MOLECULAR INTERACTIONS OF FIBRINOLYSIS

纤维蛋白溶解的分子相互作用

• 批准号: 2519321
• 负责人: JOSEPH D SHORE
• 金额: $ 25.16万
• 依托单位: HENRY FORD HEALTH SYSTEM
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2519321
• 关键词: active sites; acylation; conformation; enzyme mechanism; enzyme structure; enzyme substrate complex; fibrin; fibrinolysis; fluorescent dye /probe; fluorimetry; molecular dynamics; monoclonal antibody; plasminogen; plasminogen activator; plasminogen activator inhibitors; protein structure function; serine proteinases; urokinase

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.

描述：（改编自申请人的摘要）该项目的具体目标 项目有：（1）阐明动力学途径和反应机制 用于通过 PAI-1 抑制 tPA； (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的周转率 与纤溶酶原、其天然底物以及如何裂解单个 链抑制剂增加它； (4) 评估结构-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， 我们过去曾使用过。 快速反应动力学，特别是 将使用停流荧光测定法和快速混合化学淬火 跟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PAI-1的结构有更清晰的了解 蛋白酶复合物、其形成的动力学途径和基础 为了它的稳定性。 此信息可能与所有丝氨酸蛋白酶抑制剂相关。 我们 也希望阐明tPA的反应机理以及一些 对反应至关重要的结构特征和构象变化 链激酶的机制。 加深对 PAI-1 的了解 机制可以导致灭活剂的开发，这将提供 一种新颖的抗血栓作用。 有关 tPA 的更多知识和 链激酶机制可以导致更有效的溶栓 治疗。