Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C (APC) by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR), which is generated by metalloprotease activity, is present in plasma. The distribution of sEPCR levels in healthy populations is bimodal. Previously, we described two polymorphisms in exon 4 of the EPCR gene, 4600A/G that encodes the substitution of Ser219 by Gly in the transmembrane region of EPCR and 4678G/C in the 3'-UT region. The aim of this study was to investigate the relationship between these two polymorphisms and plasma sEPCR and APC levels and risk of venous thrombosis. We genotyped 401 healthy controls from the Spanish population and measured their plasma sEPCR and APC levels. Carriers of the 4600AG genotype had significantly higher sEPCR levels than those with the AA genotype, while the 4678CC genotype was associated, to a lesser extent, with elevated APC levels. To assess the effect of these polymorphisms on the risk of thrombosis, we genotyped 405 patients with venous thromboembolism. The frequency of the 4600AG genotype was very similar in patients and controls (p=0.975), whereas the 4678CC genotype was significantly more frequent in controls than in patients (p=0.008). In multivariate analysis, carriers of the 4678CC genotype had a decreased risk of thrombosis (OR=0.61, p=0.009). These data indicate that individuals carrying the 4600AG genotype have high sEPCR levels but do not have an increased risk of thrombosis, whereas individuals carrying the 4678CC genotype have higher APC levels and lower risk of venous thromboembolism.

BackgroundHaplotypes A1 and A3 in the endothelial protein C receptor (EPCR) gene are tagged by 4678G/C and 4600A/G respectively. We assessed whether these haplotypes modify the risk of venous thromboembolism in carriers of the prothrombin 20210A allele.

Two polymorphisms in the endothelial protein C receptor (EPCR) gene, 4600A/G and 4678G/C, have been reported to influence the risk of venous thromboembolism (VTE). The objective of this study was to assess whether these polymorphisms modify the risk of VTE in carriers of factor V (FV) Leiden. We genotyped 295 carriers of FV Leiden for these polymorphisms: 100 unrelated patients with a history of VTE (propositi) and 195 relatives (14 of them symptomatic) of 81 of the propositi. Spontaneous VTE events occurred in 71% of propositi carrying the 4678GG genotype, 65% carrying the GC, and 43% with the CC genotype. The mean age at the first onset was significantly higher in propositi carrying the 4678CC than in those with the GC or GG genotype (p = 0.046). Among the 276 carriers of FV Leiden from the 81 families studied, the 95 symptomatic members had similar 4600G allele and 4600AG genotype frequencies but significantly lower 4678C allele (p = 0.002) and 4678CC genotype (p = 0.004) frequencies than the 181 asymptomatic members. The probability of being free of thrombosis at age 40 was significantly higher in the 66 carriers of the 4678CC genotype (94%) than in the 138 carrying the GC (72%) or in the 72 with the 4678GG genotype (60%) (p < 0.001). Multivariate analysis showed that the 4678CC genotype reduced the risk of thrombosis in carriers of FV Leiden (OR = 0.31;95% CI = 0.16-0.83). The incidence of VTE was higher in the 195 relatives with FV Leiden than in the 133 without FV Leiden (OR = 4.7; CI = 1.3-7.2). These results show that carriers of FV Leiden with the 4678CC genotype have a significantly reduced risk of VTE compared with those carrying the 4678GG or GC genotype, probably due to the higherAPC levels previously observed in individuals carrying the 4678CC genotype.

Objective-To confirm the effect of the endothelial protein receptor gene (PROCR) haplotypes H1 and H3 on venous thromboembolism (VTE), to study their effect on endothelial protein C receptor (EPCR) expression in human umbilical vein endothelial cells, and to investigate the functionality of H1 tagging single-nucleotide polymorphisms in an in vitro model. Approach and Results-Protein C (PC), activated PC, and soluble EPCR (sEPCR) levels were measured in 702 patients with VTE and 518 healthy individuals. All subjects were genotyped for PROCR H1 and H3. Human umbilical vein endothelial cells isolated from 111 umbilical cords were used to study the relation between PROCR haplotypes, PROCR mRNA, cellular distribution of EPCR, and rate of PC activation. Finally, the functionality of the intragenic PROCR H1 singlenucleotide polymorphisms was analyzed using a luciferase-based method. We confirmed that individuals carrying H1 have reduced VTE risk, increased plasma activated PC levels, and reduced plasma sEPCR levels and that individuals with the H3H3 genotype have an increased VTE risk and increased plasma sEPCR levels. In cultured human umbilical vein endothelial cells, H1 is associated with increased membrane-bound EPCR, increased rate of PC activation, and reduced sEPCR in conditioned medium, but does not significantly influence PROCR mRNA levels. In contrast, H3 is associated with reduced membrane-bound EPCR and increased sEPCR in human umbilical vein endothelial cell-conditioned medium, higher levels of a truncated mRNA isoform, and a lower rate of PC activation. Finally, we identified the g.2132T>C singlenucleotide polymorphism in intron 1 as an intragenic H1-specific functional single-nucleotide polymorphism. Conclusions-These Medina et al Functional Analysis of PROCR Haplotypes 685H3 is tagged by the minor allele of g.4600A>G (Ser219Gly; rs867186) and has been associated with the risk of venous [8][9][10][11][12][13][14][15]18,19 and arterial thrombosis, [19][20][21][22][23] but with contradictory results. The presence of H3 results in increased plasma sEPCR levels, [8][9][10][11][12][13]18,20,22,24 which is largely explained by a Ser219Gly substitution, which renders EPCR more susceptible to cleavage by metalloproteinases such as tumor necrosis factor-α converting enzyme/ADAM17. 7 Another mechanism that could link H3 to high plasma levels of sEPCR is its association with a truncated form of PROCR mRNA lacking the transmembrane and intracellular domains. 25 Recently, PROCR H3 has also been found to be associated with increased plasma levels of PC. 20,26Therefore, we aimed to verify the effects of H1 and H3 on VTE risk in a case-control study to investigate their effects on EPCR expression in human umbilical vein endothelial cells (HUVECs) and to identify the functional SNP that mediates the protection of H1 against VTE using luciferase constructs.In the present study, we confirm previous reports on a protective effect of PROCR H1 against VTE 8,[11][12][13][14][15] and show, for the first time, that it is prob...

Cancer-associated venous thrombosis (VTE) increases mortality and morbidity. However, limited tools are available to identify high risk patients. Upon activation, neutrophils release their content through different mechanisms, thereby prompting thrombosis. We explored plasma microRNAs (miRNAs) and neutrophil activation markers to predict VTE in pancreatic ductal adenocarcinoma (PDAC) and distal extrahepatic cholangiocarcinoma (DECC). Twenty-six PDAC and 6 DECC patients recruited at cancer diagnosis, were examined for deep vein thrombosis and pulmonary embolisms, and were then followed-up with clinical examinations, blood collections, and biCUS. Ten patients developed VTE and were compared with 22 age- and sex-matched controls. miRNA expression levels were measured at diagnosis and right before VTE, and neutrophil activation markers (cell-free DNA, nucleosomes, calprotectin, and myeloperoxidase) were measured in every sample obtained during follow-up. We obtained a profile of 7 miRNAs able to estimate the risk of future VTE at diagnosis (AUC = 0.95; 95% Confidence Interval (CI) (0.987, 1)) with targets involved in the pancreatic cancer and complement and coagulation cascades pathways. Seven miRNAs were up- or down-regulated before VTE compared with diagnosis. We obtained a predictive model of VTE with calprotectin as predictor (AUC = 0.77; 95% CI (0.57, 0.95)). This is the first study that addresses the ability of plasma miRNAs and neutrophil activation markers to predict VTE in PDAC and DECC.

Upon activation, neutrophils release their content through different mechanisms like degranulation and NETosis, thus prompting thrombosis. The natural anticoagulant activated protein C (APC) inhibits neutrophil NETosis and, consequently, this may lower the levels of neutrophil activation markers in plasma, further diminishing the thrombotic risk exerted by this anticoagulant. We aimed to describe the status of markers of neutrophil activation in plasma of patients with venous thrombosis, their association with the thrombotic risk and the potential contribution of APC. We quantified three markers of neutrophil activation (cell-free DNA, calprotectin, and myeloperoxidase) in 253 patients with venous thromboembolism (VTE) in a stable phase (192 lower extremity VTE and 61 splanchnic vein thrombosis) and in 249 healthy controls. In them, we also quantified plasma APC, soluble endothelial protein C receptor (EPCR), and soluble thrombomodulin (TM), and we genotyped two genetic regulators of APC: the EPCR gene (PROCR) haplotypes (H) and the TM gene (THBD) c.1418C>T polymorphism. We found a significant increase in plasma cell-free DNA (p < 0.0001), calprotectin (p = 0.0001) and myeloperoxidase (p = 0.005) in VTE patients compared to controls. Furthermore, all three neutrophil activation markers were associated with an increase in the thrombotic risk. Cell-free DNA and calprotectin plasma levels were significantly correlated (Spearman r = 0.28; p < 0.0001). As expected, the natural anticoagulant APC was significantly decreased in VTE patients (p < 0.0001) compared to controls, what was mediated by its genetic regulators PROCR-H1, PROCR-H3, and THBD-c.1418T, and inversely correlated with cell-free DNA levels. This is the largest case-control study that demonstrates the increase in markers of neutrophil activation in vivo in VTE patients and their association with an increased thrombotic risk. This increase could be mediated by low APC levels and its genetic regulators, which could also increase NETosis, further enhancing thrombosis and inflammation.

Behçet's disease (BD) is a chronic inflammatory disorder in which thrombosis and posterior ocular involvement occur in about 30% of patients, whose ethiology is unknown. It has not been established whether mean platelet volume (MPV), a marker of platelet activation, is involved in the pathogenesis of thrombotic events and posterior uveitis in these patients. We aimed to analyze whether there are differences in MPV in BD patients when compared with controls and its relation with the presence of thrombosis and posterior uveitis. We determined MPV and platelet count, along with Creactive protein (CRP) and cardiovascular risk factors (because of their influence on MPV) in 89 BD patients (of which 24 had thrombosis and 23 had posterior uveitis) and 89 sex-and age-matched healthy controls. BD patients showed statistically higher MPV than controls: 10.98 ± 1.19 fL vs. 10.60 ± 1.21 fL (P = 0.044) and higher CRP: 5.9 ± 8.9 mg/L vs. 1.4 ± 1.7 mg/L (P = 0.001). The percentage of hyperlipemia and diabetes was higher in cases than in controls (P = 0.032, P = 0.013, respectively). No differences in MPV were observed when comparing: patients with and without thrombosis: 11.8 ± 1.27 fL vs. 10.94 ± 1.28 fL (P = 0.654); with and without posterior uveitis: 10.76 ± 1.18 fL vs. 11.03 ± 1.30 fL P = 0.398; with CRP and cardiovascular risk factors (P > 0.05). MPV correlated negatively with platelet count (r = -308, P < 0.01), but not with CRP (r = -0.22, P = 0.772). MPV seems to relate to neither thrombosis nor posterior uveitis in BD patients.

SummaryBehçet's disease is a multi-systemic inflammatory disorder of unknown cause. Most abnormalities have been associated with endothelial injury caused by vasculitis. Thrombosis occurs in about 25% of patients, although the mechanism is unknown. The objective of this study was to evaluate the protein C activation system in Behçet's disease and its correlation with venous thromboembolism (VTE). Thirty-nine patients (12 with VTE) and 78 age-and sex-matched controls were included in the study, and levels of protein C, protein S, activated protein C (APC), protein C inhibitor (PCI), soluble thrombomodulin (TM), antithrombin (AT), a 1 -antitrypsin, fibrinogen, factor VIII, von Willebrand factor (VWF) and C-reactive protein (CRP) were measured. APC and TM levels were significantly lower in patients than in controls, whereas protein S, AT, a 1 -antitrypsin, fibrinogen, factor VIII, VWF and CRP levels were significantly higher in patients than in controls. APC, PCI and TM levels were lower in patients with VTE (0AE65 ± 0AE19 ng/ml, 86% ± 22% and 15AE5 ± 7AE1 ng/ml respectively) than in those without VTE (0AE78 ± 0AE17 ng/ml, 100% ± 15% and 22AE1 ± 15AE3 ng/ml) (P < 0AE05). In patients, APC levels below 0AE75 ng/ml (10th percentile of the control group) increased the risk of VTE about fivefold (odds ratio ¼ 5AE1; 95% confidence interval ¼ 1AE1-23AE4). These results show that reduced APC levels are associated with the high incidence of VTE in Behçet's disease.