MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

描述：(改编自调查人员的摘要)。因子V是一种 凝血酶原酶复合体所必需的蛋白质辅因子。这 复合体由因子Va、因子Xa、钙和磷脂组成 浮出水面。凝血酶的产生是一个关键的反应 止血。凝血酶催化了一系列重要的反应 止血包括激活血小板、激活因子 V、和XIII，以及纤维蛋白原向纤维蛋白的转化。此外, 凝血酶与其内皮受体结合的凝血酶调节蛋白催化 激活蛋白C，从而使因子V和VIII失活。 为了理解激活的机制 凝血酶原是被调节的，我们已经对 因子V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并研究其蛋白水解性 激活。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，我们确定了 分子的完整初级序列。我们已经证明了 因子V是一种与铜蓝蛋白和因子同源的多结构域蛋白。 。我们目前正在使用定点突变来研究 表达的重组因子V的结构与功能关系 哺乳动物细胞。 本研究的目标是确定分子中 构成磷脂的结合部位，细胞表面受体， 凝血因子Xa和凝血酶原我们还将定义关键的蛋白水解物 该蛋白质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。利用凝血因子V基因和哺乳动物细胞 我们将表达具有特异性的重组因子V的表达系统 突变。我们还将用聚合酶链式反应(PCR)扩增从人血清白蛋白中分离到的血小板基因 因子V缺乏的患者，并确定自然突变 影响因子V的作用。 这些实验的结果将最终定义确切的分子 凝血酶原酶的组装和调节中的相互作用 很复杂。嵌合因子V/因子VIII的表达与鉴定 分子将进一步加深对结构功能的理解 在这个相关蛋白质家族中的关系。这一知识可能 为血栓性疾病的发病机制提供洞察。

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.