Factor VIIa Interaction with Endothelial Cell Protein C Receptor

说明书(申请人提供)：内皮细胞衬里构成血管组织和循环血液之间的界面，在调节血液和血管周围组织之间的分子交换方面发挥着重要作用。尽管内皮细胞处于与循环凝血因子相互作用的最佳位置，但人们对这些相互作用知之甚少。最近，我们发现VIIa因子(FVIIa)能与内皮细胞上的内皮细胞蛋白C受体(EPCR)特异性结合。Fvii和FVIIa都以真正的配体方式与EPCR结合，并且与蛋白C和活化蛋白C(APC)与EPCR的亲和力相似。目前，FVII/FVIIa与EPCR相互作用的生理意义和重要性尚不清楚。我们最近的研究表明，FVIIa与EPCR结合导致其内吞作用。这些研究还表明，EPCR介导的内吞作用和再循环可能促进FVIIa从管腔到管腔表面的运输。我们的最新研究表明，FVIIa与内皮细胞上的EPCR结合，激活了蛋白水解酶激活受体-1(PAR1)，从而提供了内皮屏障保护。在细胞模型系统中的研究表明，治疗浓度的FVIIa可能通过取代内皮上EPCR中的蛋白C而下调蛋白C的抗凝途径。所有这些发现都是新的，累积表明FVIIa与EPCR的相互作用可能在病理生理学中发挥重要作用。基于这些发现，我们假设在治疗条件下，FVIIa与内皮上的EPCR相互作用调节FVIIa的运输，提供对血管渗漏的保护，并下调蛋白C/APC抗凝途径。验证这些假说的三个具体目的是：(1)研究EPCRa在FVIIa跨细胞和清除血流中的作用；(2)阐明EPCRFVIIa介导的内皮细胞屏障保护机制(S)，并确定FVIIa信号在维持体内血管屏障完整性中的作用；(3)检验rFVIIa药物浓度下调蛋白C/APC介导的抗凝途径，从而增强rFVIIa在治疗出血性疾病中的止血作用的假说。对于这些研究，我们将同时使用细胞和动物模型系统。我们将利用具有良好特性的EPCR缺陷和EPCR过表达的小鼠以及小鼠EPCR特异性抗体来研究EPCRa在FVIIa转运和介导FVIIa诱导的细胞效应中的作用。FVIIa与内皮的相互作用及其转运将通过免疫组织化学、透射电子显微镜和检测组织中Fvii/FVIIa的活性和抗原来探讨。共聚焦和荧光共振能量转移(FRET)显微镜将被用来研究FVIIa、EPCR和PAR1的物理相互作用。这些研究将揭示EPCRa和FVIIa的新功能，并改变我们目前对FVIIa和EPCRa在止血和炎症中作用的理解。拟议的研究将对出血性疾病和脓毒症的未来治疗策略具有重大和重要的影响。 公共卫生相关性：拟议的研究将提供关于凝血因子VIIa与内皮细胞蛋白C受体(EPCR)如何相互作用调节止血和炎症的有价值的新信息。从拟议研究中获得的数据将使我们对重组因子VIIa如何在使用抑制剂治疗血友病患者中作为有效止血剂，以及预防性使用FVIIa如何即使在停止治疗后也能减少这些患者的关节出血和减缓关节破坏的现有知识有了新的理解。总体而言，拟议的研究将为提高出血性疾病的治疗效率和降低治疗成本提供关键知识，并为rFVIIa治疗炎症性疾病开辟新的治疗机会。

DESCRIPTION (provided by applicant): The endothelial cell lining constitutes the interface between vascular tissue and circulating blood, and plays an important role in regulation of molecular exchange between blood and perivascular tissues. Although endothelial cells are positioned optimally to interact with circulating clotting factors, very little is known about these interactions. Recently, we have shown that factor VIIa (FVIIa) binds specifically to endothelial cell protein C receptor (EPCR) on endothelial cells. Both FVII and FVIIa bind to EPCR in a true-ligand fashion and with a similar affinity as that of protein C and activated protein C (APC) to EPCR. At present the physiological significance and importance of FVII/FVIIa interaction with EPCR are unknown. Our recent studies showed that FVIIa binding to EPCR resulted in its endocytosis. These studies also indicate that EPCR-mediated endocytosis and recycling may facilitate the transport of FVIIa from the luminal to abluminal surface. Our latest studies show that FVIIa binding to EPCR on endothelial cells activates protease activated receptor-1 (PAR1) and provides endothelial barrier protection. Studies in cell model systems have suggested that therapeutic concentrations of FVIIa may down-regulate the protein C anticoagulant pathway by displacing protein C from EPCR on the endothelium. All of these findings are novel and cumulatively indicate that FVIIa interaction with EPCR may play an important role in pathophysiology. Based on these findings, we hypothesize that FVIIa interaction with EPCR on the endothelium modulates FVIIa transport, provides protection against vascular leakage, and down-regulates the protein C/APC anticoagulant pathway in therapeutical conditions. Three specific aims that test these hypotheses are, (1) investigate the role of EPCR in FVIIa transcytosis and clearance from bloodstream, (2) elucidate EPCR-FVIIa-mediated barrier protective mechanism(s) in endothelial cells and define the role of FVIIa-EPCR signaling in maintaining vascular barrier integrity in vivo, and (3) test the hypothesis that pharmacological concentrations of rFVIIa down-regulate the protein C/APC-mediated anticoagulant pathway and thereby potentiate the hemostatic effect of rFVIIa in the treatment of bleeding disorders. For these studies, we will use both cell and animal model systems. We will employ well characterized EPCR deficient and EPCR overexpressing mice and antibodies specific to mouse EPCR in order to investigate the role of EPCR in FVIIa transport and mediating FVIIa-induced cellular effects. FVIIa interaction with the endothelium and its transport will be probed by immunohistochemistry, transmission electron microscopy and measuring FVII/FVIIa activity and antigen in tissues. Confocal and fluorescence resonance energy transfer (FRET) microscopy will be used to investigate the physical interaction of FVIIa, EPCR and PAR1. The proposed studies will reveal novel functions of EPCR and FVIIa and revise our current understanding on the role of FVIIa and EPCR in hemostasis and inflammation. The proposed studies will have major and important implications for future treatment strategies of bleeding disorders and sepsis. PUBLIC HEALTH RELEVANCE: The proposed studies will provide valuable and novel information on how interaction of clotting factor VIIa with endothelial cell protein C receptor (EPCR) modulates hemostasis and inflammation. Data obtained from the proposed studies will provide new understanding to our existing knowledge on how recombinant factor VIIa functions as an effective hemostatic agent in treating hemophiliacs with inhibitors, and how prophylactic use of FVIIa can reduce joint bleeds and slow joint destruction in these patients even after cessation of treatment. Overall, the proposed studies will provide critical knowledge to improve efficacy of treatment of bleeding disorders and reduce treatment costs, and also open novel therapeutic opportunities for rFVIIa in treating inflammatory diseases.