MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

描述：（改编自研究者摘要）。 因子V是一种 凝血酶原酶复合物所必需的蛋白质辅因子。 这 复合物由因子Va、因子Xa、钙和磷脂组成 面 凝血酶的产生是在细胞内的关键反应。 止血。 凝血酶催化许多重要的反应， 止血，包括血小板的活化、因子的活化 V、VIII和XIII，以及纤维蛋白原向纤维蛋白的转化。 此外，本发明还提供了一种方法， 凝血酶与其内皮受体血栓调节蛋白结合， 激活蛋白C，然后灭活因子V和VIII。 为了了解激活的机制， 凝血酶原是受调节的，我们已经进行了一项研究， 第五因子分子的结构和功能。 最初，我们开发了 方法从血浆中分离人凝血因子V并研究其蛋白水解作用 activation. 随后，我们研究了因子Va作为 人血小板上的Xa因子受体。 最近，我们确定 分子的完整一级序列 我们已经证明 因子V是一种多结构域蛋白，与血浆铜蓝蛋白和因子 八. 我们目前正在使用定点突变来研究 表达重组因子V的结构与功能关系 哺乳动物细胞 本研究的目的是确定分子的区域， 构成磷脂，细胞表面受体， 因子Xa和凝血酶原。 我们还将定义关键的蛋白水解 该蛋白的激活和失活所需的事件。 我们 将使用遗传和反向遗传方法来定义功能 这种重要的蛋白质。 使用因子V cDNA和哺乳动物细胞 表达系统，我们将表达重组因子V与特异性 突变 我们还将使用PCR扩增血小板mRNA分离， 因子V缺乏症患者，并确定自然突变， 影响因子V功能。 这些实验的结果将最终确定确切的分子 参与凝血酶原酶组装和调节的相互作用 复杂. 嵌合因子V/因子VIII的表达和鉴定 分子将进一步增强对结构功能的理解 在这个相关蛋白质家族中的关系。 这些知识并 提供血栓性疾病发病机制的见解。

DESCRIPTION: (Adapted from the investigator's abstract). Factor V is a protein cofactor that is essential for the prothrombinase complex. This complex consists of factor Va, factor Xa, calcium, and a phospholipid surface. The generation of thrombin is a critical reaction during hemostasis. Thrombin catalyzes a number of important reactions during hemostasis including the activation of platelets, the activation of factors V, VIII and XIII, and the conversion of fibrinogen to fibrin. In addition, thrombin bound to its endothelial receptor thrombomodulin catalyzes activation of protein C which then inactivates factors V and VIII. In order to understand the mechanisms by which the activation of prothrombin is regulated we have undertaken an investigation of the structure and function of the factor V molecule. Initially, we developed methods to isolate human factor V from plasma and studied its proteolytic activation. Subsequently we investigated the role of factor Va as the factor Xa receptor on human platelets. More recently, we have determined the complete primary sequence of the molecule. We have demonstrated that factor V is a multidomain protein with homology to ceruloplasmin and factor VIII. We are currently using site directed mutagenesis to investigate structure function relationships in recombinant factor V expressed in mammalian cells. The goals of the present study are to identify regions of the molecule that constitute the binding sites for phospholipid, cell surface receptors, factor Xa and prothrombin. We will also define the critical proteolytic events required for the activation and inactivation of this protein. We will use both genetic and reverse genetic approaches to define the function of this important protein. Using the factor V cDNA and mammalian cell expression systems we will express recombinant factor V with specific mutations. We will also use PCR to amplify platelet mRNA isolated from patients with factor V deficiency and determine natural mutations that affect factor V function. The results of these experiments will ultimately define the exact molecular interactions involved in the assembly and regulation of the prothrombinase complex. Expression and characterization of chimeric factor V/factor VIII molecules will further enhance understanding of the structure function relationships in this family of related proteins. This knowledge may provide insight into the pathogenesis of thrombotic disorders.