PROTHROMBINASE COMPLEX--STOPPED-FLOW KINETICS

凝血酶原酶是凝血复合物， 丝氨酸蛋白酶因子Xa，蛋白辅因子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活化， 酶原凝血酶原转化为活性蛋白酶α-凝血酶。 高特异性、活性的定点荧光探针已经被广泛应用。 形成了一些凝血酶原酶成分。 的 本提案中概述的方法将利用这些可用的 荧光团来研究凝血酶原酶的动力学方面 由停流动力学复合。 因子Xa，共价键 在活性部位用丹磺酰谷氨酰甘氨酰精氨酸修饰 氯甲基酮（DEGRCK）将用于研究动力学 凝血酶原酶组装的荧光阻断机制 流 荧光α-凝血酶抑制剂丹酰精氨酸-N- （3-乙基-1，5-戊二基）酰胺（达帕）将用于停药- 流动实验研究凝血酶原酶组装在 存在的底物，并评估早期的事件， 酶的催化循环。 停流研究 凝血酶原酶在底物存在或不存在下的组装 将使用因子Va和其他因子V导出的 为了更好地定义辅因子的区域， 参与综合体的组装和功能。 研究 的动力学参数 活化蛋白C对因子Va的灭活 和缺乏蛋白S，并探讨 Xa因子和活化蛋白C的动力学和平衡 接近。 血栓的形成是一种高度放大的 仅限于血管损伤部位的反应。 的 拟议的研究将提供有关信息， 凝血酶原酶的组装、功能和调节。 这 信息将导致和增加对服装了解 在血液流动的动态条件下形成。

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.