MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

凝血酶原酶复合物调节的缺陷在 血栓形成的发病机制。 因子V调节细胞的活性， 凝血酶原酶复合物，负责凝血酶的产生 在血液凝固过程中。 因子V的遗传缺陷是导致 蛋白C抗性是最常见的遗传风险因素， 血栓形成 因子Va的产生和凝血酶原酶的组装 因此，复合物是血栓形成中的关键事件。 远景目标 本项目的目的是了解结构，功能和调节 因子V和凝血酶原酶复合物。 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， 定义蛋白质的结构-功能关系。 我们确定 因子VC 2结构域含有磷脂酰丝氨酸结合位点， 并获得了第五因子抗体 我们还发现， C2结构域的糖基化是两种形式的结构基础 不同的因子V轻链对阴离子的亲和力 磷脂 我们发现，B结构域中的大缺失导致 在表达部分辅因子活性的单链蛋白中。 最后，我们已经表征了APC抗性的分子机制。 在第二个资助期内，我们建议扩展有关 重组因子V的结构和功能。 研究将进一步确定结构域和特定的氨基酸残基 所述结合位点包含组装所述抗体所需的结合位点。 凝血酶原酶复合物 我们将使用丙氨酸扫描诱变， 确定因子V所需的轻链内的氨基酸 与磷脂和血小板膜结合。 我们将使用相同的 根据需要鉴定A1和A3结构域内氨基酸的方法 用于Xa因子结合位点。 我们将定位酪氨酸的位点 对于辅因子表达重要的硫酸化和糖基化 激活和功能。 最后，我们将研究 因子V B结构域。 我们将确定是否新的B结构域裂解， 细胞蛋白酶有助于因子V的作用。 我们还将本地化 因子XIII介导的B结构域内交联的位点。的 拟议的研究将提供新的见解，精确的分子 凝血酶原酶复合物组装中涉及的相互作用。 这 信息将是至关重要的了解监管的 凝血酶原酶复合物在生理和病理条件下的作用。

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.