PROTHROMBINASE COMPLEX--STOPPED-FLOW KINETICS

凝血酶原复合物--停流动力学

• 批准号: 3471124
• 负责人: Sriram Krishnaswamy
• 金额: $ 11.38万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-15 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3471124
• 关键词: activation product; amides; antithrombins; blood coagulation; calcium; chemical cleavage; chemical kinetics; coagulation factor V; coagulation factor X; cofactor; cow; enzyme complex; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; fluorescence spectrometry; fluorescent dye /probe; ketones; membrane lipids; oligosaccharides; peptidases; phospholipids; protein C; protein S; prothrombin; radiotracer; serine; stop flow technique; thrombin; thromboplastin

Prothrombinase is the coagulation complex composed of the serine protease factor Xa, the protein cofactor factor Va, phospholipid or cellular surface and calcium ions. This supramolecular complex catalyses the proteolytic activation of the zymogen prothrombin to the active protease alpha-thrombin. Highly specific, active site-directed fluorescent probes have been developed to some of the prothrombinase constituents. The approaches outlined in this proposal will utilize these available fluorophores to study the dynamic aspects of the prothrombinase complex by stopped-flow kinetics. Factor Xa, covalently modified at the active site with dansyl glutamylglycinylarginyl chloromethylketone, (DEGRCK), will be used to study the kinetic mechanism of prothrombinase assembly by fluorescence stopped- flow. The fluorescent alpha-thrombin inhibitor dansylarginine-N- (3-ethyl-1,5-pentanediyl)amide (DAPA), will be used in stopped- flow experiments to study prothrombinase assembly in the presence of substrate and to evaluate the early events in the catalytic cycle of the enzyme. Stopped-flow studies of prothrombinase assembly in the presence or absence of substrate will be conducted using factor Va and other factor V-derived peptides in order to better define regions of the cofactor that participate in the assembly and function of the complex. Studies are also proposed to determine the kinetic parameters for the inactivation of factor Va by activated protein C in the presence and absence of protein S and to explore the competition between factor Xa and activated protein C by kinetic and equilibrium approaches. The development of a blood clot is a highly amplified response that is confined to the site of vascular injury. The proposed studies will provide information relevant to the assembly, function and regulation of prothrombinase. This information will result in and increased understanding of clot formation under the dynamic conditions of blood flow.

凝血酶原是由以下物质组成的凝血复合物 丝氨酸蛋白酶因子Xa、蛋白质辅因子Va、 磷脂或细胞表面和钙离子。 这 超分子复合物催化蛋白水解激活 酶原凝血酶原转变为活性蛋白酶α-凝血酶。 高度特异性、活性定点荧光探针已被 发展成一些凝血酶原酶成分。 这 本提案中概述的方法将利用这些可用的 荧光团研究凝血酶原酶的动态方面 通过停流动力学复合。 Xa 因子，共价键 在活性位点用丹酰谷氨酰甘氨酰精氨酰修饰 氯甲基酮 (DEGRCK) 将用于研究动力学 荧光终止的凝血酶原组装机制 流动。 荧光 α-凝血酶抑制剂 dansylarginine-N- (3-乙基-1,5-戊二基)酰胺(DAPA)，将用于停止- 流式实验研究凝血酶原酶的组装 底物的存在并评估早期事件 酶的催化循环。 停流研究 在存在或不存在底物的情况下进行凝血酶原酶组装 将使用因子 Va 和其他因子 V 衍生的 肽，以便更好地定义辅助因子的区域 参与综合体的组装和功能。 研究 还建议确定动力学参数 存在时活化的蛋白 C 使因子 Va 失活 和蛋白质 S 的缺失并探索之间的竞争 因子 Xa 和活化蛋白 C 的动力学和平衡 接近。 血凝块的形成是高度放大的 反应仅限于血管损伤部位。 这 拟议的研究将提供与 凝血酶原酶的组装、功能和调节。 这 信息将导致并增加对血块的了解 是在血流动态条件下形成的。