Human platelets express endothelial protein C receptor,which can be utilized to enhance localization of factor VIIa activity

Essentials

• Factor VIIa binds activated platelets to promote hemostasis in hemophilia patients with inhibitors.
• The interactions and sites responsible for platelet‐FVIIa binding are not fully understood.
• Endothelial cell protein C receptor (EPCR) is expressed on activated human platelets.
• EPCR binding enhances the efficacy of a FVIIa variant and could impact design of new therapeutics.

Summary

Background

High‐dose factor VIIa (FVIIa) is routinely used as an effective bypassing agent to treat hemophilia patients with inhibitory antibodies that compromise factor replacement. However, the mechanism by which FVIIa binds activated platelets to promote hemostasis is not fully understood. FVIIa‐DVQ is an analog of FVIIa with enhanced tissue factor (TF)‐independent activity and hemostatic efficacy relative to FVIIa. Our previous studies have shown that FVIIa‐DVQ exhibits greater platelet binding, thereby suggesting that features in addition to lipid composition contribute to platelet–FVIIa interactions.

Objectives

Endothelial cell protein C receptor (EPCR) also functions as a receptor for FVIIa on endothelial cells. We therefore hypothesized that an interaction with EPCR might play a role in platelet–FVIIa binding.

Methods/results

In the present study, we used flow cytometric analyses to show that platelet binding of both FVIIa and FVIIa‐DVQ is partially inhibited in the presence of excess protein C or an anti‐EPCR antibody. This decreased binding results in a corresponding decrease in the activity of both molecules in FXa and thrombin generation assays. Enhanced binding to EPCR was sufficient to account for the increased platelet binding of FVIIa‐DVQ compared with wild‐type FVIIa. As EPCR protein expression has not previously been shown in platelets, we confirmed the presence of EPCR in platelets using immunofluorescence, flow cytometry, immunoprecipitation, and mass spectrometry.

Conclusions

This work represents the first demonstration that human platelets express EPCR and suggests that modulation of EPCR binding could be utilized to enhance the hemostatic efficacy of rationally designed FVIIa analogs.

     

Endothelial cell protein C receptor‐mediated redistribution and tissue‐level accumulation of factor VIIa

Summary. Background: Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown. Objective: The aim of the present study was to investigate the role of the FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues. Methods: Wild‐type, EPCR‐deficient or ECPR‐over‐expressing mice were injected with human recombinant (r)FVIIa (120 μg kg^?1 body weight) via the tail vein. At varying time intervals after rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR. Results: The data reveal that, after intravenous (i.v.) injection, rFVIIa rapidly disappears from the blood and associates with the endothelium in an EPCR‐dependent manner. Immunohistochemical analyses revealed that the association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. The FVIIa association with the endothelium was undetectable at time points exceeding 24 h post‐FVIIa administration. The levels of rFVIIa accumulated in tissue correlate with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days. Conclusions: The observation that an EPCR‐dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides.     

Paternal endothelial protein C receptor 219Gly variant as a mild and limited risk factor for deep vein thrombosis during pregnancy

Summary. Background: Half of all venous thromboembolism (VTE) cases during pregnancy are associated with a maternal thrombophilia. The influence of paternal genotype on the placenta and in the genesis of VTE has not been described. Objectives: To determine if the maternal and paternal Ser219Gly dimorphism of the endothelial protein C receptor (EPCR), evaluated through detection of the PROCR 6936G allele, is a risk factor for VTE during pregnancy. Methods: Using a case‐control study nested in the NOHA first cohort of primigravidae, 66 patient couples with a first episode of gestational VTE and randomly selected non‐thrombotic control couples were investigated. For each couple, factor V gene (F5) G1691A, factor II gene (F2) G20210A, factor XII gene (F12) C46T and PROCR A6936G polymorphisms were determined. Results: Only maternal F5 1691A, F2 20210A and F12 46T alleles were independently associated with iliac and infra‐iliac deep vein thromboses (DVT). The maternal PROCR 6936G allele was a mild risk factor for iliac DVT (OR = 5.5 [2.3–13.0]). The paternal PROCR 6936G allele was also a mild independent risk factor for iliac DVT (OR = 2.6 [1.1–6.2]) and only during pregnancy (rather than postpartum) among maternal carriers of the F5 1691A allele (OR = 77.6 [4.2 to > 999.9]). Conclusions: The paternal PROCR 6936G allele could be a risk factor for maternal iliac DVT. Its impact was milder than the F5 1691A and F2 20210A polymorphisms in mothers. We hypothesize that the prothrombotic effect of the paternal PROCR 6936G allele is localized. Therefore, DVT during pregnancy may be influenced by trophoblastic cell‐surface proteins inherited from both maternal and paternal alleles.     

2型糖尿病患者血浆可溶性内皮细胞蛋白C受体和血栓调节蛋白的变化

目的探讨2型糖尿病(2-DM)患者血浆可溶性内皮细胞蛋白C受体(sEPCR)和血栓调节蛋白(sTM)的变化及其临床意义.方法采用酶联免疫双抗体夹心法测定74例2-DM患者和29例正常对照组(K组)sEPCP和sTM,并根据24h尿白蛋白(24hUAE)分为A组(n=36,24hUAE＜30 mg)、B组(n=24,24hUAE30～300mg)和C组(n=14,24hUAE≥300mg).结果与K组比较,DM患者血浆sEPCR和sTM显著升高,P＜0.05和P＜0.01;分组比较,A组正sER和sTM水平无显著差异,B组显著升高(P＜0.05和＜0.01),C组更进一步升高,P值分别小于0.01和0.001;sEPCR与sTM、24hUAE和糖化血红蛋白呈显著正相关(P＜0.05),sTM与24hUAE(P＜0.01)和糖化血红蛋白(P＜0.05)呈显著正相关;sEPCR和sTM与DM病程、空腹血糖、血脂和年龄均无相关性.结论sEPCR和sTM升高与DM血管病变的发生和严重程度相关,是反映血管内皮细胞功能的良好指标,并可能参与其高凝状态的形成.     

Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin

Previous studies have shown that blocking endothelial protein C receptor (EPCR)-protein C interaction results in about an 88% decrease in circulating activated protein C (APC) levels generated in response to thrombin infusion and exacerbates the response to Escherichia coli. To determine whether higher levels of EPCR expression on endothelial cells might further enhance the activation of protein C and protect the host during septicemia, we generated a transgenic mouse (Tie2-EPCR) line which placed the expression of EPCR under the control of the Tie2 promoter. The mice express abundant EPCR on endothelial cells not only on large vessels, but also on capillaries where EPCR is generally low. Tie2-EPCR mice show higher levels of circulating APC after thrombin infusion. Upon infusion with factor Xa and phospholipids, Tie2-EPCR mice generate more APC, less thrombin and are protected from fibrin/ogen deposition compared with wild type controls. The Tie2-EPCR animals also generate more APC upon lipopolysaccharide (LPS) challenge and have a survival advantage. These results reveal that overexpression of EPCR can protect animals against thrombotic or septic challenge.     

Autoantibodies against endothelial protein C receptor and the risk of a first deep vein thrombosis

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.     

Comparative thrombotic event incidence after infusion of recombinant factor VIIa versus factor VIII inhibitor bypass activity

Thrombosis is a rare but well-recognized potential complication of Factor VIII Inhibitor Bypass Activity (FEIBA) infusion. Recombinant factor VIIa (rFVIIa) is increasingly used as an alternative to FEIBA; however, the thrombotic safety profile of rFVIIa remains incompletely characterized. To determine the incidence rates of thrombotic adverse events (AEs) after infusion of rFVIIa and FEIBA. Data from the MedWatch pharmacovigilance program of the US Food and Drug Administration, as supplemented by published case reports, were used in conjunction with estimated numbers of infusions available from manufacturers to assess comparative incidence of thrombotic AEs in patients receiving rFVIIa or FEIBA in the period from April 1999 through June 2002. Reported thrombotic AEs were rare, with incidence rates of 24.6 per 10(5) infusions (CI, 19.1-31.2 per 10(5) infusions) for rFVIIa and 8.24 per 10(5) infusions (CI, 4.71-13.4 per 10(5) infusions) for FEIBA. Thrombotic AEs were significantly more frequent in rFVIIa than FEIBA recipients (incidence rate ratio, 2.98; CI, 1.71-5.52). The most commonly documented single type of thrombotic AE after rFVIIa infusion was cerebrovascular thrombosis, while myocardial infarction was the most frequent type in patients receiving FEIBA. Contrasting AE reporting patterns between rFVIIa and FEIBA may have contributed to the observed difference in thrombotic event incidence. Nevertheless, this comprehensive pharmacovigilance assessment does not support superior thrombotic safety of rFVIIa and suggests that thrombotic AE risk may be higher in rFVIIa than FEIBA recipients.     

The tissue factor–factor VIIa complex: procoagulant activity, regulation, and multitasking

Greater understanding of the cellular interactions associated with tissue factor (TF), activated factor (F) VII and TF-FVIIa complexes is likely to provide considerable clinical benefit. This article reviews current knowledge on the function and regulation of TF and its role in a range of biological processes, including hemostasis, thrombosis and inflammation.     

内皮细胞蛋白C受体与脓毒症的研究进展

内皮细胞蛋白C受体(EPCR)是蛋白C抗凝系统的主要组成部分.EPCR不仅在蛋白C活化过程中起重要作用,而且作为蛋白C及活化蛋白C的共同受体,EPCR在脓毒症中亦介导了活化蛋白C抗炎及细胞保护作用的发挥.本文就EPCR的结构、表达调节及其在脓毒症中的作用作一综述.     

Bio‐distribution of pharmacologically administered recombinant factor VIIa (rFVIIa)

Summary. Background: Recent clinical studies suggest that the prophylactic use of recombinant factor VIIa (rFVIIa) markedly reduces the number of bleeding episodes in hemophilic patients with inhibitors. Given the short biological half‐life of rFVIIa, it is unclear how rFVIIa could be effective in prophylactic treatment. Objectives: To examine the extravascular distribution of pharmacologically administered rFVIIa to obtain clues on how rFVIIa could work in prophylaxis. Methods: Recombinant mouse FVIIa tagged with AF488 fluorophore (AF488‐FVIIa) was administered into mice via the tail vein. At different time intervals following the administration, mice were exsanguinated and various tissues were collected. The tissue sections were processed for immunohistochemistry to evaluate distribution of rFVIIa. Results: rFVIIa, immediately following the administration, associated with the endothelium lining of large blood vessels. Within 1 h, rFVIIa bound to endothelial cells was transferred to the perivascular tissue surrounding the blood vessels and thereafter diffused throughout the tissue. In the liver, rFVIIa was localized to sinusoidal capillaries and accumulated in hepatocytes. In bone, rFVIIa was accumulated in the zone of calcified cartilage and some of it was retained there for a week. The common finding of the present study is that rFVIIa in extravascular spaces was mostly localized to regions that contain TF expressing cells. Conclusions: The present study demonstrates that pharmacologically administered rFVIIa readily associates with the vascular endothelium and subsequently enters into extravascular spaces where it is likely to bind to TF and is retained for extended time periods. This may explain the prolonged pharmacological effect of rFVIIa.     

Regulation of tissue factor coagulant activity on cell surfaces

Summary. Tissue factor (TF) is a transmembrane glycoprotein and an essential component of the factor VIIa‐TF enzymatic complex that triggers activation of the coagulation cascade. Formation of TF‐FVIIa complexes on cell surfaces not only trigger the coagulation cascade but also transduce cell signaling via activation of protease‐activated receptors. Tissue factor is expressed constitutively on cell surfaces of a variety of extravascular cell types, including fibroblasts and pericytes in and surrounding blood vessel walls and epithelial cells, but is generally absent on cells that come into contact with blood directly. However, TF expression could be induced in some blood cells, such as monocytes and endothelial cells, following an injury or pathological stimuli. Tissue factor is essential for hemostasis, but aberrant expression of TF leads to thrombosis. Therefore, a proper regulation of TF activity is critical for the maintenance of hemostatic balance and health in general. TF‐FVIIa coagulant activity at the cell surface is influenced not only by TF protein expression levels but also independently by a variety of mechanisms, including alterations in membrane phospholipid composition and cholesterol content, thiol‐dependent modifications of TF allosteric disulfide bonds, and other post‐translational modifications of TF. In this article, we critically review the key literature on mechanisms by which TF coagulant activity is regulated at the cell surface in the absence of changes in TF protein levels with specific emphasis on recently published data and provide the authors’ perspective on the subject.     

血必净注射液对脓毒症大鼠血栓调节蛋白及内皮蛋白C受体基因表达的影响

目的探讨血必净注射液对脓毒症大鼠血栓调节蛋白（TM）及内皮蛋白C受体（EPCR）基因表达的影响。方法采用盲肠结扎穿孔术（CLP）制备脓毒症模型。将96只健康Wistar大鼠按随机数字表法分为正常对照组、假手术组、模型组和血必净治疗组,后两组又按处死时间分为术后2、8、24、48和72h亚组,每组8只。留取肝、肺组织,分别检查各组动物组织TM和EPCR的mRNA表达。结果正常对照组和假手术组肝、肺组织TM和EPCR的mRNA有一定表达。CLP后2h肝、肺组织TM和EPCR的mRNA表达无明显变化（P均〉0.05）,8～48h肝、肺组织TM和EPCR的mRNA表达均有不同程度的增强（P均〈0.01）,至伤后72h基本恢复到术前水平（P均〉0.05）;与模型组比较,血必净治疗组CLP后8h和24h组织TM和EPCR的mRNA表达均有不同程度下降,48h和72h的基因表达有不同程度提高。结论血必净注射液可以从基因水平影响脓毒症动物组织TM及EPCR的基因表达。     

内皮细胞蛋白C受体基因6936A/G多态性和深静脉血栓形成相关性研究

目的 通过病例对照研究,了解内皮细胞蛋白C受体(EPCR)基因6936A/G多态性和深静脉血栓形成(DVT)的相关性,进一步了解EPCR在DVT形成中的重要性.方法 用ELISA法检测65例DVT患者和71名健康体检者的外周血血浆可溶性EPCR(sEPCR)水平;提取血细胞中的DNA,PCR扩增后将目的 片段EPCR基因直接测序,分析EPCR基因第6936位点的多态性.结果 ①正常对照组中,AG基因型组血浆sEPCR水平[(0.97±0.32)ng/L]明显高于AA基因型组[(0.61±0.24)ng/L](P＜0.01);DVT患者组中AG基因型组[(0.87±0.21)ng/L]亦明显高于AA基因型组[(0.50±0.18)ng/L](P＜0.01).②DVT组的血浆sEPCR水平[(0.68±0.32)ng/L]明显高于正常对照组[(0.54±0.22)ng/L](P＜0.05).③EPCR基因6936位点AG基因型分布频率DVT组高于正常对照组(P＜0.05).④AG基因型患DVT的危险性较AA基因型高(OR=2.75,95%可信区间为1.04～7.30)(P＜0.05).结论 血浆sEPCR水平与EPCR基因6936A/G多态性有关.DVT患者血浆sEPCR水平较正常人增高.EPCR基因6936 AG基因型者可能患DVT的风险高.     

Chemotherapy-induced thrombin generation via procoagulant endothelial microparticles is independent of tissue factor activity

BACKGROUND: Cisplatin-based chemotherapy predisposes cancer patients to thromboembolic events. OBJECTIVES: To investigate whether endothelial damage, via formation of procoagulant endothelial microparticles (EMPs), contributes to cisplatin-related hypercoagulability. METHODS: Cell viability and caspase-3/7 activities were assessed in two endothelial cell (EC) lines [human umbilical vein ECs (HUVECs) and human pulmonary microvascular ECs (HMVEC-Ls)] after exposure to cisplatin (1, 2.5, 5, 10 and 20 microm) for up to 120 h. Counts and procoagulant activity of EMPs were measured by flow cytometry and a thrombin generation assay, respectively. Tissue factor (TF) antigen and TF-dependent procoagulant activity of EMP were determined by enzyme-linked immunosorbent assay and a novel functional assay. RESULTS: By inducing apoptosis, cisplatin dose- and time-dependently decreased the viability of confluent HUVECs and HMVEC-Ls. Progression of EC death was accompanied by an increased release of EMPs (relative increase at 20 microm cisplatin for 48 h vs. control: HUVECs 6.5-fold, P < 0.001; HMVEC-Ls 18.4-fold, P < 0.001). EMPs were highly procoagulant (relative increase at 20 microm cisplatin for 48 h vs. control: HUVECs 2.5-fold, P < 0.001; HMVEC-Ls 5.9-fold, P < 0.001). EMP-driven thrombin generation, however, was not dependent on TF: TF expression and TF procoagulant activity levels on microparticles were only marginal and EMP-associated thrombin generation remained unchanged when the extrinsic pathway was blocked by omission of factor VIIa and/or incubation with an anti-human TF antibody. In contrast, blocking of phospholipids by annexin V markedly diminished EMP-associated procoagulant activity. CONCLUSIONS: In vitro, cisplatin induced the release of EMPs that showed TF-independent procoagulant activity.     

Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface

BACKGROUND: Variants of recombinant factor VIIa (rFVIIa) with increased intrinsic activity have been developed to improve efficacy in the treatment of bleeding disorders in the future. The increased potency of FVIIa variants was demonstrated in limited in vitro and in vivo studies. However, further characterization of FVIIa variants is needed to evaluate their potential clinical use. METHODS: In the present study, we investigated the interactions of two FVIIa variants, FVIIa(Q) and FVIIa(DVQ), with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT), and vascular endothelium. TF-FVIIa activity or its inhibition was measured directly in an amidolytic activity assay or for its ability to activate factor X. RESULTS: Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type (WT) FVIIa in the absence of tissue factor (TF). In the presence of TF, TFPI, TFPI-Xa, and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the WT FVIIa failed to generate significant amounts of FXa on unperturbed endothelial cells, FVIIa variants, particularly FVIIa(DVQ), generated a substantial amount of FXa on unperturbed endothelium. Annexin V fully attenuated the FVIIa-mediated activation of FX on unperturbed endothelial cells. On stimulated human umbilical vein endothelial cells, FVIIa and FVIIa variants activated FX at similar rates, and annexin V blocked the activation only partly. AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. Interestingly, despite significant differences observed in FXa generation on unperturbed endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions. CONCLUSION: Overall, the present data suggest that although FVIIa variants generate substantial amounts of FXa, they do not generate excessive thrombin on the surface of endothelium.