Mechanisms of Hemostatic Protease Inhibition by Serpins

丝氨酸蛋白酶抑制剂抑制止血蛋白酶的机制

• 批准号: 7047586
• 负责人: INGRID M VERHAMME
• 金额: $ 32.46万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7047586
• 关键词: antithrombin III; antithrombins; binding sites; chondroitin sulfate B; clinical research; coronary occlusion /thrombosis; enzyme activity; enzyme complex; fibrin; fibrinogen; fluorescent dye /probe; hemostatics; human tissue; mucopolysaccharides; plasminogen activator inhibitors; protease inhibitor; protein binding; protein localization; protein protein interaction; serine proteinases; site directed mutagenesis; thrombin; tissue /cell culture

DESCRIPTION (provided by applicant): The long-term goal is to define the molecular mechanisms of thrombin (T) inhibition by the serpins, heparin cofactor II (HCII) and plasminogen activator inhibitor-1 (PAI-1), implicated in arterial thrombosis. Thrombin localized on fibrin (Fbn) and the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin, reacts with HCII and platelet PAI-1, in GAG-accelerated mechanisms different from thrombin inhibition by antithrombin (AT), accelerated by high affinity heparin, present only in trace amounts. Unlike AT, HCII is a unique inhibitor of arterial thrombosis, as only HCII in the presence of DS is capable of inhibiting Fbn-bound thrombin. Thrombin exosites I and II are hypothesized to play different roles in these processes. Exosite I binds HCII and PAI-1 directly, whereas exosite II - heparin binding may modulate HCII and PAI-1 turnover. These steps are absent in the T - AT reaction. DS bound outside exosite II is hypothesized to act as template for inhibition by HCII of exosite ll-blocked thrombin, meizothrombin (MzT), and MzT(desFI). The identity of this site; the HCII and PAI-1 substrate pathways; the mechanisms of DS-selective inhibition of Fbn-bound thrombin by HCII, and of fibrinogen (Fbg) and Fbn regulation of thrombin inhibition by PAI-1 are all unknown. The studies will resolve these significant gaps, by using fluorescence equilibrium binding, steady-state and rapid kinetics with native thrombin, HCII and PAI-1, and specific loss-of- function mutants. They will test the hypotheses: that the exosite roles in the GAG-catalyzed thrombin inactivation mechanisms by HCII, PAI-1 and AT are distinctly different; that GAG binding outside exosite II on thrombin mediates inhibition by HCII; and that Fbg and Fbn regulate thrombin inhibition by these serpins differentially. Specific aims are: (1) To quantitate binding and chemical steps in the sequence of molecular events in the GAG-catalyzed thrombin inactivation and substrate pathways of HCII and PAI-1, compared to AT; (2) To characterize the DS-binding site outside exosite II in thrombin and MzT, and its role in thrombin and MzT inhibition; and (3) To determine the contributions of Fbg and Fbn binding to exosite I and GAGs in thrombin protection from HCII, PAI-1, and AT. These mechanism-based studies are relevant to understanding the selective, localized regulation of thrombin activity by HCII and PAI-1, and serpin turnover, in arterial clots. They may facilitate development of novel anticoagulants based on HCII and DS specifically targeted to arterial thrombosis

描述（由申请人提供）：长期目标是确定与动脉血栓形成有关的蛇形蛋白、肝素辅助因子II （HCII）和纤溶酶原激活物抑制剂-1 （PAI-1）抑制凝血酶(T)的分子机制。凝血酶定位于纤维蛋白（Fbn）、糖胺聚糖（GAGs）、皮肤硫酸酯（DS）和肝素，与HCII和血小板PAI-1发生反应，其加速机制不同于抗凝血酶（AT）对凝血酶的抑制，由高亲和力肝素加速，仅微量存在。与AT不同，HCII是一种独特的动脉血栓形成抑制剂，因为只有DS存在时HCII才能抑制fbn结合的凝血酶。据推测，凝血酶外源I和II在这些过程中起着不同的作用。外源体I直接结合HCII和PAI-1，而外源体II -肝素结合可能调节HCII和PAI-1的转换。这些步骤在T - AT反应中不存在。假设外源II外结合的DS作为HCII抑制外源II阻断凝血酶、减数凝血酶（MzT）和MzT（desFI）的模板。该网站的身份；HCII和PAI-1底物途径；HCII对ds选择性抑制Fbn结合凝血酶的机制，以及PAI-1对纤维蛋白原（Fbg）和Fbn调节凝血酶抑制的机制都是未知的。这些研究将通过荧光平衡结合、稳态和快速动力学与天然凝血酶、HCII和PAI-1以及特异性功能丧失突变体的结合来解决这些重要的空白。他们将验证以下假设：外源位点在HCII、PAI-1和AT催化的gag催化凝血酶失活机制中的作用明显不同；GAG结合凝血酶外外源II介导HCII的抑制；Fbg和Fbn对凝血酶抑制的调节是不同的。具体目的是：(1)与AT相比，定量测定gag催化的HCII和PAI-1凝血酶失活和底物途径中分子事件序列的结合和化学步骤；(2)研究凝血酶和MzT中外源II外ds结合位点及其在凝血酶和MzT抑制中的作用；(3)确定Fbg和Fbn结合外源位点I和GAGs对HCII、PAI-1和AT的凝血酶保护作用的贡献。这些基于机制的研究有助于理解HCII和PAI-1对动脉血栓凝血酶活性的选择性、局部调节以及serpin的转换。它们可能促进基于HCII和DS的新型抗凝剂的开发，这些抗凝剂专门针对动脉血栓形成