MOLECULAR BIOLOGY OF HUMAN COAGULATION FACTOR V

描述：（研究者摘要）在美国， 每30秒就有一人死亡。临床疾病，如 心肌梗死、深静脉血栓形成和肺栓塞以及中风 通常由血栓形成事件引起。虽然基础研究在 血栓形成已经导致在诊断和治疗方面的显著进步， 血栓性疾病目前的方法仍然是次优的。一代 凝血酶原酶复合物的凝血酶在 静脉血栓形成的发病机制。凝血酶原酶复合物由 酶因子Xa、辅因子因子Va和磷脂膜表面。 因子Xa与因子Va的相互作用需要辅因子活化 用于表达因子Xa结合位点。因子Va与 血小板膜需要磷脂酰丝氨酸在血小板表面的表达。 活化的血小板或内皮细胞。Xa因子和 磷脂膜是不连续的，位于几个不同的位置， 域.这些结合位点的复杂性可以允许精细的 凝血酶原酶复合物的调节。这些的分子基础 相互作用仍然知之甚少。该项目的长期目标是 使用综合的分子、结构和生物物理方法来了解 Va因子与生物膜的相互作用。前一 在资助期间，使用昆虫细胞表达因子C2结构域， 对两种晶型的结构进行了鉴定。因子Va的表达 哺乳动物细胞中的突变体证明，C2结构域的糖基化 调节膜结合，并且两个双链体位于移动的溶剂中 暴露的环在因子V的高亲和力结合中起关键作用， 含有低浓度磷脂酰丝氨酸的磷脂膜。的 本提案的具体目的是进一步确定结合位点 在磷脂膜和细胞膜的因子Va轻链中。 将使用重组因子Va突变体定位结合位点， 重组轻链结构域、结构域特异性和单克隆抗体。 实验将使用现有的晶体结构或分子设计 个别领域的模型。结合相互作用将使用 表面等离子体共振和荧光结合测定。这些信息 将为凝血酶原酶的调节提供重要的新见解 复杂的，并可能确定网站，可以利用作为新的目标， 抗血栓治疗。

DESCRIPTION: (Investigator's abstract) In the United States, cardiovascular disease results in one death every 30 seconds. Clinical disorders such as myocardial infarction, deep vein thrombosis and pulmonary embolism, and stroke are usually precipitated by thrombotic events. Although basic research in thrombosis has lead to significant advances in the diagnosis and treatment of thrombotic disorders current approaches remain sub optimal. Generation of thrombin by the prothrombinase complex plays a particularly important role in the pathogenesis venous thrombosis. The prothrombinase complex consists of the enzyme factor Xa, the cofactor factor Va and a phospholipid membrane surface. The interaction of factor Xa with the factor Va requires cofactor activation for expression of factor Xa binding sites. The interaction of factor Va with platelet membranes requires expression of phosphatidylserine on the surface of activated platelets or endothelial cells. The binding sites for factor Xa and phospholipid membranes are discontinuous and are located in several different domains. The complexity of these binding sites may allow for the fine regulation of the prothrombinase complex. The molecular bases for these interactions remain poorly understood. The long-term goal of this project is to use integrated molecular, structural and biophysical approaches to understand the interaction of factor Va with biological membranes. During the previous funding period the factor C2 domain was expressed using insect cells and the structures of two crystal forms were elucidated. Expression of factor Va mutants in mammalian cells demonstrated that glycosylation of the C2 domain modulates membrane binding and that two tryptophans located in a mobile solvent exposed loop play a critical role in high affinity binding of factor V to phospholipid membranes containing low concentrations of phosphatidylserine. The specific aims of the present proposal are to further define the binding sites in the factor Va light chain for phospholipid membranes and cellular membranes. Binding sites will be localized using recombinant factor Va mutants, recombinant light chain domains, domain specific and monoclonal antibodies. Experiments will be designed using available crystal structures or molecular models for individual domains. Binding interactions will be characterized using surface plasmon resonance and fluorescence binding assays. This information will provide important new insights into regulation of the prothrombinase complex and may identify sites that could be exploited as novel targets for anti-thrombotic therapy.