Processed Defining Megakaryocyte Endocytosis of Factor V

描述(由申请人提供)：血小板和血浆衍生的凝血因子Va在凝血酶原酶催化的凝血酶生成中起着重要作用，凝血酶原酶由1：1的化学计量比的钙依赖的辅因子Va和结合在激活的血小板上的丝氨酸蛋白酶Xa组成。从复合体中去除凝血因子Va可使凝血酶生成率降低10,000倍，这一生理效应体现在凝血因子V缺乏症中观察到的严重出血。最近的研究表明，整个血小板源因子V来源于血浆，通过血小板前体细胞、巨核细胞对血浆因子V的内吞。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，这些差异使血小板衍生辅助因子具有更强的促凝血活性。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，与其血浆相比较，该分子在物理和功能上都是独一无二的。这项提议详细说明了检验以下假设的策略：血浆衍生因子V被巨核细胞内吞，运输到跨高尔基网络，翻译后零售，包装成α颗粒，并进行蛋白水解性处理，产生整个血小板衍生辅因子池。第一个目标是利用CD34+骨髓细胞体外扩增产生的巨核细胞、原代骨髓来源的巨核细胞和适当的巨核细胞样细胞系，将因子V内吞作用与巨核细胞分化和成熟相关联。第二个目标是确定调节因子V内吞作用的细胞事件，它在细胞内向α颗粒的运输(可能通过跨高尔基网络)，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在维持正常止血方面比其血浆因子更重要，这些研究将增加我们对巨核细胞如何获取、处理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.