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: 7837515
• 负责人: INGRID M VERHAMME
• 金额: $ 21.01万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7837515
• 关键词: Acylation; Address; Affinity; Anticoagulant therapy; Anticoagulants; Antithrombin III; Antithrombins; Arterial Fatty Streak; Binding; Binding Sites; Blood Platelets; Cell surface; Chemicals; Coagulation Process; Competitive Binding; Complex; Dependence; Dermatan Sulfate; Development; Enzymes; Equilibrium; Event; Fibrin; Fibrinogen; Fluorescence; Glycosaminoglycans; Goals; Hemostatic Agents; Heparin; Heparin Binding; Heparin Cofactor II; Injury; Kinetics; Label; Ligands; Mediating; Molecular; Pathway interactions; Peptide Hydrolases; Plasminogen Activator Inhibitor 1; Play; Process; Proteins; Reaction; Regulation; Research Personnel; Role; Rupture; Serpins; Site; Site-Directed Mutagenesis; Surface; Testing; Therapeutic Embolization; Thrombin; Thrombosis; Thrombus; Time; Variant; arginyllysine; base; chemical binding; chemical reaction; deacylation; inhibitor/antagonist; insight; loss of function; meizothrombin; mutant; novel; percutaneous coronary intervention; programs; stopped-flow fluorescence

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) Tocharacterize 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.

长期目标是确定蛇类抑制凝血酶(T)的分子机制， 肝素辅助因子II(HCII)和纤溶酶原激活物抑制物-1(PAI-1)，与动脉血栓形成有关。 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 肝素与HCII和血小板PAI-1以不同于凝血酶的GAG加速机制反应 由高亲和力肝素加速的抗凝血酶(AT)抑制作用仅在微量存在。不像 在AT，HCII是一种独特的动脉血栓形成抑制物，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被假设在这些中扮演不同的角色 流程。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周转率。这些步骤在T-AT反应中缺失。在Exosite II外部结合的DS是 假设用作HCII抑制外切酶11-封闭的凝血酶，甲硫唑凝血酶(MZT)的模板， 和MZT(DesFI)。该位点的同源性；HCII和PAI-1底物通路；DS-1的作用机制。 HCII选择性抑制纤维蛋白原结合凝血酶及纤维蛋白原和纤维蛋白原对凝血酶的调节 PAI-1的抑制作用尚不清楚。这些研究将通过使用荧光来解决这些显著的差距 与天然凝血酶、HCII和PAI-1的平衡结合、稳态和快速动力学，以及比损失 功能缺陷突变体。他们将检验这样的假设：外植体在GAG催化的凝血酶中起作用 HCII、PAI-1和AT的失活机制明显不同；Exosite II外的GAG结合 凝血酶介导HCII抑制；Fbg和Fbn通过这些途径调节凝血酶抑制 不同的蛇类。具体目标是：(1)量化以下序列中的结合步骤和化学步骤 GAG催化的凝血酶失活和HCII和PAI-1底物途径的分子事件 与AT比较；(2)表征凝血酶和MZT中Exosite II外DS结合部位及其作用 抑制凝血酶和MZT；以及(3)确定FBG和FBN结合对外膜的贡献 HCII、PAI-1和AT对凝血酶的保护作用 这些基于机制的研究与理解选择性的、局部性的调控有关 动脉血栓中HCII和PAI-1的凝血酶活性，以及蛇针的周转率。它们可能会促进发展 以HCII和DS为基础的针对动脉血栓形成的新型抗凝剂。