MOLECULAR INTERACTIONS OF FIBRINOLYSIS

描述：（改编自申请人的摘要） 主要工作有：（1）阐明反应动力学途径和反应机理 派-1抑制tPA; (2)to构象研究 影响稳定性、活性和 派-1的特异性;（3）确定限制tPA周转的因素 与纤溶酶原，其天然底物，以及如何切割的单一 链抑制剂使其增加;（4）评价结构-功能 作用机制的方面和构象变化 链激酶 这些研究将依赖于某些独特的方法， 我们过去使用过的。 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猝灭法 来追踪中间体的形成和衰变。 荧光 光谱学;包括偏振或各向异性和共振能量 转移，将被广泛用于评估相互作用和不同的 蛋白质的构象状态。 在这项工作结束时， 我们希望对派-1的结构有更清楚的了解， 蛋白酶复合物，其形成的动力学途径，和基础 因为它的稳定性。 该信息可能与所有的丝氨酸蛋白酶抑制剂相关。 我们 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 加强对派-1的了解 机制可能导致灭活剂的发展， 一种新的抗血栓作用。 更多关于tPA的知识， 链激酶机制可导致更有效的血栓溶解 疗法

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.