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：
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
项目类别：
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

项目摘要

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）：长期目标是确定丝氨酸蛋白酶抑制剂、肝素辅因子 II (HCII) 和纤溶酶原激活剂抑制剂 1 (PAI-1) 抑制凝血酶 (T) 的分子机制，这些机制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) 和肝素上，与 HCII 和血小板 PAI-1 发生反应，其加速机制与抗凝血酶 (AT) 的凝血酶抑制不同，由高亲和力肝素加速，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 结合凝血酶。推测凝血酶外切位点 I 和 II 在这些过程中发挥不同的作用。外部位点 I 直接结合 HCII 和 PAI-1，而外部位点 II - 肝素结合可能调节 HCII 和 PAI-1 周转。 T - AT 反应中不存在这些步骤。假设结合在外部位点 II 外部的 DS 可以作为 HCII 抑制外部位点 II 阻断的凝血酶、甲凝血酶 (MzT) 和 MzT(desFI) 的模板。本网站的身份； HCII 和 PAI-1 底物途径； HCII 对 Fbn 结合凝血酶的 DS 选择性抑制机制，以及 PAI-1 对纤维蛋白原 (Fbg) 和 Fbn 调节凝血酶抑制的机制均未知。这些研究将通过使用天然凝血酶、HCII 和 PAI-1 以及特定功能丧失突变体的荧光平衡结合、稳态和快速动力学来解决这些重大差距。他们将测试以下假设：HCII、PAI-1 和 AT 在 GAG 催化的凝血酶失活机制中的外位点作用明显不同； GAG 与凝血酶外位点 II 外部的结合介导 HCII 的抑制作用； Fbg 和 Fbn 不同地调节这些丝氨酸蛋白酶抑制剂对凝血酶的抑制作用。具体目标是： (1) 与 AT 相比，定量 HCII 和 PAI-1 的 GAG 催化凝血酶失活和底物途径中分子事件序列中的结合和化学步骤； (2) 表征凝血酶和 MzT 中外位点 II 外部的 DS 结合位点，及其在凝血酶和 MzT 抑制中的作用； (3) 确定与外位点 I 和 GAG 结合的 Fbg 和 Fbn 在凝血酶保护中免受 HCII、PAI-1 和 AT 影响的贡献。这些基于机制的研究与了解动脉血栓中 HCII 和 PAI-1 以及丝氨酸蛋白酶抑制剂转换对凝血酶活性的选择性、局部调节有关。它们可以促进基于 HCII 和 DS 的新型抗凝剂的开发，专门针对动脉血栓形成