Functional Domains of Coagulation Factor V

凝血因子 V 的功能域

• 批准号: 6876625
• 负责人: Michael Kalafatis
• 金额: $ 22.65万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6876625
• 关键词: binding sites; chemical models; coagulation factor V; coagulation factor X; enzyme biosynthesis; enzyme induction /repression; fluorescent dye /probe; molecular assembly /self assembly; platelets; point mutation; protein binding; protein structure function; prothrombin; thrombin; tissue /cell culture

DESCRIPTION (provided by applicant): Prothrombinase is composed of the protein cofactor, factor Va, and the enzyme, factor Xa, associated on a cell surface in the presence of divalent metal ions. Incorporation of factor Va into prothrombinase and its interaction with factor Xa results in a 300,000-fold acceleration of the catalytic efficiency of the enzyme as compared to the catalysis of the reaction by factor Xa alone. The procofactor, factor V, does not participate in prothrombinase. Following activation of factor V by thrombin, factor Va is composed of heavy and light chains associated via divalent metal ions. Both chains of the cofactor interact with factor Xa while only the heavy chain of the cofactor binds prothrombin. The factor Va cofactor activity is efficiently down-regulated following proteolysis of the heavy chain by activated protein C (APC) only in the presence of a membrane surface and results in the inability of the cofactor to bind factor Xa. Thus, the positive and negative regulatory processes associated with factor V activation and its inactivation are directly associated with the capability of the cofactor to be incorporated into prothrombinase and to bind factor Xa. The amino acids responsible for the interaction of factor Va with factor Xa and prothrombin remain to be identified. We have data demonstrating that the heavy chain of the cofactor possesses a binding region for factor Xa within amino acid region 323-331, whereas the NH2-terminal portion of the light chain (amino acid residues 1546-1558) also interacts with factor Xa. In addition, we have data suggesting that the COOH-terminal portion of the heavy chain contain an interactive site for prothrombin while previous data have suggested that a binding site for thrombin is located on the B region of the procofactor. The specific aims of this grant proposal are: (1) to identify and characterize the factor Xa-binding domain(s) on factor Va light chain; (2) to identify and characterize the thrombin and prothrombin-binding domain(s) on the factor V molecule; (3) to test the physiological relevance of our findings by studying the assembly and function of prothrombinase on platelets. To achieve these goals we have designed a series of experiments that are prioritized and integrated with complementary molecular and structural approaches. Characterization of the specific amino acid regions of factor V that are critical for its function will allow for a profound understanding of the macromolecular interactions that control prothrombinase and are required for its assembly, function, and specificity. We have established a system to study phospholipids-driven macromolecular complex formation, which may be a model for the generation of complexes that form extra-and intra-cellularly.

描述（由申请人提供）：凝血酶原由蛋白辅因子Va和酶Xa组成，在二价金属离子存在下结合在细胞表面。在凝血酶原中加入因子Va及其与因子Xa的相互作用，使酶的催化效率比单独使用因子Xa的催化效率提高了30万倍。原因子，因子V，不参与凝血酶原。凝血酶激活因子V后，因子Va由重链和轻链组成，并通过二价金属离子相结合。辅助因子的两条链都与Xa因子相互作用，而只有辅助因子的重链与凝血酶原结合。仅在膜表面存在的情况下，活化蛋白C （APC）对重链进行蛋白水解后，Va辅因子活性有效下调，导致辅因子无法结合Xa因子。因此，与因子V激活及其失活相关的正调控和负调控过程与辅因子并入凝血酶原和结合因子Xa的能力直接相关。负责因子Va与因子Xa和凝血酶原相互作用的氨基酸仍有待鉴定。我们有数据表明，辅助因子的重链在323-331氨基酸区具有因子Xa的结合区，而轻链的nh2末端部分（氨基酸残基1546-1558）也与因子Xa相互作用。此外，我们有数据表明，重链的cooh末端部分含有凝血酶原的相互作用位点，而先前的数据表明凝血酶的结合位点位于原因子的B区。该拨款提案的具体目标是：(1)识别和表征因子Va轻链上的因子xa结合结构域；(2)鉴定和表征因子V分子上的凝血酶和凝血酶原结合域；(3)通过研究凝血酶原在血小板上的组装和功能来检验我们发现的生理学相关性。为了实现这些目标，我们设计了一系列的实验，这些实验被优先考虑并与互补的分子和结构方法相结合。表征因子V的特定氨基酸区域对其功能至关重要，将允许对控制凝血酶原的大分子相互作用有深刻的理解，这是其组装、功能和特异性所必需的。我们已经建立了一个系统来研究磷脂驱动的大分子复合物的形成，这可能是细胞外和细胞内形成复合物的一个模型。