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Summary. Background: The endothelial protein C receptor (EPCR) binds protein C and enhances its activation. Anti-EPCR autoantibodies are found in patients with antiphospholipid syndrome and may explain the increased risk of thrombosis in these patients. Anti-EPCR autoantibodies have been associated with fetal death and myocardial infarction in young women. Objectives: To determine whether anti-EPCR autoantibodies are associated with deep vein thrombosis (DVT). Patients/methods: We measured plasma anti-EPCR autoantibody levels in the Leiden Thrombophilia Study (LETS), a population-based case–control study consisting of 474 patients with a first DVT and 474 control subjects. Results: The estimated risk of DVT was increased approximately 2-fold in the presence of elevated IgA, IgG or IgM anti-EPCR autoantibodies (i.e. levels above the 90th percentile as measured in the control subjects). The risk conferred by anti-EPCR increased in a dose-dependent manner for IgA and IgG. When anti-EPCR autoantibodies were considered in the co-presence of lupus anticoagulant (LAC) the odds ratio (OR) was 6.1 [95% CI 1.3–27.9]. Anti-EPCR without LAC remained associated with DVT (OR 1.6; 95% CI 1.2–2.1). Anti-EPCR autoantibodies were associated with high levels of D-dimer and soluble EPCR in controls, suggestive of a prothrombotic status induced by the autoantibodies. Conclusions: This study demonstrates that the presence of anti-EPCR autoantibodies is a moderate risk factor for DVT in the general population.
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Deep vein thrombosis (DVT) is a common disorder with an annual incidence in the general population of 1–3 in 1000 individuals [1,2]. Although numerous risk factors for venous thrombosis have been discovered over the past decades, it is clear that still a substantial number of factors that lead to the development of DVT remain unknown.
Protein C is a vitamin K dependent glycoprotein that is synthesized in an inactive form in the liver. Upon activation, activated protein C (APC) is an important natural anticoagulant that, together with its co-factor protein S, suppresses thrombin generation by inhibition of coagulation factor (F) Va and FVIIIa. Several studies have shown that deficiencies of protein C or protein S are risk factors for DVT [3,4], as is resistance to the inhibitory action of APC, which is present in factor V Leiden (FV Leiden) . Protein C is activated by thrombin bound to thrombomodulin on cell surfaces. Binding to the endothelial cell protein C receptor (EPCR), a type I transmembrane protein, which is highly expressed on the endothelium of large blood vessels, enhances protein C activation more than tenfold [6,7]. After being released from EPCR, APC is fully activated. Therefore, EPCR plays an important role in the activation of protein C. Soluble EPCR (sEPCR) is released from the endothelium by a metalloprotease. This release is stimulated by thrombin and inflammatory proteins . sEPCR can still bind APC and as a result inhibit the anticoagulant function of APC .
Individuals with antiphospholipid syndrome are at increased risk of thrombosis and pregnancy complications. A previous study showed the presence of anti-EPCR autoantibodies in patients with antiphospholipid syndrome, which may explain the increased risk of thrombotic complications in these patients . Anti-EPCR autoantibodies have also been shown to be associated with an increased risk of a first fetal death episode and an increased risk of acute myocardial infarction in young women [10,11]. The effect of the anti-EPCR autoantibodies is possibly via impaired protein C activation on cell surfaces as low levels of circulating APC were reported to be associated with an increased risk of thrombosis [12,13]. In fact, inhibitory anti-EPCR autoantibodies have been described in two patients with combined venous and arterial thrombosis [10,14]. It is also plausible that antibodies against EPCR lead to an increased risk of thrombophilic complications as a result of an endothelial injury.
In this study, we investigated the role of autoantibodies against EPCR (i.e. IgA anti-EPCR, IgG anti-EPCR and IgM anti-EPCR) in the development of a first DVT in the Leiden Thrombophilia Study (LETS), a population-based case–control study into the etiology of venous thrombosis.
Analyses were performed in the LETS. This study has been described in detail previously [15,16]. In brief, 474 patients with a first objectively confirmed episode of DVT, who were younger than 70 years old at the time of the event, were enrolled in this study. As control subjects, 474 individuals without a history of DVT, who were friends or partners of the patients, were included. Control subjects were sex and age (± 5 years) matched to the patients. Individuals with a history of malignant disease were excluded. This study was approved by the institutional review board. Informed consent was obtained from all participants.
Blood was collected from the antecubital vein into Sarstedt Monovette® tubes, in 0.1 volume 0.106 m trisodium citrate. Plasma was prepared by centrifugation for 10 min at 2000 × g at room temperature and stored at −70 °C until used. Patients and control subjects were seen concurrently and all samples were analyzed with the same batch of reagents within a 6-week period. The median time between thrombosis and venepuncture in this study was 18 (range: 6–56) months.
For 21 individuals (16 patients and five control subjects) no data on the anti-EPCR autoantibodies were available.
Expression and purification of soluble EPCR
sEPCR fused with myc epitope and a six histidines tag at its C-terminal end was expressed in Pichia pastoris strain X-33 using the EasySelect Pichia expression kit (Invitrogen, Paisley, UK). Recombinant sEPCR was purified from culture supernatants using a three-step purification essentially as described .
Assay for anti-EPCR autoantibodies detection
Anti-EPCR autoantibodies of IgA, IgG and IgM isotypes were measured using a previously described enzyme-linked immunosorbent assay (ELISA) with minor changes. Results were expressed as arbitrary units (U) of absorbance [8,9]. The assay was performed without knowledge of whether the sample was from a patient or a control subject. The intra-assay coefficients of variation were 5.2%, 5.8% and 2.8% for IgA, IgM and IgG anti-EPCR autoantibodies, respectively. The inter-assay coefficients of variation were 11.0%, 5.1% and 11.4% for IgA, IgM and IgG anti-EPCR autoantibodies, respectively.
Measurement of lupus anticoagulant, D-dimer and sEPCR levels
The measurement of lupus anticoagulant (LAC) has been described in detail previously . In brief, LAC was measured with a dilute Russell’s viper venom time (dRVVT, Gradipore, Australia). Samples were diluted 1:1 with pooled normal plasma. A sample was considered positive when the clotting time was longer than the mean of 40 healthy controls + 3 SD (LAC screen). For those samples that showed a prolonged clotting time, a confirmation assay was performed with added phospholipids (LAC confirm). If addition of extra phospholipids resulted in a clotting time reduction of more than 20%, the sample was considered LAC positive [LAC ratio: LAC screen (1:1)/LAC confirm (1:1) > 1.2].
D-dimer levels were measured by ELISA using two monoclonal antibodies against non-overlapping antigenic determinants . With this technique, the fragment D-dimer of cross-linked fibrin is detected.
Plasma sEPCR levels were measured by ELISA, as described previously . D-dimer and sEPCR levels were expressed in ng mL−1.
Putative determinants of the anti-EPCR autoantibodies were studied in the control subjects as reflecting the general population. The determinants were mainly established by comparing means and 95% CIs. We investigated whether elevated levels of the anti-EPCR autoantibodies were associated with an increased risk of DVT by calculating odds ratios (ORs) and their 95% CIs according to the method of Woolf . As a cut-off point we used the 90th and 95th percentiles of the control group. The levels of the anti-EPCR autoantibodies were also divided into quartiles to assess the association between the levels of the autoantibodies and the risk of DVT (dose–response relation)
To adjust for possible confounders (i.e. age, sex, oral contraceptive use at the time of the thrombosis as well as at the time of the venepuncture) and the presence of the FV Leiden and prothrombin 20210A mutation, we used a logistic regression model. All computations were carried out with the spss for Windows Version 12.0.1 (SPSS Inc., Chicago, IL, USA) statistical package.
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The mean age of the patients at the time of the thrombosis and the control subjects was 45 years (range: patients 15–69; control subjects 15–72). Among patients and control subjects alike, 58% were women. In both patients and control subjects, the levels of anti-EPCR autoantibodies were detectable in more than 95% of individuals.
The mean levels of all anti-EPCR autoantibodies were higher in patients compared with control subjects: IgA, 54.2 U vs. 44.5 U, mean difference 9.7 (95% CI: 2.7–16.8); IgG, 34.2 U vs. 28.3 U, mean difference 5.8 (95% CI: 1.9–9.8); IgM, 27.1 U vs. 20.7 U, mean difference 6.3 (95% CI: 0.8–11.9).
Determinants of the anti-EPCR autoantibodies
Table 1 shows the putative determinants of the anti-EPCR autoantibody levels assessed in control subjects. The levels of the anti-EPCR autoantibodies were equal in men and women except for the levels of IgG autoantibodies, which were markedly higher in men compared with women: mean difference 8.3 U (95% CI: 2.1–14.5). Only the levels of IgA autoantibodies were associated with age (i.e. the levels of the IgA autoantibodies increased with increasing age). No relevant differences could be seen between users and non-users of oral contraceptives. Furthermore, the presence of the anti-EPCR autoantibodies was not related to the presence of FV Leiden mutation or the prothrombin 20210A mutation.
Table 1. Anti-endothelial protein C receptor (anti-EPCR) autoantibody levels in arbitrary units (95% CI) in healthy control subjects
| ||n||IgA anti-EPCR||IgG anti-EPCR||IgM anti-EPCR|
|All||469||44.5 (39.6–49.4)||28.3 (25.6–31.1)||20.7 (18.0–23.5)|
| Men||199||47.4 (41.8–53.0)||33.1 (27.3–38.9)||19.1 (15.6–22.6)|
| Women||270||42.4 (34.9–49.9)||24.8 (22.6–27.1)||22.0 (17.9–26.0)|
| ≤ 35||117||37.9 (31.5–44.4)||29.7 (25.5–33.9)||24.9 (20.5–29.4)|
| 35–45||118||42.0 (34.7–49.3)||22.9 (19.4–26.4)||20.2 (16.6–23.9)|
| 45–55||117||46.2 (35.0–57.3)||29.3 (24.7–33.9)||17.5 (13.7–21.3)|
| > 55||117||52.0 (38.8–65.2)||31.5 (22.9–40.2)||20.3 (11.5–29.1)|
| Yes||54||40.1 (29.2–51.0)||27.3 (21.3–33.2)||17.0 (14.7–19.4)|
| No||98||35.1 (23.5–46.8)||22.5 (19.4–25.5)||23.0 (18.9–27.2)|
| Yes||14||46.4 (22.6–70.3)||22.9 (10.4–35.5)||15.2 (6.7–23.7)|
| No||455||44.5 (39.4–49.5)||28.5 (25.7–31.4)||20.9 (18.1–23.8)|
| Yes||10||38.2 (24.2–52.2)||30.7 (17.4–44.1)||17.9 (3.6–32.2)|
| No||459||44.7 (39.6–49.7)||28.3 (25.4–31.1)||20.8 (18.0–23.6)|
Risk of DVT associated with elevated levels of anti-EPCR autoantibodies
Elevated levels of all measured anti-EPCR autoantibodies (i.e. IgA, IgG, and IgM anti-EPCR) were separately associated with an increased risk of DVT (Table 2). Individuals with IgA anti-EPCR autoantibodies above the 90th percentile measured in control subjects (85.8 U) had a 1.7-fold increased risk of DVT compared with individuals with an IgA anti-EPCR autoantibody level below this cutoff value (95% CI: 1.2–2.6). The thrombotic risk increased 1.8-fold (95% CI: 1.2–2.6) and 1.6-fold (95% CI: 1.1–2.4) for IgG and IgM anti-EPCR autoantibody levels, respectively. Adjustment for age and sex did not affect these risk estimates. When the 95th percentile, as measured in the control subjects, was used as a cutoff point, the risk estimates for IgG and IgM anti-EPCR autoantibodies were slightly higher: OR 2.0 (95% CI: 1.2–3.3) and 2.0 (95% CI: 1.2–3.3), respectively. Stratification for men and women yielded similar risk estimates. Adjusting the overall association between the autoantibodies and thrombosis for age, sex, FV Leiden, prothrombin 20210, and oral contraceptive use at the time of thrombosis as well as at the time of the venepuncture gave similar results: IgA P90, OR = 1.8 (95% CI: 1.2–2.8); IgA P95, OR = 2.2 (95% CI: 1.3–3.9); IgG P90, OR = 1.6 (95% CI: 1.1–2.5); IgG P95, OR = 1.9 (95% CI: 1.1–3.5); IgM P90, OR = 1.8 (95% CI: 1.2–2.9); IgM P95, OR = 2.2 (95% CI: 1.2–3.9).
Table 2. Risk of deep-venous thrombosis after dichotomization of the anti-endothelial protein C receptor (anti-EPCR) autoantibody levels at the 90th and 95th percentiles measured in control subjects
| ||Cases, n (%)||Controls, n (%)||Odds ratio (95% CI)|
| ≥ P90||73 (15.9)||46 (9.8)||1.7 (1.2–2.6)|
| ≥ P95||38 (8.3)||23 (5.0)||1.8 (1.0–3.0)|
| ≥ P90||72 (15.7)||45 (9.6)||1.8 (1.2–2.6)|
| ≥ P95||42 (9.2)||23 (5.0)||2.0 (1.2–3.3)|
| ≥ P90||68 (14.8)||46 (9.8)||1.6 (1.1–2.4)|
| ≥ P95||42 (9.2)||23 (5.0)||2.0 (1.2–3.3)|
Performing a pooled analyses of the anti-EPCR autoantibodies (i.e. the risk of having an IgA, IgM, or IgG anti-EPCR autoantibody level above the 90th percentile) yielded an OR of 1.7-fold (95% CI: 1.3–2.2). The risk of DVT was 1.8-fold increased when one or more of the anti-EPCR autoantibodies were above the 95th percentile (OR = 1.8; 95% CI: 1.3–2.6).
After stratifying the levels of the anti-EPCR autoantibodies into quartiles, a clear dose–response relation could be seen for IgA and IgG anti-EPCR autoantibody levels, as shown in Table 3. For IgA anti-EPCR autoantibodies, the risk of DVT increased from 1.2-fold (95% CI: 0.8–1.8) for individuals with levels in the second quartile up to 2.0-fold (95% CI: 1.4–2.9) for individuals with levels in the fourth quartile (all compared with individuals with levels in the first quartile; P-value for trend< 0.001). In a similar analysis for IgG anti-EPCR autoantibodies, the risk estimates increased from 0.9-fold (95% CI: 0.6–1.3) for individuals with levels in the second quartile up to 1.6-fold (95% CI: 1.1–2.3) for individuals with levels in the fourth quartile (P-value for trend 0.002). For IgM anti-EPCR autoantibodies, the risk was only increased when levels were above the 90th percentile measured in control subjects.
Table 3. Dose–response relation between anti-endothelial protein C receptor (anti-EPCR) levels and risk of deep vein thrombosis
| ||IgA anti-EPCR||IgG anti-EPCR||IgM anti-EPCR|
|P25–P50||1.2 (0.8–1.8)||0.9 (0.6–1.3)||1.1 (0.7–1.6)|
|P50–P75||1.5 (1.0–2.3)||1.1 (0.8–1.6)||1.2 (0.8–1.8)|
|> P75||2.0 (1.4–2.9)||1.6 (1.1–2.2)||1.2 (0.9–1.8)|
|> P90||2.4 (1.5–3.7)||1.9 (1.2–2.9)||1.7 (1.1–2.7)|
|> P95||2.4 (1.4–4.4)||2.1 (1.2–3.7)||2.1 (1.2–3.8)|
Combination of anti-EPCR autoantibodies and lupus anticoagulant
The association between LAC and the risk of DVT was studied previously in LETS . Individuals positive for LAC (ratio > 1.2) had a 3.6-fold increased risk of DVT compared with LAC negative individuals . High titers of anti-EPCR autoantibodies were found in a previous study in individuals with LAC . We adjusted the risk estimates for the anti-EPCR autoantibodies for LAC (ratio, dichotomized at 1.2, i.e. < 1.2 negative LAC, > 1.2 positive LAC) to assess whether LAC and high levels of anti-EPCR autoantibodies are independent risk factors for DVT. In the adjusted analysis, the risk estimates for the anti-EPCR autoantibodies were all similar to the crude risk estimates. The risk estimate for LAC only marginally changed
Furthermore, we analyzed the effect on the risk of thrombosis of combinations of LAC and elevated anti-EPCR autoantibodies. As shown in Table 4, individuals with LAC more often had elevated levels of anti-EPCR autoantibodies than individuals negative for LAC [50% of individuals LAC positive in the control group also displayed elevated anti-EPCR autoantibodies (2/4) whereas only 26% of those LAC negative displayed elevated anti-EPCR autoantibodies (123/463)]. Although an elevated level of one or more of the anti-EPCR autoantibodies and LAC both contributed to the risk of DVT, the risk was highest when both risk factors were present (OR = 6.1; 95% CI: 1.3–27.9; Table 4).
Table 4. Combined effect of high levels of the anti-endothelial protein C receptor (anti-EPCR) autoantibodies and lupus anticoagulant (LAC)
| Anti-EPCR*|| LAC†||Patients||Controls||Odds ratio (95% CI)|
Anti-EPCR autoantibodies, sEPCR, and D-dimer levels
Looking for an explanation for the increased risk of DVT associated with elevated levels of anti-EPCR autoantibodies, we assessed whether there was an association between anti-EPCR and coagulation status. The levels of the anti-EPCR autoantibodies were positively associated with D-dimer levels in the group of healthy control subjects, as shown in Table 5. The levels of the anti-EPCR autoantibodies were also positively associated with the levels of sEPCR in the control group (Table 5), which could be related with endothelial injury induced by the autoantibodies.
Table 5. Mean (95% CI) levels of D-dimer and soluble endothelial protein C receptor (sEPCR) in control subjects with high or low levels of anti-EPCR autoantibodies
| ||D-dimer (ng mL−1)||sEPCR (ng mL−1)|
| > P90||157.7 (83.6–231.7)||145.1 (114.3–175.9)|
| ≤ P90||101.1 (90.2–112.0)||134.4 (126.3–142.5)|
| > P90||114.5 (76.1–152.9)||142.5 (112.0–173.0)|
| ≤ P90||105.9 (93.0–118.8)||134.7 (126.6–142.8)|
| > P90||141.2 (92.2–190.2)||142.2 (114.5–169.8)|
| ≤ P90||102.9 (90.5–115.4)||134.7 (126.5–143.0)|
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To our knowledge, this is the first study on the association between anti-EPCR autoantibodies and the risk of DVT. We have shown that elevated levels of IgA, IgG, and IgM anti-EPCR autoantibodies are associated with a moderate increased risk of DVT. Furthermore, a clear dose–response relation can be demonstrated for IgA and IgG anti-EPCR autoantibodies.
Previously, it was hypothesized that LAC could just be a marker for a more complex autoimmune disorder . In this context, the high titers of anti-EPCR could also be seen as a mere part of a more complex picture. However, the current results show clearly that anti-EPCR autoantibodies and LAC are both risk factors for DVT, as their thrombotic risks remained elevated after adjustment for each other. Furthermore, when high anti-EPCR autoantibody levels and LAC occurred simultaneously, the risk increased notably, which indicates a synergistic effect. Provided that further studies confirm these findings, screening for anti-EPCR autoantibodies in LAC positive patients may help to identify individuals at high thrombotic risk.
Anti-EPCR autoantibodies can reasonably be thought to play a causative role in thrombosis. Firstly, as has previously been demonstrated [10,14], the anti-EPCR autoantibodies may impair the APC generation, which may lead to increased thrombin generation. Indeed, low levels of circulating APC are associated with an increased risk of venous and arterial thrombosis [12,13]. Secondly, an association between elevated levels of the anti-EPCR autoantibodies and high levels of D-dimers was demonstrated. This association was not influenced by the thrombosis, which could obscure the interpretation of the result, as it was shown in the control subjects. Thus, coagulation is more active, or less inhibited, in individuals with elevated levels of anti-EPCR autoantibodies. Thirdly, the association between elevated levels of the anti-EPCR autoantibodies and high levels of sEPCR, again shown in the healthy control subjects, indicates that endothelial damage, leading to the generation of a prothrombotic surface, is taking place upon exposure to high levels of anti-EPCR autoantibodies. Additionally, the higher levels of sEPCR associated with anti-EPCR might also reflect a higher basal thrombin generation in these subjects, which in turn would increase the shedding of EPCR . Finally, in a murine model some autoantibodies induce thrombosis by triggering the activation of the complement system , which could also be a mechanism through which anti-EPCR autoantibodies could be involved in the development of DVT. Once bound to EPCR on the endothelial cell surface, anti-EPCR would be capable of triggering the complement system leading to a vascular injury and, eventually, to the development of a thrombosis.
It is possible that some anti-EPCR autoantibodies bind to sEPCR by the same epitope recognized by the antibodies used to determine the plasma sEPCR levels. Subsequently, measured sEPCR levels could be lower than the actual sEPCR levels. However, already a positive association between anti-EPCR and sEPCR levels was found and this artefact would only result in an even stronger association between anti-EPCR and sEPCR.
A possible limitation of this study is that the elevated levels of the anti-EPCR autoantibodies could be the result rather than the cause of the venous thrombotic event. However, this seems unlikely as the venepuncture took place at least 6 months after the event, and it is not easily imagined how such a post hoc event would have taken place.
In conclusion, high levels of anti-EPCR autoantibodies are a moderate risk factor for DVT in patients without a known underlying autoimmune disease. The association between high levels of anti-EPCR and high D-dimer concentrations reflects that coagulation status can be influenced by anti-EPCR autoantibodies.