MOLECULAR INTERACTIONS OF FIBRINOLYSIS

描述：(改编自申请者的摘要) 主要工作有：(1)阐明反应的动力学途径和反应机理 PAI-1对tPA的抑制作用；(2)构象研究 影响稳定性、活性和稳定性的变化和相互作用 PAI-1的特异性；(3)确定是什么限制了tPA的周转 与纤溶酶原，其天然底物，以及如何单裂解 链抑制剂使其增加；(4)评价结构-功能 黄曲霉毒素作用机制的方面及构象变化 链激酶素。这些研究将依赖于某些独特的方法 我们在过去已经利用过。快速反应动力学，特别是 将使用停流荧光法和快速混合化学淬火 跟踪中间体的形成和腐烂。荧光 光谱学；包括极化或各向异性和共振能量 迁移，将被广泛用于评估交互和不同 蛋白质的构象状态。在这项工作结束时 我们希望对PAI-1的结构有更清楚的了解 蛋白水解酶复合体及其形成的动力学途径和基础 因为它的稳定性。这一信息可能与所有的蛇类有关。我们 同时也希望阐明tPA的反应机理和一些 对反应至关重要的结构特征和构象变化 链激酶的作用机制。加深对PAI-1的了解 机制可以导致灭活剂的发展，这将提供 一种新的抗血栓作用。更多关于tPA和 链激酶机制可导致更有效的溶栓作用 心理治疗。

DESCRIPTION: (adapted from applicant's abstract) The specific aims of the project are: (1) to elucidate the kinetic pathway and reaction mechanism for the inhibition of tPA by PAI-1; (2)to study the conformational changes and interactions which affect the stability, activity and specificity of PAI-1; (3) to determine what limits the turnover of tPA with plasminogen, its natural substrate, and how cleavage of the single chain inhibitor increases it; and (4) to evaluate structure-function aspects and conformational changes in the mechanism of action of streptokinase. The studies will depend on certain unique methods which we have utilized in the past. Rapid reaction kinetics, specifically stopped-flow fluorimetry and rapid mixing chemical quenching will be used to follow the formation and decay of intermediates. Fluorescence spectroscopy; including polarization or anisotropy and resonance energy transfer, will be used extensively to evaluate interactions and different conformational states of the proteins. At the conclusion of this work we hope to have a clearer understanding of the structure of the PAI-1 protease complex, the kinetic pathway for its formation, and the basis for its stability. This information may be relevant to all serpins. We also hope to elucidate the reaction mechanism of tPA and some of the structural features and conformational changes critical to the reaction mechanism of streptokinase. Enhanced understanding of the PAI-1 mechanism can result in development of inactivators which would provide a novel antithrombotic action. More knowledge about the tPA and streptokinase mechanisms can result in more effective thrombolytic therapy.