Factor VIIa interaction with Endothelial Cell Protein C Receptor

 描述(申请人提供)：最近的研究表明，凝血因子FVIIa(FVIIa)在血管损伤后与促凝剂辅因子组织因子(Tf)结合时启动凝血级联反应，也与抗凝辅因子内皮细胞蛋白C受体(EPCR)结合。EPCR通过促进蛋白C的活化在蛋白C抗凝途径中发挥重要作用，同时也促进活化蛋白C(APC)诱导的细胞保护信号转导。在上一个资助周期中进行的研究表明，FVIIa与内皮细胞上的EPCr结合促进FVII/FVIIa从循环到血管外部位的运输，而与EPCr结合的FVIIa激活蛋白酶激活受体1(PAR1)介导的细胞信号并提供屏障保护作用。药理浓度的rFVIIa被发现取代EPCR中的内源性蛋白C。RFVIIa常规用于抗FVIII和FIX的抑制性抗体治疗血友病患者。它是 也被用作儿科和成人患者的紧急止血剂。尽管rFVIIa被广泛而成功地使用，但对rFVIIa在出血性疾病中的作用机制的了解还不完全。清楚地了解rFVIIa在治疗中的作用机制对于提高rFVIIa的临床疗效、更好地管理患者和开发基于rFVIIa的新一代疗法至关重要。我们假设rFVIIa的药理浓度与内源性蛋白C竞争内皮上有限的EPCR位点，从而减少依赖于EPCR的APC的产生，从而下调APC的抗凝途径。APC抗凝途径的下调将增强FVIIa诱导的凝血酶生成，因为降低的APC水平不容易抑制Va因子的生成。我们还假设，在rFVIIa预防治疗中，EPCR介导的FVIIa转运和FVIIa-EPCR介导的屏障保护和抗炎作用在防止关节损伤和骨丢失方面发挥了关键作用。以下目标将检验上述假设。目的1：探讨FVIIa：ePCR相互作用在rFVIIa治疗出血性疾病中止血作用的重要性，并阐明其作用机制；目的2：研究FVIIa在预防血友病中的生物分布和组织滞留，评价FVIIa-EPCR介导的止血、屏障保护和抗炎作用在预防血友病关节病中的作用；目的3：阐明在没有rFVIIa治疗的情况下，EPCR与EPCRmAb结合提供血友病血管屏障保护作用的机制。为了进行这些研究，我们将产生新的mFVIIa变种和独特的转基因小鼠，并使用大隐静脉出血、针刺膝关节损伤以及血管内皮生长因子和脂多糖诱导的血管渗漏模型系统。拟议研究的结果将使我们对rFVIIa的药理浓度如何在血友病患者和其他出血性疾病患者中提供止血效果的理解发生范式转变。

 DESCRIPTION (provided by applicant): Recent studies showed that clotting factor FVIIa (FVIIa), which initiates the coagulation cascade upon binding to procoagulant cofactor tissue factor (TF) following vascular injury, also binds to anticoagulant cofactor endothelial cell protei C receptor (EPCR). EPCR plays a critical role in the protein C anticoagulant pathway by promoting the activation of protein C. EPCR also promotes activated protein C (APC)-induced cytoprotective signaling. Studies conducted in the last funding cycle revealed that FVIIa binding to EPCR on the endothelium facilitates the transport of FVII/FVIIa from the circulation to extravascular sites and FVIIa bound to EPCR activates the protease-activated receptor 1 (PAR1)-mediated cell signaling and provides the barrier protective effect. Pharmacological concentrations of rFVIIa were found to displace endogenous protein C from EPCR. rFVIIa is routinely used to treat hemophilia patients with inhibitory antibodies against FVIII and FIX. It is also being used as an emergency hemostatic agent in both pediatric and adult patients. Despite its widespread and successful use, the understanding of the mechanism of rFVIIa action in bleeding disorders is incomplete. A clear understanding of the mechanistic action of rFVIIa in therapy is essential to improve the clinical efficacy of rFVIIa, and to better manage patients and develop a new generation of FVIIa-based therapeutics. We hypothesize that pharmacological concentrations of rFVIIa compete with endogenous protein C for the limited EPCR sites on the endothelium, which diminishes EPCR-dependent APC generation, and thus the down-regulation of the APC anticoagulant pathway. The down-regulation of the APC anticoagulant pathway would enhance FVIIa-induced thrombin generation as factor Va is not readily inhibited by the reduced APC levels. We also hypothesize that EPCR-mediated FVIIa transport and FVIIa-EPCR-mediated barrier protective and anti-inflammatory effects play crucial roles in preventing the joint damage and bone loss in rFVIIa prophylaxis. The following aims will test the above hypotheses. Aim 1: Investigate the importance of FVIIa:EPCR interaction in the hemostatic effect of rFVIIa in treating bleeding disorders and elucidate its mode of action; Aim 2: Characterize FVIIa bio-distribution and tissue retention in prophylaxis and evaluate the contribution of FVIIa-EPCR-mediated hemostatic, barrier protective and anti-inflammatory effects to the prophylactic effect of preventing hemophilic arthropathy; and Aim 3: Elucidate the mechanism by which EPCR ligation with EPCR mAb, in the absence of rFVIIa treatment, provides the vascular barrier protective effect in hemophilia. To perform these studies, we will generate novel mFVIIa variants and unique transgenic mice and employ saphenous vein bleeding, the needle puncture knee injury, and VEGF-and LPS- induced vascular leakage model systems. The outcome of the proposed studies will introduce a paradigm shift in our understanding of how the pharmacological concentration of rFVIIa provides the hemostatic effect in hemophilia patients and patient with other bleeding disorders.