Abstract
Elevated soluble thrombomodulin (sTM) levels are an accepted marker of endothelial damage. The physiological significance of plasma endothelial protein C receptor (sEPCR) levels is not known. To assess the relevance of this plasma protein in Wegener’s granulomatosis (WG), sEPCR levels were measured in sera obtained from WG patients and related to disease activity, sTM levels, and other known markers of disease activity. In total, 129 sera (37 at active disease, 92 during follow-up) from 31 WG patients were tested. During active disease, eight (22%) and 17 (46%) out of 37 active sera had elevated levels of sEPCR and sTM, respectively (NS); sEPCR (r = 0·39; P = 0·02) and sTM (r = 0·53; P < 0·01) levels correlated with disease activity (Birmingham Vasculitis Activity Score). Analysis of longitudinal sera revealed a significant increase in sEPCR (P = 0·01) and sTM (P = 0·04) levels prior to the moment of a relapse. Corrected for renal function, the increase in sEPCR remained significant (P = 0·04) whereas sTM did not (NS). Levels of sEPCR correlated with sTM levels (r = 0·32; P < 0·001). Plasma levels of sEPCR respond to changes in the disease in patients with WG
Keywords: Wegener’s granulomatosis, vasculitis, disease activity, AECA, ANCA, thrombomodulin, endothelial cell protein C receptor
INTRODUCTION
Factors reflecting endothelial cell damage or response to injury are of considerable interest in patients with Wegener’s granulomatosis (WG), since they may be potential markers of vasculitic disease activity due to the central role of vascular endothelial cells in the pathology of this disease [1]
Thrombomodulin (TM) is an endothelial cell transmembrane co-factor for thrombin-mediated protein C activation [2]. Immunohistochemistry studies have shown that membrane-bound TM is expressed on both large vessels and in the microcirculation [3]. While TM expression is not restricted to endothelium [4], the surface area of endothelium exposed to blood is much greater than that for other cell types, with greatest exposure in capillary beds. Membrane-bound TM exposed to elastase from activated neutrophils is degraded [5], and the plasma level of soluble TM antigen (sTM) is an accepted marker of endothelial cell damage [6]. Elevated levels of sTM in relation to disease activity have been measured in autoimmune disorders, such as systemic lupus erythematosis (SLE), Churg–Strauss syndrome and WG [7,8].
The endothelial cell protein C receptor (EPCR) is a recently described member of the protein C anti-coagulant pathway [9]. EPCR accelerates formation of activated protein C [10], a potent anti-coagulant and anti-inflammatory agent [11]. Immunohistochemistry studies have shown that membrane-bound EPCR is expressed exclusively on endothelial cells, with greater expression corresponding to increasing vessel diameter [3,12]. A soluble form of EPCR (sEPCR) exists in plasma and retains its ability to bind both protein C zymogen and activated protein C [13]. Recently, it was demonstrated that sEPCR binds to activated neutrophils, partly via proteinase 3 (PR3) [14]. PR3 is a neutrophil granule protein expressed on the membrane of activated neutrophils [15] and is the primary target antigen of anti-neutrophil cytoplasmic antibodies (PR3-ANCA) in WG [16]. Elevation of sEPCR levels may result either from vascular injury or through a regulated proteolytic release of sEPCR [17]. Regulated release of sEPCR, possibly through thrombin stimulation of the endothelium and subsequent metalloproteinase activity [18], may modulate inflammation through interaction with activated neutrophils. Consistent with this is an in vitro study showing PR3-ANCA-induced increases in tissue factor expression [19], the physiological initiator of the coagulation cascade that results in thrombin production. This concept is further supported by the fact that significantly elevated levels of circulating sEPCR are observed in patients with SLE and sepsis [20], conditions associated with systemic inflammation of the vasculature. However, the clinical relevance of sEPCR in patients with WG is still unknown.
The aim of the present study was to determine whether sEPCR levels are elevated and/or correlate with disease activity in patients with WG. Secondly, we compared this marker with other known markers of endothelial damage and/or disease activity, such as sTM, ANCA, anti-endothelial cell antibodies (AECA), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)
METHODS
Patients
Patients with WG who had been positive for PR3-ANCA [21] during an active phase of the disease were eligible for this study. The diagnosis of WG was based on the presence of classical symptoms and histological findings. All patients fulfilled the American College of Rheumatology 1990 criteria for WG [22] and met the definition for WG of the 1992 Chapel Hill Consensus Conference [23]. Patients are followed and treated according to a previously described protocol [24,25] at the vasculitis outpatient clinic, University Hospital Groningen. Patients were evaluated for signs and symptoms of active vasculitis or infections at least every 3 months. Patients were treated with prednisolone and cyclophosphamide, with or without plasma exchange, according to our standard protocol [25]. At each visit, disease activity was scored using the Birmingham Vasculitis Activity Score (BVAS) [26]. Complete remission was defined as the total absence of symptoms or signs attributable to active vasculitis (BVAS = 0). A relapse was defined as described previously [27]. The moment of a clinical relapse was defined as the time at which immunosuppressive treatment was started or intensified. The study was carried out in accord-ance with the 1997 Declaration of Helsinki of the World Medical Association [28].
At each visit, plasma was collected and stored at – 20°C until further use. Samples from consecutive patients with WG, who were diagnosed in our hospital between October 1992 and March 1997, were collected. When available, samples of these patients were studied at the time of a complete remission when immunosuppressive treatment was stopped. In addition, samples from consecutive patients with WG who had had a relapse between August 1996 and March 1998 were studied. From these patients also, samples obtained 3 and 6 months prior to, and samples obtained 3 and 6 moths after relapse were studied.
Laboratory parameters and kidney dysfunction
At each visit, standard laboratory evaluation included determination of ESR, serum creatinine level, microscopic analysis of urine sediment, and 24 h urine collection for protein determination. In addition, CRP levels were measured by nephelometry (Behring, Marburg, Germany) [29], and ANCA levels by IIF and by direct PR3-ANCA ELISA [25]. The intra-and interassay variations of the ELISA were <10%.
Detection of soluble EPCR
An ELISA for detection of sEPCR antigen in plasma was performed as previously described [20]. Briefly, microtitre plates (Maxisorp; NUNC NS, Roskilde, Denmark) were coated with 50 μl of 2 μg/ml anti-EPCR 1494 monoclonal antibody [10] overnight at 4°C. The wells were blocked with assay buffer containing 0·1% (w/v) gelatin for at least 1 h at room temperature. The plates were washed and 50 μl of 1:50 diluted plasma samples were added in duplicate wells and incubated for 1 h. After washing, 50 μl 4 μg/ml biotinylated anti-EPCR 1495 MoAb [10] were added and incubated for 1 h, followed by detection with streptavidin-alkaline phosphatase and Blue Phos substrate (KPL, Gaithersburg, MD, USA). The optical density was measured at 650 nm, and sEPCR antigen levels were calculated by reference to a standard curve determined with recombinant soluble EPCR [30] on the same plate. Values of 289 ng/ml (mean +2 s.d. of 19 normal controls) or more were considered to be above normal. Inter-assay variation was 5% and intra-assay variation was <5%. Since little is known about the clearance of sEPCR, both adjusted (divided by the patient’s serum creatinine value) and unadjusted sEPCR levels are given.
Detection of soluble TM
An ELISA for detection of sTM in plasma was performed essentially as described previously [20]. ELISA plates were coated with 50 μl 5 μg/ml anti-TM 1029 MoAb overnight at 4°C, and subsequently blocked with 200 μl Tris-HCl, pH 7·5 (TBS), containing 0·1% (w/v) gelatin for 1 h at room temperature. After washing, plates were incubated with 50 μl 1:20 diluted plasma samples, in duplicate, for 1 h at 37°C. After washing, 50 μl 1·0 μ/ml biotinylated anti-TM 1009 MoAb were added and incubated for 1 h at 37°C. Bound antibody was detected with 50 μl 0·1 μg/ml streptavidin-alkaline phosphatase (Gibco BRL, Grand Island, NY, USA), and substrate amplification with an ELISA amplification system (Gibco BRL). The optical density was measured at 490 nm. For each plate, a standard curve was included using recombinant soluble TM [31]. Values of 26·9 ng/ml (mean +2 s.d. of 19 normal controls) or more were considered to be above normal. Inter-assay variation was 10% and intra-assay variation was <5%. sTM is cleared from the circulation by the kidneys, and marked kidney dysfunction is known to result in accumulation of plasma sTM [32]. Therefore, both adjusted (divided by the patient’s serum creatinine value) and unadjusted sTM levels are given.
Detection of AECA
Human umbilical vein endothelial cells (HUVEC) were isolated from normal term umbilical cord vein by collagenase perfusion and cultured as previously reported [33]. In all the experiments, endothelial cells from at least three different donors were used. AECA were detected by a cell solid-phase ELISA as previously described [33].
The results of the tested plasma samples were expressed as percentage of a positive serum arbitrarily chosen as 100% of endothelial binding activity. Values of 27% for IgG-AECA and 11% for IgM-AECA (mean +3 s.d. of 35 normal controls) or more were considered positive. The inter-and intra-assay variations were <10%.
Statistical analysis
To determine differences in levels of serological markers between disease and control groups, the Fischer’s exact test for categorical quantities, and the Mann–Whitney test (unpaired observations) and Wilcoxon test (paired observations), were used when appropriate for continuous quantities. Univariate associations of serological factors with disease activity were evaluated using Spearman’s correlation coefficients for continuous quantities. A Friedman test was used to evaluate difference in the serial samples. A two-sided P-value of < 0·05 was used to indicate statistical significance.
RESULTS
Two different cohorts were studied. (i) Sixteen patients with newly diagnosed WG; from eight of these patients, a sample at complete remission was available (median [range] interval presentation–remission, 22 months [16–37]). (ii) Twenty-one patients from whom sequential samples were collected at 6 and 3 months prior to relapse, at the moment of relapse, and 3 and 6 months after relapse. Furthermore, the composite cohort of 37 samples from patients with active disease (16 samples at presentation from the first cohort and 21 samples at relapse from the second cohort) were analysed to study the possible relation of the different markers with disease activity.
Samples at presentation and at remission
Clinical characteristics. The clinical characteristics of the 16 patients from whom a sample at the time of presentation was obtained are listed in Table 1
Table 1.
Clinical characteristics of patients with Wegener’s granulomatosis at presentation and at relapse of disease (median [range])
| Patient at time of presentation | Patient at time of relapse | P-value | |
|---|---|---|---|
| n | 16 | 21 | |
| Sex, male/female | 11/5 | 14/7 | N.S. |
| Age, years | 57 [25–79] | 53 [26–77] | N.S. |
| BVAS score | 14 [3–21] | 8 [2–15] | 0·05 |
| Organ involvement* | |||
| Systemic | 81% | 76% | N.S. |
| Cutaneous | 31% | 24% | N.S. |
| Mucous | 56% | 33% | N.S. |
| Ear, nose and throat | 88% | 67% | N.S. |
| Chest | 50% | 43% | N.S. |
| Cardiac | 6% | 0% | N.S. |
| Abdominal | 6% | 5% | N.S. |
| Renal | 69% | 67% | N.S. |
| Nervous system | 44% | 19% | N.S. |
Systemic: e.g. fever, arthralgia, myalgia, malaise; Mucous: genital/mouth ulcers or red eyes/proptosis; Renal: positive biopsy and/or decreased creatinine clearance in combination with proteinuria and erythrocyturia; N.S: not significant.
Soluble EPCR. At presentation of disease, elevated sEPCR levels were present in three out of 16 patients (19%). Remarkably, levels of sEPCR at presentation tended to be lower compared with levels at remission (P = 0·11) (Table 2). This difference became significant when sEPCR levels were corrected for renal function (P = 0·04). At the moment of remission, elevated sEPCR levels were present in four out of eight patients (50%)
Table 2.
Serological disease activity parameters in patients with Wegener’s granulomatosis (median [range])
| Patients at time of presentation | Patients in remission | P-value | |
|---|---|---|---|
| n | 16 | 8 | |
| sEPCR (ng/ml) | 235 [113–373] | 277 [127–580] | 0·11 |
| sTM (ng/ml) | 28 [13–148] | 19 [6–61] | 0·08 |
| ANCA (ELISA) (arbitrary units) | 822 [62–6597] | 123 [14–429] | 0·02 |
| ANCA (IIF) (dilution 1/) | 320 [80-> 640] | 80 [0–320] | 0·02 |
| IgG-AECA (%) | 27 [11–73] | 31 [4–59] | 0·46 |
| IgM-AECA (%) | 14 [0–31] | 8 [0–20] | 0·38 |
| CRP (mg/l) | 101 [12–315] | 14 [<3–39] | 0·008 |
| ESR (mm/h) | 83 [42–122] | 31 [4–86] | 0·03 |
sEPCR, soluble endothelial protein C receptor; sTM, soluble thrombomodulin; ANCA, antineutrophil cytoplasmic antibodies; ELISA, enzyme linked immunosorbent assay; IIF, indirect immunofluorescence; AECA, antiendothelial cell antibodies; CRP, C-reactive protein; ESR, erythrocyte sediment rate.
Soluble TM. At presentation of disease, elevated sTM levels were present in eight out of 16 patients (50%). Levels of sTM in patients with WG at presentation of disease were higher than at remission (difference not significant, P = 0·08) (Table 2). When sTM levels were corrected for renal function, levels did not appear to be different (P = 0·31). At the moment of remission, elevated sTM levels were present in one out of eight patients (13%).
Other markers. CRP, ESR and PR3-ANCA antibodies closely followed clinical disease activity (Table 2). However, IgG-AECA and IgM-AECA levels at presentation did not differ significantly from levels at remission (Table 2). Either IgG-AECA, IgM-AECA or both antibodies were present in 10 out of 16 patients (63%) at presentation, and in six out of eight patients (75%) at the time of remission.
Sequential samples at relapse
Clinical characteristics. The clinical characteristics of the 21 patients from whom samples prior to, at the time of, and following relapse were obtained are listed in Table 1.
Soluble EPCR. sEPCR levels rose prior to relapse and dropped to baseline (–6 months) levels after treatment (P = 0·01) (Fig. 1a). Correction of sEPCR levels for renal function did not affect the results (P = 0·04). In five out of 21 patients (24%), levels of sEPCR were above the normal range at the time of relapse.
Fig. 1.
Box plots indicating the overall range (error bars), 25–75% range (boxes) and median value (horizontal lines) of levels of soluble endothelial protein C receptor (sEPCR) (a), soluble thrombomodulin (sTM) (b), levels of C-reactive protein (CRP) (c), erythrocyte sedimentation rate (ESR) (d), anti-neutrophil cytoplasmic antibody (ANCA) as detected by indirect immunofluorescence (IIF) (e) and ANCA as detected by enzyme-linked immunosorbent assay (f), IgG-anti-endothelial antibodies (AECA) (g), and IgM-AECA (h), at 6 months, 3 months prior to relapse, at the time of relapse, and 3 months and 6 months after relapse, in 21 Wegener’s granulomatosis patients studied longitudinally.
Soluble TM. sTM levels rose prior to relapse and dropped below baseline (–6 months) levels after treatment (P = 0·04) (Fig. 1b). When sTM levels were corrected for renal function, these changes in sTM levels were no longer significant (P = 0·23). In nine out of 21 patients (43%), levels of sTM were above the normal range at the time of relapse.
Other markers. CRP levels (P < 0·001) and ESR (P < 0·01) rose at relapse and dropped to baseline (–6 months) levels after treatment (Fig. 1c–d). PR3-ANCA levels, as detected by both IIF and ELISA, rose prior to relapse and dropped below baseline (–6 months) levels after treatment (P < 0·0001) (Fig. 1e,f). In all patients, PR3-ANCA detected by either IIF or ELISA were present at relapse. No significant change was noted in IgG-AECA and IgM-AECA levels during relapse (Fig. 1g,h). At the moment of relapse, 10 out of 21 patients (48%) had either IgG-AECA, IgM-AECA or both antibodies present.
Correlation of markers with disease activity
The correlation between the different markers and BVAS scores of 37 patients with active disease (16 at presentation and 21 at relapse) is shown in Table 3. Spearman’s correlation of the sEPCR (r = 0·39; P = 0·02), sTM (r = 0·53; P < 0·01) and CRP (r = 0·59; P < 0·001) levels revealed a strong association with the BVAS score. When sEPCR (r = –0·14; P = 0·39) and sTM (r = 0·21; P = 0·20) levels were corrected for renal function, these correlations were no longer significant.
Table 3.
Spearman’s correlation between serological disease activity parameters and disease activity in patients with active Wegener’s granulomatosis
| Disease activity (BVAS score) | P-value | |
|---|---|---|
| sEPCR (ng/ml) | 0·39 | 0·02 |
| sTM (ng/ml) | 0·53 | <0·01 |
| ANCA (ELISA) (arbitrary units) | 0·27 | 0·11 |
| ANCA (IIF) (dilution 1/) | 0·31 | 0·06 |
| IgG-AECA (%) | 0·05 | 0·76 |
| IgM-AECA (%) | –0·02 | 0·91 |
| CRP (mg/l) | 0·59 | <0·001 |
| ESR (mm/h) | 0·29 | 0·11 |
sEPCR = soluble endothelial protein C receptor; sTM = soluble thrombomodulin; BVAS = Birmingham Vasculitis Activity Score; ANCA = antineutrophil cytoplasmic antibodies; ELISA = enzyme linked immunosorbent assay; IIF = indirect immunofluorescence; AECA = antiendothelial cell antibodies; CRP = C-reactive protein; ESR = erythrocyte sediment rate.
In Fig 2, sEPCR and sTM levels are displayed for 37 samples from patients with active disease (21 samples at relapse, 16 samples at presentation). There was a significant correlation between sEPCR and sTM levels (r = 0·40; P = 0·01). Elevated sEPCR levels correlated with greater age, while elevated sTM levels were associated with higher creatinine levels, renal involvement, BVAS scores and age (Table 4).
Fig. 2.
Levels of soluble endothelial protein C receptor (sEPCR) and soluble thrombomodulin (sTM) in 37 Wegener’s granulomatosis patients studied at active disease (16 samples at presentation, 21 samples at relapse). Dotted lines indicate the upper limit of the normal range.
Table 4.
Soluble TM and EPCR in patients with active Wegener’s granulomatosis (median [range])
| Elevated sTM | Normal sTM | P-value | Elevated sEPCR | Normal sEPCR | P-value | |
|---|---|---|---|---|---|---|
| n | 17 | 20 | 8 | 29 | ||
| Sex, male/female | 14/3 | 11/9 | 0·09 | 6/2 | 19/10 | 1·00 |
| Serum creatinine (μmol/l) | 226 [73–659] | 94 [65–419] | <0·001 | 194 [73–570] | 108 [65–659] | 0·12 |
| Renal | 88% | 50% | 0·02 | 88% | 62% | 0·23 |
| BVAS | 13 [6–21] | 7 [2–21] | <0·01 | 13 [6–18] | 9 [2–21] | 0·20 |
| Age, years | 59 [26–79] | 50 [24–76] | 0·05 | 62 [50–71] | 52 [24–79] | 0·04 |
| CRP (mg/l) | 81 [12–307] | 52 [<3–315] | 0·30 | 47 [<3–191] | 67 [6–315] | 0·58 |
| Leukocytes (x109/l) | 7·9 4·4–24·7] | 7·2 4·0–17·4] | 0·57 | 7·2 4·4–12·1] | 7·9 4·0–24·7] | 0·45 |
| IgG-AECA (%) | 19 [0–90] | 24 [4–51] | 0·99 | 18 [9–71] | 25 [0–90] | 0·92 |
| IgM-AECA (%) | 4 [0–36] | 8 [0–26] | 0·61 | 3 [0–26] | 10 [0–36] | 0·06 |
| ANCA (ELISA) | 726 [54–6597] | 411 [10–14370] | 0·32 | 638 [26–6201] | 424 [10–14370] | 0·87 |
| ANCA (IIF) | 640 [80-> 640] | 320 [40-> 640] | 0·17 | 640 320[-> 640] | 640 [40-> 640] | 0·57 |
| Atheroscleroticevents, yes/no | 4/13 | 5/15 | 1·00 | 3/5 | 6/23 | 0·37 |
sEPCR = soluble endothelial protein C receptor; sTM = soluble thrombomodulin; BVAS = Birmingham Vasculitis Activity Score; CRP = C-reactive protein; AECA = antiendothelial cell antibodies; ANCA = antineutrophil cytoplasmic antibodies; ELISA = enzyme linked immunosorbent assay (arbitrary units); IIF = indirect immunofluorescence (dilution 1/); Atherosclerotic events =, e.g. angina, myocardial infarction, stroke, etc.
DISCUSSION
In this study, we found that levels of soluble EPCR in plasma samples from patients with WG were related to clinical disease activity. Consistent with previous findings, we found that sTM, which is an established marker of endothelial cell damage [6], also correlates with disease activity in WG [7]. Only a minority (22%) of patients with active WG had elevated sEPCR levels compared with healthy controls, whereas sTM levels were increased in 46% of these patients. The reason for this difference is not known, but there are several possible explanations. Since WG patients with active disease express PR3 on their neutrophils [15] and PR3 supports sEPCR binding to activated neutrophils [14], plasma sEPCR levels might be underestimated in patients with active disease due to binding of sEPCR to activated circulating neutrophils. It is also possible that the plasma sTM and sEPCR levels are reflecting different mechanisms of release from their endothelial-bound parents. sTM arises from elastase proteolytic degradation and endothelial cell damage. In contrast, thrombin interaction with its receptor on endothelial cells induces subsequent metalloproteolytic activity to give rise to sEPCR. Therefore, sEPCR levels may be reporting a response to injury, rather than cellular damage per se. This possibility may provide an insight into the apparent paradox that sEPCR levels correlate with WG disease activity, despite the fact that membrane-bound EPCR is expressed primarily on larger vessels and WG is primarily a disease of small-to medium-sized vessels by clinical criteria.
In patients with WG, levels of sEPCR, before correction for renal function, correlated not only with disease activity scores but also with sTM levels. This contrasts with an earlier study in which no correlation was observed between sEPCR and sTM levels in patients with sepsis or SLE [20]. The simplest explanation for this is that it results from differences in the sites of vascular injury between WG, SLE and sepsis. We also found that patients with active disease and elevated sEPCR levels tended to be older than patients with active disease but normal sEPCR levels. Thus, there may be multiple, chronic, pathological processes contributing to sEPCR levels, such as atherosclerotic damage of the larger vessels. This is, however, purely speculative and additional studies in different patient populations are needed.
We also found that sTM levels, before correction for renal function, correlated with disease activity. These findings are in agreement with a previous study in which elevated levels of sTM were demonstrated in patients with active WG [7]. In our study, patients with active disease and elevated sTM levels had a higher prevalence of renal involvement, higher serum creatinine levels and greater age compared with patients with active disease but normal sTM levels. These observations are consistent with the known renal damage associated with WG pathology, and with previous studies in a variety of clinical populations demonstrating that impaired renal function is associated with elevated levels of sTM [32,34].
The second part of our study addressed the relevance of serial measurements of sEPCR for monitoring disease activity during follow-up of patients with WG. We found that serum levels of sEPCR rose prior to a diagnosed relapse and dropped to baseline levels after treatment. These results remained significant even after correction of sEPCR levels for renal function. Therefore, our results indicate that sEPCR levels respond to changes in the disease during relapse. Interestingly, sEPCR levels at diagnosis were lower than the levels obtained during remission, when patients had less severe active disease, reflected by lower CRP levels, and lower BVAS scores at remission, compared with at diagnosis. We hypothesize that sEPCR levels at presentation were lower because of binding of sEPCR to circulating activated neutrophils. In line with this hypothesis, we previously demonstrated that circulating neutrophils in patients with newly diagnosed active disease are activated, since CD11b (an adhesion molecule that is used as a marker of neutrophil activation) expression is increased only in patients at diagnosis and not in relapsing patients [35].
sTM levels also rose prior to the moment of a relapse and dropped below baseline levels after treatment. However, when corrected for renal function, these changes in sTM levels were no longer significant. Longitudinal data from other clinical studies are not available.
Previous in vitro studies have shown that autoantibodies such as PR3-ANCA activate cytokine-primed neutrophils and monocytes, leading to the release of proteolytic enzymes and the production of oxygen radicals and pro-inflammatory cytokines reviewed in 36], and that AECA may play an important role in vascular injury by attracting leucocytes to the inflammatory site reviewed in 37]. Interaction of both ANCA and AECA with their target antigens may aggravate an inflammatory pro-cess. These effects may contribute to vascular injury and by these means, induce sTM and sEPCR production. Consistent with this observation, ANCA and AECA are often detected in patients with active WG, and PR3-ANCA levels mirror disease activity [24,25]. Although it was previously reported that AECA levels show a relationship with disease activity in patients with systemic small-vessel vasculitides similar to that for PR3-ANCA [38], we could not confirm this finding in the present study.
In conclusion, we found that levels of sEPCR respond to changes in the disease in WG. However, only in a minority of patients with active WG were significantly elevated plasma levels of sEPCR found. Therefore, more clinical studies are required to establish the use of sEPCR as a marker of vascular disease processes.
Acknowledgments
This study was supported by a grant from the University Hospital Groningen (AZG-stimuleringsgelden), the Ricerca Corrente 2000 IRCCS Istituto Auxologico Italiano, and the National Institutes of Health (HL64787 and AI47575 to S.K.), and by an Initial Investigator Award (D.S.K.) and a Scientist Development Grant (S.K.) from the American Heart Association. The antibodies and recombinant proteins used in the sTM and sEPCR ELISAs were a kind gift of Dr Charles Esmon (Howard Hughes Medical Institute, Oklahoma Medical Research Foundation, Oklahoma City, OK). We wish to thank Roelie van Wijk and Marcel van der Leij (University Hospital Groningen), and Kandice Swindle (Oklahoma Medical Research Foundation), for technical support.
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