Studies on anticoagulant properties of thrombin

凝血酶抗凝特性的研究

• 批准号: 8368991
• 负责人: Enrico Di Cera
• 金额: $ 37.5万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8368991
• 关键词: Address; Affect; Anticoagulant therapy; Anticoagulants; Attention; Binding; Blood; Blood Platelets; Cardiovascular Diseases; Cause of Death; Cessation of life; Cleaved cell; Clinic; Coagulation Process; Complex; Data; Development; Employee Strikes; Engineering; Enzyme Precursors; Enzymes; Fibrin; Fibrinogen; Fibrinolytic Agents; Generations; Homeostasis; Individual; Kinetics; Laboratories; Life Expectancy; Life Style; Mediating; Molecular; Molecular Conformation; Mutation; PAR-1 Receptor; Physiological; Platelet aggregation; Productivity; Property; Protein C; Protein Engineering; Proteins; Prothrombin; Reporting; Research Project Grants; Residual state; Roentgen Rays; Role; Site; Site-Directed Mutagenesis; Snake Venoms; Solvents; Specificity; Structure; Substrate Specificity; Surface; Testing; Thrombin; Thrombomodulin; Trypsin; United States National Institutes of Health; Ursidae Family; activated Protein C; base; clinical application; cofactor; disability; in vivo; mutant; prethrombins; prothrombinase complex; receptor; statistics; structural biology; success

DESCRIPTION (provided by applicant): The proposed research project addresses basic and translational aspects of the function of thrombin as an anticoagulant, which has received far less attention than the procoagulant and prothrombotic roles mediated by cleavage of fibrinogen and the platelet receptor PAR1, respectively. Specifically, the project deals with the molecular mechanism of protein C activation by thrombin and its connection with the mechanism of prothrombin activation, as well as with the possibility of turning thrombin into an exclusive activator of protein C for clinical applications. We will address the following basic questions: What is the mechanism of protein C activation by thrombin? Is this mechanism related to that of prothrombin activation? Can thrombin be converted into an exclusive activator of protein C by selectively abrogating activity toward fibrinogen and PAR1? The project builds upon recent exciting developments and consists of the following specific aims: 1. Elucidate the molecular mechanism of prethrombin-2 activation and auto-activation; 2. Elucidate the molecular mechanism of protein C activation by thrombin; 3. Convert thrombin into an exclusive activator of protein C. In specific aim 1, we will build on a recent breakthrough structure of the thrombin precursor prethrombin-2 in the free form where R15 at the site of cleavage in the activation domain is buried inside the protein, in ionic interaction with E14e, D14l and E18. This observation is unique among existing structures of trypsin-like zymogens and bears on the molecular mechanism of prothrombin activation. Remarkably, mutation of E14e, D14l and E18 to Ala generates a prethrombin-2 mutant that auto-activates to thrombin without the need for the snake venom ecarin or the physiological prothrombinase complex. We will elucidate the mechanism and factors that control this remarkable property of prethrombin-2 in the context of prothrombin activation using site-directed mutagenesis, kinetics and X-ray structural biology. In specific aim 2, we will exploit the striking sequence similarity between protein C and prethrombin-2 in the activation domain, with E14e, D14l and E18 around the site of cleavage at R15 in prethrombin-2 replaced by E160, D167 and D172 around the site of cleavage at R169 in protein C. We will test the hypothesis that R169 of protein C is not exposed to solvent and that the action of thrombomodulin is to induce exposure of R169 to enable thrombin cleavage. We also hypothesize that mutations of protein C in the activation domain result in constructs with enhanced activation by thrombin or even capable of auto-activation. In specific aim 3, we will use a combination of existing mutations that produce a shift in substrate specificity in favor of protein C to generate a new class of therapeutically relevant thrombin mutants that have completely lost activity toward the procoagulant substrate fibrinogen and the prothrombotic substrate PAR1, but retain activity toward the anticoagulant substrate protein C in the presence of the cofactor thrombomodulin. PUBLIC HEALTH RELEVANCE: Recent statistics indicate that cardiovascular disease and its thrombotic complications will remain the leading cause of death and disability and will represent a major burden to productivity in the US and worldwide well into the year 2020. Because of its involvement in thrombotic deaths, thrombin remains a major target of antithrombotic and anticoagulant therapies. Progress in the understanding of the anticoagulant properties of thrombin and how they can be exploited to engineer a new class of anticoagulants has the potential to influence the life-style and life expectancy of millions of people in the US and worldwide.

描述（由申请人提供）：拟议的研究项目涉及凝血酶作为抗凝剂的功能的基本和翻译方面，其受到的关注远远低于分别由纤维蛋白原和血小板受体PAR 1裂解介导的促凝血和促血栓形成作用。具体而言，该项目涉及凝血酶激活蛋白C的分子机制及其与凝血酶原激活机制的联系，以及将凝血酶转化为蛋白C的专用激活剂用于临床应用的可能性。我们将解决以下基本问题：凝血酶激活蛋白C的机制是什么？这种机制是否与凝血酶原激活有关？凝血酶能通过选择性地消除对纤维蛋白原和PAR 1的活性而转化为蛋白C的唯一激活剂吗？该项目建立在最近令人兴奋的发展，并包括以下具体目标：1。阐明凝血酶原-2激活和自激活的分子机制; 2.阐明凝血酶激活蛋白C的分子机制; 3.将凝血酶转化为蛋白C的唯一激活剂。在具体目标1中，我们将建立在游离形式的凝血酶前体凝血酶前体-2的最近突破性结构的基础上，其中活化结构域中切割位点处的R15被埋在蛋白质内部，与E14 e、D14 l和E18进行离子相互作用。这一观察结果在胰蛋白酶样酶原的现有结构中是独特的，并且与凝血酶原激活的分子机制有关。值得注意的是，E14 e、D14 l和E18突变为Ala产生凝血酶前体-2突变体，其自动激活为凝血酶，而不需要蛇毒ecarin或生理凝血酶原酶复合物。我们将阐明的机制和因素，控制这一显着的性质的凝血酶原-2的凝血酶原激活的背景下，使用定点诱变，动力学和X-射线结构生物学。在具体目标2中，我们将利用蛋白C和凝血酶前体-2在活化结构域中的惊人序列相似性，其中凝血酶前体-2中R15处切割位点周围的E14 e、D141和E18被蛋白C中R169处切割位点周围的E160、D167和D172取代。我们将检验蛋白C的R169不暴露于溶剂以及血栓调节蛋白的作用是诱导R169暴露以使凝血酶裂解的假设。我们还假设，蛋白C在激活结构域中的突变导致构建体具有增强的凝血酶激活或甚至能够自激活。在具体目标3中，我们将使用产生有利于蛋白C的底物特异性转变的现有突变的组合，以产生一类新的治疗相关的凝血酶突变体，其完全丧失了对促凝血底物纤维蛋白原和促血栓底物PAR 1的活性，但在辅因子血栓调节蛋白存在下保留了对抗凝底物蛋白C的活性。 公共卫生相关性：最近的统计数据表明，心血管疾病及其血栓性并发症仍将是死亡和残疾的主要原因，并将成为美国和全球生产力的主要负担，直至2020年。由于凝血酶与血栓性死亡有关，它仍然是抗血栓和抗凝治疗的主要靶点。对凝血酶抗凝特性的理解以及如何利用它们来设计新一类抗凝剂的进展有可能影响美国和全球数百万人的生活方式和预期寿命。