ENDOTHELIAL CELL PROTEIN C RECEPTOR

本项目的目的是确定内皮细胞蛋白C受体（EPCR）的功能机制。这些目标是从我们随后的观察中产生的。EPCR在大血管内皮上优先表达，增强蛋白C的活化。它存在于细胞表面和包括小泡在内的离散细胞器中，并在细胞因子和凝血酶诱导下以高度调节的方式脱落。凝血酶也上调体内EPCR mRNA水平。阻断EPCR-蛋白C的相互作用会导致灵长类动物对大肠杆菌的反应急剧加剧，导致细胞因子水平升高、DIC、毛细血管渗漏和EPCR表达少的部位血栓形成。EPCR在不携带活化蛋白C作为货物的情况下，也可以在质膜上进行核易位。在培养的293细胞中，EPCR转染导致有限数量基因的表达改变，而不会改变绝大多数基因的表达。本项目的目的是确定膜中EPCR的EPCR的横向迁移是否对EPCR加速蛋白C激活和EPCR对蛋白C激活的浓度依赖性很重要；确定EPCR和蛋白C相互作用所需的位点；通过测量APC与EPCR结合时与直接与磷脂结合时APC活性位点到膜表面的距离来确定复合物的形貌；并确定血栓患者中发现的EPCR启动子突变对培养细胞表达的影响。EPCR的结构要求和EPCR介导的基因表达改变的细胞机制将被研究。这些研究旨在提高我们对EPCR功能机制的理解，为EPCR突变如何影响血栓形成风险提供见解，并提高我们对EPCR在血栓形成疾病病理生理学中的作用的理解。

The goal of this project is to determine the mechanisms by which the endothelial cell protein C receptor (EPCR) functions. The aims grow out of our observations which follow. EPCR is expressed preferentially on the large vessel endothelium and enhances protein C activation. It is found on the cell surface and in discrete organelles include caveolae and is shed in a highly regulated fashion induced by cytokines and thrombin. Thrombin also up-regulates EPCR mRNA levels in vivo. Blocking EPCR-protein C interactions leads to a dramatic exacerbation of the primate response to E. coli resulting in a elevated cytokine levels, DIC, capillary leak, and thrombosis at sites where little EPCR is expressed. EPCR can also undergo nuclear translocation form the plasma membrane with our without carrying activated protein C as cargo. In cultured 293 cells, EPCR transfection results in an altered expression of a limited number of genes without changing the expression of the vast majority of genes. The aims of the present project are to determine whether lateral mobility of EPCR of EPCR in the membrane is important for EPCR acceleration of protein C activation and the EPCR concentration dependence of protein C activation: to determine the sites on EPCR and protein C required for interaction; to determine the topography of the complex by measuring the distance from the active site of APC to the membrane surface when APC is bound to EPCR vs. directly to phospholipid; and to determine the impact of EPCR promoter mutations identified in thrombotic patients on expression in cultured cells. The structural requirements within EPCR and the cellular mechanisms involved in EPCR mediated alteration of gene expression will be investigated. These studies are designed to improve our understanding of the mechanisms by which EPCR functions, provide insights into how EPCR mutations might influence thrombotic risk, and improve our understanding of the role of EPCR in the pathophysiology of thrombotic disease.