Molecular Basis of Blood Coagulation Regulation

Serpin家族蛋白蛋白酶抑制剂是凝血蛋白酶的关键抗凝调节剂。两种蛇形蛋白，抗凝血酶和蛋白z依赖性蛋白酶抑制剂（ZPI），已知以一种由辅助因子调节的方式抑制促凝蛋白酶，并依赖于蛋白酶的功能状态，但这种复杂调节机制的分子细节知之甚少。这些蛇形蛋白在调节凝血蛋白酶方面的生理学重要性是通过观察得到的，当小鼠在ZPI的情况下在因子V Leiden背景下繁殖时，敲除小鼠基因会导致胚胎死亡，这是由于抗凝血酶的情况下的消耗性凝血功能障碍和血栓前表型。我们提出的研究试图建立在我们之前的研究基础上，以推进对这些蛇蛋白对凝血蛋白酶的辅因子依赖性调节的分子理解。关于抗凝血酶，我们最近的研究提出了对肝素激活蛇蛋白的变构机制的重要修正，表明激活主要是通过抗凝血酶外源内与因子Xa和因子IXa的排斥相互作用的缓解而不是通过提供有吸引力的外源来介导的。我们提出的后续研究旨在描述蛋白酶结合位点抗凝血酶的分子决定因素，以及参与将激活的构象变化从肝素结合位点传递到蛋白酶结合位点的变构通信网络。在ZPI方面，我们的研究旨在阐明ZPI及其辅助因子Z蛋白靶向的Xa因子生理复合物，确立ZPI独特的n端尾部在Xa因子抗凝调节中的作用，并发现破坏ZPI-蛋白Z复合物并下调ZPI抗凝机制的小分子，作为血友病出血性疾病恢复止血的潜在新疗法。利用α - 1蛋白酶抑制剂的蛇形蛋白支架将蛋白Z结合位点和蛋白酶结合位点的分子决定因子移植到ZPI上的功能研究成果，将为该蛇形蛋白对辅因子和蛋白酶识别的最小决定因子提供严格的测试。

Serpin family protein protease inhibitors function as key anticoagulant regulators of blood coagulation proteases. Two serpins, antithrombin 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 antithrombin and a prothrombotic phenotype when mice are bred on a factor V Leiden background in the case of ZPI. Our proposed studies seek to build on our prior studies to advance molecular understanding of the cofactor-dependent regulation of blood coagulation proteases by these serpins. With respect to antithrombin, 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 mitigation of repulsive interactions with factor Xa and factor IXa within an antithrombin exosite rather than by provision of an attractive exosite. Our proposed continuation studies seek to characterize the molecular determinants in antithrombin of protease binding exosites and of the allosteric communication network involved in transmitting activating conformational changes from the heparin binding site to the protease binding site. With respect to ZPI, our studies seek to elucidate the physiologic complexes of factor Xa that are targeted by ZPI and its cofactor, protein Z, establish the role of the unique N-terminal tail of ZPI in anticoagulant regulation of factor Xa and discover small molecules that disrupt the ZPI-protein Z complex and downregulate the ZPI anticoagulant mechanism as a potential novel therapy for restoring hemostasis in hemophilia bleeding disorders. Gain of function studies in which the serpin scaffold of α1-protease inhibitor is used to graft the molecular determinants of the protein Z binding site and protease binding sites onto ZPI will provide a stringent test of the minimal determinants of cofactor and protease recognition by this serpin.