the registry up to 7 years
under LA; low CVE rates were constantly seen throughout these years.
As an example, the graphs for HLP Group B are depicted (Fig. 23.4; with per-
mission of the Lipid-League).

Table 23.4 Percent reductions of MACE (major adverse cardiac events) and of MANCE (major
adverse noncardiac events) in the HLP Groups A, B, and C comparing the 2 years before the start
of LA with those during the first 2 years under LA (Schettler et al. 2020)

HLP group Reduction of MACE (%) Reduction of MANCE (%)

A 61 25
B 83 64
C 72 65
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 389

Fig. 23.4 MACE (a) and MANCE rates (b) in HLP Group B (with permission of Lipid-League).
(a) MACE rate (per patient and year) in HLP Group B. (b) MANCE rate (per patient and year) in
HLP Group B
390 U. Julius and S. Tselmin

Many patients had suffered from CVEs in the years anteceding the years included
in these graphs.
These data underline the already previously reported higher efficiency of LA
with respect to the reduction rate of CVEs in patients with elevated Lp(a) when
compared to patients without this feature.

 ussian-Specific Columns Against Lp(a) [Lp(a) Lipopak®

R
Adsorption Columns]

The commonly available LA systems decrease both LDL-C and Lp(a) concentra-
tions. The Russian POCARD Ltd. company offers antibody-coated columns which
specifically decrease Lp(a) only (Pokrovsky et al. 2017; Safarova et al. 2013). A
slight decrease in LDL-C does not represent the removal of LDL but only the
removal of cholesterol in Lp(a) particles. In a prospectively carried out angiographic
study over 18 months, it could be shown that a weekly Lp(a) reduction was associ-
ated with decrease in the mean percent diameter stenosis by 5.05% and increase in
minimal lumen diameter by 14%; mean total atheroma volume was reduced by
4.60 mm3 (p < 0.05 for all). These data were compared to those seen in the control
group which was treated with atorvastatin only. This small study points to the effec-
tiveness of a specific elimination of Lp(a) as a tool to combat atherosclerotic lesions.
Unfortunately, these specific columns are not used in a large scale anywhere.

MultiSELECt Study

MultiSELECt, a prospective European multicenter study on the effect of Lp(a)

elimination by LA on cardiovascular outcomes, was designed to directly compare
subjects with significantly elevated Lp(a) approved for LA subsequently undergo-
ing apheresis treatment versus a continuation of maximal medical therapy
(Hohenstein et al. 2017). The study aims at establishing matched pairs; it is still
ongoing (NCT02791802).

 ther Effects of LA in Patients with High Lp(a): A Study

O
with Sham Control

A British group conducted a prospective randomized, sham-controlled, single-­

blinded, crossover study involving 20 patients with refractory angina and elevated
Lp(a) >500 mg/L and LDL-C <4 mmol/L (Khan et al. 2017). Patients were random-
ized to a treatment arm with weekly LA for 3 months (12 sessions) or a control
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 391

group with placebo “sham” sessions weekly for 3 months. Treatments were per-
formed using the DX21 DHP (Direct Hemo Perfusion) Lipoprotein Apheresis
machine (Kaneka Corporation, Osaka, Japan) with the Liposorber DL-75 column,
which uses dextran sulfate to covalently bind ApoB-containing lipoproteins.
Baseline tests were repeated after treatment periods for both groups. After a 1-month
washout period, patients crossed over to the opposite treatment arm.
Patients underwent cardiovascular magnetic resonance imaging at baseline
assessing quantitative first-pass stress/rest perfusion and assessment of carotid ath-
erosclerosis with measurement of total carotid wall volume. Patients had exercise
capacity tested using the Six Minute Walk test (6MWT) and assessment of their
angina symptoms using the Seattle Angina Questionnaire (SAQ) and quality of life
(QoL) with the SF-36 questionnaire.
The results indicate that an improvement in myocardial perfusion rate was pri-
marily driven by improvements in stress perfusion, with insignificant change in rest
perfusion. In terms of secondary endpoints, improvements with apheresis compared
with sham also occurred in carotid atherosclerotic burden as assessed by total
carotid wall volume (p < 0.001), exercise capacity measured by the 6MWT
(p = 0.001), four of five domains of the SAQ (all p < 0.02), and quality of life physi-
cal component summary assessed by the SF-36 survey (p = 0.001).

Selected Case Reports

Some patients with elevated Lp(a) levels develop CVEs in several arterial regions.
We report two patients who were treated with LA at our center. Both suffered from
new CVEs despite being treated extracorporeally and were switched to two LA ses-
sions per week (Figs. 23.4 and 23.5).
Despite the intensive therapeutic regimen starting in 2018, the patient under-
went new interventions of her carotids, leg, and visceral arteries. Before being
referred to our center, she was smoking and it was not easy to persuade her to stop
this habit.
In Fig. 23.4, the lipid data are also shown. Lp(a) was effectively reduced, but in
all these years, LDL-C remained above the requested level (1.0 mmol/L). That is
why we started inclisiran in 04/2021. Since then, no new CVEs were observed. Her
actual lipid concentrations are as follows (before/after an LA session in 2022/02):
LDL-C 0.66/0.14 mmol/L, Lp(a) 153/26 nmol/L, HDL cholesterol
1.43/1.12 mmol/L, and triglycerides 1.68/0.56 mmol/L. Now LDL-C is optimal;
Lp(a) level is still high.
Another patient suffered from severe atherosclerosis of his coronaries
(Fig. 23.6).
At the age of 39 years, he had an acute myocardial infarction. His Lp(a) concen-
tration was found to be extremely high (≈593 nmol/L) only in 2006 (23 years after
his MI). He was also suffering from an increasing statin intolerance. From 1994 to
392 U. Julius and S. Tselmin

Fig. 23.5 A 56-year-old female patient with atherosclerotic affections of all vessel territories; LA
was started in 2017, switched to two sessions per week in 10/2018 (patient agreed that her data
could be included into this manuscript)

2011, he needed several interventions at his coronaries—in 1998, a fourfold coro-

nary bypass was performed. After 4 years of weekly LA therapy, he came to the
sessions twice per week. Nevertheless, he needed further interventions, though his
Lp(a) levels were clearly reduced. On the background of LDL-C concentrations of
more than 2 mmol/L before the LA sessions, we initiated an evolocumab injection
therapy in 2019. Since then, no new CVEs occurred.
His current lipid levels are as follows (before/after an LA session in 10/2022):
LDL-C 2.09/0.45 mmol/L, Lp(a) 215/31 nmol/L, HDL cholesterol 1.34/1.19 mmol/L,
and triglycerides 2.48/0.45 mmol/L.
At our department, we treat several patients whose indication for an LA therapy
was an elevation of Lp(a) which was likely, after the exclusion of cardiac reasons,
responsible for strokes (Table 23.5). In the literature, this association had been
described (Nave and von Eckardstein 2019; Arnold et al. 2021). Lp(a) seems first of
all to be responsible for large artery atherosclerosis stroke. In a Russian paper, it was
reported that in logistic regression analysis adjusted for age, sex, hypertension, type
2 diabetes, smoking, and Lp(a) concentration, the hyperlipoproteinemia(a) was
associated with ischemic stroke and isolated stenotic carotid atherosclerosis
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 393

Fig. 23.6 A 77-year-old male patient with multiple events at his coronary arteries; LA was started
in 2012 and switched to two LA sessions per week in 2016 (patient agreed that his data could be
included into this manuscript)

(Tmoyan et al. 2020). In the group with severe carotid atherosclerosis, 16 patients
(24%) had ischemic stroke. Lp(a) concentration in these patients was higher 36 [20;
59] mg/dL than in the patients with isolated carotid atherosclerosis without stroke
15 [7; 54] mg/dL (p = 0.04).
All patients listed in Table 23.5 had an elevation of Lp(a) and a (mostly) mod-
est increase of LDL-C. Only in two patients atherosclerotic lesions of their carot-
ids were documented. In one patient, coronary atherosclerosis was observed.
One patient does not take a statin (statin intolerance, he started evolocumab
in 2019).
Our patients did not develop any further strokes after they started to be treated
with LA, though Lp(a) interval mean values were not optimal.
In the literature, a genetically lowered Lp(a) concentration predicted a decreased
risk of stroke (Kamstrup 2021). An elevated Lp(a) level was associated with unfa-
vorable functional outcomes in patients with ischemic stroke (Jiang et al. 2021).
Thus we are convinced that the lowering of Lp(a) levels by LA is a beneficial con-
tribution to the further follow-up situation of stroke patients.
 394

Table 23.5 Male patients who are treated with LA because of an elevation of Lp(a) and following cerebrovascular events (patients agreed that their data could
be included into this manuscript)
LDL-C Lipoprotein(a)
Stroke events (mmol/L) (nmol/L)
Actual/
Before Before interval Statins: name/
ID Born Year Localization Atherosclerosis ApoE LA start LA Actual LA mean daily dose (mg)
RB 1967 2019 Left A. Cerebri Mild in Aa. carotis 3/3 08/04/20 2.0 1.7 363 321/236 A/80
nk Media Subcortical arteriosclerotic
Old lacunar encephalopathy
infarctions in white One-vessel-CAD, no
matter interventions
MG 1980 2017 Caput nuclei caudate, No 3/3 02/27/19 1.56 1.46 125 138/90 S/30
on the left side
HK 1962 2003 Cerebellum TEA in the left A. car int in 4/3 03/23/20 1.41 1.43 137 95/62 No
2007 Left A.cer media left 2007
2008 A.cer ant Actual—severe plaques Aa.
2009 Left A.cer ant parietal carotis without relevant stenosis
2009 operculum
F-PK 1968 2020 Cerebellum No 3/3 08/25/20 4.76 1.83 428 287/214 A/60
HS 1986 2018 Left A.cer media No 4/3 05/16/19 1.94 1.39 120 121/56 A/40
U-OS 1978 nk Cerebellum: small old No Nk 01/07/21 2.5 1.22 346 227/151 A/60
2020 defect
Vertebral-basilar TIA
JW 1980 nk Cerebellum: old No 3/3 01/28/20 3.21 2.3 152 122/87 R/10
2017 lacunar defect
Right basal ganglia
U. Julius and S. Tselmin
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 395

 ctions Which Should Be Taken in Patients Who Develop

A
Cardiovascular Events (CVEs) Despite Being Treated with LA

Of course, these patients should be continuously advised to follow the rules of a

healthy lifestyle (no smoking, diet, regular intake of drugs). The permanent contact
with physicians and the medical staff offers opportunities to regularly discuss these
aspects.
In very few patients with an extremely high cardiovascular risk (see selected
cases above), we decided to perform two LA sessions per week.
In patients whose LDL-C concentrations remained clearly above the internation-
ally recommended target (1.4 mmol/L in high-risk patients, 1.0 mmol/L in those
with repeated CVEs) (Mach et al. 2019) despite taking statins and ezetimibe (when
tolerated), we started an injection therapy with PCSK9 antibodies (evolocumab,
alirocumab). Presession Lp(a) levels were decreased between 0 and 44% after
12 weeks injecting these drugs (Julius et al. 2019). In the last months, we also initi-
ated a therapy with inclisiran.
As shown in the chapter on selected cases, the addition of injections of PCSK9
inhibitors stopped the progression of CVEs in extremely high-risk patients.

Unresolved Problems

Though LA therapy is performed, especially in Germany, for more than 30 years

now, some questions still remain unanswered.

Which LA Method Is the Best?

At the Dresden Center for Extracorporeal Therapy, we have a long-lasting experi-

ence with six different LA methods (Julius 2016). When we observe an insufficient
acute decrease of Lp(a) in a given patient, we try to optimize the situation, for
example, by increasing the treated plasma/blood volume. If the result is not satisfy-
ing, we usually switch to another system.
In a paper which appeared in 2013, we recommended that each apheresis center
should work with more than one LA system (Julius et al. 2013). The calculations
presented in this paper are based on laboratory data measured at the last three avail-
able apheresis sessions before switching to another method and at the end of the
observation period, respectively. With respect to the reduction of LDL-C, DALI and
Liposorber D appeared to be the most effective LA methods, for reduction of Lp(a),
Liposorber D. In any comparisons between the LA methods in the following years,
we did no longer observe any differences with respect to lipid lowering data—in
other words, we are treating our patients quite effectively.
396 U. Julius and S. Tselmin

Other differences between LA systems have been described, for example, for
proteins, PCSK9 levels, and coagulation factors (Julius et al. 2002, 2015a, b). The
significance of these differences for the prognosis of the patients remains still to be
clarified.
Moreover, additional pleiotropic effects of LA (removal of C-reactive protein,
complement, of apolipoprotein CIII, TNF-α, interleukin 6, and adhesion molecules
like ICAM-1 or VCAM-1) may have an impact on the course of atherosclerotic
lesions (Waldmann and Parhofer 2016; Makino et al. 2019). LA leads to vascular
tone reduction, reduced thrombogenesis, increased neo-angiogenesis, and impor-
tantly plaque stabilization (Poller et al. 2017). No data comparing different LA
methods with regard to these parameters have been published.

 ow Low Should Lp(a) Be to Effectively Prevent New CVEs

H
During LA Therapy

For LDL-C in the last years, target values have been defined with the aim to effec-
tively reduce the cardiovascular risk. The major message is “the lower the better.”
In the absence of randomized, controlled trial data demonstrating reduced car-
diovascular risk with reduction in Lp(a), no such targets have been proclaimed for
this parameter.
Usually, in lab reports, a normal range for Lp(a) is given below 30 mg/dL (about
75 nmol/L). It has been discussed in a paper by Boffa et al. that lowering Lp(a)
below this threshold would ameliorate the atherogenic risk (Boffa et al. 2018).
We think that in order to obtain an optimal effect of LA therapy with respect to
CVEs, interval mean values should be normalized [probably below 30 mg/dL (about
75 nmol/L)]. As shown in Fig. 23.1, the reality is far from this request. In only 21%
of our patients, this goal was reached. About half of them had an interval mean
Lp(a) concentration higher than 120 nmol/L. Though as a matter of fact, we did not
see a relationship between these concentrations and the incidence of CVEs during
LA therapy (Julius et al. 2020). And in the Russian prospective study, using specific
anti-Lp(a) columns, with coronary angiography, a beneficial effect on coronary ath-
erosclerosis was observed, though the mean interval value in the apheresis group
was 73 mg/dL (about 175 nmol/L) (Pokrovsky et al. 2020).
In the literature, two papers suggested, based on data with Mendelian randomiza-
tion, that a decrease of Lp(a) by about 100 mg/dL (about 240 nmol/L) (Burgess
et al. 2018) or 65 mg/dL (about 156 nmol/L) (Lamina et al. 2019) will induce a
similar reduction of CVEs as a decrease of 1 mmol/L of LDL-C, for example, by
about 22%. Populations included into these meta-analyses had much lower median
Lp(a) concentrations (approximately 30 nmol/L, maximally in one study
104 nmol/L) than those who are usually treated extracorporeally. Moreover, both
studies were population based. In contrast, a Danish group looked at patients with a
history of cardiovascular disease who were followed after their initial event (Madsen
et al. 2020). The authors calculated that plasma Lp(a) should be lowered by 50
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 397

(about 120 nmol/L) and 99 mg/dL (about 240 nmol/L) for 5 years to achieve 20%
and 40% MACE risk reduction in secondary prevention. Accordingly, for a 22%
MACE reduction, a reduction of Lp(a) by 55 mg/dL (about 132 nmol/L) would be
required.
From the viewpoint of LA data, these publications are not conclusive at all.
In the HPS2-THRIVE Study, niacin laropiprant reduced mean Lp(a) by
12 nmol/L overall and by 34 nmol/L in the top quintile by baseline Lp(a) level
≥128 nmol/L (Parish et al. 2018). The authors write that estimates from genetic
studies suggest that these Lp(a) reductions during the short term of the trial might
yield proportional reductions in coronary risk of ≈2% overall and 6% in the top
quintile by Lp(a) levels.
In studies using PCSK9 antibodies (evolocumab, alirocumab), a small decrease
of Lp(a) concentrations was seen (Kronenberg 2022; Julius et al. 2019). When
excluding the impact of the reduction of LDL-C on outcome data by mathematical
modeling, the decrease of Lp(a) was effective with respect to a certain lowering of
CVEs when compared with the placebo groups.
A comparison between PCSK9 antibodies and pelacarsen [an antisense oligo-
nucleotide against Apo(a)] showed an interesting difference: Pelacarsen reduced
Lp(a) by 47% and as a consequence the pro-inflammatory gene expression in mono-
cytes of cardiovascular disease patients with elevated Lp(a), which coincided with a
functional reduction in transendothelial migration capacity of monocytes ex vivo
(Stiekema et al. 2020). In contrast, PCSK9 antibody treatment lowered Lp(a) by
16% and did not alter transcriptome nor functional properties of monocytes, despite
an additional reduction of 65% in low-density lipoprotein cholesterol (LDL-C). The
effect of Lp(a) lowering by LA is in the same range as described for pelacarsen in
this manuscript.

Should We Calculate “True” LDL-C?

When LDL-C is measured, both LDL and Lp(a) particles are included (Yeang et al.
2015). In order to calculate the LDL-C mass transported with LDL, the following
steps are required: (1) The Lp(a) mass should be in mg/dL—we measure Lp(a) in
nmol/L—the conversion into the Lp(a) mass is not correct (and no longer recom-
mended). (2) The estimated percentage of LDL-C in the Lp(a) particles usually is
set to be 30%. Data have shown that this percentage may be variable interindividu-
ally. (3) Due to these problems, in some patients, negative “true” LDL-C levels are
seen. The British colleagues do not recommend to calculate “true” LDL-C because
(1) it is not validated with isoform-independent assays in treated and untreated
patients, (2) it is not validated in large epidemiological studies for cardiovascular
risk prediction or in RCTs of lipid-lowering therapies, and (3) it is not in clinical use
(Cegla et al. 2019). Recently, a novel method for quantification of Lp(a) cholesterol
had been suggested (Yeang et al. 2021). This problem is relevant for patients who
are treated with LA and with PCSK9 inhibitors.
398 U. Julius and S. Tselmin

 A in Children with Ischemic Stroke and Patients

L
on Hemodialysis with High Lp(a) Levels

In children, a highly elevated Lp(a) concentration may be associated with an

increased risk for ischemic stroke (deVeber et al. 2019). The arterial ischemic
stroke-free survival in children with elevated Lp(a) was lower compared with that in
the remaining children with normal Lp(a) levels. The authors do not mention LA as
a therapeutic option.
Higher Lp(a) values and LDL-unbound Apo(a) particles were found in patients
with end-stage renal disease; their LDL has different chemical and structural prop-
erties as compared to control (Trenkwalder et al. 1997). Apheresis would be an
optimal tool to remove all these atherogenic lipoproteins. In reality, a few patients
who are treated with hemodialysis due to renal insufficiency have started an LA
treatment on the background of severe atherosclerotic complications.
No outcome data for the combination of hemodialysis and LA are available.

Future of LA in Patients with High Lp(a) Levels

Pros and Cons of LA

LA allows the treatment of high-risk patients with elevated Lp(a) levels who have
suffered from CVEs. It is tolerated very well. In patients with familial hypercholes-
terolemia, which is not seldom associated with elevated Lp(a) levels, tendon xan-
thomas usually disappear under a year-long LA therapy. This points to the fact that
the body cholesterol pool is diminished.
The extracorporeal removal of Lp(a) particles significantly decreases their con-
centration in blood, especially in the days immediately after LA sessions. The
increase of Lp(a) thereafter makes it necessary to perform LA sessions weekly. In
some countries, a 2-week interval is the prevailing therapeutic approach, mainly
because of financial problems.
Interval mean values reflect approximately the averaged Lp(a) level in the days
between LA sessions and in this way the atherogenic burden. With the available LA
methods, it is possible to reach optimal Lp(a) concentrations (<75 nmol/L) only in
a small part of the patients. But extremely high Lp(a) levels which confer a very
high atherogenic risk can be reduced a lot.
In LA patients, all other risk factors (hypertension, diabetes mellitus, hyperurice-
mia, smoking habit, hypothyroidism, obesity) should be optimized.
The usual way to describe the effect of LA therapy on outcome data—by com-
paring the incidences before the start of the extracorporeal treatment with those
during this treatment—has been criticized (Waldmann and Parhofer 2016). It should
be noted that the observational studies suffer from potential confounding due to the
selection bias for survivors inherent in their design, as well as the lack of the ability
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 399

to rule out the effect of apheresis on other drivers of events such as fibrinogen (Boffa
et al. 2018). Hopefully, the MultiSELECt study will offer a clarification of this dis-
pute. Clearly, placebo-controlled apheresis studies are not feasible. The British
study which had an individual sham control lasted only a few months.
Under extracorporeal therapy, some patients will develop new CVEs. We did not
detect any difference in lipid concentrations, including Lp(a), before or after LA
sessions or in interval mean values between patients with or without CVEs during
LA therapy (Julius et al. 2020). According to our data, older age at the start of the
LA therapy and a higher number of CVEs before LA started playing a role. Both
these aspects point to the fact that atherosclerosis has progressed in patients who
suffer from CVEs during the extracorporeal therapy.
Thus, in order to be on the safe side, an LA therapy should not be initiated too
late. But on the other hand, LA may be lifesaving in high-risk patients. The number
of patients who die when they are undergoing LA therapy is rather low; no reliable
data on these numbers are available.
In general, an LA therapy should not be started in patients who are (biologically)
older than 70 years. On the other hand, LA is a lifelong treatment and should not be
discontinued even in very old patients who started LA years ago. The diagnosis of a
malignant tumor, a severe cardiac insufficiency, or a poor compliance may be rea-
sons for stopping the extracorporeal therapy.
In the British sham-controlled apheresis study, a regression of atherosclerosis
was seen at the neck vessels. In some angiographic studies, a certain percentage of
regression at the coronary arteries was found in LA-treated patients. In our experi-
ence, we are already happy when in a given patient a nonprogression of the lesions
(no new stenoses, no new CVEs) is observed. Of course, patients should regularly
be checked by a cardiologist and/or angiologist.
LA is expensive and time-consuming (2–3 h) and needs the work of a qualified
staff. But by avoiding new CVEs, money can be saved in the long run. In Germany,
nephrologists may apply for the permission to perform LA. Taking into attention the
fact that LA can be optimally performed only in centers with sufficient experience
in this field, the number of patients at each center should not be too low (probably
not less than ten patients, at least two LA systems should be offered).

 iet, Statins, Ezetimibe, Bempedoic Acid, PCSK9 Inhibitors,

D
and Evinacumab

Each patient who is taking lipid-lowering drugs should be advised to adhere to a

healthy diet as well. This rule is also valid for patients with high Lp(a) concentra-
tions though nutrition does not exert any effect on Lp(a). In patients with familial
hypercholesterolemia, the effectiveness of diet on LDL-C is rather limited. Despite
these restrictions, in Germany, insurance companies demand that a consultation
about diet should be documented; this also concerns all LA patients.
400 U. Julius and S. Tselmin

If in a given patient with elevated Lp(a) concentration the LDL-C level exceeds
the internationally accepted targets (1.4 mmol/L for high-risk patients, 1.0 mmol/L
for patients with repeated CVEs) (Mach et al. 2019), usually a statin therapy should
be started. More effective statins (atorvastatin, rosuvastatin) are to be preferred.
When the effect is not satisfactory, ezetimibe can be added. Bempedoic acid can be
administered either together with a statin (in order to improve LDL-C) or instead of
a statin when the latter is not tolerated. Ezetimibe can be continued in these cases.
When the result seen after the introduction of these first steps is not optimal, after
several months, the indication to use PCSK9 inhibitors is given.
All these measures are the prerequisite before an LA is allowed to be started, at
least in Germany.
An LA therapy may be commenced in the following LDL-C ranges in patients
with Lp(a) concentrations exceeding 60 mg/dL or 120 nmol/L:
1. The LDL-C target has been reached. That is the purpose of the official
regulations.
2. The LDL-C target was not reached despite the patient regularly took the lipid-­
lowering drugs. This indication is not officially covered by the existing rules, but
this situation is not a very rare one.
3. Patients experience a drug intolerance—this may be the case with statins or
PCSK9 inhibitors, very seldom with ezetimibe. LDL-C is still too high.
Of course, a progression of atherosclerosis has to have been documented
(repeated CVEs or shown by imaging techniques). Exceptions from this demand are
made in very young patients (aged under 40 years) with extremely high Lp(a) levels
who survived a life-threatening acute myocardial infarction and who have a positive
family history for CVEs among first-degree relatives in younger ages.
During the LA therapy, the administration of lipid-lowering therapy should
always be continued. Some adjustment of doses may be necessary depending on the
measured LDL-C level.
In our hands, the addition of PCSK9 inhibitors to LA procedures may further
improve LDL-C and—at least in a majority of patients—Lp(a) concentrations. The
combined treatment with PCSK9 monoclonal antibodies and apheresis may be pref-
erable in certain hypercholesterolemic patients with high Lp(a), because of the com-
bined benefits of both approaches in lowering LDL-C, triglyceride-rich lipoproteins,
inflammation, hemorheology, and Lp(a) (Ruscica et al. 2019).
We saw the end of a series of cardiovascular interventions in high-risk patients
after the start of this intensive injection therapy. In some patients, we then switched
to a biweekly LA regimen—provided the cardiovascular situation remains stable.
Evinacumab, a fully human monoclonal antibody against angiopoietin-like 3, is
a new drug which can be applied in therapy-resistant hypercholesterolemia
(Rosenson et al. 2020). But this drug does not decrease Lp(a). In an ApoE*3-Leiden
CETP mouse model, a triple therapy with atorvastatin, alirocumab, and evinacumab
has been successfully performed (Pouwer et al. 2020). The future role of intrave-
nously infused evinacumab in the daily routine has still to be defined.
23 Lipoprotein Apheresis for Reduction of Lipoprotein(a) 401

Pleiotropic effects of LA differ essentially from those described for lipid-­

lowering drugs. The combination of drug and LA therapy promises to obtain the
best clinical results.

Inhibitors of Apo(a) Synthesis

The antisense oligonucleotide AKCEA-APO(a)-LRx (pelacarsen) effectively

reduces Lp(a) levels (up to 80%) by impairing the synthesis of Apo(a) (Tsimikas
et al. 2020). Since 2020, a prospective, placebo-controlled Phase III HORIZON
study (ClinicalTrials.gov Identifier: NCT04023552) is ongoing. The tolerability of
pelacarsen is described to be very good. A special focus in this study is on obtaining
low LDL-C values, even PCSK9 inhibitors are allowed. It will be interesting to see
what will be the effect of pelacarsen on outcome data.
Moreover, two other companies are currently testing small interfering RNA
drugs against Apo(a) (Amgen: Olpasiran (AMG 890); ClinicalTrials.gov Identifier:
NCT04270760) and Silence Therapeutics plc (SLN360; ClinicalTrials.gov
Identifier: NCT04606602). Olpasiran was already used in a phase 1 dose escalation
trial (Koren et al. 2022).
These drugs will be a competitor to LA with regard to the indication “isolated
elevation of Lp(a),” provided the outcome data of the HORIZON study will be
convincing.
At present, two problems with these drugs are evident: (1) They will be r expen-
sive. (2) They do not show any effect on LDL-C—drugs to lower LDL-C are not
always effective enough to reach target levels; they are not well tolerated in a sub-
stantial number of patients.
It can be supposed that some patients with an extremely high atherogenic risk
will still need the extracorporeal therapy in order to save their life. The commonly
used LA procedures decrease both Lp(a) and LDL-C and exert some beneficial
pleiotropic effects.
Ideally, a study comparing prospectively Apo(a) synthesis inhibitors with LA
with respect to the occurrence of CVEs could answer the question what will be the
best way to treat high-risk patients with elevated Lp(a).

Conclusions

LA is at present the only accepted therapy to decrease highly elevated Lp(a) concen-
trations in high-risk patients with the aim to revert a progressive course of Lp(a)-
associated cardiovascular disease to a stable course and to prevent future CVEs.
Most probably, pleiotropic (anti-inflammatory, antithrombotic, rheologic) effects
exert an additional benefit. Up to now only observational studies documented a high
efficiency of LA with respect to reduction of the incidence of CVEs. LA requires a
402 U. Julius and S. Tselmin

high qualification of the medical staff, is time-consuming, and is expensive but is

associated with good tolerability.
LA therapy should only be initiated when dietary efforts and all conventional
lipid-lowering drugs (when tolerated) have been brought into play and patients
experienced CVEs. When Lp(a) will be measured more often in high-risk patients
(which can already be observed today), the indication of an LA therapy will have to
be considered more often.
Unfortunately, at present, the role of LA for reduction of Lp(a) is not accepted
everywhere. In a new review about elevated Lp(a) levels in persons with familial
hypercholesterolemia—this association is described to be highly atherogenic—the
Danish scientists do not mention apheresis at all (Langsted and Nordestgaard 2022).
LA was shown to decrease both Lp(a) and LDL-C—this combined effect should be
especially beneficial in patients who are resistant to the usual lipid-lowering therapy
or who do not tolerate these drugs.
Drugs inhibiting the synthesis of Apo(a) will represent a competitor to LA for
Lp(a) patients in the future. For these drugs, outcome data showing an advantage in
comparison with LA will be required. A major advantage of these drugs is that
Lp(a) levels remain permanently low, while on LA therapy, they are fluctuating.
Nevertheless, some patients with an extremely high atherogenic risk (severe affec-
tion of several vessel territories on the background of a positive family history for
early CVEs, progression despite an optimal therapy with lipid-lowering drugs) will
still need extracorporeal therapy in order to survive in the future. This will espe-
cially be the case in patients who did not show a sufficient decrease of LDL-C
concentrations.
Finally, there is still another important aspect which is cited here literally from
Thompson and Parhofer (2019): “Patients treated by regular apheresis have the
advantage of being seen by the same medical team on a very regular (weekly or
biweekly) basis. This tight control and guidance improves compliance (generally
speaking) and allows medical issues to be discussed regularly in a familiar setting.
Although this effect is hard to quantify, it would be surprising if it did not also affect
the cardiovascular event rate. Obviously, drug therapy gives the patient more “free-
dom” but maybe at the cost of less strict medical surveillance.”

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Chapter 24
Elevated Lp(a): Why Should I Test For It,
If I Cannot Treat It? A Patient’s
Perspective

Sandra Revill Tremulis

“ How Can I Have Normal LDL-Cholesterol and Almost Die

of a Heart Attack?”

Cardiovascular diseases (CVDs) are the leading cause of death globally (World
Health Organization 2021). Lp(a), pronounced “Lp little a,” is an LDL (low-density
lipoprotein)-like, fatty, sticky lipoprotein particle with an additional protein
apolipoprotein(a) [apo(a)] wrapped around it and found in blood serum. It is an
inherited atherogenic lipoprotein and an independent risk factor for atherosclerotic
cardiovascular disease, vascular thrombosis, stroke, and calcific aortic stenosis
(Nordestgaard et al. 2010; Bennet et al. 2088; Erqou et al. 2009; Kampstrup et al.
2009; Boffa and Koschinsky 2016; Rogers and Aikawa 2015; Langsted et al. 2019).
Lp(a) is one of the strongest genetically determined risk factors for cardiovascular
disease (Kronenberg and Utermann 2013; CARDIoGRAMplusC4D Consortium
and Deloukas 2013; Thanassoulis et al. 2013). Approximately 1 in 5 people have
inherited high Lp(a), more than 1 billion people globally and 63 million in the
United States who are unaware they have up to a 60% increased risk for coronary
artery disease (Nordestgaard et al. 2010; Kamstrup et al. 2009). High Lp(a) is
80–90% genetically determined (Schmidt et al. 2016). Diet and exercise have little
to no impact on high Lp(a) (Mackinnon et al. 1997). Most people fully express the
LPA gene by the time they are 2 years old, reach adult levels by five, and for the
most part maintain the same Lp(a) levels for a lifetime, although Lp(a) levels tend
to increase with age in females after menopause (Wilson et al. 2019; Bittner 2002).
Unfortunately, there are no visible symptoms, such as xanthomas, to indicate high
Lp(a), and traditional cholesterol tests miss 8% of people who have a cardiovascular
event whose only risk factor is high Lp(a) (Mortensen et al. 2015; Bittner 2015).

S. R. Tremulis (*)
Redwood City, CA, USA

© The Author(s), under exclusive license to Springer Nature 409

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_24
410 S. R. Tremulis

The traditional cardiovascular lipid screening does not include high Lp(a). However,
a simple blood test performed once in a person’s lifetime could be the first step in
preventing up to 120,000 cardiovascular events every year (Wilson et al. 2019;
Mortensen et al. 2015). Unfortunately, the first sign of cardiovascular disease often
is a heart attack or a stroke. In 2003, I almost died of a heart attack despite having a
healthy lifestyle, annual preventative health screenings, and no significant risk fac-
tors except my family history of cardiovascular disease. Bloodwork ordered after
my heart event identified high Lp(a) as the only potentially significant contributing
risk factor. I, therefore, propose expanding the current standard cardiovascular pre-
vention lipid screening panel to include high Lp(a) testing for everyone to provide
patients, families, and their healthcare providers with a more accurate prediction of
their overall risk for premature cardiovascular disease and death (Wilson et al. 2019;
Mortensen et al. 2015; Bittner 2015).

 eveal Lp(a): “You Must Have Had Some Really Bad Habits
R
When You Were Younger”

Heart disease is the leading cause of death for men, women, and people of most
racial and ethnic groups in the United States (Table 24.1) (Centers for Disease
Control and Prevention 2018) and costs the United States about $363 billion each
year from 2016 to 2017 (Virani et al. 2021). This includes the cost of healthcare
services, medicines, and lost productivity due to death. There is no cure for cardio-
vascular disease; it is a chronic, systemic disease.
These daunting heart disease statistics have provoked fear in me for decades. My
father died young of a heart attack; he had his first heart event at age 30, femoral
bypass surgery at age 40, and a fatal heart attack at age 50. I was 22 years of age
when he died. I never got over it; I just adjusted to it. Do you have a family history
of cardiovascular disease? What are your personal inherited cardiometabolic risk
factors for cardiovascular disease? I thought, heart attack, it was never going to

Table 24.1 2020 Top causes of death in the United States—Centers for Disease Control

2020 Top causes of death in the United States—Centers for Disease Control
Heart disease: 696,962
Cancer: 602,350
COVID-19: 350,831
Accidents: 200,955
Stroke (cerebrovascular diseases): 160,264
Alzheimer’s disease: 134,242
The table was created from data in Mortality in the United States, 2020, data table for Figure 4
Murphy SL, Kochanek KD, Xu JQ, Arias E. Mortality in the United States, 2020. NCHS Data
Brief, no 427. Hyattsville, MD: National Center for Health Statistics. 2021. DOI: https://doi.
org/10.15620/cdc:112079. Copyright 2020 CDC/National Center for Health Statistics
24 Elevated Lp(a): Why Should I Test For It, If I Cannot Treat It? A Patient’s Perspective 411

happen to me. I was a young female, and I thought heart disease primarily impacted
older men, like my father. I was in the medical device industry, knowledgeable
about heart disease, and working on cutting-edge technology to help families faced
with a cardiovascular disease diagnosis and helping to make a difference. I was
proactive about my cardiovascular health. Because of my family history, I had a
lifelong commitment to fitness and health, never missing my annual checkups. I
thought I was doing everything right, and according to my Framingham and
Reynold’s risk score, I was!
Table 24.2 below is my Framingham Risk Score from 2003; it gave me a 1%
chance of having a cardiovascular event.
Furthermore, Table 24.3 is my Reynolds Risk Score, an assessment that predicts
cardiovascular disease, gave me a 1% chance of having a cardiovascular event, and
includes family history in their risk calculation algorithm.
However, based on the Bruneck Study, if you add Lp(a) to the Framingham and
Reynolds Risk Score, I would have been reclassified with my high Lp(a) into a
higher risk category like 20% of patients (Willeit et al. 2014).
In 2003, at 39 years of age, I went out for my usual 5-mile run, got one block
down the road, and physically felt I could not go any further. I experienced fatigue
and mild tingling in my chest upon exertion. Even still, like many people, I rational-
ized away my symptoms. I taught fitness classes as a hobby and had run a marathon
the year before. I thought my tiredness was due to a potential thyroid issue, early

Table 24.2 Framingham Risk Score including Sandra Revill’s data from 2003
Information about your risk score
Age 39
Gender Female
Total 136 mg/dL (3.52 mm/L)
cholesterol
HDL 30 mg/dL (0.77 mm/L)
cholesterol
Smoker No
Systolic blood 117 mm/Hg
pressure
On medication No
for HBP
Risk scorea Less than 1%
The score means less than 1 in 100 people with this level of risk will have a
heart attack in the next 10 years
a
Your risk score was calculated using an equation. Other NCEP products, such
as printed ATP III materials, use a point system to determine a risk score close
to the equation score
The table was created from the Framingham Heart Study https://www.framinghamheartstudy.org/
fhs-­risk-­functions/cardiovascular-­disease-­10-­year-­risk/using the 2003 personal medical data from
Sandra Revill. Copyright for the Framingham Risk Score Calculator, D’agostino RB, Vasan RS,
Pencina MJ, Wolf PA, Cobain M, Massaro JM, Kannel WB. General cardiovascular risk profile for
use in primary care. Circulation. 2008 Feb 12;117:743–53. PMID:18212285
412 S. R. Tremulis

Table 24.3 Reynold’s Risk Score including Sandra Revill’s data from 2003
Information about your risk score
Age 39a
Gender Female
Total cholesterol 136 mg/dL (3.52 mm/L)
HDL cholesterol 30 mg/dL (0.77 mm/L)
Smoker No
Systolic blood pressure 117 mm/Hg
High-sensitivity C-reactive 0.16 mg/L
protein (hsCRP)
Did your mother or father Yes
have a heart attack before age
60?
Risk score a
Note the value you entered for age is outside the lower range.
The result is based on age 45
As shown in the graph below, age 45, your chance of having a
heart attack, stroke, or other heart disease event at some point in
the next 10 years is 1%
a
The table was created from the Reynolds Risk Calculator, Calculating Heart and Stroke Risk for
Men and Women http://www.reynoldsriskscore.org/Default.aspx using the 2003 personal medical
data from Sandra Revill. Copyright for Reynolds Risk Calculator, Journal of the American Medical
Association (Ridker PM, Buring JE, Rifai N, Cook NR. Development and validation of improved
algorithms for the assessment of global cardiovascular risk in women: The Reynolds Risk Score.
JAMA 2007;297:611–619)

menopause, or the flu. It never occurred to me that I might have a heart problem.
However, I knew something was wrong, so I scheduled an appointment with my
family practice physician, who had a comprehensive overview of my family history
of cardiovascular disease. The physician I saw wanted me to have a stress test in the
emergency room, but they were too busy that day. Not thinking it was emergent, he
suggested I was doing too much and recommended scheduling a treadmill test with
the cardiology group. A couple of weeks later, I passed the treadmill test and got
approval to teach my indoor cycling class that evening. Nevertheless, something
was still wrong; I had to stop riding three times during class due to an overwhelming
feeling of fatigue.
I still thought I had the flu! Later that week, I left on a business trip to Washington,
DC, to attend a major medical device conference with all the top cardiologists in the
world. I exercised in the hotel, as I usually did, but this time I felt terrible. I had the
same fatigue and mild tingling in my chest as I ran on the treadmill, and when I
slowed down, the tingling went away. I had swollen feet, so I started to take aspirin
to try and reduce the swelling in my feet. I was aware of the risk of blood clots when
flying long distances and that aspirin could help reduce that risk.
After I arrived home, compelled to seek a second opinion, I referred myself to an
interventional cardiologist I knew in my professional role as a product marketing
manager in the vascular business of a major medical device company. He listened to
my history and informed me that he would be conservative and order a Nuclear
24 Elevated Lp(a): Why Should I Test For It, If I Cannot Treat It? A Patient’s Perspective 413

Stress Test because of my family history and symptoms. He felt I might have
exercise-­induced angina. I completed the Nuclear Stress Test, and I could not
believe his diagnosis of potential cardiovascular disease. I had been physically fit
my whole life and watched my diet because of losing my father at such an early age
and diligent about my annual medical screenings, so what had I done wrong? He
wanted me to take blood thinners overnight. He stated, “There are several abnor-
malities on your test, and I need you in hospital first thing tomorrow morning for a
heart catheterization.” I arrived at the hospital and was shocked when they asked me
if I had a Will. I was a young, single female who owned property and had never
dreamed I would need a Will at my age. I felt a sense of impending doom as I signed
the consent papers at check-in to immediately convert me to bypass surgery should
it be necessary if they could not stent the potential blockages in my heart arteries. I
knew there might be limitations reaching the blockages with the current portfolio of
stents because of my smaller female anatomy. I was facing this life crisis alone; my
family was overseas without time to reach me. Petrified, they wheeled me down to
the catheterization lab for the procedure.
Upon injection of dye into my coronary arteries, I heard a collective expression
of surprise in the Cath Lab as it revealed a 95% occluded proximal left anterior
descending coronary artery. This type of blockage is commonly referred to as the
“widow maker” in the cardiology world. I remember asking, “Can you fix it?” The
doctor said, “I think your father is sitting on your shoulder because I am not sure
how you are still here.” They inserted a drug-coated stent to open the blockage.
Interestingly, after my procedure, the Cath Lab nurses asked me, “Strong family
history?” As I think about my journey with heart disease, they were a few of the
people with empathy who instinctively, based on their experience, realized the
inherited nature of my premature heart disease. During my follow-up visit with my
cardiologist, I cried, and I asked him, “What did I do wrong?”
I felt this way because of the public misconception that a poor lifestyle is the
only reason people get cardiovascular disease.

TEST Lp(a): “Why Test for It If You Cannot Treat It?”

The cardiologist said he wanted to understand the cause of my premature heart dis-
ease and ran more cardiometabolic bloodwork. That is the moment I discovered I
had high Lp(a). He told me I did not do anything wrong, and I inherited this from
my parents. I had an uncontrollable genetic risk factor for my premature heart dis-
ease, but surprisingly, it was a huge relief not to carry the shame that I could have
prevented this event somehow. Finally, I could give my disorder a name. I was able
to have a sense of control over an uncontrollable situation. He also assured me there
would be significant advances in heart disease and not worry about my future.
Some would argue that testing for high Lp(a) is pointless in that “why test for it
if you cannot treat it.” Others would argue the benefit that testing for high Lp(a)
uncovers a hidden genetic risk factor. I am one of the faces of high Lp(a) who had
414 S. R. Tremulis

an inaccurate prediction of my cardiovascular disease risk and feel that Lp(a) testing
provides patients and their families a better risk prediction for premature cardiovas-
cular disease and death. A personalized prescription for more aggressive primary or
secondary prevention, including optimizing all cardiometabolic risk factors, can be
initiated for an at-risk individual. Due to the thrombogenic nature of Lp(a), the doc-
tor thought the aspirin I took when I felt my symptoms may have saved my life on
the plane journey home by reducing the risk of a flow-limiting coronary thrombosis
and a heart attack. In the Women’s Health Study, carriers of the rare LPA gene vari-
ant (rs3798220) had a relative 56% risk reduction in ASCVD risk in carriers on
aspirin therapy versus noncarriers (Chasman et al. 2009). More research should be
conducted to improve the risk calculators and on aspirin use for primary and sec-
ondary prevention for patients with high Lp(a) (Mortensen et al. 2015; Zheng and
Roddick 2019). I would also support risk-based versus trial-based calculators
because many other factors decide enrollment criteria in randomized controlled
clinical trials.
Over the next 10 years after my stent procedure, I would go through the grieving
process for my former self as I recovered and returned to my new normal. I became
an advocate for women’s heart disease, but little information was shared about
inherited cardiovascular disease. During these years, I had a child, and just as there
is a 30-year deficit of data on women and heart disease, there was a total deficit of
data to manage a woman through the reproductive years of her life with diagnosed
heart disease and high Lp(a). Nevertheless, this was the beginning of my journey to
learn about high Lp(a) and become educated and empowered and protect my own
life. I lived with the trauma from my father’s death and from my event, but it
appeared nothing had advanced in the field of Lp(a) research in the 10 years since
my heart event, and I wanted to know why. I later learned that the lack of implemen-
tation of a US and global standardized Lp(a) assay had hampered the progression of
Lp(a) research, but now there are exciting new developments with a mass
spectrometry-­based approach for Lp(a) measurement. I would support the rapid
adoption of a global standardized Lp(a) assay because time is measured in lives for
patients.
A pivotal moment occurred when I went to see a leading lipid researcher at a
major medical institution for a consultation. I wanted to know about the latest
research on Lp(a). The appointment with the consultant took 2 h and cost $600. He
concluded with a very clinical and dogmatic statement, “You have a malignant fam-
ily history; there is no treatment for what you have. It is prohibitively expensive to
research because each different ethnic group has a different normal level of Lp(a),
and the child you risked your life having has a 50% chance of inheriting it!” I was
motivated to make a difference and said to my husband, “I have nothing to lose
except my life and everything to gain; I want us to make a difference. I do not want
another family to suffer. I want to save lives by educating everyone about the health
consequences of high Lp(a) and empower them to take action to reduce their risk
and save lives.” So, in 2013, I founded the Lipoprotein(a) Foundation.
24 Elevated Lp(a): Why Should I Test For It, If I Cannot Treat It? A Patient’s Perspective 415

 ducate, Empower, and Save Lives: “I Wish All My Patients

E
Were Like You and Engaged in Their Care”

I reviewed the published research focusing on evidence-based data and found that
20% (one in five people) have inherited high Lp(a), the most prevalent genetic risk
factor for cardiovascular disease; more than one billion people worldwide are
unaware they have at least a 60% increased risk for cardiovascular disease or death
(Nordestgaard et al. 2010; Kampstrup et al. 2009; Emerging Risk Factors
Collaboration et al. 2009; Patel et al. 2021). This lack of awareness was an unac-
ceptable situation that could not continue. The first sign of the disease, for some
people, is a heart attack or stroke. More than one billion families worldwide are
unaware of their actual risk. I thought I was rare and an outlier, but, as I discovered,
high Lp(a) is not a rare disorder (Nordestgaard et al. 2010).
When I mention these statistics, most people are shocked. Repeatedly, I hear
heart disease is 80% preventable, but what about the 20% who have inherited
an uncontrollable risk factor such as high Lp(a), familial hypercholesterolemia,
hypertriglyceridemia, and homocysteinemia and their age or gender? During
my research, it became increasingly clear we needed a dedicated charity to
raise awareness and help educate families about their genetic risk from the
fatty, sticky, Lp(a) particle in their blood on which diet and exercise have little
to no impact. These families, including friends, neighbors, or loved ones, may
not die from this inherited Lp(a) risk if we fund more awareness, advocacy,
community support, and research programs. Very few people talk about inher-
ited cardiometabolic disease, and even fewer people are diagnosed with it. I
would support adding comprehensive cardiometabolic genetic testing to the
risk calculators.
Our promise of value to our members was as follows:
“Guided by evidenced-based data on Lp(a), we help educate and empower our members to
save lives.”

Our vision was as follows:

“To live in a world where high Lp(a) is routinely diagnosed, treated, and family screened.”

Our mission was as follows:

“To reveal high Lp(a) as an inherited lipid risk for premature cardiovascular disease; edu-
cate and empower patients and save lives.”

Our inaugural 5-year strategy in 2013 was as follows:

“To save lives by increasing awareness, advocating for routine testing, and a specific treatment
for high Lp(a).”
416 S. R. Tremulis

What We Know: The Facts

• Fifty percent of hospital admissions for coronary artery disease have a normal
LDL-C <100 mg/dL (Sachdeva et al. 2009).
• Lp(a) is currently the strongest, single genetic risk factor for coronary heart dis-
ease and aortic stenosis (Kronenberg and Utermann 2013).
• Increasing evidence reveals that high Lp(a) is a genetic, independent, and causal
risk factor for coronary artery heart disease, atherosclerosis, thrombosis, stroke,
and aortic stenosis (Nordestgaard et al. 2010).
• Approximately sixty-three million people in the United States are unaware of
their risk from high Lp(a), one in five Americans and more than a one billion
people globally (Nordestgaard et al. 2010).
• The Lp(a) blood test is not part of the regular lipid panel.
• Traditional lifestyle preventative measures including diet and exercise have little
or no impact on Lp(a) levels (Mackinnon et al. 1997).
• High Lp(a) levels occur in all ethnic groups, but it is more common among
African Americans, South Asians, and Hispanics (Paré et al. 2019).

What We Know: The Clinical Evidence

The increasing clinical evidence high Lp(a) is a causal risk factor for cardiovascular
disease and calcific aortic stenosis.
• Epidemiological studies/meta-analyses (Emerging Risk Factors Collaboration
et al. 2009)
• Mendelian randomized studies (Kampstrup et al. 2009)
• Genetic association studies (Clarke et al. 2009)
• Insights from UK Biobank (Patel et al. 2021)
Randomized controlled clinical trials (RCT)—patients with high Lp(a) levels are
randomized to potential therapy. As of 2019, there are now at least three clinical
trials underway for a specific therapy to lower Lp(a) (Viney et al. 2016).
Kare Berg discovered Lp(a) in human serum in 1963. After 60 years, there still
is no FDA-approved therapy for lowering high Lp(a). With the launch of recent
Lp(a) clinical trials, there is hope on the horizon for patients with high Lp(a).
The Lipoprotein(a) Foundation was a patient-founded and patient-focused organi-
zation that helped reveal the impact of high Lp(a). It was supported by a team of
researchers, healthcare practitioners, and patient advocates who volunteered their
knowledge and passion for helping others. We were honored to have Lp(a) key opin-
ion leaders, both from the US and international arena, sharing their expertise and
research insights with the Lipoprotein(a) Foundation. Our success was rooted in pas-
sion, empathy, innovation, and commitment. The foundation took pride in its innova-
tive and grassroots approach to making real change for 20% of the global population
24 Elevated Lp(a): Why Should I Test For It, If I Cannot Treat It? A Patient’s Perspective 417

living with or at risk of cardiovascular disease or death due to high Lp(a). Based on
feedback from our member community, I identified a set of strategic program areas
for the foundation. Specific objectives were set within each program to help overcome
the barriers to adoption for Lp(a) testing, which included executing an integrated mar-
keting and communications plan within 5 years prioritizing grassroots efforts due to
minimal funding and resources. The Lipoprotein(a) Foundation and its community
made a measurable impact in these key strategic areas. Since 2013, the Lipoprotein(a)
Foundation has delivered impactful programs driving awareness, advocacy, commu-
nity support, and research to effect change and address unmet needs (Table 24.4).

Table 24.4 Overview of the foundation’s key accomplishments from 2013 to 2020 (The
Lipoprotein(a) Foundation 2020)
Then (2013) Now (2020)
No ICD codes for ICD-CM Codes E78.41 and Z83.430 approved—56% increase in
Lp(a) individuals and 71% increase in families diagnoseda
No Lp(a) contact 8000+ enrolled; helped enroll three phase 1 and one phase 2 clinical
registry trials. Published market research study on participating in clinical trials
during COVID-19 (Swerdlow et al. 2021)
No Lp(a) awareness 500+ million impressions from PR activities, 500+ online headline
postings, top Google ranking, featured in New York Times, USA Today,
Fox News, American Airlines, Martha Stewart Living, plus others.
Community outreach focused on high-priority gender, ethnic, and
disease state groups
No Lp(a) support Growing community online and in person—social media, patient forum
community program, community events, and support phone line
No Lp(a)-focused 140,000+ visitors from 166 countries to the website each year offering
website Lp(a) expert physician location services
No professional Lp(a) in ACC/AHA, NLA, ESC/EAS, and cholesterol guidelines as a
guidelines risk factor (Virani et al. 2022)
No group advocating Seven years of Lp(a) advocacy with NIH, CDC, and others
for Lp(a)
Little attention and NIH strategic research proposal (Tsimikas et al. 2018)—$400K grant
funding for Lp(a) awarded to Columbia University Medical Center
No gathering of Thirty top Lp(a) experts on SAB and CAB advisory board, including
experts representation from all our prioritized groups
No treatment options Five potential innovative treatments in development, three in clinical
trials; helped enroll three phase 1 (Akcea/Ionis, Amgen, Silence
Therapeutics) and one phase 2 clinical trial (Akcea/Ionis/Novartis)
No directory of Lp(a) 600+ physicians registered with the foundation
specialists
No standardized NHLBI/CDC working group conducted on global standardization of
Lp(a) blood test Lp(a) assay in humans (Lijuan et al. 2019)
The table was created from data from the 2019 Impact Report for the Lipoprotein(a) Foundation
L00012US 6/20. Copyright 2020 Lipoprotein(a) Foundation EIN: 46-3024812 a nonprofit, 501(c)3
patient advocacy organization
a
Data provided by Vladimir Polony from the Green Button team to the Lipoprotein(a) Foundation,
led by Nigam Shah at the Stanford Center for Biomedical Informatics Research from a representa-
tive sample database
418 S. R. Tremulis

Patients and their families need an accurate prediction of their risk for premature
cardiovascular disease to prevent the first symptom from being death. A 2016 study
by Mortensen et al. looked at statin eligibility and 5-year cardiovascular disease
outcomes in 37,892 individuals (57% women) aged 40–75 years of age in the
Copenhagen General Population Study (Mortensen et al. 2015). The study limita-
tions include that it only looked at Caucasian subjects and was limited to a 5-year
follow-up. It would have been more informative to include higher-risk Hispanic,
Black, and South Asian populations with high Lp(a). The study used the 2013
American College of Cardiology/American Heart Association (ACC/AHA) risk
prediction tool. In the results of their study, as noted by Dr. Vera Bittner (the
University of Alabama at Birmingham) in an accompanying editorial, “The study
suggests that Lp(a) levels might help identify the 8% of individuals who had an
event despite being ineligible for statins.” She noted, “Comprehensive risk factor
control is associated with improved prognosis, and our challenge is to develop care
models that will allow us to achieve such control.” Another perspective accompany-
ing the article notes, “Future research should be directed toward developing more
accurate risk prediction tools.” In the editorial accompanying this study, Dr. Valentin
Fuster, JACC editor-in-chief (Icahn School of Medicine at Mount Sinai, New York),
suggested, “Let’s begin to pay attention to high Lp(a) because it may explain cardio-
vascular events in patients who otherwise do not have a significant risk factor pro-
file.” (Bennet et al. 2088) There are 1.5 million people in the United States who have
a cardiovascular event every year; 8% of that number is 120,000 people (Erqou
et al. 2009).
The public appears to have little empathy for cardiovascular disease because it is
often perceived as self-inflicted. But would not it be good to reduce the emotional
and financial impact on US society of an estimated 120,000 people with only iso-
lated high Lp(a) having a cardiovascular event every year and many more globally?
Individuals can be diagnosed with a simple inexpensive Lp(a) blood test, but you
can also identify a family potentially at risk for generations to come. It is a simple,
blood test once in a person’s life and annual bloodwork is not required. We encour-
age healthcare practitioners to pursue continued education about this inherited lipid
risk. In medical practices, one in five individuals and their families already have
high Lp(a) and face at least a 60% increased risk of a cardiovascular event
(Nordestgaard et al. 2010; Kamstrup et al. 2009). Educating and empowering
patients about high Lp(a) does save lives. In 2019, the Lipoprotein(a) Foundation
was named a Top-Ranked Nonprofit by the leading platform for community-sourced
stories about nonprofits. The foundation received this award for successfully achiev-
ing the objectives of our 5-year strategic plan and because of community feedback
on our programs. A patient advocate, stated, “The Lipoprotein(a) Foundation has
helped save my life! It was the beginning of my journey, guiding me through what
I needed to test to identify what ended up being significant heart disease.” This tes-
timonial was just one of the many mission moments that occurred as we fulfilled our
objectives for the foundation.
24 Elevated Lp(a): Why Should I Test For It, If I Cannot Treat It? A Patient’s Perspective 419

Conclusion

Awareness

So, how can I have had normal LDL-cholesterol and almost died of a heart attack
despite having a healthy lifestyle, annual preventative health screening, and no sig-
nificant risk factors except my family history of cardiovascular disease? It is my
opinion that, unfortunately, there is low public awareness for personal inherited
cardiovascular disease risk (Sanderson et al. 2011). The data to generate that aware-
ness for high Lp(a) has been inconsistent and largely missing due to the lack of a
standardized Lp(a) assay for research purposes, drug target development, and level
1 data from clinical trials for a therapy to improve outcomes for patients. Level 1
data is the trigger to include a risk target into the global cholesterol guidelines if
they are trial-based versus risk-based, and those guidelines are periodically updated
(Marcovina et al. 2003). Often, the public perception is that cardiovascular disease
is entirely self-inflicted, and the stigma attached to it is similar to AIDS and lung
cancer, which reduces the funding and empathy that often drives awareness of a
disease state (Benson 2021). This is also the case for women’s heart disease due to
a 30-year deficit of women and heart disease data (Garcia et al. 2016). I was aware
of my family history of heart disease but not my risk as a young woman with a
strong family history. The global focus and funding imperative for COVID-19 vac-
cines show how an enormous-focused response might finally eliminate the insur-
mountable global burden of cardiovascular disease. All stakeholders involved in the
cardiometabolic disease industry should focus on driving awareness for personal-
ized, inherited cardiometabolic disease.

Women’s Heart Disease Data

In addition, we must do better for women in healthcare and recognize the unique
and important differences between men and women; women’s more subtle symp-
toms may be ignored or treated less aggressively than male patients (Garcia et al.
2016). I was one of those women with subtle symptoms treated less aggressively.
Many biases can impair diagnostic accuracy by humans. Availability bias, a cogni-
tive bias, can lead to diagnosis errors (Yagoda 2018).

Access to Latest Top-Quality Evidenced-Based Data

Without having the latest medical research data available on-demand to healthcare
providers at the point of care within their institution, a physician cannot be informed
about the latest evidence-based data to aid in care (Lenaerts et al. 2021). With the
420 S. R. Tremulis

advent of artificial intelligence (AI), there is promise for AI tools such as the Human
Diagnosis Project, also known as Human Dx, aiding in diagnosis if the human
biases do not become embedded in the AI tools (Human Diagnosis Project 2022).

Clinical Centers of Excellence for High Lp(a)

It would benefit patients and their families to establish focused clinical centers of
excellence providing equal access to specialized treatment and care for inherited
cardiometabolic disease with a priority given to underserved minority groups at
increased risk from high Lp(a). Developing a standard of care to direct families with
inherited cardiometabolic disorders to these clinical centers of excellence would
simplify access to state-of-the-art research and care (Elrod and Fortenberry Jr. 2017).

Lack of Standard of Care for High Lp(a)

There was no standard of care to manage me through my life or reproductive years

with high Lp(a). Including reproductive risk factors as part of cardiovascular risk
assessment in clinical guidelines would help identify women at risk. Identifying
reproductive risk factors such as amenorrhea, polycystic ovary syndrome, thyroid
disorders, pregnancy loss, and pregnancy complications at an early stage in a wom-
en’s life might provide a more accurate prediction of risk and facilitate the initiation
of strategies to modify potential risks. Including gynecologists on the care team for
women attending a clinical center of excellence for inherited cardiovascular disease
would provide a more comprehensive view of a women’s potential lifelong risk
(Garcia et al. 2016).

 apid Deployment of a Globally Standardized Assay

R
for High Lp(a)

We have known about high Lp(a) for 60 years and still do not have a globally stan-
dardized Lp(a) assay, which has hampered research and the progression of the body
of scientific evidence in this area (Marcovina et al. 2003). The bloodwork identify-
ing my only inherited, hidden, significant contributing risk factor, high Lp(a), was
performed after my life-threatening event. Including a globally standardized test for
high Lp(a) in the standard preventative lipid screening and risk calculators would
provide a more accurate prediction of risk for patients and their families, initiating
more aggressive primary and secondary prevention, which otherwise may not have
been identified (Mortensen et al. 2015).
24 Elevated Lp(a): Why Should I Test For It, If I Cannot Treat It? A Patient’s Perspective 421

Improved Risk Calculators

There is no path of vigilance for inherited high Lp(a) as there is for other diseases
from birth onward. The risk calculators that guide the standard of care for cardiovas-
cular disease are designed for population medical care and not personalized medi-
cine. They do not include inherited cardiometabolic risk markers such as high Lp(a)
or factor in premature cardiovascular disease at an age younger than 45 (Semaev
and Shakhtshneider 2020). I was one of those young people with high Lp(a) missed
by the risk calculators.

Precision Medicine

A prescription for cardiovascular disease prevention specifically tailored for the

individual and their genes is needed. The healthcare industry should remove finan-
cial penalties for patients diagnosed with an inherited risk. Instead, reward patients
for seeking to understand their risk for inherited cardiometabolic disease and take
action to optimize all their cardiometabolic risk factors. Patients pay for the testing
and care to build their natural history data in institutional databases. Allowing
patients to own and monetize their natural history data and be informed of the
research developments derived from that data would help expedite research partici-
pation. It would make the patient a true stakeholder in the research development
process.

Aspirin for Event Prevention for Patients with High Lp(a)

Personalized preventative medical care is costly to adopt for the general population.
Researching the benefit of aspirin use for high Lp(a), including aspirin resistance
and other forms of blood clotting disorders, could provide a cost-effective preventa-
tive solution for patients with high Lp(a) (Greving et al. 2008). It will not prevent
cardiovascular disease due to high Lp(a) but might save lives. By chance, I took
aspirin during my heart event due to public awareness information about blood clots
when flying.

National Database for Cardiovascular Disease

Developing a comprehensive national database of premature cardiovascular events,

as there is for cancer, accessible to all researchers would also help expedite research,
improve standards of care, and draw public attention to the emotional and financial
422 S. R. Tremulis

burden of premature cardiovascular disease in the United Sates and help prioritize
funding (Bilimoria et al. 2008). The healthcare industry could invest savings gener-
ated by preventing cardiovascular events and heart damage for 20% of the global
population with high Lp(a) into the cost of precision, preventative cardiometabolic
care. Improving the survival rate of a heart event is good but often moves the costs
along to managing the chronic condition of heart failure after the heart is damaged
(Heidenreich et al. 2013).

Measurable Impact But Still More Work to Be Done

The Lipoprotein(a) Foundation provided impactful programs to effect change to

prevent families from suffering the same fate as my family. There is still much work
to be done to improve the healthcare process for patients with high Lp(a) and our
knowledge of the overall health consequences of high Lp(a). However, the founda-
tion made a measurable impact on awareness, advocacy for the rapid deployment of
a standardized assay, improvements in the risk calculators, screening, and diagnosis
for inherited high Lp(a), expediting the development of therapies and educating and
empowering the healthcare community and the public to help save lives with very
limited funding and resources (The Lipoprotein(a) Foundation 2020).
Unfortunately, on July 31, 2020, the Lipoprotein(a) Foundation dissolved after a
90% reduction in donations due to COVID-19 and increasing costs. I was honored
to represent the more than one billion people globally living with or at risk of car-
diovascular disease due to high Lp(a). My educational journey with high Lp(a) con-
tinues as more research identifies new insights into high Lp(a) and the origins of my
inherited immuno-cardiometabolic risk. I am an educated and empowered individ-
ual who actively participates in their care. I dedicate this chapter to my family and
all those families with a history of inherited cardiometabolic disease and Dr.
Tomoaki Hinohara for saving my life.

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Chapter 25
Unresolved Questions

Gerhard M. Kostner and Karam Kostner

It is now almost exactly 60 years since Kare Berg first described an extra pre-β band
found in lipid electrophoresis that later was named sinking pre-β and finally Lp(a).
There was a continuous up and down in Lp(a) research that was mainly driven by
actual research findings related to Lp(a) function, metabolism, correlation to cardiac
risk, and epidemiology. The following are four key findings that caused a major
boost in the interest for Lp(a) that led to a flurry in publications:
1. Cloning of LPA by McLean and Lawn demonstrating homology of the apo(a)
protein and the LPA gene with plasminogen (McLean et al. 1987; Utermann 2001).
2. The unique size polymorphism caused by variations in the number of K-IV2
repeats that paved the way for consecutive genetic epidemiological studies by
the group of Utermann [reviewed in (Utermann 2001; Kamstrup et al. 2009)].
3. The demonstration of the causal relationship of elevated Lp(a) levels with ath-
erosclerosis and coronary heart diseases by Mendelian randomization in the
Copenhagen Heart Study (Kamstrup et al. 2009; Graham et al. 2016).
4. The development of a very efficient therapy for elevated-Lp(a) with antisense
oligonucleotide (ASO) therapy by the group of Tsimikas (Graham et al. 2016).
All these exciting milestones in Lp(a) research cannot change the fact that our
knowledge in all areas of Lp(a) research is still fragmented. This is due to a lack of
knowledge in the following areas:
1. Function
2. Metabolism

G. M. Kostner (*)
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria
e-mail: [email protected]
K. Kostner
Department of Cardiology, Mater Hospital and University of Queensland, Brisbane, Australia

© The Author(s), under exclusive license to Springer Nature 425

Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6_25
426 G. M. Kostner and K. Kostner

3. Pathophysiology
4. Lp(a) measurement in clinical laboratories
5. Significance in diseases other than related to atherosclerosis
6. Therapy
Many of these points have been already discussed in the previous chapters and
will therefore only be summarized here:

I s There a Function of Lp(a) in Longevity and Suppression

of Malignant Growth?

Nature rarely designs complex structures such as apo(a) without any physiological
function in mind. This may not necessarily be true for the whole human population
but may be only for some ethnicity. Thus, it has been speculated that individuals
exposed to dangerous parasites or bacterial and viral infections may have an advan-
tage if they have high Lp(a) plasma concentrations. The actual mechanism behind
this is far from being clear, yet it may explain why populations originating from
African countries where such diseases prevail have significantly higher Lp(a) levels
than Europeans and Asians (Schmidt et al. 2006; Sandholzer et al. 1992).
Another function of Lp(a) might relate to aging and longevity. In fact, lipids and
lipoproteins have been implicated in life span regulation (Joshi et al. 2017), and in
the list of genes suggested in previous research to code for such factors, LPA, APOE,
and APOAI are found. We addressed this question in early investigations in view of
age-related diseases and hypothesized that assuming that Lp(a) might be a signifi-
cant risk factor for atherosclerosis and MI, individuals with high Lp(a) should die
earlier than individuals with low Lp(a). Thus, we first measured Lp(a) in a family
kindred within three generations and anticipated that Lp(a) values in the older gen-
eration might be lower than in the younger one (Pagnan et al. 1982). In fact, the
opposite turned out to be the case. In another study, we measured Lp(a) in octo-­
nonagenarians (Zuliani et al. 1995), and although we could not confirm a correla-
tion of Lp(a) with age, to our surprise, the plasma Lp(a) concentration of “very old”
individuals and more importantly the apo(a) isoform distribution did not differ sig-
nificantly from that of young individuals (Zuliani et al. 1995). Comparable studies
have been also published from other investigators (Wood and Schumacher 1995).
The question obviously arises about the physiological relevance of these obser-
vations—or in other words—does this relate to a physiological function of Lp(a).
We studied this possibility by asking whether apo(a) might be involved in angiogen-
esis (Schulter et al. 2001). Angiogenesis has been found to be important not only for
tumor growth but also for cancer metastasis. O’Reilly et al. (1994) were first to
demonstrate that angiostatin, a proteolytic cleavage product of plasminogen secreted
into urine, has very high angiostatic properties. Since proteolytic fragments from
apo(a) are found in urine as well, we purified these fragments and tested their angio-
static properties in vitro in a tube forming assay: indeed apo(a) from urine that
25 Unresolved Questions 427

consist mainly of N-terminal fragments exhibited a significant reduction of tubes in

the Matrigel assay. In another study, transgenic apo(a) mice and control mice were
injected with 107 Ehrlich ascite cells that form solid tumors within 4 months. The
number and size of solid tumors in tg-APOA mice were significantly lower as com-
pared to control mice. Whether or not these findings are applicable to humans
in vivo remains to be demonstrated.
Another physiological function of Lp(a) might relate to its high binding capacity
of secretory phospholipases (sPL-A2), PAF acetyl hydrolase (PAF-AH), and oxi-
dized phospholipids (OxPhos). We were among the first to demonstrate that Lp(a)
carries a three times higher activity of phospholipase-A2 as compared to LDL
(Gorges et al. 1995). In addition, a manifold higher PAF-AH activity compared to
LDL has been demonstrated in Lp(a) (Blencowe et al. 1995). This is probably one
reason why Lp(a) is less susceptible to oxidation than LDL (Sattler et al. 1991). The
transport of OxPhos by Lp(a) was suggested to be the major culprit for its patho-­
mechanism in atherogenesis: OxPhos Lp(a) complexes that enter the arterial
intima—particularly when other risk factors such as high LDL are abundant—trig-
ger inflammatory processes, recruitment of lymphocytes and cytokines, foam cell
formation, and all the well-described features of atherosclerosis and heart diseases.
On the other hand, nature seldom produces pathogenic substances for fun, and we
hypothesize that the absence of atherogenic bystanders Lp(a) might be beneficial
and counteracts the development of cancer. This might relate to the mentioned inter-
ference with angiogenesis on one hand and to the Lp(a)-OxPhos-phospholipase
pathway on the other hand. Phospholipase-A2—and in particular PAF-AH—cleave
and then inactivate free radicals and hydroperoxides found on short-chain fatty
acids of phospholipids. The latter substances have been found to trigger cancero-
genesis by creating an inflammatory milieu, chemokine attraction, signaling, and
cell growth (Hermann et al. 2014). Alternatively, PL-A2 receptor-1 that is found on
the surface of numerous cancer cells has been suggested to possess tumor suppres-
sor activity by interaction with certain phospholipases (Sukocheva et al. 2019).
Taken together, there is a great deal of speculations about the physiological func-
tion and possible beneficial roles of Lp(a), and this needs to be addressed in future
research.

Metabolism

Biosynthesis and assembly: The chapter authored by Dan Rader and John Miller in
this book gives an excellent overview on the current concepts of Lp(a) metabolism.
As these authors point out, our research group was first to demonstrate that the
Lp(a) metabolism is distinct from that of LDL: other than for LDL, VLDL is not a
precursor of Lp(a) (Krempler et al. 1979). We also published that other than for
LDL, plasma Lp(a) concentrations are governed by the rate of biosynthesis—or in
other words, individuals with high Lp(a) concentrations show a high rate of apo(a)
expression (Krempler et al. 1980). The expression of apo(a) is driven by
428 G. M. Kostner and K. Kostner

transcription factors and nuclear receptors. In silico search in the APOA promoter
revealed more than 70 binding regions for known transcription factors; two of such
response elements, ETS −1630 to −1615 and DR-1 −826 to −814, turned out to be
of particular importance as they are strongly turned off by FXR signaling
(Chennamsetty et al. 2011). We also identified several cAMP response elements that
were responsible for the Lp(a) lowering effect of nicotinic acid (Chennamsetty et al.
2012). But how about the role of all the other response elements that we identified
in the apo(a) promoter? This is an ample research field that deserves much further
attention.
Following APOA transcription, translation, and glycosylation, the mature apo(a)
protein assembles with LDL to form Lp(a). The individual steps in assembly have
been addressed in numerous studies in the past, yet there is currently no general
agreement on the site where this might occur. Whereas some data favor an intrahe-
patic assembly, other data point toward an assembly on the surface of liver cells, and
even others suggest an assembly in circulating blood. Undoubtedly there are further
studies needed to clarify the location of the assembly of Lp(a).
Another fully open field is the role of the APOA expression in the brain and testes
(McLean et al. 1987): does this have any physiological relevance? Nobody has ever
studied this rather interesting phenomenon in detail.
Catabolism: In our early experiments in man, we found that FH patients lacking
LDL receptors catabolize Lp(a) to the same extent than healthy controls. The cata-
bolic rate in both, however, was markedly slower as compared to LDL (Krempler
et al. 1980). This led us to conclude that LDL receptor-mediated catabolism plays
little role in Lp(a) removal from circulation. Since then, a wealth of publications
appeared that found Lp(a) binding to almost any specific lipoprotein receptor
including the apoE receptor, the VLDL receptor, the remnant receptor, LRP recep-
tor, the asialoglycoprotein receptor, the plasminogen receptor, several scavenger
receptors, and possibly others. Fact is that in all animal studies even in hedge hogs,
approximately 50% of intravenously injected Lp(a) is taken up by the liver (Kostner
et al. 1997). In addition to the liver, also the kidney plays an important role in Lp(a)
metabolism (see chapter of H. Dieplinger in this book). Apo(a) is fragmented in the
blood by Ca2+-dependent proteases, and even large fragments are secreted into urine
(Frank et al. 2001). The significance of this pathway has never been clarified so far.

 athophysiology: What Are the Most Important Determinants

P
of Lp(a) Pathogenicity?

We know more about the pathophysiology of Lp(a) than about its physiology. Lp(a)
consists of an LDL particle with all its proatherogenic properties. In addition, Lp(a)
gets into atherosclerotic plaques by interacting with proteoglycans, which causes
foam cell formation and inflammation; Lp(a) also carries OxPhos that trigger
inflammation. Due to the homology of apo(a) with plasminogen, Lp(a) has also
25 Unresolved Questions 429

been connected to fibrinolysis and thrombosis [reviewed in (Boffa 2022)]: on one

hand, it was suggested that Lp(a) interferes with the conversion of plasminogen to
plasmin by plasminogen activator and urokinase, and on the other hand, it interferes
with the action of PAI on the endothelial surface. Further studies revealed that Lp(a)
is incorporated into fibrin clots and aggravates the action of plasmin in fibrinolysis.
Considering the complexity of hemostasis and fibrinolysis that involve numerous
significant components working in a concerted action to prevent bleeding on one
hand and uncontrolled blood clotting on the other, it seems questionable that Lp(a)
plays a significant role in these pathways—and if it does—this certainly needs fur-
ther clarification.

Lp(a) Analysis in Clinical Laboratories

The early laboratory methods for measuring Lp(a) were based on immunochem-
istry. Almost all immunochemical methods including radial immune-diffusion
(Ouchterlony test), rocket electrophoresis, radioimmunoassay (RIA), ELISA
(enzyme-linked immunosorbent assay), DELFIA (dissociation-enhanced lantha-
nide fluorescence immunoassay), and more methods have been applied. Today,
high-throughput methods for Lp(a) quantitation are based on immune-turbidime-
try or immune-nephelometry. Unfortunately, commercial methods are far from
being harmonized and subject to drastic improvements. There is currently no vali-
dated reference material commercially available, and reference methods for typ-
ing Lp(a) standards are still under development. All the problems with Lp(a)
quantitation in the clinical laboratory are impressively documented in the chap-
ters from S. Marcovina and C. Cobbaert and D. Sullivan in this book. Another
article that highlights this thematic was recently published by F. Kronenberg
(Kronenberg 2022). Kronenberg looks at this topic from practical point of view
and stresses the point that due to the great genetic heterogeneity of LPA, it will be
hardly possible to have a validated routine method for high-throughput Lp(a)
measurement at reasonable costs available that fulfills all standard requirements
of ISO 17511:2020. Former studies where commercial assays for Lp(a) were
evaluated revealed a significant number of outliers that at present time cannot be
explained (Scharnagl et al. 2019). C. Cobbaert established an IFCC (International
Federation of Clinical Chemistry)-sponsored working group with experts in the
field of mass spectrometry and laboratory medicine with the goal to develop a
reference method based on LC-MS (liquid chromatography-mass spectrometry)
http://www.ifcc.org/ifcc-­scientific-­division/sd-­working-­groups/wg-­apo-­ms/. In
addition, this group works on the preparation of a harmonized SI-traceable refer-
ence material that shall be used by industry to standardize their commercial
assays. Although the IFCC working group has been operational for more than 5
years, it may take another 2–3 years to come up with a practicable reference
method and a reference material.
430 G. M. Kostner and K. Kostner

Major hurdles in this study are the following

1. SI units in clinical chemistry need to be expressed in molar units, and this is hard
to achieve for Lp(a) because of the large size heterogeneity ranging from 300 to
800 kD for apo(a). Also, apo(a) contains a variable number of identical K-IV2
repeats and in addition other homologous K-IV’s cross reacting with polyclonal
antibodies that are normally used for nephelometric Lp(a) assays. In order to
overcome the bias created by these properties of apo(a), commercial assays use
an algorithm for correcting the measured values—yet this is just an approxima-
tion and not correct for a large number of samples.
2. Even using a standardized reference method such as ELISA with monoclonal
antibodies or a validated LC-MS method, there are numerous outliers observed
in bias plots by comparing two or more methods. The reason behind is unknown
and needs further research.
3. Not all apo(a) is complexed with LDL, and there is an appreciable amount of
free apo(a) and apo(a) fragments found in plasma that might vary in concentra-
tion particularly in kidney disease and sepsis. These fractions are differentially
measured in various assays but need to be considered for different assays.
4. A further question that needs to be answered beyond any doubt is the atheroge-
nicity of large versus small apo(a) isoforms. There are quite a few papers pub-
lished on this issue, but they are partly controversial mainly due to problems
mentioned in (2) and (3). It is also not clear whether polymorphic or mutant
forms of apo(a) are to the same extent atherogenic than “wild-type” apo(a).
5. What is the significance of the variation in lipid composition of Lp(a) from indi-
vidual donors? As pointed out in the chapter of G. Kostner in this book, it turned
out that Lp(a) in reality is far from being homogenous in its lipid part. This is
corroborated by data from novel Lp(a) cholesterol assay stressed by C. Yeang
(Yeang et al. 2021) who showed that Lp(a) cholesterol content in percent relative
to the Lp(a) mass varies from 5.8 to 57.3% in his study group (Yeang et al.
2021). An interesting question would be whether the atherogenicity of Lp(a)
relates to its cholesterol content or lipid composition.
6. Is there an easy way to quantify LDL-C without Lp(a)-C? So far, corrections for
Lp(a)-C have mostly done—if at all—by subtracting 30% of the Lp(a) mass
from LDL-C—yet considering the results from C. Yeang (Yeang et al. 2021),
this gives quite striking erroneous results.
Summing up the open questions related to laboratory methods, our knowledge in
this field is limited and deserves intensive future research.

Does Lp(a) Lowering Reduce Hard CV Endpoints?

Apart from LDL apheresis therapy, it is currently not clear whether lowering of
Lp(a) reduces hard cardiovascular endpoints. Several phase 2 and 3 trials with anti-
sense and siRNA-targeted therapies are exploring this currently. Most lipidologists
25 Unresolved Questions 431

and clinicians recommend to lower LDL cholesterol more aggressively to levels

below 100 mg/dL in case of elevated Lp(a) levels, even though the hard evidence for
this is also lacking.
Pelacarsen is an antisense oligonucleotide that targets apo(a) mRNA. It has
shown Lp(a) reductions of more than 80% and is being tested in a large phase 3 trial
called HORIZON (Viney et al. 2016).
Olpasiran is an example of an siRNA targeting apo(a) mRNA that has shown to
also reduce Lp(a) by more than 80% and is also being tested in a phase 2 study, with
a phase 3 study planned for later this year (Koren et al. 2020).
These trials will help answer the question whether Lp(a) reduction leads to CV
endpoint reduction.

How Much Should Lp(a) Be Lowered?

Mendelian randomization studies suggest that to achieve significant CV risk reduc-

tion, similar to what has been seen with LDL reduction of 1 mmol/L, Lp(a) would
have to be reduced by 250 nmol/L. However, these studies were population based
and included many patients with low Lp(a) levels, which would usually not be con-
sidered for Lp(a) lowering. In addition, if Lp(a) is more atherogenic than LDL,
smaller reductions in Lp(a) may prove more important clinically. Data from recent
PCSK9 inhibitor trials indicate that smaller reduction in Lp(a) may have a signifi-
cant effect on CV endpoints.
In the FOURIER (Cardiovascular Outcomes Research With PCSK9 Inhibition in
Subjects With Elevated Risk) trial, reduction in risk of MACE (major adverse car-
diac event) with evolocumab was associated with baseline and change in Lp(a) lev-
els (Gencer et al. 2021).
In the ODYSSEY OUTCOMES (Evaluation of Cardiovascular Outcomes After
an Acute Coronary Syndrome During Treatment With Alirocumab) trial, reduction
in risk of total cardiovascular events with alirocumab was also associated with base-
line and change in Lp(a) levels (Bittner et al. 2020).
Reduction in risk of major adverse limb events (MALE) with alirocumab was
also associated with baseline and change in Lp(a) levels.
In our opinion, Lp(a) should be lowered as low as possible in high-risk individuals.

Which Is the Most Effective Therapy to Lower Lp(a)?

LDL apheresis is effective and leads to significant Lp(a) reductions as well as HR

reductions in observational studies but has not been tested in CV outcome trials.
Apheresis is FDA approved and can be considered in patients with elevated Lp(a) at
very high risk of ASCVD (atherosclerotic cardiovascular disease), but it is expen-
sive, inconvenient, and not widely available (Moriarty et al. 2019). Emerging
432 G. M. Kostner and K. Kostner

antisense and RNA technologies are more specific and show much larger Lp(a)
reductions. Clinical outcome trials are currently underway (HORIZON,
NCT04023552; OCEAN(a)-DOSE; NCT04270760).
Monoclonal PCSK9 antibodies, as well as RNA-based inhibitors of PCSK9,
which lower LDL-C, can also reduce Lp(a) by up to 35%. While they are not reim-
bursed through Medicare for Lp(a) treatment, elevated Lp(a) is often treated coinci-
dentally in patients with FH or progressive ASCVD whose LDL-C remains elevated
despite maximal statin and ezetimibe therapy. Sub-analysis of the major outcomes
trials for PCSK9 inhibitors has shown greater relative and absolute risk reduction in
patients with elevated Lp(a). In Cardiovascular Outcomes Research With PCSK9
Inhibition in Subjects With Elevated Risk (FOURIER) trial, reduction in risk of
major acute coronary events (MACE) with evolocumab was associated with base-
line and change in Lp(a) levels (O’Donoghue et al. 2019). In the Evaluation of
Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment
With Alirocumab (ODYSSEY OUTCOMES) trial, reduction in risk of total cardio-
vascular events with alirocumab was also associated with baseline and change in
Lp(a) levels (Szarek et al. 2020). Reduction in risk of major adverse limb events
(MALE) with alirocumab was also associated with baseline and change in Lp(a)
levels (Schwartz et al. 2020). These trials support the conclusion that elevated Lp(a)
is a major driver of residual risk.

The Effect of Statins on Plasma Lp(a) Levels

Statins do have a variable effect on plasma Lp(a). Although most statins are able to
lower Lp(a) to some extent, there are numerous patients who do not respond to
statins at all or even show an increase of Lp(a) on statin therapy (Kostner et al.
1989). The mechanisms responsible have never been defined. The important fact to
remember is that statins are beneficial in patients with elevated Lp(a) by removing
LDL, which reduces some of the CV risks associated with elevated Lp(a).

Who Should We Screen for Lp(a) and How?

Knowledge of Lp(a) could be particularly valuable in reclassification of patients at

intermediate risk of ASCVD, as assessed by established risk algorithms. Most
societies recommend that Lp(a) should be measured in individuals with a personal
or family history of premature ASCVD (or aortic valve stenosis) and familial
hypercholesterolemia (FH) or in those with recurrent coronary events despite opti-
mal LDL cholesterol on diet and statins, with or without ezetimibe. Information on
Lp(a) may guide more aggressive treatment of conventional risk factors or the
need to assess subclinical atherosclerosis with cardiac CT scanning (Kostner
et al. 2018).
25 Unresolved Questions 433

We recognize the importance of elevated Lp(a) as a cardiovascular risk enhancer,

particularly in light of the significant residual risk that remains despite reduced
LDL. The precise value of cascade testing first-degree relatives of an index case
with elevated Lp(a) has not been demonstrated. However, it could help define and
consolidate the family history of ASCVD and improve adherence to existing thera-
pies in secondary prevention, as well as to healthy lifestyle and behavior in primary
prevention in family members. Elevated Lp(a) with a coexistent polygenic hyper-
cholesterolemia or familial combined hyperlipidemia may mimic FH and should
always be considered in patients who return a negative genetic test for FH.
There is a great deal of information available on Lp(a) physiology and patho-
physiology that is published in more details, that is, >9000 10,000 scientific publi-
cations. This should not mislead that there are still numerous open questions and
gaps in our knowledge, and we should capitalize the current hype in Lp(a) to address
this topic more rigorously.

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Index

A protein structures, 45–47

Acute coronary syndrome (ACS), structural features, 47
150–151, 276 APOA transcription, 428
Acute lymphoblastic lymphoma, 140 ApoE*3-Leiden CETP mouse model, 400
Ag system, 1, 40 Apolipoprotein(a) (apo(a)), 8–15,
Alirocumab, 351 17–21, 23–25
All Lab Total Mean (ALTM), 335 Apolipoprotein B (ApoB), 382
American College of Cardiology (ACC), 278 antisense oligonucleotides, 102
American College of Cardiology/American lowering, 199–201
Heart Association (ACC/AHA) risk synthesis/secretion, 85
prediction tool, 418 Arterial vascular disease, 22
American Heart Association (AHA), 278 Artificial intelligence (AI), 420
Angiogenesis, 141, 142, 147 ASCVD, see Atherosclerotic cardiovascular
Anticoagulation, 193, 199, 201 disease (ASCVD)
Anti-fibrinolytic mechanisms, 144 Asialoglycoprotein surface receptor (ASGPR),
Antiplatelet agents, 199 130, 215, 363, 428
Antisense oligonucleotide (ASO), 85, 86, 367 Aspirin, 414
antisense oligonucleotide pharmaceutical Associations of Statutory Health Insurance
activity, 366 Physicians, 379
development of, 363, 365 Atherosclerosis, 262
generations of, 362, 363 aortic stenosis, 163
with IONIS-APO(a)LRx/AKCEA-APO(a) ASCVD, 160
LRx,/TQJ230/Pelacarsen, 370 genetic studies, 165
ISIS-APO(a)Rx/IONIS-APO(a)Rx, 367 inflammation, 162
Lp(a) HORIZON trial, 373, 374 mortality, 164
pre-clinical proof of, 360 myocardial infarctions, 163
Aortic leaflet valve interstitium, 328 PAD, 164
Aortic stenosis (AS), 163 stroke, 163
Apheresis, 352 treatment, 166
Apo B100-containing particles, 283 Atherosclerotic cardiovascular disease
Apo(a), 40, 42–53 (ASCVD), 160, 162, 173, 180, 191,
carbohydrate moiety, 47 192, 199–201, 275, 409
gene variants, 49 Atherosclerotic profile, 161
isoforms, 57 Atherothrombosis, 191, 195, 199–201
mutations, 51

© The Editor(s) (if applicable) and The Author(s), under exclusive license to 437
Springer Nature Switzerland AG 2023
K. Kostner et al. (eds.), Lipoprotein(a), Contemporary Cardiology,
https://doi.org/10.1007/978-3-031-24575-6
438 Index

Atherothrombosis Intervention in Metabolic Commercial immunonephelometric assay, 334

syndrome with low HDL/high Continuous ambulatory peritoneal dialysis
triglycerides: Impact on Global (CAPD), 218–221
Health outcomes (AIM-HIGH) Copenhagen City Heart Study, 251, 253
study, 276 Copenhagen cohorts, 255
Autoimmune disease, 147, 148 Copenhagen General Population Study
Awareness, 419 (CGPS), 242, 251, 347
Coronary artery disease, 135, 145, 148–151,
162, 165
B Coronary calcium scores, 275
ß-lipoprotein, 275 Coronary heart disease (CHD), 160, 165, 192,
Bone morphogenetic protein 2 (BMP2), 163 199, 200
British sham-controlled apheresis study, 399 COVID-19, 152–153, 197, 198, 419, 422
CVD, see Cardiovascular disease (CVD)

C
CAD, see Coronary artery disease (CAD) D
Calcific aortic stenosis, 409 Danger associated molecular patterns
Calcific aortic valve disease (CAVD), 173, (DAMPs), 263
275, 290 Deep vein thrombosis, 145
Calcific aortic valve stenosis (CAVS), 148, DELFIA, 429
149, 162 Denka Seiken based assays, 307
Candidate reference measurement procedure The Denka Seiken reagents, 306
(cRMP), 338, 339 Direct assays, 235
Cardiovascular disease (CVD), 135, 138, 148, Dresden Center for Extracorporeal Therapy,
150, 162–166, 220, 221, 328 386, 395
Cardiovascular mortality, 164
Cardiovascular outcomes trials, 354
Cardiovascular risk management, 349 E
Catabolism, 428 Electrospray differential ion mobility
CD14++CD16+ intermediate monocyte analysis, 310
subpopulation, 266 Endogeneous fibrinolytic system, 190, 195
CD16+ monocytes, 264 Enzyme-linked immunosorbent assay
CDC-Clinical Standardization Program (ELISA), 304, 328, 429, 430
(CSP), 342 E-selectin, 162
Cell surface receptors European ancestry, 235
kidney receptors, 130 European Society of Cardiology (ESC)/
liver receptors, 127–130 European Atherosclerosis Society
Lp(a) (EAS) guidelines, 278, 347
assembly, 127 European Union, 362
catabolism, 127 Evinacumab, 400
structure, 125–127 Evolocumab, 351
macrophages receptors, 131 Extracorporeal therapy, 269, 381
Center for Disease Control (CDC), 303 Ezetimibe, 350
Cerebral venous thrombosis, 191, 193
CETP inhibitors, 352
Cholesterol Reference Method Laboratory F
Network, 298 Familial hypercholesterolaemia (FH), 235,
Cholesteryl ester transfer protein 276, 289, 432
(CETP), 85, 352 Fibrinolysis, 141, 142, 145, 147, 152,
Clot lysis, 195 179–181
Coagulation, 195–196
Index 439

Fractional catabolic (or clearance) rate International Lp workshop, 3

(FCR), 93, 382 International Organization for Standardization
Framingham Risk Score, 411 (ISO), 300
Intracellular adhesion molecule
(ICAM), 162
G In-vitro diagnostic manufacturers, 299
GalNac antisense, 353 In vivo kinetic studies, 216
GalNAc-modified ASOs, 370 Ionis pharmaceuticals, 360
Genetic Mendelian randomization studies, 347 IONIS-APO(a)-LRx trial, 371
Genetics Iron deficiency, 385
KIV-2 VNTR, 63 Ischemic stroke, 246
Lp(a) concentration, 55–61, 68 ISIS-APO(a)Rx complementary binding
Lp(a), structure of, 57 sites, 364
LPA KIV-2 VNTR, 57–62, 67
simple repeats or restriction site
polymorphisms, 63 J
SNPs, 63–66 Joint Committee for Guides in Metrology
German Lipoprotein Apheresis Registry, 388 (JCGM), 299
Glomerular filtration rates (GFR), 209, Justification for the Use of Statins in
217, 218 Prevention: An Intervention Trial
Guidelines and position statements, 291 Evaluating Rosuvastatin (JUPITER)
trial, 348, 349
Justification for the Use of Statins in the
H JUPITER study, 276
Healthcare system policies, 291
HEART UK consensus statement, 278, 382
Hemodialysis, 218–219 K
Hemorrhagic strokes, 195 Kexin type 9 (PCSK9) inhibition, 166
Hepatocellular cancer, 140 Kidney disease
Hepatocellular carcinoma (HCC), 142 early stages, 217, 218
High-density lipoprotein (HDL) hemodialysis, 218–219
metabolism, 130 high Lp(a) concentrations, 221, 222
High-density lipoprotein cholesterol Lp(a), catabolism of, 221
(HDL-C), 351 nephrotic syndrome, 217
HMG-CoA-reductase inhibitors, 24 pathogenesis of, 222
Homeostasis, 189 peritoneal dialysis, 218–219
HORIZON, 252 proteinuria, 217
HPS2-THRIVE Study, 397 transplantation, 220
Human antisense oligonucleotides, 363 Kidney receptors, 130
Human LPA gene, 58 Kringle 4 type 2 (KIV2), 231
Hyperlipoproteinemia, 392 Kringle domains, 9, 141
Kupffer cell-specific glycoprotein
receptor, 48
I
Ile4399Met polymorphism, 145
Imaging modalities, 275 L
Inflammation, 135, 137–140, 142, 147–150, LCAT deficiency (LCAT-D), 52
152, 153, 191, 192, 196, 197, 200 LDL-cholesterol, 409–415
INTERHEART study, 236 LDL receptor (LDLR), 214, 215
International Federation of Clinical Chemistry LDLR-related protein 1 (LPR1), 118
and Laboratory Medicine Ld system, 40
(IFCC), 300 Lipid-modifying agents, 351, 352
440 Index

Lipoprotein primary and secondary reference

accurate test results, 343 materials, 340
age and sex and adjusted Cox proportional regression dilution bias, 251
hazard ratios, 253 risk thresholds for, 278
ASCVD pathogenesis, 264, 265 safety and quality, 286, 287
and ASCVD risk assessment, 276, 277 science and metrology, 326, 327
assay methods, 285 scientific publications on, 252
benefit of, 347, 348 WHO reference material for, 342
cardiovascular risk management, 335, 336 Lipoprotein apheresis (LA)
characteristics and design of, 328–330 adverse effects and contraindications of,
clinical application, 289 385, 386
on clinical utility, 334 apo(a) synthesis, 401
contemporary immunoassay-based cardiovascular events, 386–390, 395–398
methods, 344 case reports, 391, 393
in contemporary primary prevention in clinical laboratories, 429, 430
studies, 252, 255 effective therapy, 431
in contemporary secondary prevention efficiency of, 380–382
studies, 256, 258 hard CV endpoints, 430
continuum of apoB-containing lipoprotein high Lp(a), 390, 420
classes, 327, 328 history of, 378, 380
current therapies on, 348–353 longevity and suppression, 426, 427
and CVD, 231, 233 metabolism, 427, 428
aortic valve stenosis by, 244 oxidized phospholipids, 385
concentration distribution, 242 pathophysiology, 428
elevated Lp(a) levels, 233, 234 plasma Lp(a) levels, 432
familial hypercholesterolemia, 235 pros and cons of, 398, 399
FOURIER trial, 233 reveal Lp(a), 410–413
ischemic stroke in, 246 screen for, 432
primary and secondary prevention TEST Lp(a), 413, 414
cohorts, 252 Lipoprotein receptor-related protein-1
race and ethnicity on, 235, 236 (LRP1), 129
determinants of inaccuracy, 343 Lipoprotein(a) Foundation, 422
dyslipidaemias, 232 Lipoprotein(a)(Lp(a)), 40
epidemiological and genetic animal studies, 12
evidence, 231 antisense therapy, 24
experimental drugs apo(a), 9, 10
antisense oligonucleotides, 353 assembly, 127
small interfering RNA, 354 catabolism, 127
future perspectives, 258 CHD, 17, 18, 20, 21
healthcare systems, 290 chemical composition, 44, 45
and IgG and IgM autoantibodies, 262 definition, 207
inflammatory mediators, 267, 268 discovery of, 1–4
and innate immunity cells, 263, 264 epidemiological and genetic studies, 208
interim solutions, 342 evolution, 151, 152
International Guidelines, 278 functional studies, 10, 11
interpretation, 289 genetics, 12–16, 92
measurement for clinical purposes, historical review, 1
281, 282 HORIZON trial., 374
mechanisms of, 268 isolation and characterization, 6–8
method selection, 282, 284 LDL structure, impact of, 51–53
metrological traceability of, 326 Mendelian randomization, 18–21
next generation reference measurement metabolism, 8, 9, 47
procedure, 340, 341 non-genetic effects, 22
next generation SI-traceable apo(a), plasma concentrations, 49–51
337, 338 preparation, 43, 44
Index 441

protein structures, 45 Lysophosphatidic acid, 268

PubMed search, 1
purification, 42
renal disease, 22, 23 M
structure, 57, 92, 125–127 Macrophage receptors, 131
transgenics, 12 Major adverse cardiovascular events (MACE),
type 2 diabetes mellitus, 22 257, 265, 350
Liposorber D, 395 Major adverse limb events (MALE), 431
Liver receptors, 127–130 Malignancies, impact of, 140
Low density lipoprotein receptor-related Mendelian randomization, 17–21, 23, 64, 195,
protein 1 (LRP-1), 267 232, 251, 289, 431
Low-density lipoprotein (LDL) Mendelian randomization-phenome-wide
cholesterol, 257 association approach, 194
Low-density lipoprotein (LDL) Metastatic breast cancer, 140
particle, 92, 283 Metrological traceability, 299, 326
Low-molecular weight (LMW) isoforms, Mighty Medic Group, 278
208, 222 MOE-gapmers, 362
Lp(a) metabolism Monoclonal PCSK9 antibodies, 432
clearance and catabolism, 82–84, Monocyte/macrophage phenotype, 178
95, 96, 209 Monocytes, 263, 264
immune-histochemical studies, MTP inhibitor lomitapide, 352
209–211, 214 MultiSELECt study, 390, 399
in vivo kinetic studies, 216 Myocardial infarctions (MIs), 161, 163,
LDL receptor, 214, 215 165, 254
concentration, 79–81
drug effects
apoB synthesis/secretion, 85 N
niacin, 85 National Association of Statutory Health
PCSK9 inhibitors, 84 Insurance Physicians, 380
pelacarsen, 86 National Database for Cardiovascular
statins, 84 Disease, 421
Volanesorsen, 86 National Institute of Standards and
LDL receptor, 80–82 Technology (NIST), 298
lipid-regulating agents National Lipid Association (NLA), 278
apoB antisense oligonucleotides, 102 Natural IgM antibodies, 262
aspirin, 103 Nephrotic syndrome (NS), 216, 217
CETP inhibitors, 102 Netherlands the Dutch External Quality
lomitapide, 103 Assessment (EQA-) organizer, 335
niacin, 100 Neutrophil extracellular traps (NETs), 264
PCSK9 inhibitors, 100, 101 Neutrophil granulocytes, 264
statins, 99, 100 Niacin, 85, 352
THR agonists, 103 Non-apoB-directed therapies, 353
pharmacological interventions, 99 Nonsense mutation (R21X), 64, 65
synthesis, assembly and secretion, 94–95, Northwest Lipid Research Laboratories
208, 209 (NWRL), 304
VLDL, LDL, and Lp(a) Notch1-NFκB pathway, 178
interconversion, 77–79 Nuclear stress test, 412–413
Lp(a)-mediated plaque instability, 197 Null allele, 13, 65, 66
Lp(a)-OxPhos-phospholipase Null mutations, 25
pathway, 427 NWRL ELISA assay, 312
LPA-directed antisense oligonucleotides, 366
LPA gene, 57, 231
LPA genotyping, 286 O
LPA screening, 363 ODYSSEY OUTCOMES trial, 431
Lung cancer, 140 Olpasiran, 354, 431
442 Index

Oral anticoagulants, 386 Proteomic analysis, 267

Ouchterlony test, 40 Proteotypic peptides, 339
Ouchterlonys double diffusion, 1, 2
Oxidized LDLs (OxLDLs), 139, 262
Oxidized phospholipids (OxPL), 125, Q
135–137, 139, 140, 149, 151, 162, Quality of life (QoL), 391
174–176, 178, 179, 182, 196,
385, 427
Oxitope, 374 R
Radial immune-diffusion, 429
Radio-immuno assay (RIA), 429
P Randomized controlled clinical trials
PAF acetyl hydrolase (PAF-AH), 427 (RCT), 416
Papillary thyroid cancer, 142 Remnant receptor, 428
PCSK9 antibodies, 397 Reverse causation, 251
PCSK9 inhibitors, 24, 84, 350, 351, 400, 431 Reynold’s Risk Score, 411, 412
PCSK9-LDLR-Lp(a) axis, 117–119 RNA-induced silencing complex (RISC), 354
Pearson correlation, 310 Roche immunoassay, 340
Pelacarsen, 353, 370, 397, 431 Rocket electrophoresis, 429
Pentanucleotide repeat (PNRP), 64 Runt-related transcription factor 2
Peptide-calibrated reference measurement (RUNX2), 163
procedure, 340
Pericellular plasminogen activation, 144
Peripheral artery disease (PAD), 164 S
Peripheral vascular disease, 192 Scavenger receptor BI (SR-BI), 117
Peritoneal dialysis, 218–219 SDS-polyacrylamide gel
Personalized preventative medical care, 421 electrophoresis, 7, 310
Phosphodiester (PO) bonds, 362 Seattle angina questionnaire (SAQ), 391
Phosphodiester (PO) linkages, 365 Selected reaction monitoring, 308
Phospholipase-A2, 427 Selective thyroid hormone receptor (THR)
Phosphorothioate (PS), 362 agonists, 103
Plasma lipoprotein(a) concentrations Sib pair linkage studies, 13
apo(a) isoform size, 97, 98 Single nucleotide polymorphisms (SNPs),
production rate vs fractional catabolic 165, 232
rate, 96–97 Sinking pre-ß lipoprotein, 41, 42
Plasminogen, 142–144, 147, 151, 152 SI units, 430
Plasminogen activator inhibitor 1 (PAI-1), Six minute walk test (6MWT), 391
189, 193 Small interfering RNA, 354
Platelets, 195–196 Southern blotting, 13, 14, 20
Pre-beta-lipoprotein, 22 Stable isotopic tracer methodologies, 93, 94
Precision medicine, 421 Standardization
Premature cardiovascular disease, 418 clinical relevance of, 315, 316, 318
Production rate (PR), 382 current harmonization system, 305, 306
Pro-inflammatory phenotype, 263 history of, 303–305
Proprotein convertase subtilisin kexin in clinical laboratory medicine, 298
9 (PCSK9) LC-MS/MS, 308–312
elevated plasma Lp(a) levels, 114, 116, 117 measuring Lp(a), 301–303
LDL cholesterol-lowering drug target, metrological traceability, 299, 301
115, 116 Statins, 349, 350
LDL-C metabolism, 113, 114 Statistical analysis, 283
PCSK9-LDLR-Lp(a) axis, 117–119 Stroke, 163–164, 409
Prostate cancer, 142 Strong lysine-binding site (sLBS), 174,
Proteinuria, 217 175, 177
Index 443

T Vascular smooth muscle cells, 178

Thrombogenesis, 142 Vascular thrombosis, 409
Thrombolysis, 179–182 Venous thromboembolism (VTE), 192,
Thrombosis, 142–145, 175, 179–182, 193–195 194–196, 198, 200, 201
Tissue factor pathway inhibitor (TFPI), 144, Venous thrombosis, 193–195, 200
145, 189, 193 Very-low-density lipoprotein (VLDL),
Tl system, 40 130, 428
Triglyceride-rich lipoproteins, 287
Type 2 diabetes, 145–147
W
Western blotting, 13, 23, 57–59, 64
U Wilcoxon rank-sum test, 371
UK biobank, 254, 255 Women’s Heart Disease Data, 419
World health organization (WHO), 300
Wound healing, 147, 152
V
Valve interstitial cell phenotype, 179
Vascular cell adhesion molecule Y
(VCAM)−1, 162 Yeast artificial chromosomes (YACs), 12
Vascular endothelial cell layer, 177