Processed Defining Megakaryocyte Endocytosis of Factor V

描述（由申请人提供）：血小板和血浆衍生因子Va在凝血酶原酶催化的凝血酶生成中起重要作用，凝血酶原酶由1：1、化学计量、钙依赖性的辅因子因子Va和丝氨酸蛋白酶因子Xa的复合物组成，与活化的血小板结合。从复合物中去除因子Va导致凝血酶生成速率降低10，000倍，在因子V缺乏症中观察到的严重出血证明了生理效应。最近的研究表明，血小板衍生因子V的整个池来源于血浆，通过血小板祖细胞，巨核细胞内吞血浆因子V。然而，血小板衍生因子Va表现出明显区别于血浆衍生因子Va的生物化学和物理差异，这些差异赋予血小板衍生辅因子增加的促凝血潜力。这个项目的目标是了解产生血小板衍生辅因子分子的细胞内过程，该分子与其血浆对应物相比在物理和功能上是独特的。该提案详细说明了策略，以测试的假设，即血浆衍生因子V是由巨核细胞内吞，贩运到trans-Golgi网络，重新定制postperformancally，包装成α-颗粒和蛋白水解加工，以产生整个池的血小板衍生辅因子。第一个目的是关联因子V的内吞作用与巨核细胞的分化和成熟，使用巨核细胞产生的体外扩增的CD 34+骨髓细胞，原代骨髓衍生的巨核细胞，和适当的巨核细胞样细胞系。第二个目的是定义调节因子V内吞作用的细胞事件，其细胞内运输到α-颗粒（可能通过trans-Golgi网络），以及这些相互作用导致的因子V的表型变化。由于血小板衍生因子Va在维持正常止血中起着比其血浆对应物更重要的作用，这些研究将增加我们对巨核细胞如何获得，加工和包装这种关键凝血因子的理解。

DESCRIPTION (provided by applicant): Platelet- and plasma-derived factor Va serve an essential role in thrombin generation catalyzed by Prothrombinase, which consists of 1:1, stoichiometric, calcium-dependent complex of the cofactor factor Va and the serine protease factor Xa bound to activated platelets. Removal of factor Va from the complex results in a 10,000-fold decrease in the rate of thrombin generation, the physiologic effect being demonstrated by the severe hemorrhage observed in factor V deficiency. Recent studies indicate that the entire pool of platelet-derived factor V originates from plasma through endocytosis of plasma factor V by platelet progenitor cells, megakaryocytes. However, platelet-derived factor Va exhibits biochemical and physical differences that clearly distinguish it from plasma-derived factor Va, differences that impart an increased procoagulant potential to the platelet-derived cofactor. The goal of this project is to understand the intracellular processes that produce a platelet-derived cofactor molecule that is physically and functionally unique when compared to its plasma counterpart. This proposal details strategies to test the hypothesis that plasma-derived factor V is endocytosed by megakaryocytes, trafficked to the trans-Golgi network, retailored posttranslationally, packaged into alpha-granules and processed proteolytically to yield the entire pool of the platelet-derived cofactor. The first aim is to correlate factor V endocytosis with megakaryocyte differentiation and maturity using megakaryocytes generated by ex vivo expansion of CD34+ bone marrow cells, primary bone marrow-derived megakaryocytes, and appropriate megakaryocyte-like cell lines. The second aim is to define the cellular events that regulate factor V endocytosis, its intracellular trafficking to alpha-granules (perhaps through the trans-Golgi network), and the phenotypic changes in factor V resulting from these interactions. Since platelet-derived factor Va plays a more essential role in maintaining normal hemostasis than does its plasma counterpart, these studies will increase our understanding of how megakaryocytes acquire, process and package this critical coagulation factor.