PROTHROMBINASE COMPLEX--STOPPED-FLOW KINETICS

凝血酶原酶是一种凝血复合体，由 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 超分子络合物催化蛋白水解酶活性 酶原凝血酶原转变为活性蛋白酶α-凝血酶。 高度特异、活性的位点定向荧光探针已经被 发展成一些凝血酶原酶组分。这个 本提案中概述的方法将利用这些可用的 荧光载体研究凝血酶原酶的动力学特性 复杂的停流动力学。因子Xa，共价 丹磺酰谷氨酰甘氨酸精氨基修饰活性中心 氯甲基酮(DEGRCK)将用于研究动力学 荧光阻断法组装凝血酶原酶的机制 流。荧光α-凝血酶抑制剂丹辛精氨酸-N- (3-乙基-1，5-戊二基)酰胺(DAPA)，将用于阻止- 流动实验研究凝血酶原酶在人体内的组装 底物的存在，并评估早期事件在 酶的催化循环。停流研究 凝血酶原酶在底物存在或不存在时的组装 将使用因子Va和其他因子V衍生进行 为了更好地定义辅因子的区域 参与建筑群的组装和运作。研究 也被建议用来确定动力学参数 活化蛋白C对Va因子的灭活作用 和缺少蛋白质的S之间的竞争 Xa因子与活化蛋白C的动力学与平衡 接近了。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。这个 建议的研究将提供与 凝血酶原酶的组装、功能和调节。这 信息将导致并增加对血栓的了解 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.