Studies on anticoagulant properties of thrombin

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功能的基础和转化方面，其受到的关注远远少于分别由纤维蛋白原和血小板受体PAR1切割介导的促凝剂和促血栓作用。具体而言，该项目涉及凝血酶活化蛋白C的分子机制及其与凝血酶原活化机制的联系，以及将凝血酶转化为临床应用蛋白C的专用激活剂的可能性。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？这一机制是否与凝血酶原活化有关？凝血酶能否通过选择性地取消对纤维蛋白原和PAR1的活性而转化为蛋白C的唯一激活剂？该项目建立在最近令人兴奋的发展基础上，包括以下具体目标：阐明凝血酶-2活化和自活化的分子机制；2. 阐明凝血酶活化蛋白C的分子机制3. 将凝血酶转化为蛋白c的独家激活剂。在具体目标1中，我们将以最近的凝血酶前体凝血酶-2自由形式的突破性结构为基础，其中激活区域切割位点的R15与E14e， D14l和E18离子相互作用，被埋在蛋白质内部。这一观察结果在现有的胰蛋白酶样酶原结构中是独一无二的，并与凝血酶原活化的分子机制有关。值得注意的是，E14e、D14l和E18突变为Ala会产生一个凝血酶2前突变体，该突变体可以自动激活凝血酶，而不需要蛇毒毒素或生理凝血酶原复合物。在凝血酶原激活的背景下，我们将利用定点诱变、动力学和x射线结构生物学来阐明控制凝血酶前2这一显著特性的机制和因素。针对性2中,我们将利用蛋白质C之间惊人的相似序列和prethrombin-2激活域,与E14e D14l和E18网站R15的乳沟prethrombin-2 E160所取代,D167和D172网站的乳沟R169蛋白质C .我们将测试假设R169蛋白质C不暴露在溶剂的作用,诱发接触thrombomodulin R169使凝血酶的乳沟。我们还假设激活域的蛋白C突变导致构建体被凝血酶激活增强，甚至能够自激活。在特定目标3中，我们将使用现有突变的组合，使底物特异性向有利于蛋白C的方向转变，以产生一类新的治疗相关的凝血酶突变，这些突变完全失去了对促凝底物纤维蛋白原和促凝底物PAR1的活性，但在辅因子血栓调节蛋白存在时，仍保留对抗凝底物蛋白C的活性。

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.