Molecular Basis of Blood Coagulation Regulation

描述（由申请人提供）：丝氨酸蛋白酶抑制剂家族蛋白酶抑制剂作为凝血蛋白酶的关键调节剂发挥作用。已知两种丝氨酸蛋白酶抑制剂（抗凝血酶和蛋白Z依赖性蛋白酶抑制剂（ZPI））以受辅因子调节并依赖于蛋白酶的功能状态的方式抑制促凝血蛋白酶，但对这种复杂调节机制的分子细节知之甚少。这些丝氨酸蛋白酶抑制剂在调节凝血蛋白酶中的生理学重要性通过以下观察得到证实：在抗凝血酶的情况下，敲除小鼠基因导致胚胎死亡，这是由于消耗性凝血病，而在ZPI的情况下，当给予小鼠血栓形成攻击或在因子V莱顿背景下繁殖时，血栓形成增加。我们研究的长期目标是了解凝血蛋白酶如何被这两种关键的丝氨酸蛋白酶抑制剂调节。我们提出的研究旨在建立在我们以前的研究，以推进这种辅因子依赖性调节的详细分子理解。关于抗凝血酶，我们最近的研究表明，肝素激活丝氨酸蛋白酶抑制剂的变构机制的重要修订表明，激活主要是介导的排斥性相互作用与因子Xa和因子IXa的救济和救济的反应性位点的结构限制，促进反应性位点和exosite与蛋白酶的相互作用。我们提出的研究旨在表征排斥相互作用的分子决定因素，建立某些突变如何可以解耦伴随激活的结构变化，以产生中间激活状态，并显示肝素结合的诱导适应机制是如何耦合到变构激活。关于ZPI，我们最近的ZPI与其辅因子蛋白（蛋白Z）的复合物的X射线结构提供了对辅因子促进膜相关因子Xa的特异性抑制的机制的新见解，尽管ZPI的P1 Tyr是不利的。我们的研究旨在映射假定的exosites对ZPI因子Xa和因子XIa，验证ZPI网站的蛋白Z结合，已揭示的X-射线结构，并评估的作用，独特的N-末端尾巴的ZPI在膜相关因子Xa的抑制。对于这两种丝氨酸蛋白酶抑制剂，其中11-蛋白酶抑制剂的丝氨酸蛋白酶抑制剂支架用于移植抗凝血酶中的外位点相互作用和ZPI中的蛋白Z和外位点相互作用的分子决定簇的功能研究的获得将提供对这些丝氨酸蛋白酶抑制剂的蛋白酶和辅因子识别的最小决定簇的严格测试。

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.)