Processed Defining Megakaryocyte Endocytosis of Factor V

描述（由申请人提供）：血小板和血浆来源的因子Va在凝血酶原催化的凝血酶生成中起重要作用，凝血酶原由1:1的化学计量，钙依赖的辅因子Va和丝氨酸蛋白酶因子Xa结合到活化的血小板上。从复合体中去除因子Va导致凝血酶生成速率降低10,000倍，在因子V缺乏中观察到的严重出血证明了生理效应。最近的研究表明，整个血小板衍生因子V池起源于血浆，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表现出与血浆衍生因子Va明显不同的生化和物理差异，这些差异赋予血小板衍生辅助因子更高的促凝潜能。该项目的目标是了解产生血小板衍生的辅助因子分子的细胞内过程，该辅助因子分子在物理和功能上与血浆对应物相比是独特的。该提案详细说明了验证以下假设的策略：血浆衍生因子V被巨核细胞内吞，运输到反式高尔基网络，翻译后重新修饰，包装成α颗粒，并进行蛋白水解处理，以产生整个血小板衍生的辅助因子库。第一个目的是利用CD34+骨髓细胞、原代骨髓来源的巨核细胞和适当的巨核细胞样细胞系体外扩增产生的巨核细胞，将因子V内噬作用与巨核细胞分化和成熟联系起来。第二个目标是定义调节因子V内吞作用的细胞事件，其细胞内转运到α颗粒（可能通过反高尔基网络），以及这些相互作用导致因子V的表型变化。由于血小板衍生因子Va在维持正常止血方面比血浆衍生因子Va起着更重要的作用，这些研究将增加我们对巨核细胞如何获取、加工和包装这一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.