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毒、肝素辅助因子II(HCII)和纤溶酶原激活物抑制物-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)和糖胺多聚糖(GAG)、硫酸皮肤素(DS)和肝素上，与HCII和血小板PAI-1以不同于抗凝血酶(AT)抑制凝血酶的GAG加速机制发生反应，高亲和力肝素仅以微量存在。与AT不同，HCII是一种独特的动脉血栓形成抑制剂，因为只有在DS存在的情况下，HCII才能抑制FBN结合的凝血酶。凝血酶外切酶I和II被认为在这些过程中扮演不同的角色。Exosite I直接与HCII和PAI-1结合，而Exosite II-肝素结合可能调节HCII和PAI-1的周转。这些步骤在T-AT反应中缺失。结合在Exosite II外的DS被假设为HCII抑制Exosite II阻断的凝血酶、甲硫氧基凝血酶(MZT)和MZT(DesFI)的模板。该位点的同源性；HCII和PAI-1底物途径；HCII对FBN结合的凝血酶的DS选择性抑制机制；以及纤维蛋白原(FBG)和纤维蛋白原(FBN)对PAI-1抑制凝血酶的调节机制都是未知的。这些研究将通过使用荧光平衡结合、与天然凝血酶、HCII和PAI-1以及特定的功能丧失突变体的稳态和快速动力学来解决这些重大差距。他们将检验以下假设：Exosite在HCII、PAI-1和AT催化的凝血酶失活机制中的作用明显不同；Exosite II外部与凝血酶的GAG结合介导了HCII的抑制；以及FBG和FBN不同地调节这些蛇对凝血酶的抑制。具体目标是：(1)与AT相比，定量GAG催化的凝血酶失活和底物通路中HCII和PAI-1的分子事件序列中的结合和化学步骤；(2)表征凝血酶和MZT中Exosite II外的DS结合部位，及其在凝血酶和MZT抑制中的作用；以及(3)确定FBG和FBN结合到Exosite I和GAG在HCII、PAI-1和AT保护凝血酶中的作用。这些基于机制的研究有助于理解HCII和PAI-1对动脉血栓中凝血酶活性的选择性、局部性调节以及蛇毒蛋白的周转。它们可能会促进基于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