MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

凝血酶原酶复合体的调节缺陷在 血栓形成的发病机制。因子V调节血管活性。 凝血酶原酶复合体，负责凝血酶的产生 在血液凝固过程中。因子V中的遗传缺陷是导致 蛋白C抵抗，这是最常见的遗传风险因素 血栓形成。凝血因子Va的产生及凝血酶原酶的组装 因此，复合体在血栓形成中是一个关键事件。长期目标 这个项目的目的是了解细胞的结构、功能和调节。 凝血因子V和凝血酶原酶复合体。在第一个资助期内 我们鉴定了因子V基因，并使用因子V表达系统 定义蛋白质中的结构-功能关系。我们决定 因子V C2结构域含有磷脂酰丝氨酸结合部位 和获得性因子V抗体。我们还发现了另一种选择 C2结构域的糖基化是这两种形式的结构基础 对阴离子亲和力不同的因子V轻链 磷脂。我们发现在B-域结果中有很大的缺失 在表达部分辅因子活性的单链蛋白中。 最后，我们对APC抗性的分子机制进行了研究。 在第二个资助期内，我们建议延长我们对 重组因子V的结构和功能 研究将进一步定义结构域和特定的氨基酸残基 组成组装所需的结合位点 凝血酶原酶复合体。我们将使用丙氨酸扫描诱变来 确定因子V所需的轻链中的氨基酸 与磷脂和血小板膜结合。我们将使用相同的 根据需要识别A1和A3结构域中的氨基酸的方法 用于凝血因子Xa结合位点。我们将定位酪氨酸的位置 对辅因子表达起重要作用的硫酸化和糖基化 激活和发挥作用。最后，我们将研究 因子V B结构域。我们将确定新的B结构域是否通过以下方式切割 细胞内的蛋白水解酶参与因子V的作用。我们还将本地化 凝血因子XIII在B结构域内介导了交联性。这个 拟议的研究将为精确的分子提供新的见解 参与组装凝血酶原酶复合体的相互作用。这 信息将是理解对 生理和病理条件下的凝血酶原酶复合体。

Defects in regulation of the prothrombinase complex play a central role in the pathogenesis of thrombosis. Factor V regulates the activity of the prothrombinase complex which is responsible for the generation of thrombin during blood coagulation. Genetic defects in factor V are the cause of protein C resistance which is the most common inherited risk factor for thrombosis. Generation of factor Va and assembly of the prothrombinase complex is therefore a critical event in thrombosis. The long-term goal of this project is to understand the structure, function and regulation of factor V and the prothrombinase complex. During the first funding period we characterized the factor V gene and used factor V expression systems to define structure-function relationships in the protein. We determined that the Factor V C2 domain contains binding sites for phosphatidylserine and acquired factor V antibodies. We also found that alternative glycosylation of the C2 domain is the structural basis for the two forms of the factor V light chain that differ in their affinity for anionic phospholipid. We found that a large deletion within the B-domain results in a single chain protein that expresses partial cofactor activity. Finally, we have characterized molecular mechanisms of APC resistance. During the second funding period we propose to extend our studies on the structure and function of recombinant factor V. The goals of these studies will be to further define domains and specific amino acid residues that comprise the binding sites required for assembly of the prothrombinase complex. We will use alanine scanning mutagenesis to identify the amino acids within the light chain required for factor V binding to phospholipid and platelet membranes. We will use the same approach to identify amino acids within the A1 and A3 domains as required for the factor Xa binding site. We will localize the sites of tyrosine sulfation and glycosylation that are important for expression of cofactor activation and function. Finally we will investigate the functions of the factor V B-domain. We will determine whether novel B-domain cleavages by cellular proteases contribute to factor V action. We will also localize the sites of factor XIII mediated crosslinking within the B-domain. The proposed studies will provide new insights into the precise molecular interactions involved in assembly of the prothrombinase complex. This information will be critical for understanding the regulation of the prothrombinase complex under physiological and pathological conditions.