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的基本和翻译方面的功能，与分别由纤维蛋白原裂解和血小板受体PAR1介导的促凝血和促血栓作用相比，这一功能受到的关注要少得多。具体地说，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，以及将凝血酶转变为蛋白C的独有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？这一机制是否与凝血酶原激活有关？凝血酶能否通过选择性地消除对纤维蛋白原和PAR1的活性而转化为蛋白C的独占激活剂？该项目建立在最近激动人心的发展基础上，包括以下特定目标：1.阐明凝血酶前激活和自身激活的分子机制；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的专属激活剂。在特定目标1中，我们将建立在凝血酶前体凝血酶原-2的自由形式的突破性结构上，其中激活区域中裂解部位的R15埋入蛋白中，与E14E、D14l和E18发生离子相互作用。这一观察结果在现有的胰酶样酶原结构中是独一无二的，并与凝血酶原激活的分子机制有关。值得注意的是，E14e、D14l和E18突变为ALA会产生凝血酶前-2突变体，该突变体可以自动激活凝血酶，而不需要蛇毒ecarin或生理凝血酶原酶复合体。我们将在凝血酶原激活的背景下，利用定点突变、动力学和X射线结构生物学来阐明控制凝血酶原-2这一显著特性的机制和因素。在特定的目标2中，我们将利用蛋白C和凝血酶前2在激活区的惊人序列相似性，在凝血酶前2的R15处的裂解位点附近的E14e、D141和E18被蛋白C的R169处的裂解位点附近的E160、D167和D172所取代。我们将检验蛋白C的R169不暴露于溶剂中的假设，即血栓调节蛋白的作用是诱导R169暴露以使凝血酶被切割。我们还假设，蛋白C在激活区域的突变导致凝血酶增强激活的构建体，甚至能够自动激活。在具体目标3中，我们将使用现有突变的组合，这些突变产生有利于蛋白C的底物特异性转变，以产生一类新的治疗相关凝血酶突变体，这些突变体已经完全失去了对促凝血底物纤维蛋白原和血栓前底物PAR1的活性，但在辅因子血栓调节蛋白存在的情况下，对抗凝底物蛋白C保持活性。 与公共健康相关：最近的统计数据表明，心血管疾病及其血栓并发症仍将是导致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。由于与血栓死亡有关，凝血酶仍然是抗血栓和抗凝治疗的主要靶点。在了解凝血酶的抗凝血剂特性以及如何利用它们来设计一种新型抗凝血剂方面的进展，可能会影响美国和世界各地数百万人的生活方式和预期寿命。

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.