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: 7540399
• 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/7540399
• 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.

长期目标是定义塞旋蛋白抑制凝血酶（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。 凝血酶位于纤维蛋白（FBN）和糖胺聚糖（GAGS）硫酸盐（DS）和 肝素与HCII和血小板PAI-1反应，在与凝血酶不同的GAG加速机制中 抗凝血酶（AT）抑制，高亲和力肝素加速，仅以痕量为单位。与众不同 AT，HCII是一种独特的动脉血栓形成抑制剂，因为仅在DS存在下HCII才能 抑制FBN结合的凝血酶。假设凝血酶外生I和II在这些中扮演不同的角色 过程。 Exosite I直接结合HCII和PAI -1，而Exosite II-肝素结合可能调节 HCII和PAI-1营业额。在反应时，这些步骤不存在。 DS绑定外部II是 假设可以作为HCII抑制的模板 和MZT（Desfi）。该网站的身份； HCII和PAI-1底物途径； DS-的机制 通过HCII选择性抑制FBN结合凝血酶，纤维蛋白原（FBG）和FBN调节凝血酶 PAI-1抑制都是未知的。研究将通过使用荧光来解决这些显着的差距 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特定损失 - 功能突变体。他们将检验假设：凝结催化凝血酶中的外部作用 HCII，PAI-1和AT的灭活机制截然不同。插科打结合在外部II之外 凝血酶介导HCII抑制； FBG和FBN调节这些调节凝血酶的抑制 Serpins差异化。具体目的是：（1）定量结合和化学步骤。 HCII和PAI-1的GAG催化凝血酶灭活和底物途径中的分子事件， 与AT相比（2）将Exosite II外II外的DS结合位点在凝血酶和MZT中及其作用 在凝血酶和MZT抑制中； （3）确定FBG和FBN与Exosite的贡献 我和插科打'in hcii，pai-1和at。 这些基于机制的研究与理解选择性的局部调节有关 HCII和PAI-1的凝血酶活性以及动脉凝块中的SERPIN转换。他们可能会促进发展 基于HCII和DS专门针对动脉血栓形成的新型抗凝剂。