Volume 14 Supplement 4
Clinical implications of serum thrombomodulin in PR3-ANCA-associated vasculitis
© I. Holzapfel Publishers 2009
Published: 7 December 2009
Vascular injury is the main mechanism in pathophysiology of PR3-ANCA-associated vasculitis. Soluble serum thrombomodulin (sTM) is a membrane-bound receptor for thrombin expressed by vascular endothelial cells.
The aim of study was to determine the blood levels of sTM in patients with PR3-ANCA-associated vasculitis.
Material and methods
Twenty five patients with Wegener's granulomatosis (WG), 13 with generalized WG and 12 with limited WG, with histologically proven disease, and 15 healthy subjects as a control were investigated. An ELISA for detection of sTM and PR3-ANCA was performed. The disease activity was evaluated according to BVAS and DEI indexes.
Significant increases in sTM were found in both active generalized and limited active WG compared with control values: 108 ± 12, 56 ± 2, and 12 ± 4 ng/ml, respectively. Elevated ANCA titer correlated with disease activity, but more weakly than sTM levels did. Elevated sTM concentration is a result of vascular endothelial injury in the course of PR3-ANCA associated vasculitis.
Soluble serum thrombomodulin is a promising, both diagnostic and therapeutic, marker of endothelial cell injury in relation to disease activity and progression in autoimmune disorders, reflecting the degree of endothelial cell damage.
Endothelial cell damage, probably mediated by polymorphonuclear cells (PMN) and PR3-ANCA, plays the main role in the pathogenesis of Wegener's granulomatosis (WG). Elevated soluble thrombomodulin (sTM) is an accepted marker of endothelial damage . Factors reflecting endothelial cell damage, or response to injury, are of considerable interest in patients with PR3-ANCA positive vasculitis. They may be potential markers of vasculitic disease activity and progression . Thrombomodulin (TM) is an integral membrane protein made up of 557 aminoacids that bears some structural resemblance to the LDL receptor. Expressed by endothelial cells and functioning as a thrombin receptor, TM is one of the factors responsible for the anticoagulant properties of the vascular endothelium [3, 4]. TM is an endothelial cell transmembrane co-factor for thrombin-mediated protein C activation. There are 30000-50000 thrombomodulin molecules expressed on an endothelial cell, representing 50-60% of all thrombin binding sites. This glycoprotein is present on all endothelial cells, except the sinusoidal hepatic lining cells and postcapillary endothelial cells in lymph node veins [4, 5]. It is also found in the mucosal mesothelium and lining of the body cavities, blood plasma, platelets, neutrophils, monocytes, urine, and placenta. Membranes and large arteries respond to exposed TM much more strongly than veins and capillaries. A strongly positive response in blood vessels does not depend on the investigated organ, but rather is related to the quality of blood flow . TM plays a major role in maintaining blood in a liquid state and preventing intravascular coagulation. Furthermore, thrombin complexed with thrombomodulin loses its pro-coagulation properties in the conversion of fibrinogen into fibrin, activation of factors V, VII, and XIII, inactivation of protein S, and induction of platelet aggregation .
A soluble form of thrombomodulin (sTM) found in plasma and urine is released mainly due to endothelial call damage, but not due to physiological activation [6, 7]. The aim of the study was to determine the relevance of sTM as a predictor of endothelial cell damage in Wegener's granulomatosis (WG) and as a marker of disease activity (progression and remission).
Materials and methods
The study was approved by a local Ethics Committee. Twenty five serum samples obtained from 13 patients with active generalized biopsy-proven WG and from 12 patients with limited WG were tested. The patients' mean age was 46.8 ± 12.5 SD. All patients fulfilled the American College of Rheumatology criteria for classification of WG and the Chapel Hill Consensus Conference definition, and also EUVAS ANCA-associated vasculitis definition for WG. Disease activity was confirmed by clinical scoring, laboratory variables, and imaging procedures. DEI and BVAS indexes were determined to measure organ involvement and disease activity. An infective reason of inflammation was ruled out in the patients by routine blood and urine culture, bronchoalveolar lavage fluid culture, and serological tests. Another fifteen serum samples obtained from 8 women and 7 men, aged 32-68, without any clinical symptoms of vasculitis, were tested as control. CRP was measured with a routine turbidimetry assay (ILAD-900): a value greater than 10 mg/l was considered to be abnormally high. An ELISA test for detection of PR3-ANCA and sTM in plasma was performed. The concentration of TM was determined using the Imubind® Thrombomodulin ELISA Kit assay that is able to recognize the intact and partially degraded forms of TM (reference values: women-agedependent: 2.73 ng/ml for the age 21-30, than increased up to 4.79 ng/ml for the age 61-79 ng/ml; men-age-independent: 4.00-5.35 ng/ml).
All data were expressed as means ± SE. The Wilcoxon and Spearman tests were used for statistic analysis. P < 0.05 was considered statistically significant.
Baseline characteristics in generalized and limited Wegener's granulomatosis (WG).
Generalized WG (n = 13)
Limited WG (n = 12)
Control (n = 15)
Median age (yr)
Disease duration (mo)
Correlation table in Wegener's Granulomatosis group.
Laboratory data in generalized and limited Wegener's granulomatosis (WG).
108 ± 12
56 ± 2
12 ± 4
79.1 ± 26.6
27.3 ± 10.7
8.4 ± 4.2
13.7 ± 7.8
1.5 ± 0.98
1.5 ± 0.98
426.5 ± 164.2
212.6 ± 21.6
160.6 ± 22.6
9900 ± 3500
5400 ± 1500
4400 ± 1500
398.7 ± 134.1
223.4 ± 57.5
130.4 ± 59.5
11.4 ± 2.4
12.2 ± 1.8
13.2 ± 1.8
64.21 ± 16.8
76.8 ± 9.4
96.8 ± 8.4
640 ± 160
80 ± 40
160 ± 80
80 ± 20
100 ± 80
8.2 ± 4.6
40 ± 14
9.4 ± 2.1
Clinical studies have shown elevated levels of soluble thrombomodulin in various pathological conditions such as: diffuse intravascular coagulation (DIC) syndrome, pulmonary embolism, chronic renal failure (MIA-syndrome), diabetic microangiopathy, systemic lupus erythematosus (SLE), peripheral and coronary atherosclerosis, Schönlein-Henoch purpura and malignancies (lymphomas, leukemias, carcinomas) [3, 8]. Endothelial call activation and damage also seem to be a pathophysiological mechanism of PR3-ANCA positive vasculitis .
Previous in vitro studies have shown that autoantibodies, such as PR3-ANCA, activate cytokine-primed neutrophils and monocytes leading to release of proteolytic enzymes and the production of the oxygen radicals and pro-inflammatory cytokines. PR3-ANCA may play a role in vascular injury by attracting leucocytes to the inflammatory site. Interaction of PR3-ANCA with their target antigens may aggravate an inflammatory process [9–11]. In the present study, we demonstrated a significant correlation between Wegener's granulomatosis disease activity and sTM levels. This finding is in accord with a study by Boehme et al  in which 197 serum samples obtained from 102 patients with WG, of different disease activity, and 41 samples from patients with other active vasculitides: 12 patients with Takayasu arteritis (TA), 7 with giant cell arteritis (GCA), 10 with polyarteritis nodosa (PAN), and 12 with Behcet's disease (BD) were assessed .
In the present study, in limited WG, sTM levels were elevated in more patients than the PR3-ANCA titers were. This findings is in agreement with other studies in which elevated levels of sTM were demonstrated in patients with active WG [13, 14]. In our study, patients with active disease and elevated sTM levels had a higher prevalence of renal involvement, and higher serum creatinine levels, the findings being similar to those of Boomsma et al's . The expression of TM is decreased by inflammatory cytokines, such as TNF-α, due to suppression of TM transcription and translation. Moreover, in vitro granulocyte-derived proteases, such as cathepsin, elastase, and hydrogen peroxide have been show to induce the release of TM from the endothelial cell surface as a result of endothelial cell damage. Thus, this pathway would explain a good correlation between the disease activity in PR3-ANCA positive vasculitis and thrombomodulin concentration, as a predictor of vascular damage and injury.
We conclude that patients with PR3-ANCA positive vasculitis, such as Wegener's granulomatosis, had elevated sTM levels, which may result from vascular endothelium damage and injury. Higher sTM concentrations were observed in patients with active generalized Wegener's granulomatosis. Thus, sTM may be considered as a serological marker and clinically useful predictor of disease activity and course in vasculitis and angiitis directly correlating with the degree of endothelial cell damage. On the other hand, determination of sTM may be helpful in assessing the disease activity in patients who are negative for c-ANCA or PR3-ANCA, or in whom ANCA titers do not reflect clinical disease activity.
Conflicts of interest
The authors declare that they have no competing interests.
- Kallenberg CG: Pathogenesis of PR3-ANCA associated vasculitis. J Autoimmun 2008,30(1–2):29–36. 10.1016/j.jaut.2007.11.005PubMedView ArticleGoogle Scholar
- Pagnoux C, Teixeira L: Wegener's Granulomatosis. Presse Med 2007,36(5):860–887. 10.1016/j.lpm.2007.02.015PubMedView ArticleGoogle Scholar
- Widerkiewicz K, Dzianott-Pabian D, Maslowska B, Kotschy M: Thrombomodulin in the blood of patients with acute cerebral ischemia. Adv Clin Exp Med 2006,15(2):271–277.Google Scholar
- Ditman WA, Majerus PW: Structure and function of thrombomodulin: a natural anticoagulant. Blood 1990, 75: 329–336.Google Scholar
- Isermann B, Hendrikson SB, Zogg M, Wing M, Cummiskey M, Kisanuki YY, Yanagisawa M, Weiler H: Endotheliumspecific loss of murine thrombomoduline disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis. J Clin Invest 2001,108(4):537–546.PubMed CentralPubMedView ArticleGoogle Scholar
- Ohlin AK, Norlund L, Marlar RA: Thrombomodulin gene variations and thromboembolic disease. Thromb Haemost 1997,78(1):396–400.PubMedGoogle Scholar
- Van der Wouwer M, Collen D, Conway EM: Thrombomodulin-protein C-EPCR system: integrated to regulate coagulation and inflammation. Aterioscler Thromb Vasc Biol 2005,24(8):1374–1383.View ArticleGoogle Scholar
- Salomaa V, Salomaa V, Matei C, Aleksic N, Sansores-Garcia L, Folsom AR, Juneja H, Chambless LE, Wu KK: Soluble thrombomodulin as a predictor of incident coronary heart disease and symptomless carotid artery atherosclerosis in the Atherosclerosis Risk in Communities (ARIC) Study: a case-cohort study. Lancet 1999, 353: 1729–1734. 10.1016/S0140-6736(98)09057-6PubMedView ArticleGoogle Scholar
- Nienhuis HL, de Leeuw K, Smit AJ, Bijzet J, Stegeman CA, Kallenberg CG, Bijl M: Enhanced endothelium-dependent microvascular responses in patients with Wegener's granulomatosis. J Rheumatol 2007,34(9):1875–1881.PubMedGoogle Scholar
- Jayne D: Progress of treatment in ANCA-associated vasculitis. Nephrology 2009,14(1):42–48. 10.1111/j.1440-1797.2009.01101.xPubMedView ArticleGoogle Scholar
- Seo P: Vasculitis: lessons learned. Curr Opin Rheumatol 2009,21(1):1–2. 10.1097/BOR.0b013e32831e4250PubMed CentralPubMedView ArticleGoogle Scholar
- Boehme MWJ, Schmitt WH, Youinou P, Stremmal WR, Gross W: Clinical relevance of elevated serum thrombomodulin and soluble E-selectin in patients with Wegener's granulomatosis and other systemic vasculitides. Am J Med 1996, 101: 387–393. 10.1016/S0002-9343(96)00230-6PubMedView ArticleGoogle Scholar
- De Leeuw K, Sanders JS, Stegeman C, Smit A, Kallenberg CG, Bijl M: Accelerated atherosclerosis in patients with Wegener's granulomatosis. Ann Rheum Dis 2005,64(5):753–759. 10.1136/ard.2004.029033PubMed CentralPubMedView ArticleGoogle Scholar
- Knight A, Ekbom A, Brandt L, Askling J: What is the significance in routine care of c; ANCA/PR3ANCA in the absence of systemic vasculitis? A case series. Clin Exp Rheumatol 2008,26(3 Suppl 49):S53-S56.PubMedGoogle Scholar
- Boomsma MM, Stearns-Kurosawa DJ, Stegeman CA, Raschi E, Meroni PL, Kurosawa S, Tervaert JW: Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener's granulomatosis. Clin Exp Immunol 2002,128(1):187–194. 10.1046/j.1365-2249.2002.01803.xPubMed CentralPubMedView ArticleGoogle Scholar