Studies on the anticoagulant properties of thrombin

描述(由申请人提供)：建议的研究项目集中在凝血酶作为抗凝剂在生理上的重要作用的一些基本的、尚未解决的方面。该项目建立在以前NIH支持的基础上，包括以下具体目标：1.阐明Na+对凝血酶切割纤维蛋白原和蛋白C的不同作用；2.阐明W215在识别纤维蛋白原和蛋白C中的作用；3.解决野生型和突变型凝血酶与血栓调节蛋白形成的复合体的结构。我们将结合动力学、定点突变和X射线结构研究。在具体目标1中，我们将解决凝血酶功能的一个基本悬而未决的问题，即为什么Na+促进凝血酶对纤维蛋白原的切割，但对蛋白C的激活没有影响。对小鼠凝血酶的平行突变研究将检验这样一种假设，即它对Na+结合形式的分子模拟转化为对扰乱Na+结合和稳定抗凝剂无Na+形式的突变的抗性。这些研究将对人类凝血酶特异性的钠离子依赖开关的分子基础以及小鼠酶用来优化其促凝血活性的分子策略产生迫切需要的理解。在特定的目标2中，我们将对残基W215进行饱和突变，并确定凝血酶的特异性可以改变到什么程度有利于纤维蛋白原或蛋白C。产生显著抗凝作用的W215突变将与其他突变相结合，产生新一代抗凝血酶突变体，它对纤维蛋白原完全失去活性，但对蛋白C保持活性。这些研究将加深我们对凝血酶如何识别活性部位的生理底物的理解，并将产生具有潜在药理意义的新试剂。在具体目标3中，我们将解决人和鼠的野生型和突变型凝血酶的X射线晶体结构，这些结构与凝血酶抗凝活性的分子基础特别相关。我们将优先解决野生型和精选突变体与血栓调节蛋白复合体的结构。抗凝血酶突变体可以自由结晶，并与活性部位抑制物结合。这些研究将极大地拓宽我们目前关于凝血酶如何与血栓调节蛋白在分子水平上相互作用的知识，以及该酶的突变如何导致体外和体内研究中记录的显著抗凝作用。

DESCRIPTION (provided by applicant): The proposed research project focuses on some basic, unresolved aspects of the physiologically important role of thrombin as an anticoagulant. The project builds upon developments from previous NIH support and consists of the following specific aims: 1. elucidate the differential effect of Na+ on the cleavage of fibrinogen and protein C by thrombin; 2. elucidate the role of W215 in the recognition of fibrinogen and protein C; and 3. solve the structures of wild-type and mutant thrombins in complex with thrombomodulin. We will use a combination of kinetic, site-directed mutagenesis and X-ray structural studies. In specific aim 1, we will address a basic unresolved issue of thrombin function, i.e., why Na+ promotes fibrinogen cleavage by thrombin but has no effect on protein C activation. Parallel mutagenesis studies on murine thrombin will test the hypothesis that its molecular mimicry of the Na+-bound form translates into resistance to mutations that perturb Na+ binding and stabilize the anticoagulant Na+-free form. These studies will produce a much needed understanding of the molecular basis of the Na+-dependent switch in specificity of human thrombin and of the molecular strategy utilized by the murine enzyme to optimize its procoagulant activity. In specific aim 2, we will carry out saturation mutagenesis of residue W215 and establish to what extent the specificity of thrombin can be altered in favor of fibrinogen or protein C. Mutations of W215 that produce significant anticoagulant effects will be combined with other mutations to produce a new generation of anticoagulant thrombin mutants that has completely lost activity toward fibrinogen, but retain activity toward protein C. These studies will deepen our understanding of how thrombin recognizes physiologic substrates at the active site and will produce new reagents of potential pharmacological relevance. In specific aim 3, we will solve the X-ray crystal structures of wild-type and mutant thrombins, human and murine, that are particularly relevant to the molecular basis of thrombin anticoagulant activity. We will give top priority to solution of the structure of wild-type and selected mutants in complex with thrombomodulin. Anticoagulant thrombin mutants will be crystallized free and bound to active site inhibitors. These studies will significantly broaden our current knowledge of how thrombin interacts with thrombomodulin at the molecular level, and how mutations of the enzyme result in the remarkable anticoagulant effects documented in vitro and in vivo studies.