|Year : 2014 | Volume
| Issue : 2 | Page : 52-57
High levels of soluble thrombomodulin may be a marker of arterial disease and peripheral ischemia in Egyptian patients with diabetes mellitus
Walaa A Elsalakawy1, Botheina A.T. Farweez2, Mohamed T.H. Sallam2, Mohamed A Hamza3
1 Department of Internal Medicine, Clinical Hematology and Bone Marrow Transplant Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Clinical and Chemical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
3 Department of Cardiology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||27-Aug-2013|
|Date of Acceptance||29-Jan-2014|
|Date of Web Publication||30-Aug-2014|
Walaa A Elsalakawy
Clinical Hematology and Bone Marrow Transplant Unit, Internal Medicine Department, Faculty of Medicine, Ain Shams University, Block 9, Building 1, Apart. 303,
Source of Support: None, Conflict of Interest: None
Background Thrombomodulin is an endothelial cell membrane protein acting as a cofactor for the activation of plasma protein C. It was found that soluble forms of thrombomodulin (sTM) exist in plasma and is considered a marker of endothelial dysfunction.
Materials and methods The study included 160 individuals: 40 healthy controls and 120 diabetic patients (40 patients with type 1 diabetes and 80 patients with type 2 diabetes). sTM concentrations were measured by an enzyme-linked immunosorbent assay-based assay employing monoclonal antithrombomodulin antibodies.
Results The diabetic patients had highly significantly increased plasma sTM concentrations compared with the control group (P < 0.001). Type 2 diabetic patients with peripheral limb ischemia showed a highly significant increase in plasma sTM levels when compared with type 2 or type 1 diabetic patients without ischemia (P < 0.001). sTM concentrations were positively correlated with age, duration of diabetes, and urinary albumin concentration, with highly significant P value (P < 0.001). A cutoff value of greater than 9500 ng/ml of sTM was found to be of 96.9% diagnostic accuracy in differentiating type 2 diabetic patients with and without peripheral limb ischemia.
Conclusion We recommend including sTM in the follow-up panel of diabetic patients because of its potential ability to predict the risk for ischemic manifestations.
Keywords: diabetes mellitus, peripheral ischemia, thrombomodulin
|How to cite this article:|
Elsalakawy WA, Farweez BA, Sallam MT, Hamza MA. High levels of soluble thrombomodulin may be a marker of arterial disease and peripheral ischemia in Egyptian patients with diabetes mellitus. Egypt J Haematol 2014;39:52-7
|How to cite this URL:|
Elsalakawy WA, Farweez BA, Sallam MT, Hamza MA. High levels of soluble thrombomodulin may be a marker of arterial disease and peripheral ischemia in Egyptian patients with diabetes mellitus. Egypt J Haematol [serial online] 2014 [cited 2020 Jan 17];39:52-7. Available from: http://www.ehj.eg.net/text.asp?2014/39/2/52/139759
| Introduction|| |
Thrombomodulin (TM), a key protein in the generation of activated protein C, is an important regulator of thrombotic and inflammatory processes. The major physiological location of TM is thought to be on the surface of endothelial cells. Smaller heterogeneous soluble thrombomodulin (sTM) fragments, collectively termed sTM, are found in plasma and urine of healthy individual  . Elevated sTM levels have been detected in various clinical conditions, including cardiovascular disease and diabetes  . Consequently, sTM is traditionally thought to serve as a marker of endothelial cell damage, and a number of cross-sectional studies have demonstrated a positive association with atherosclerotic disease and diabetes , .
The aim of this study was to determine sTM level in diabetic patients with and without ischemic manifestations to evaluate its importance as a marker of peripheral ischemia in diabetic patients.
| Materials and methods|| |
A total of 160 participants were enrolled in this study. They were divided into two groups:
- Group I [diabetes mellitus (DM) group] included 120 patients with DM fulfilling the WHO criteria of diagnosis of DM. They were referred to the outpatient clinics of the Internal Medicine Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt, during the period from September 2012 to May 2013. They were divided into three subgroups as follows:
- Group Ia included 40 patients with type 2 DM complaining of ischemic manifestations.
- Group Ib included 40 patients with type 2 DM, with no ischemic manifestations.
- Group Ic included 40 patients with type 1 DM, with no ischemic manifestations.
- Group II (the control group) included 40 apparently healthy individuals of matched age and sex.
All patients were clinically assessed for symptoms of peripheral limb ischemia (claudication pains, rest pain, ischemic ulcers, and gangrene), previous amputation due to limb ischemia, and previous peripheral ischemia diagnosed by arterial duplex or angiography.
None of the participants had clinical or laboratory evidence of liver or autoimmune diseases. Patients with serum creatinine levels above 1.3 mg/dl were excluded from the current study. Patients with ischemic heart disease (had history of myocardial infarction, ECG alterations, and enzymatic changes) were not included. The study protocol was approved by the scientific ethical committee of Faculty of Medicine, Ain Shams University. Informed consent was obtained from each patient and healthy control participant before study entry.
Peripheral blood samples (4-5 ml) were aseptically withdrawn from diabetic patients and healthy controls by routine vein puncture.
Fasting plasma glucose was measured by the glucose oxidase method (Beckman Synchron CX9; Beckman-Coulter Inc., Hialeah, California, USA), and HbA1c was determined by high-performance liquid chromatography (Bio-Rad D-10; Bio-Rad Laboratories, California, USA).
Serum TM level was determined by enzyme-linked immunosorbent assay (ELISA) (Quantikine-Human Thrombomodulin/BDCA-3; R&D Systems, Minneapolis, MN, USA) in the patients and control groups according to the manufacturer's instructions.
First, morning urine samples were collected in clean and sterile containers. The urinary concentrations of microalbumin were measured using an ELISA-based assay (Orgentec Diagnostika GmbH, Mainz, Germany). Microalbuminuria was defined by urinary albumin concentration as a level from 30 to 150 μg/ml, whereas macroalbuminuria was defined by urinary albumin concentration above 300 μl/ml.
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS software version 17; SPSS Inc., Chicago, Illinois, USA). A probability less than 0.05 was statistically significant and less than 0.001 was statistically highly significant. Differences between groups were assessed by one-way analysis of variance.
A receiver operator characteristic curve was constructed. Sensitivity and specificity were profiled by curves.
| Results|| |
Clinical characteristics and laboratory data of patients and controls are presented in [Table 1].
Soluble thrombomodulin level among the studied groups
We assessed sTM in the patients and control groups. The concentrations of sTM were significantly increased in the three studied DM subgroups compared with the control group (P < 0.001) [Figure 1].
|Figure 1: Soluble thrombomodulin (sTM) levels in different studied groups.|
Click here to view
When comparing sTM concentrations in group Ia (type 2 DM with associated symptoms of ischemia) with that in group Ib (type 2 DM without symptoms of ischemia) and in group Ic (type 1 DM without symptoms of ischemia), a highly significant increase was detected in group Ia patients (13 875 ± 5033, 6840 ± 1065, and 6498 ± 3706, respectively) (P < 0.001).
Statistical positive correlation was detected between sTM and age, duration of DM, and microalbuminuria (P < 0.001) but not with fasting blood glucose level or HbA1c (P > 0.05).
A cutoff value of 5000 ng/ml sTM was found to be 82.0% sensitive and 81.6% specific with 90.1% positive predictive value, 69.0% negative predictive value, and 85.9% diagnostic accuracy in differentiating patients from controls [Table 2] and [Figure 2]a and b.
|Table 2: Diagnostic performance of soluble thrombomodulin in differentiating diabetic patients from healthy controls|
Click here to view
|Figure 2: (a) Receiver operating characteristic curve for soluble thrombomodulin (sTM) in differentiating diabetic patients from healthy controls, (b) scatter plots of sTM in different diabetics and control groups. The dashed line indicates the cutoff value of sTM (5000 ng/ml).|
Click here to view
Furthermore, a cutoff value of 9500 ng/ml sTM was found to be 95.0% sensitive and 92.9% specific with 96.9% diagnostic accuracy in differentiating diabetic patients with ischemic manifestations from diabetic patients without ischemic manifestations [Table 3] and [Figure 3]a and b.
|Table 3: Diagnostic performance of soluble thrombomodulin in predicting ischemic manifestations in diabetic patients|
Click here to view
|Figure 3: (a) Receiver operating characteristic curve for soluble thrombomodulin (sTM) in ischemic manifestations in diabetic patients, (b) scatter plots of sTM in different diabetics groups. The dashed line indicates the cutoff value of sTM (9500 ng/ml).|
Click here to view
| Discussion|| |
DM is a group of disorders characterized by hyperglycemia and associated with microvascular (i.e. retinal, renal, neuropathic) and macrovascular (i.e. coronary, cerebrovascular, peripheral vascular) complications  .
Eighty percent of patients with DM die a thrombotic death. Seventy-five percent of these deaths are because of cardiovascular complications and the remainder is because of cerebrovascular events and peripheral vascular complications. The abnormal metabolic state in diabetes affects both structure and function of the vessels, resulting in accelerated atherogenesis  .
TM is a glycoprotein that can bind to thrombin and activate protein C, thus mitigating the effects of cytokines produced by inflammatory and immunological processes. The molecule exerts a protective function on the endothelial cells  .
TM is not only found bound to endothelial cells, but also sTMs are present in the circulation  .
TM is cleaved to its soluble form (sTM) by neutrophil elastase and by other substances produced during acute and chronic inflammatory responses, immunologic reactions, and complement activation. sTM is traditionally thought to serve as a marker of endothelial cell damage , .
ELISA technique is traditionally used to measure sTM concentrations , . It was reported that an increase in sTM was observed in patients with diseases associated with vascular injury, such as disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, systemic lupus erythematosus, chronic myelogenous leukemia, and diabetic nephropathy , .
In this study, a highly significant increase in sTM concentration was detected in group I diabetic patients when compared with group II controls. In addition, sTM levels showed highly significant increase in group Ia patients compared with groups Ib and Ic.
The increase in sTM concentration in diabetic patients was detected by several investigators ,,,. Elevated levels of sTM in DM is an expected finding due to ongoing endothelial injury seen in those conditions induced by hyperglycemia and/or diabetic perimicroangiopathy. sTM levels are thus correlated with the extension of the vascular pathology  .
Elevated serum glucose levels can cause endothelial cell damage leading to elevated sTM levels. Moreover, Mclaren et al.  stated that TM increases in adult diabetic patients and also in patients with juvenile diabetes, in the absence of vascular disease.
Wojtkielewicz et al.  and Mezaki et al.  also found that chronic diseases that are related to inflammation and endothelial dysfunction, such as atherosclerotic arterial diseases in general and atherosclerotic diabetic patients, specially have increased level of sTM.
A significantly higher sTM level among diabetic patients with and without ischemia is in concordance with many previous reports ,,,,.
Aso et al.  reported that plasma TM might reflect endothelial damage better than plasma von Willebrand Factor (vWF) in the state of insulin resistance in patients with type 2 diabetes.
An Indian study published in 2012 reported a TM mutation (TM Ala455Val substitution) that was linked to increased coronary artery disease risk in patients aged 49 years or below  .
In this study, sTM values showed a significant positive correlation with patients' ages (P < 0.05). This was consistent with the study by Inukai et al.  but inconsistent with the studies by others , . Califano et al.  also noted that there was a tendency for an increase in sTM with age. It is therefore suggested that the sTM level may be partially influenced by atherosclerosis caused by aging.
In this study, no significant correlation was detected between sTM and fasting blood sugar (FBS) and HbA1c. This is consistent with the findings of several researchers who found sTM to be not associated with the relatively short-term controlled state of diabetes because no correlation was found between sTM and markers of glycemic control, such as FBS and HbA1c ,,, .
Yudkin et al.  reported that sTM level in type 2 diabetes was not influenced by improved glycemic control over 16 weeks. Improved metabolic control with insulin could, however, be associated with reduced concentrations of acute-phase marker C-reactive protein.
Our results showed that sTM level was significantly correlated to the presence of microalbuminuria, with highly significant P value (P < 0.001). Several previous reports confirming the association between sTM level and the presence of microalbuminuria have been published , .
In addition to the strong correlation between sTM and microalbuminuria, Hirano et al.  found another strong correlation between serum creatinine and TM levels in total participants. To eliminate the influence of renal function, they used TM index - TM (fU/ml)/serum creatinine (mg/dl) - instead of sTM concentration as an endolethial marker. The TM index was elevated in diabetic patients with microalbuminuria and markedly elevated in patients with overt diabetic nephropathy, whereas this was not observed in patients with primary renal disease. Thus, they suggested that increased TM index found in diabetic nephropathy is less associated with kidney damage per se but mainly attributable to systemic vascular damage.
In 2010, Kubisz et al.  investigated the relationship between serum vascular endothelial growth factor level and parameters of endothelial injury and/or dysfunction in patients with type 2 DM with or without microalbuminuria. They investigated 84 diabetic patients (42 normoalbuminuric and 42 microalbuminuric patients). They compared their results with another 42 blood donors as a control group. They found that the difference between microalbuminuric patients and controls was not statistically significant, but there was a trend toward significance.
In this study, the sTM concentration in diabetic patients with associated ischemia was significantly greater than in patients without ischemic manifestations.
In previous study by Tanaka et al.  , the sTM values in patients with diabetic nephropathy or retinopathy were significantly greater than in those without these complications. They concluded that there may be a close relationship between these higher sTM values and the genesis of diabetic nephropathy or retinopathy. This may be due to the release of sTM from the surface of the endothelial cells after damages related to metabolic abnormalities in diabetes. TM also exists in the endothelial cells of renal and retinal capillaries, which are the sites for pathological changes in patients with microangiopathy  .
Nephropathy is frequently observed in diabetic patients with retinopathy, which may reflect the pathogenesis of diabetic complications  .
It is hypothesized that sTM levels are related to diabetic complications. Hence, progression of diabetic retinopathy, nephropathy, or arterial occlusive disease can be predicted by monitoring of sTM levels that increase with progression of the disease , .
Although measurement of endothelial dysfunction in vivo presents a major challenge, it has important implications and represents a clinically relevant tool to predict the overall vascular risk in diabetic patients, as it may identify the clinical need for therapeutic intervention.
We conclude that raised sTM may be an indicator of a poor prognosis in patients with diabetes. This may aid in the identification of patients at greater risk for disease complications, and hence allow medical resources to be targeted with greater efficiency.
We recommend including sTM in the follow-up panel of diabetic patients because of its potential ability to predict the risk for ischemic manifestations. Further prospective studies are needed to explore the alterations in sTM with respect to other ischemic risk factors in diabetic patients.
| Acknowledgements|| |
| References|| |
|1.||Konstantoulas C, Cooper J, Ohlin A, Humphries E, Goodall A, Toh C, Mather H. Low soluble thrombomodulin activity and antigen is associated with a family history of heart disease while a high level is associated with a personal history of heart disease in type 2 diabetes. Thromb Haemost 2007; 97 :161-164. |
|2.||Thorand B, Baumert J, Döring A, Schneider A,Chambless L, Löwel H, et al. Association of cardiovascular risk factors with markers of endothelial dysfunction in middle-aged men and women. Results from the MONICA/KORA Augsburg Study. Thromb Haemost 2006; 95 :134-141. |
|3.||Inukai T, Fujiwara Y, Tayama K. Clinical significance of measurements of urinary and serum thrombomodulins in patients with non-insulin-dependent diabetes mellitus. Diabetes Res Clin Pract 1996; 33 :99-104. |
|4.||Blann A, Seigneur M, Steiner M. Circulating endothelial cell markers in peripheral vascular disease: relationship to the location and extent of atherosclerotic disease. Eur J Clin Invest 1997; 27 :916-921. |
|5.||Wolfs M, Hofker M, Wijmenga C, Van Haeften T. Type 2 diabetes mellitus: new genetic insights will lead to new therapeutics. Curr Genomics 2009; 10 :110-118. |
|6.||Huysman F, Mathieu C. Diabetes and peripheral vascular disease. Acta Chir Belg 2009; 109 :587-594. |
|7.||F Califano, T Giovanniello, P Pantone, E Campana, C Parlapiano, F Alegiani, et al. Clinical importance of thrombomodulin serum levels. Eur Rev Med Pharmacol Sci 2000; 4 :59-66. |
|8.||Thorand B, Baumert J, Herder C, Meisinger C, Koenig W. Soluble thrombomodulin as a predictor of type 2 diabetes: results from the MONICA/KORA Augsburg case-cohort study, 1984-1998. Diabetologia 2007; 50 :545-548. |
|9.||Constans J, Conri C. Circulating markers of endothelial function in cardiovascular disease. Clin Chim Acta 2006; 368 :33-47. |
|10.||Raitakari OT, Celermajer DS. Testing for endothelial dysfunction. Ann Med 2000; 32 :293-304. |
|11.||Tohda G, Oida K, Okada Y, Kosaka S, Okada E, Takahashi S, Ishii H, Miyamori I. Expression of thrombomodulin in atherosclerotic lesions and mitogenic activity of recombinant thrombomodulin in vascular smooth muscle cells. ArteriosclerThromb Vasc Biol 1998; 18 :1861-1869. |
|12.||Blann AD, Seigneur M, Steiner M, Boisseau MR, Mccollum CN. Circulating endothelial cell markers in peripheral vascular disease: relationship to the location and extent of atherosclerotic disease. Eur J Clin Invest 1997; 27 :916-921. |
|13.||Mclaren M, Elhadd TA, Greene SA, Belch JJ. Elevated plasma vascular endothelial cell growth factor and thrombomodulin in juvenile diabetic patients. Clin Appl Thromb Hemost 1999; 5 :21-24. |
|14.||Wojtkielewicz K, Urban M, Peczyñska J, G³owiñska B. Is soluble thrombomodulin a molecular marker of endothelial cell injury in children and adolescents with arterial hypertension? Med Wieku Rozwoj 2006; 10 :893-902. |
|15.||Mezaki T, Matsubara T, Hori T, Higuchi K, Nakamura A, Nakagawa I, et al. Plasma levels of soluble thrombomodulin, C-reactive protein, and serum amyloid A protein in the atherosclerotic coronary circulation. Jpn Heart J 2003; 44 :601-612. |
|16.||Aso Y, Inukai T, Takemura Y. Mechanisms of elevation of serum and urinary concentrations of soluble thrombomodulin in diabetic patients: possible application as a marker for vascular endothelial injury. Metabolism 1998; 47 :362-365. |
|17.||Moussavi N, Renier G, Roussin A, Mamputu J, Buithieu J, Serri O. Lack of concordance between plasma markers of cardiovascular risk and IMT in patients with type 2 diabetes. Diabetes Obes Metab 2004; 6 :69-77. |
|18.||Shah SA, Ashavaid TF, Mankeshwar R, Ponde CK, Rajani R. Role of thrombomodulin gene in Indian population with coronary artery disease. Biomarkers 2012; 17 :610-617. |
|19.||Aso Y, Fujiwara Y, Tayama K, Takanashi K, Inukai T, Takemura Y. Relationship between plasma soluble thrombomodulin levels and insulin resistance syndrome in type 2 diabetes: a comparison with von Willebrand factor. Exp Clin Endocrinol Diabetes 2001; 109 :210-216. |
|20.||Blann A, Amiral J, McCollum C. Prognostic value of increased soluble thrombomodulin and increased soluble E-selectin in ischaemic heart disease. Eur J Haematol 1997; 59 :115-120. |
|21.||De Angelis L, Marfella MA, Siniscalchi M, Marino L, Nappo F, Giugliano F, et al. Erectile and endothelial dysfunction in type II diabetes: a possible link. Diabetologia 2001; 44 :1155-1160. |
|22.||Dietrich S, Falk CS, Benner A, Karamustafa S, Hahn E, Andrulis M, et al. Endothelial vulnerability and endothelial damage are associated with risk of graft-versus-host disease and response to steroid treatment. Biol Blood Marrow Transplant 2013; 19 :22-27. |
|23.||Yudkin JS, Panahloo A, Stehouwer C, Emeis JJ, Bulmer K, Mohamed-Ali V, Denver AE. The influence of improved glycaemic control with insulin and sulphonylureas on acute phase and endothelial markers in type II diabetic subjects. Diabetologia 2000; 43 :1099-1106. |
|24.||Iwashima Y, Sato T, Watanabe K, Ooshima E, Hiraishi S, Ishii, H, et al. Elevation of plasma thrombomodulin level in diabetic patients with early diabetic nephropathy. Diabetes 1990; 39 :983-988. |
|25.||Sumida Y, Wada H, Fujii M, Mori Y, Nakasaki T, Shimura M, et al. Increased soluble fibrin monomer and soluble thrombomodulin level in non-insulin dependent diabetes mellitus. Blood Coagul Fibrinolysis 1997; 8 :303-307. |
|26.||Hirano T, Ookubo K, Kashiwazaki K, Tajima H , Yoshino G , Adachi M. Vascular endothelial markers, von Willebrand factor and thrombomodulin index, are specifically elevated in type 2 diabetic patients with nephropathy: comparison of primary renal disease. Clin Chim Acta 2000; 299 :65-75. |
|27.||Kubisz P, Chudý P, Stasko J, Galajda P, Hollý P, Vysehradský R, Mokán M. Circulating vascular endothelial growth factor in the normo- and/or microalbuminuric patients with type 2 diabetes mellitus. Acta Diabetol 2010; 47 :119-124. |
|28.||Tanaka A, Ishii H, Hiraishi S, Kazama M, Maezawa H. Increased thrombomodulin values in plasma of diabetic men with microangiopathy. Clin Chem 1991; 37 :269-272. |
|29.||Gabat S, Keller C, Kempe HP, Amiral J, Ziegler R, Ritz E, et al. Plasma thrombomodulin: a marker for microvascular complications in diabetes mellitus. Vasa 1996; 25 :233-241. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]