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

基本信息

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
项目类别：
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

项目摘要

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.

长期目标是确定丝氨酸蛋白酶抑制剂抑制凝血酶 (T) 的分子机制，肝素辅因子 II (HCII) 和纤溶酶原激活剂抑制剂-1 (PAI-1)，与动脉血栓形成有关。凝血酶定位于纤维蛋白 (Fbn) 和糖胺聚糖 (GAG) 硫酸皮肤素 (DS) 和肝素，与 HCII 和血小板 PAI-1 发生反应，其 GAG 加速机制与凝血酶不同抗凝血酶 (AT) 的抑制作用，高亲和力肝素的加速作用，仅以微量存在。不像 AT、HCII 是一种独特的动脉血栓形成抑制剂，因为只有 HCII 在 DS 存在的情况下才能够抑制 Fbn 结合凝血酶。凝血酶外切位点 I 和 II 被假设在这些过程中发挥不同的作用流程。外部位点 I 直接结合 HCII 和 PAI-1，而外部位点 II - 肝素结合可能会调节 HCII 和 PAI-1 周转率。 T - AT 反应中不存在这些步骤。 DS 结合在外位点 II 之外是假设作为 HCII 抑制外位点 ll 阻断的凝血酶、酶联凝血酶 (MzT) 的模板，和MzT(desFI)。本网站的身份； HCII 和 PAI-1 底物途径； DS-的机制 HCII 选择性抑制 Fbn 结合的凝血酶，以及纤维蛋白原 (Fbg) 和 Fbn 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重大差距天然凝血酶、HCII 和 PAI-1 的平衡结合、稳态和快速动力学，以及特异性损失功能丧失突变体。他们将测试以下假设：外位点在 GAG 催化的凝血酶中的作用 HCII、PAI-1和AT的灭活机制明显不同；外位点 II 外部的 GAG 结合对凝血酶介导 HCII 的抑制； Fbg 和 Fbn 通过这些调节凝血酶抑制丝氨酸蛋白酶抑制剂有差异。具体目标是：（1）定量结合和化学步骤的顺序 GAG 催化的凝血酶失活以及 HCII 和 PAI-1 底物途径中的分子事件，与AT相比； (2) 表征凝血酶和MzT中exosite II外部的DS结合位点及其作用凝血酶和 MzT 抑制； (3) 确定 Fbg 和 Fbn 与外部位点结合的贡献 I 和 GAG 在凝血酶保护中免受 HCII、PAI-1 和 AT 的影响。这些基于机制的研究与理解选择性、局部调节有关 HCII 和 PAI-1 的凝血酶活性以及动脉血栓中的丝氨酸蛋白酶抑制剂转换。它们可以促进发展基于 HCII 和 DS 的新型抗凝剂，专门针对动脉血栓形成。