Molecular Basis of Blood Coagulation Regulation

说明(申请人提供)：丝氨酸氨基转移酶家族蛋白酶抑制剂是凝血酶的关键调节剂。抗凝血酶和蛋白Z依赖的蛋白水解酶抑制物(ZPI)是两种已知的抑制促凝血酶的蛋白，其抑制方式受辅酶因子的调节，并且依赖于蛋白水解酶的功能状态，但这种复杂调控机制的分子细节尚不清楚。这些蛇类在调节凝血蛋白水解酶方面的生理重要性得到了以下观察的证实：敲除小鼠的基因会导致胚胎死亡，这是由于抗凝血酶的消耗性凝血障碍和当小鼠受到血栓形成的挑战或在ZPI的情况下在因子V莱顿背景下繁殖时增加的血栓形成。我们研究的长期目标是了解凝血酶是如何被这两种关键的丝氨酸抑制物调节的。我们建议的研究试图建立在我们之前的研究基础上，以促进对这种辅因子依赖调控的详细的分子理解。在抗凝血酶方面，我们最近的研究对肝素激活这种丝氨酸的变构机制进行了重要的修改，表明激活主要是通过解除与因子Xa和因子IXa的排斥相互作用，其次是通过解除反应部位的结构限制来促进反应部位和外切酶与酶的相互作用。我们的研究试图描述排斥作用的分子决定因素，确定某些突变如何解偶联伴随激活的结构变化以产生中间激活状态，并展示肝素结合的诱导-匹配机制如何耦合到变构激活。关于ZPI，我们最近对ZPI及其辅因子蛋白Z的复合体的X射线结构提供了新的见解，揭示了辅因子促进膜相关因子Xa的特异性抑制的机制，尽管ZPI存在不利的P1 Tyr。我们的研究试图绘制ZPI上Xa和Xia因子的推测外显子图，验证X-射线结构揭示的蛋白Z结合的ZPI位点，并评估ZPI独特的N端尾巴在抑制膜相关因子Xa中的作用。对于这两种蛇酶，使用11-蛋白酶抑制剂的蛇针支架嫁接抗凝血酶中的胞外相互作用的分子决定因素以及ZPI中的蛋白Z和胞外相互作用的分子决定因素的功能研究将对这些蛇的最小决定因素和辅因子识别提供严格的测试。)

DESCRIPTION (provided by applicant): Serpin family protein protease inhibitors function as key regulators of blood coagulation proteases. Two serpins, anti-thrombin and protein Z-dependent protease inhibitor (ZPI), are known to inhibit procoagulant proteases in a manner that is regulated by cofactors and dependent on the functional state of the proteases, but the molecular details of this complex regulatory mechanism are poorly understood. The physiologic importance of these serpins in regulating coagulation proteases is borne out by the observations that knocking out the mouse genes results in embryonic lethality due to a consumptive coagulopathy in the case of anti-thrombin and increased thrombosis when mice are given a thrombotic challenge or are bred on a factor V Leiden background in the case of ZPI. The long-term goal of our studies is to understand how coagulation proteases are regulated by these two critical serpin inhibitors. Our proposed studies seek to build on our prior studies to advance the detailed molecular understanding of this cofactor-dependent regulation. With respect to anti-thrombin, our recent studies have suggested an important revision of the allosteric mechanism of activation of this serpin by heparin in showing that activation is mediated principally by the relief of repulsive interactions with factor Xa and factor IXa and secondarily by the relief of structural constraints of the reactive site that promote reactive site and exosite interactions with the protease. Our proposed studies seek to characterize the molecular determinants of the repulsive interactions, establish how certain mutations can decouple the structural changes accompanying activation to produce an intermediate activated state and to show how an induced-fit mechanism of heparin binding is coupled to allosteric activation. With respect to ZPI, our recent X- ray structure of a complex of ZPI with its cofactor protein, protein Z, has provided new insights into the mechanism by which the cofactor promotes the specific inhibition of membrane-associated factor Xa, despite the unfavorable P1 Tyr of ZPI. Our studies seek to map putative exosites on ZPI for factor Xa and factor XIa, validate the ZPI sites for protein Z binding that have been revealed by the X-ray structure, and assess the role of the unique N-terminal tail of ZPI in the inhibition of membrane-associated factor Xa. For both serpins, gain of function studies in which the serpin scaffold of 11-proteinase inhibitor is used to graft the molecular determinants of exosite interactions in anti-thrombin and of both protein Z and exosite interactions in ZPI will provide a stringent test of the minimal determinants of protease and cofactor recognition by these serpins.)