Molecular Basis of Blood Coagulation Regulation

Serpin家族蛋白质蛋白酶抑制剂是凝血蛋白酶的关键抗凝调节剂。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重要性由以下观察结果证实：在抗凝血酶的情况下，敲除小鼠基因导致消耗性凝血病，而在ZPI的情况下，当小鼠在因子V莱顿背景下繁殖时，敲除小鼠基因导致血栓形成前表型。我们提出的研究旨在建立在我们以前的研究，以推进这些丝氨酸蛋白酶抑制剂的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，激活主要是介导的抗凝血酶外位点内，而不是通过提供一个有吸引力的外位点与因子Xa和因子IXa的排斥性相互作用的缓解。我们提出的继续研究的目的是表征抗凝血酶的蛋白酶结合exosites和变构通信网络的分子决定簇参与传输激活的构象变化从肝素结合位点的蛋白酶结合位点。关于ZPI，我们的研究旨在阐明ZPI及其辅因子蛋白Z靶向的因子Xa的生理复合物，建立ZPI独特的N-末端尾在因子Xa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α1-蛋白酶抑制剂的丝氨酸蛋白酶抑制剂支架将蛋白Z结合位点和蛋白酶结合位点的分子决定簇移植到ZPI上的功能研究的获得将提供该丝氨酸蛋白酶抑制剂对辅因子和蛋白酶识别的最小决定簇的严格测试。

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.