Original Article

Thrombomodulin and Lactate Dehydrogenase as Potential Predictors of

Leg Ulcers in Sickle Cell Disease
Helen C. Okoye1, Sunday Ocheni1, Lisa I. Eweputanna2, Theresa U. Nwagha1, Chioma S. Ejezie1, Charles E. Nonyelu1,
Oji A. Nnachi3, Onochie I. Obodo1, Omolade A. Awodu4

Department of Background: Endothelial dysfunction, hypercoagulability, hemolysis, and

Abstract
Haematology and vasculopathy are involved in the pathogenesis of sickle cell leg ulcers (SCLUs).
Immunology, College of Understanding these processes is crucial for developing effective strategies to manage
Medicine, University of
and prevent SCLU. Objectives: To evaluate the plasma levels of thrombomodulin
Nigeria, Enugu, 1University
of Nigeria Teaching (TM) in patients with SCLU, determine if TM levels have any association with
Hospital, Ituku-Ozalla, SCLU and its severity, and determine if TM has any association with markers of
Enugu, 2Department of hemolysis. Materials and Methods: This was a cross-sectional analytical study
Radiology, Abia State carried out among patients with SCLU and their age-matched sickle cell disease
University Teaching (SCD) patients without leg ulcers. Venous blood samples were collected for the
Hospital, Aba, Abia determination of TM and lactate dehydrogenase (LDH) levels. Statistical significance
State, 3Department of
across means was determined using independent t tests and the one-way analysis of
Haematology and Blood
Transfusion, Alex Ekwueme variance (ANOVA). Pearson’s correlation and linear regression analysis were used to
Federal University determine the relationship between variables. Results: Of the 82 patients with SCD,
Teaching Hospital, 41 had SCLU (test group), and 41 had no SCLU (control group). Their mean age was
Abakaliki, 4Department of 33.3 ± 9.0 and 33.0 ± 8.5 (P = 0.30), respectively. There was no significant statistical
Haematology, University of difference between plasma levels of TM and number (P = 0.191) and severity
Benin Teaching Hospital, (P = 0.148) of SCLU. Seventy-three (89%) SCD patients had LDH concentration
Benin City, Nigeria above the reference limit of normal, and 9 (11%) were within normal limits (P =
0.002). None of the test (SCLU) group patients had normal LDH values. Conclusion:
SCLU is associated with higher levels of LDH, whereas plasma TM levels showed no
association with the occurrence and severity of SCLU.
Keywords: LDH, sickle cell leg ulcer, thrombomodulin

Introduction hemolytic anemia, SCD is marked with increase in the

markers of hemolysis in circulation, including lactate
S ickle cell disease (SCD) is one of the most
common inherited hemolytic anemias globally.[1] It
manifests as a heterogenous multisystem disease with a
dehydrogenase (LDH).
Sickle cell leg ulcer (SCLU) is one of the hemolysis-
variety of clinical phenotypes ranging from pulmonary endothelial dysfunction manifestations of SCD. It is
hypertension, priapism, leg ulcers, sudden death, stroke, a debilitating and long-term complication of SCD.[3]
acute chest syndrome, osteonecrosis, vaso-occlusive People with SCD have a ten-fold higher risk of
crises, and possibly asthma. These clinical features developing leg ulcers than in the general population, thus
are described in the two spectrums of the disease –
the hemolytic-endothelial dysfunction phenotype
Address for correspondence: Prof. Sunday Ocheni,
and the vaso-occlusive phenotype.[2] As a chronic Department of Haematology and Immunology,
College of Medicine, University of Nigeria,
Submission: 12-Aug-2024, First revision: 01-Oct-2024, Accepted: 05-Dec-2024, Ituku-Ozalla Campus, Enugu 400001, Nigeria.
Published: 15-Jan-2025. E-mail: [email protected]
This is an open access journal, and articles are distributed under the terms of the
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows
Access this article online others to remix, tweak, and build upon the work non-commercially, as long as
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Website:
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How to cite this article: Okoye HC, Ocheni S, Eweputanna LI, Nwagha TU,
DOI: Ejezie CS, Nonyelu CE, et al. Thrombomodulin and lactate dehydrogenase
10.4103/ijmh.ijmh_75_24 as potential predictors of leg ulcers in sickle cell disease. Int J Med Health
Dev 2025;30:6-14.

6 © 2024 International Journal of Medicine and Health Development | Published by Wolters Kluwer - Medknow
 Okoye, et al.: Thrombomodulin, lactate dehydrogenase, and hemogram levels in patients with sickle cell leg ulcers

making SCD a significant risk factor in the development among others, have all been linked to elevated plasma
of leg ulcers.[3] It is one of the commonest cutaneous TM levels resulting from shedding of TM from the
manifestations of SCD, typically observed in areas of endothelium due to endothelial damage from these
thin skin, little subcutaneous fat, and reduced blood conditions.[13]
flow like the malleoli, anterior tibial areas, dorsum of
Endothelial dysfunction and vasculopathy have been
the foot, and the Achilles tendon.[3,4] They can be single
implicated in the pathogenesis of SCLUs.[7] Thrombosis
or multiple at the site.[3] The incidence of SCLU varies
and inflammation are also notable contributions to
with the hemoglobin phenotype, age, gender, and one
the development of leg ulcers in sickle cell patients.[7]
geographical location to the other, ranging from as low
Endothelial protection is a feature of TM as well as
as 1% to as high as 43%.[3,5] In Nigeria, the prevalence
anticoagulation and antiinflammation, since TM in
ranges from 3.1% to 9.6% with a recurrence rate of
the endothelium performs various functions such
95.0%.[5,6] It is thought to be commoner among the male
as anticoagulation and antiinflammation, thereby
gender, older age, those with sickle cell anemia (HbSS),
protecting the endothelium. Inadequate expression of
and those with low socioeconomic status. The hallmark
TM will lead to reduced levels of TM, while damage to
of SCLU is in its indolent clinical course, recalcitrant
the endothelium will lead to the shedding of fragments of
pain, and recurrence despite multiple approaches to its
TM, making it lose its protective potential.[7] Emerging
treatment.[3,4]
data suggest that the synthesis and secretion of TM are
The cause of SCLUs is completely unknown, their controlled by reactive oxygen species produced in the
prevention is impractical, and their management, process of hemolysis in this disease entity.[14] Hypoxia,
once present, is often difficult. Many factors have been cell-free hemoglobin, and oxidative stress are already
proposed to potentially contribute to the formation established factors that downregulate TM expression.[8]
of leg ulcers in SCD. Impaired oxygen delivery to It may then be suspected that TM may be involved in
tissues as a result of vaso-occlusion, vasoconstriction the pathogenesis of leg ulcers in SCD, given that this
from depleted nitric oxide by free hemoglobin from protein possesses the qualities that may counteract the
haemolyzed red cells, venous inconsistency, endothelial pathways involved in the development of leg ulcers in
dysfunction, thrombosis, and genetics has all been SCD. We, therefore, designed this study to investigate
reported to contribute significantly to the pathogenesis the predictive role of TM and LDH in SCLUs. To
of leg ulcers.[7] Trauma, infection, and inflammation ascertain the relationship with the severity of leg ulcers
are thought to play a role in the pathogenesis as in SCD, this study assessed the plasma levels of TM,
well.[4] People with SCLUs may have a significant LDH, and hemogram in individuals with SCLUs.
decline in their quality of life. They are also exposed
to the risk of social isolation, increased disability, time Materials and Methods
away from work, and excessive healthcare resource The study was conducted in accordance with the
consumption.[3,4] Declaration of Helsinki (as revised in 2013) with
Thrombomodulin (TM), although historically the ethical approval number NHREC/05/01/2008B-
thought of being an anticoagulant, has recently FWA0000245B-1RB00002323 dated April 15, 2021,
been demonstrated to control the endothelium and issued by the Health Research Ethics Committee of
to have some anti-inflammatory characteristics.[8] the University of Nigeria Teaching Hospital, Ituku-
TM has been studied in different disease states, and Ozalla, Enugu, Nigeria. Written informed consent was
changes in the plasma levels have been associated with obtained from each participant or their legal guardians
endothelial dysfunction, organ failure, and mortality. or wards before enrollment into the study.
Increased levels of TM may result from damage to the The study was a cross-sectional hospital-based analytical
endothelium where TM is mostly stored. Decreased study. All adult SCD patients with high-performance
levels of TM in circulation may mean reduced liquid chromatography (HPLC) confirmed hemoglobin
expression of TM.[9,10] The continuous elevation in variants and in a steady state with no acute exacerbation
circulating TM levels during diseases is now widely of SCD features other than the chronic complications
acknowledged as a crucial circulatory biomarker for were eligible for the study. The SCD patients with
endothelial dysfunction and vascular risk assessment, active leg ulcers served as the test group, while SCD
even if these TM levels are likely too low to significantly patients without active leg ulcers and previous history
affect coagulation processes.[11] Atherosclerosis, of leg ulcers served as the control group. SCD patients
cardioembolic stroke, obesity, preeclampsia, and sepsis- whose diagnoses were not made with HPLC, and
associated disseminated intravascular coagulation,[11,12] those with co-morbidities such as stroke, pulmonary

International Journal of Medicine and Health Development ¦ Volume 30 ¦ Issue 1 ¦ January-March 2025 7
 Okoye, et al.: Thrombomodulin, lactate dehydrogenase, and hemogram levels in patients with sickle cell leg ulcers

hypertension, diabetes mellitus, chronic renal disease, the correlation of plasma levels of TM with markers
and other inflammatory conditions were excluded. of hemolysis which include LDH, reticulocyte count,
The socio-demographic variables and the clinical data and hemogram. For this study, the normal plasma level
of the patients were extracted from the patient’s case of TM will be taken as 0.14 ng/dL; normal LDH will
notes with the help of a proforma. For the test group be taken as follows: <248 U/L for males and <247 U/L
(those with SCLU), the ulcers were examined using for females.
the following criteria: site (s), sizes, and depth of the
Data management was performed using Microsoft
ulcer(s) on the lower limb as well as the stage of the
Excel 2013. Statistical analysis was performed using
ulcers. Observed details were documented as well.
the statistical package for social sciences (SPSS)
The sample size was calculated using the following version 21.0. Complications related to SCLU (ulcer
formula,[15] n = (z2pq)/d2; where n = desired sample duration, stage, number) were summarized using
size in a population greater than 1000; z = standard medians and range and also presented as frequencies
deviation usually set at 1.96, corresponding to the and proportions. Biological values were summarized
95% confidence interval; p = proportion of the using means and standard deviations. Statistical
target population estimated to have a particular significance between means was determined using
characteristic; q = 1 − p; d = degree of precision used t tests for two categories and analysis of variance
(0.05). In this study, p was taken as 3.1% (0.031), (ANOVA)/F-test for more than two categories.
representing the prevalence of SCLU in the previous Proportions were compared using Fisher’s exact
Nigerian as documented by Hassan et al.[5] Following statistics. Pearson’s correlation and linear regression
the substitution of appropriate values, 41 subjects analysis were used to explore the relationship between
(n = 42) were found to be appropriate for each group, the dependent variable (TM) and independent
and a total of 82 subjects were needed for the study. hematological parameters. Bivariate analysis was
conducted with a significance level of P < 0.25.
Cases and controls were selected using a consecutive
Variables showing statistical significance in bivariate
sampling method, whereby study participants were
analysis were included in a multivariate analysis
recruited as they presented to the clinic. Recruitment
model. Multiple linear regression analysis was used
was from the pool of SCD patients registered at the
for the multivariate analysis with a significance level
Hospital’s Adult Sickle Cell Clinic who had come for
of 0.05. Confidence intervals were determined at the
routine visits.
95% level.
Sample collection and processing
Peripheral venous blood was collected from recruited Results
subjects under standard aseptic conditions. The venous Socio-demographic characteristics of study
blood was obtained from an antecubital vein or any participants: A total of 82 SCD patients participated
other visible vein on the forearm using a dry, sterile, in the study, comprising 41 patients with SCLU and
disposable 10 mL syringe, and a 21G needle. A total of another 41 SCD without leg ulcers. Both groups were
8 mL venous blood samples were collected from each matched for age with a mean age of 33.3 ± 9.0 and
participant. The first 4 mL was mixed with EDTA in a 33.0 ± 8.5 (P = 0.30). A total of 28 (68.4%) of our
sample bottle for a full blood count using the Orphee study participants were males, while 13 (31.6%) were
Swiss Mythic 22 blood counter. The reticulocyte females. Only 2 (4.9%) were married, while 38 (92.7%)
count was performed manually. The remaining 4 mL were married, and 1 (2.4%) was divorced. Twenty-eight
was processed within 30 min by centrifuging at 4000 (68.4%) had secondary education, while 13 (31.6%)
RPM for 15 min at 2–8°C. The platelet-poor plasma had a university education. The study was conducted
obtained was stored in plastic tubes at −80°C until for 3 months, from May to August 2022. The data
analysis for plasma levels of TM using the enzyme- were found to be parametric after testing for normality
linked immunosorbent assay (ELISA) method (Kits using Kolmogorov–Smirnov statistics. The study group
from Elabscience Biotechnology Inc Texas, USA). The had a statistically significantly lower body mass index
other 4 mL was collected in a plain sample bottle for (BMI) of 15.39 ± 7.60 kg/m2 compared to the control
LDH estimation. group’s BMI of 21.17 ± 2.67 kg/m2 (P = < 0.001). Most
The main outcome measures were as follows: of the participants (68.4%) were males. Among the
determination of the mean levels of TM in the plasma SCLU patients, 27 (66.7%) were either artisans or self-
of SCD patients with leg ulcers; the correlation of employed, while in the control group, 22 (52.6%) were
plasma levels of TM with the severity of SCLU; and employed in the private or public sector. See Table 1.

8 International Journal of Medicine and Health Development ¦ Volume 30 ¦ Issue 1 ¦ January-March 2025
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Table 1: Distribution of socio-demographic characteristics Table 2 : Characteristics of leg ulcer among SCLU (cases)
in the cases and controls of the study Variables Frequency Percentage
Variable Cases Controls Number of ulcer sites
(SCLU) N (%)  1 20 48.8
n (%)  2 9 22.0
Gender  3 9 22.0
 Males 28 (68.4) 25 (61.0)  4 3 7.3
 Females 13 (31.6) 16 (39.0) Median number (range) = 2.00 (1–4)
Marital status Stage of ulcer
 Married 2 (4.9) 4 (9.8)  Stage 1 9 22.0
 Single 38 (92.7) 37 (90.2)  Stage 2 10 24.4
 Divorced 1 (2.4) 0 (0.0)  Stage 3 16 39.0
Highest level of education  Stage 4 6 14.6
 Secondary 28 (68.4) 17 (42.1) Median stage (range) = 3.00 (1–4)
 Tertiary 13 (31.6) 24 (57.9) Duration of ulcer (years)
Occupation/employment status  1–3 26 63.4
 Artisan/self-employed 27 (66.7) 2 (4.9)  4–6 9 22.0
 Public/private employed 2 (4.9) 22 (52.6)  7–10 2 4.9
 Student 1 (2.4) 14 (35.2)  >10 4 9.8
 Unemployed 11 (26.0) 8 (7.3) Median duration (range) = 2.00 (0–15)
Religion
 Christianity 40 (97.6) 39 (95.1)
difference in the plasma levels of TM between males
 Islam 1 (2.4) 2 (4.9)
Previous history of leg ulcer
and females (P = 0.202) as well as across different age
 Yes 37 (90.2) 0 (0.0) groups (P = 0.629). Seventy-three (89%) had LDH
 No 4 (9.8) 41 (100.0) concentration above the reference limit of normal, and
SCLU: sickle cell leg ulcer 9 (11%) were within normal limits (P = 0.002). None
of the test (SCLU) groups had normal LDH values.
Characteristics of the leg ulcers
The mean plasma TM levels of those with normal and
abnormal LDH were 0.1 ± 0.02 ng/mL and 0.1 ± 0.1 ng/
The leg ulcers were unilateral in 21 (51.2%) and
mL, respectively (t = −0.616; P = 0.539). The mean
bilateral in 20 (48.8%) patients with SCLU. The ulcer
TM ± SD plasma levels did not differ statistically
was commonly located on the right medial malleolus
(t = 0.312; P = 0.755) between groups. See Table 4
followed by the left medial malleolus. The ulcer was
recurrent in 37 (90.2%) cases. Evaluation of the association between plasma levels of
TM with SCLU and severity – There was no statistically
The median (range) of ulcer site was 2.0 (1–4), and
significant difference between plasma levels of TM and
the majority of the patients 20 (48.8%) had an ulcer
the number (P = 0.191) and severity (P = 0.148) of the
at a single site. The median (range) stage of the ulcers
leg ulcers as shown in Table 5.
was 3.0 (1–4), and most of the patients 16 (39.0%) had
ulcers at stage 3. The ulcers ranged from 0.5 to 8.5 cm2 To determine the association (if any) between
and had a depth of 0.1–1.0 cm The median (range) plasma levels of TM with markers of hemolysis
duration of the ulcers was 2.0 (0–15) years. See Table 2. (LDH levels and reticulocyte count)
A bivariate analysis was initially performed by raising
Hematological parameters between the two groups
the P value to select possible predictors to determine
Hemoglobin concentration (Hb), white blood cell count any association between TM and markers of hemolysis.
(WBC), absolute lymphocyte count (ALC), and most TM was significantly associated with LDH only in
of the red cell indices were significantly higher in the the SCLU group. A multivariate analysis was then
test group when compared to the control group, while performed using multiple linear regression to determine
the absolute basophil count was significantly higher in predictors and control for confounders. Plasma TM
the control group than in the test group. See Table 3. levels failed to show any significant association with
The plasma levels of TM, LDH, and reticulocyte LDH and reticulocyte count. See Table 6.
count of the study participants
The mean TM ± SD was 0.1 ± 0.1 ng/mL, with a Discussion
median (range) level of 0.1 (0.1–0.5) ng/mL for the entire SCLU is a common and challenging musculocutaneous
study participants. There was no statistically significant complication of SCD with a complex pathophysiology

International Journal of Medicine and Health Development ¦ Volume 30 ¦ Issue 1 ¦ January-March 2025 9
 Okoye, et al.: Thrombomodulin, lactate dehydrogenase, and hemogram levels in patients with sickle cell leg ulcers

Table 3: Hematological parameters between the two groups

Variables Study groups t** P value
Cases Control
Mean ± SD Mean ± SD
Hb (g/dL) 6.5 ± 1.5 8.4 ± 1.5 −5.935 <0.001*
WBC (×10 /L) 9
12.0 ± 6.9 8.8 ± 2.9 2.763 0.007*
ANC (×10 /L) 9
8.5 ± 11.8 5.0 ± 2.0 1.883 0.063
ALC (×109/L) 4.1 ± 2.9 3.0 ± 1.4 2.098 0.039*
AMC (×109/L) 0.8 ± 1.4 0.5 ± 0.5 1.377 0.172
AEC (×109/L) 0.3 ± 0.5 0.5 ± 1.6 0.625 0.534
ABC (×109/L) 0.0 ± 0.0 0.0 ± 0.0 4.068 <0.001*
PLT (×109/L) 324.7 ± 113.0 307.8 ± 144.0 0.594 0.554
MCV (fL) 84.4 ± 11.2 81.0 ± 8.7 0.928 0.361
MCH (pg) 30.7 ± 2.8 27.0 ± 3.5 3.047 0.005*
MCHC (g/dL) 35.7 ± 0.3 33.2 ± 1.6 4.355 <0.001*
SD: standard deviation, Hb: hemoglobin concentration, WBC: white cell count, ANC: absolute neutrophil count, ALC: absolute
lymphocyte count, AMC: absolute monocyte count, AEC: absolute eosinophil count, ABC: absolute basophil count, PLT: platelet
count, MCV: mean cell volume, MCH: mean cell hemoglobin, MCHC: mean cell hemoglobin concentration
*
Statistically significant; **independent t test

Table 4: Comparison of mean TM, LDH, and reticulocyte count between cases and controls
Group T P value
Cases Controls
Mean ± SD Mean ± SD
TM (ng/mL) 0.3 ± 0.0 0.1 ± 0.1 −0.312 0.755
LDH (U/L) 585.5 ± 210.7 440.2 ± 219.8 3.06 0.003*
RETIC (%) 3.4 ± 1.9 3.4 ± 1.6 −0.084 0.934
SD: standard deviation, TM: thrombomodulin, LDH: lactate dehydrogenase, RETIC: reticulocyte count
Statistically significant P < 0.05

with several factors implicated, including inflammation

Table 5: Comparison of mean TM values by ulcer
characteristics of cases (SCLU) and thrombosis. TM has been proven to have some anti-
Variables TM inflammatory and antithrombotic effects.[8] This study
Mean ± SD was conducted to determine the plasma level of TM
Number of ulcer cases and evaluate its association with the severity of SCLU
 1 0.1 ± 0.061 as well as with hemograms and markers of hemolysis in
 2 0.1 ± 0.009 patients with SCLU. A key finding in this research was
 3 0.1 ± 0.048 that the plasma levels of TM in all study participants
 4 0.1 ± 0.028 were low, but there was no significant association
Analysis of variance (ANOVA) = 0.40; P = 0.753 between plasma TM levels, markers of hemolysis, and
Stage of ulcer SCLU.
 Stage 1 0.1 ± 0.091
 Stage 2 0.1 ± 0.014 Plasma levels of TM will entirely depend on what
 Stage 3 0.1 ± 0.037 the soluble TM fragments are because each TM
 Stage 4 0.1 ± 0.023 domain has a unique activity that can be produced
ANOVA = 0.48; P = 0.702 under particular circumstances.[16] It is not known,
Duration of ulcer (years) nevertheless, whether the release of soluble TM is a
 1–3 0.1 ± 0.054
strictly controlled procedure in which only certain
 4–6 0.1 ± 0.021
domains are cleaved and released. The plasma levels
 7–10 0.1 ± 0.009
 >10 0.2 ± 0.063
of TM in this study (0.14 ng/dL) were found to be
ANOVA = 0.86; P = 0.472 lower than values in the normal population,[17] and
SD: standard deviation, TM: thrombomodulin, SCLU: sickle previously, reported mean values obtained in studies
cell leg ulcer conducted among SCD patients[18,19] and non-SCD

10 International Journal of Medicine and Health Development ¦ Volume 30 ¦ Issue 1 ¦ January-March 2025
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Table 6: Correlation (bivariate analysis) between TM and hemolytic markers in the cases and controls in the study
Variable Cases Controls
TM (ng/mL) TM (ng/mL)
Pearson correlation co-efficient (r) P value Pearson correlation co-efficient (r) P value
RETIC (%) 0.06 0.73 −0.14 0.386
LDH (U/L) 0.24 0.14* 0.00 0.999
*
Statistically significant (P < 0.250)

patients.[19,20] The reason for this difference may not thrombin.[24] Additionally, since the plasma level
be farfetched. SCD is a chronic hemolytic anemia represents the soluble TM fragment generated following
characterized by intravascular hemolysis with the proteolytic cleavage of the integral membrane protein,[8]
release of cell-free hemoglobin and heme into the the varying plasma levels may be a reflection of varying
circulation, leading to oxidative injury with tissue degrees of clearance of the different fragments as
hypoxia.[2] Hypoxia, cell-free hemoglobin, and suggested by Loghmani and Conway[8]. Notable renal
oxidative stress are already established factors manifestations of SCD are glomerular hyperfiltration
that downregulate TM expression.[8,21] When and tubular hyperfunction.[25] Renal excretory function
TM expression is downregulated, there may be a also has a significant impact on plasma soluble
concomitant reduction in plasma TM levels with TM levels.[26] According to a report, plasma levels
a loss of the protective effects of TM on vascular of endothelium indicators are determined by renal
endothelium.[8] This may contribute to vasculopathy function.[27] It was discovered that patients with renal
in SCD. It is important to note that samples for this disease receiving conservative treatment and showing
study were drawn from patients in a steady state, and no signs of liver failure experienced a considerable rise
there was no acute exacerbation of SCD features other in their soluble TM levels. Therefore, the lower plasma
than the chronic complications. This may indicate TM levels in SCD patients in this study may indicate a
that the elevated intravascular hemolysis may be a higher clearance in this cohort of TM in SCD patients;
function of the chronic hemolytic state, which might however, the renal function of these patients was not
have suppressed TM expression, making TM levels in assessed in this study, to confirm these speculations.
SCD patients lower than in the normal population. These notwithstanding, the higher plasma level of
As such, it may not be related to leg ulcers in SCD TM in the work by Ruiz et al.[18] might have been a
patients. result of the characteristics of the study participants.
While all the study participants in the index study were
The role of leukocytes has been described in the
SCD patients apparently in steady state, Ruiz et al.[18]
pathophysiology of vaso-occlusion in SCD in which
conducted their study among SCD patients hospitalized
there are increased levels of leukocytes and adhesion
for the vaso-occlusive crisis, a condition which may
molecules.[22] Additionally, SCD is described as a
contribute to vascular injury in SCD. Mori et al.[20]
chronic inflammatory condition that is heightened
have demonstrated that plasma TM levels are increased
during a vaso-occlusive crisis.[22] It has been observed
when there is an injury to the vascular endothelium.
that endothelial TM can undergo lysis by various
leukocyte-derived proteases, such that a number of Studies have shown four to seven different fragments of
inflammatory events are linked to a mild but statistically TM in plasma samples, indicating diverse cleavage sites
significant rise in plasma levels of soluble TM (elastase and various mechanisms, resulting from endothelial
and cathepsin).[23] Ruiz et al. studied TM in SCD cell injury in a variety of illnesses.[28,29]
patients with the vaso-occlusive crisis who could have
TM levels are often extensively evaluated in disease
more cleavage and release of TM by leukocyte-derived
monitoring and diagnosis. However, it is important
proteases secondary to the vaso-occlusion, while the
to know that after being released from vascular
SCD patients in this study are in a steady state. This
endothelial injury, preexisting/coexisting diseases
may explain the higher levels of TM in the former.
like liver or renal disorders, or both, are capable of
Another explanation for the low TM levels is that altering plasma soluble TM levels.[30] Experimental
of a consumptive state. Because SCD is regarded as animals have established that the liver is a major site
a hypercoagulable state with chronic activation of for TM elimination, and this mechanism has not been
coagulation proteins and increased generation of studied exhaustively in people.[31] As a result, assessing
thrombin,[2] there may be consumption of TM through the indicators of liver function in addition to renal
its interaction and the formation of a complex with function is required for the clinical application, and

International Journal of Medicine and Health Development ¦ Volume 30 ¦ Issue 1 ¦ January-March 2025 11
 Okoye, et al.: Thrombomodulin, lactate dehydrogenase, and hemogram levels in patients with sickle cell leg ulcers

the interpretation of soluble TM levels in clinical the SCLU group because SCLU is an SCD phenotype
research should pay special attention to liver and renal marked by more intense hemolysis.[35] Between the two
function. Unfortunately, the liver functions of the study study groups, there was no difference in the plasma TM
participants were not assessed. This would have helped levels, and TM showed no association with the severity
to control the possibility that the renal and hepatic or stage of SCLU. Most participants with SCLU were
status may affect the plasma concentration of TM. in stage 3, but there was no association with TM levels.
The plasma level of TM in SCD patients is thought to
Another aspect of this is the knowledge that various increase with increasing intravascular hemolysis.[19] There
techniques can be employed to determine the is evidence of intravascular hemolysis in both groups
concentration of soluble TM in biological materials. with the mean LDH levels in both groups significantly
The most popular techniques for measuring soluble TM higher than reference values. This may explain why there
when an anti-TM antibody is employed are the enzyme was no difference in the plasma TM levels since it was
immunoassay and (ELISA) procedures. The index dependent on the presence of intravascular hemolysis.
study used the ELISA method to determine the plasma The Hb concentration of the group with leg ulcers was
concentration of TM. There are multiple types of lower than those of the control group. The previous
soluble TM in the biofluids. The main query is whether studies have shown a higher prevalence of SCLU in
a single antibody can capture all of the fragments. In individuals with lower steady-state Hb.[5] This may
other words, it is not certain if the antibodies in the stem from increased hemolysis, reduced nutrients, and
kit used were able to capture all the TM fragments oxygen delivery to the tissues with decreased healing.[5]
circulating in plasma, given that different domains of There is evidence that an increase in Hb concentration
TM have distinct activities and numerous fragments of through the use of chronic transfusion therapy or
soluble TM exist.[32] Plasma levels of TM are found to erythropoiesis-stimulating agents has led to improved
be higher in the pediatric population and gradually drop healing and a reduction in the prevalence of SCLU.[36-38]
toward adulthood.[32] Results from this study suggest Poor supply of nutrients and oxygen to the malleolar
that the plasma level of TM may not be dependent area may also explain the site of leg ulcers observed
or determined by gender or age (in adults) because in this cohort of patients. All the study participants
no significant difference in values between males and in this study had ulcers on the malleoli as reported by
females as well as across different age groups was other researchers.[3,5] The malleolus is an area with thin
found. The reason for this is not clear but noteworthy skin, less subcutaneous fat, and reduced blood flow. In
was that there was no age difference between the two addition, most of the ulcers are located on the right leg.
study groups; besides, the study was conducted among The reason for this is not clear but was also reported
adult population. However, more male participants by Hassan et al.[5] As similarly observed by Babalola
were observed in the SCLU group than controls, a et al.,[37] mean corpuscular hemoglobin and mean
finding similar to that reported in other studies.[5,33] corpuscular hemoglobin concentration were higher in
The higher frequency of SCLU among males may be the SCLU group than in the control group, and this
due to the pattern of distribution of fat between males may be a function of hemolysis with supplemental use
and females. Females, perhaps due to estrogen, have of folic acid. The WBCs were higher in patients with
more subcutaneous fat around the malleolar area when SCLU than in those without SCLU. This may be a
compared to males, and ulcers are more common on marker of chronic inflammation and vasculopathy
areas with less subcutaneous fat.[33] The mean levels of which are implicated in the pathogenesis of SCLU.[3,2]
both LDH and reticulocyte counts were higher than the With increased WBC, there may be increased cell-cell
normal reference values in both groups of participants. and cell-endothelium adhesion with an increase in
SCD is characterized by a chronic hemolytic state which circulating adhesion molecules leading to vaso-occlusion
is exacerbated in acute events.[2] LDH is an enzyme with and subsequently tissue hypoperfusion and ischemia.[22]
five isoforms that catalyze the conversion of lactate to These promote leg ulceration, recurrence, and poor
pyruvate.[34] Isoforms 1 and 2 are mainly expressed in the wound healing,[5,37] explaining the high recurrence rate
red cells and red cell hemolysis (especially intravascular) of 90.2%, chronic course of up to 15 years, and mostly
causes elevated plasma levels.[34] Reticulocytes on the with stage 3 ulcers, as observed in this study. The higher
other hand are markers of bone marrow hemopoietic WBC may also be due to the infective process.[39] When
activity whose peripheral blood levels are also increased the white cell differentials were reviewed, the mean
in hemolytic conditions but may also be affected by values of the absolute neutrophil counts did not differ
other factors of erythropoiesis such as folic acid.[34] between groups; however, the mean ALC of the SCLU
Expectedly, the mean levels of LDH were higher in group was significantly higher than those without leg

12 International Journal of Medicine and Health Development ¦ Volume 30 ¦ Issue 1 ¦ January-March 2025
 Okoye, et al.: Thrombomodulin, lactate dehydrogenase, and hemogram levels in patients with sickle cell leg ulcers

ulcers. Lymphocytes are activated and recruited in Author contributions

the presence of inflammation and chronic infection, HCO and SO conceptualized and designed the
and in SCLU, there is an interplay of many factors study. HCO, LIE, TUN, and CSE were involved in
including infection and inflammation.[2] Platelet counts data collection/acquisition and statistical analysis.
were higher in the SCLU group than the control group HCO, CEN, OAN, OIO, and OA interpreted the
though not to a level of statistical significance. Platelets results and together, with SO, LIE, and TUN, were
play a role in vaso-occlusion by adhering to other blood involved in the writing and revising the manuscript for
cells and the endothelium, leading to tissue ischemia. intellectual content. All authors read and approved
Platelets also contribute to the hypercoagulability of the final manuscript and agreed to be accountable
blood which complicates tissue ischemia.[37] Higher for all aspects of the work. All authors have accepted
platelet counts were observed in similar studies among responsibility for the entire content of this manuscript
participants with SCLU than those without leg ulcers.[8] and consented to its submission to the journal,
Study limitations
reviewed all the results, and approved the final version
of the manuscript.
Ulcers were measured using the subjective method
which is encumbered by inter- and intra-variability in Ethical approval
measurements and so not as reliable as the objective The study was conducted in accordance with the
methods. The measurement of ulcers can be more Declaration of Helsinki (as revised in 2013) with
reliably made using objective methods of measurement the ethical approval number NHREC/05/01/2008B-
such as stereophotogrammetry. This was not available FWA0000245B-1RB00002323 dated April 15, 2021,
in the study center, and the technique is quite expensive. issued by the Health Research Ethics Committee of the
However, care was taken to see that the measurements University of Nigeria Teaching Hospital, Ituku-Ozalla,
were consistently performed to avoid individual Enugu, Nigeria
variations in measurements.
Informed consent
It could not be proven that the reduced plasma TM
Informed consent was obtained from all individuals
was a result of the downregulation of endothelial TM
included in this study, or their legal guardians or
expression because TM function was not assessed
wards.
concurrently in this study. Knowledge of this might
have corroborated the fact that chronic hypoxia and Declaration of Helsinki
oxidative stress may downregulate the expression of TM The study was conducted in accordance with the
in the endothelium. Data on the use of hydroxyurea by Declaration of Helsinki (as revised in 2013).
these SCD patients were not collected. It is not certain if Availability of research data
the use of hydroxyurea contributed to the development
Authors are available and ready to supply the data upon
of the SCLU as is reported in the literature.[6]
any request through the corresponding author.
Conclusion Financial support and sponsorship
This study has demonstrated that plasma levels of TM Nil.
in patients with SCD are low which may be a result of Conflict of interest
the presence of hypoxia and oxidative stress in these
There are no conflict of interest.
patients from chronic hemolysis. TM may not have
any association with the severity of SCLU. SCLU is References
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14 International Journal of Medicine and Health Development ¦ Volume 30 ¦ Issue 1 ¦ January-March 2025