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: 7338327
• 负责人: INGRID M VERHAMME
• 金额: $ 29.78万
• 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/7338327
• 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; Coronary; Dependence; Dermatan Sulfate; Development; Endopeptidases; Enzymes; Equilibrium; Event; Fibrin; Fibrinogen; Fluorescence; Glycosaminoglycans; Goals; Hemostatic Agents; Heparin; Heparin Binding; Heparin Cofactor II; Injury; Intervention; Kinetics; Label; Ligands; Localized; Mediating; Molecular; Pathway interactions; Peptide Hydrolases; Plasminogen Activator Inhibitor 1; Play; Process; Proteins; Rate; 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; 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）和纤溶酶原激活物抑制剂-1（派-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）， 肝素与HCII和血小板派-1反应，其GAG加速机制不同于凝血酶 抗凝血酶（AT）抑制，高亲和力肝素加速，仅以痕量存在。不像 AT，HCII是动脉血栓形成的独特抑制剂，因为只有在DS存在下的HCII能够 抑制Fn结合的凝血酶。假设凝血酶外泌位点I和II在这些细胞中发挥不同的作用。 流程.外切位点I直接结合HCII和派-1，而外切位点II -肝素结合可以调节HCII和PAI-1的结合。 HCII和派-1周转。这些步骤在T-AT反应中不存在。外部位点II外结合的DS为 假设其作为通过HCII抑制外位点II封闭的凝血酶，美佐凝血酶（MzT）， 和MzT（desFI）。该位点的身份; HCII和派-1底物途径; DS-1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调节凝血酶 派-1抑制作用均未知。这些研究将通过使用荧光来解决这些重大差距 与天然凝血酶、HCII和派-1的平衡结合、稳态和快速动力学以及特异性损失- 功能缺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、派-1和AT的失活机制明显不同;外部位点II外的GAG结合 凝血酶介导的抑制HCII;和Fbg和Fbn调节凝血酶抑制这些 Serpins差异化。具体目标是：（1）定量结合和化学步骤的顺序， GAG催化的凝血酶失活和HCII和派-1的底物途径中的分子事件， （2）研究凝血酶和MzT中外切位点II外的DS结合位点，并探讨其在凝血酶和MzT中的作用 凝血酶和MzT的抑制作用;（3）确定Fbg和Fbn与胞外位点结合的贡献 I和GAG在凝血酶保护免受HCII、派-1和AT中的作用。 这些基于机制的研究与理解选择性的、局部的调节有关， 在动脉凝块中，通过HCII和派-1测定凝血酶活性，以及丝氨酸蛋白酶抑制剂周转。它们可以促进发展 基于HCII和DS的新型抗凝血剂专门针对动脉血栓形成。