Factor VIIa Interaction with Endothelial Cell Protein C Receptor

项目摘要/摘要 内皮细胞衬里构成了血管组织和循环血液之间的界面，并发挥了 在调节血液和血管周围组织之间的分子交换方面发挥重要作用。虽然 内皮细胞处于与循环凝血因子相互作用的最佳位置，人们对此知之甚少 这些互动。最近，我们发现VIIa因子(FVIIa)能与内皮细胞蛋白特异性结合。 内皮细胞上的C受体(EPCR)。Fvii和FVIIa都以真正的配体方式与EPCR结合，并与 与蛋白C和活化蛋白C(APC)对EPCR的亲和力相似。目前生理学上 FVII/FVIIa与EPCR相互作用的意义和重要性尚不清楚。我们最近的研究表明， FVIIa与EPCR结合后可发生内吞作用。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。我们最新的 研究表明，FVIIa与内皮细胞EPCR结合可激活蛋白水解酶激活受体1(PAR1) 并提供内皮屏障保护。对细胞模型系统的研究表明，治疗性 FVIIa的浓度可能通过取代蛋白C而下调蛋白C的抗凝途径 内皮细胞上的EPCR值。所有这些发现都是新的，累积表明FVIIa与 内源性聚合酶链式反应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FVIIa 与内皮上的EPCR相互作用调节FVIIa的运输，提供血管保护 在治疗条件下，蛋白C/APC抗凝途径被下调。三 检验这些假说的具体目的是：(1)研究EPCR在FVIIa跨细胞和 血流清除，(2)阐明EPCRFVIIa介导的屏障保护机制(S) 并确定FVIIa-EPCR信号在维持血管屏障完整性中的作用 以及(3)检验rFVIIa的药物浓度下调 蛋白C/APC介导的抗凝途径，从而增强rFVIIa的止血作用 用于治疗出血性疾病。对于这些研究，我们将同时使用细胞和动物模型系统。 我们将使用具有良好特性的EPCR缺陷和EPCR过表达的小鼠和特异性抗体 小鼠EPCR在FVIIa转运和介导FVIIa诱导的细胞中的作用 效果。FVIIa与内皮细胞的相互作用及其转运将通过免疫组织化学来探讨， 并测定组织中FVII/FVIIa活性和抗原。共焦和 将使用荧光共振能量转移(FRET)显微镜来研究物理相互作用 FVIIa、EPCR和PAR1。拟议的研究将揭示EPCR和FVIIa的新功能，并修订我们的 目前对FVIIa和EPCR在止血和炎症中作用的了解。建议进行的研究 将对未来出血性疾病和败血症的治疗策略产生重大而重要的影响。

PROJECT SUMMARY/ABSTRACT 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.