Proteinase Allostery and the Regulation of Blood Coagulation

描述（申请人提供）：凝血酶是血液凝血级联的末端丝氨酸蛋白酶，在血栓形成和调节中起关键作用。凝血酶在凝血过程中的多重作用，有时是相反的作用，受到酶的两个相对表面的外源位点（ABE1和ABE2）和辅因子或配体的影响，辅因子或配体结合到这些位点或其他位点以变构调节蛋白酶。在凝血酶变构是如何实现并转化为调节功能的机制理解方面，主要的差距和不一致仍然存在。我们以意想不到的发现提出了这个问题，即凝血酶可以根据与酶结合的配体的补体容易地和可逆地沿着酶原样和蛋白酶样状态的连续体相互转化。我们认为这些相互转换是凝血酶变构的核心。我们的研究集中在发现片段1.2 (F12)， ABE2的真实蛋白质配体，在热力学上倾向于酶原样形式。我们现在将采用滴定量热法来建立与ABE1结合的血栓调节蛋白（TM）选择性稳定并倾向于蛋白酶样形式以对抗F12的热力学基础。我们还发现在凝血酶形成过程中作为中间体产生的减数凝血酶（mIIa），由于F12和蛋白酶结构域之间的共价连接，特别具有酶原样。我们将采用一种停止流动的动力学方法来检查mIIa在酶原样和蛋白酶样形式之间的分布。虽然F12内的F2区域预计会与ABE2接触，但我们现在提出了一种新的功能，用于推测远处F1的Ca2+稳定结构，以增强F12有利于酶原样形式的能力。在这些机制研究的基础上，我们将研究不同配体结合ABE1和F12结合ABE2的相反作用对凝血酶功能的调节，以解释mIIa和各种抗凝血凝血酶在特异性上的明显差异。我们假设它们改变的特异性在于它们显著的酶原样特征，这种特征可以通过配体和底物的结合而可变地获救。这些概念也为新型适体探针的开发提供了合适的框架，这种探针可以调节凝血酶在酶原样状态和蛋白酶样状态之间的分布，从而调节其具有治疗潜力的特异性。最后，基于我们最近成功进入结构生物学领域，我们提出了解决一系列新结构的长期目标，以建立F12酶化功能的结构基础。我们的策略为凝血酶变构的重要问题带来了新鲜和统一的概念。我们期望我们的发现能够揭示在正常止血和疾病状态下调节凝血酶功能的机制。我们的发现有可能揭示这种酶在血栓性和血管疾病中的靶向治疗的新策略。

DESCRIPTION (provided by applicant): Thrombin, the terminal serine proteinase of the blood coagulation cascade, plays a pivotal role in thrombus formation as well as its regulation. These multiple and sometimes opposing roles of thrombin in coagulation are influenced by two exosites (ABE1 and ABE2) on opposite faces of the enzyme and cofactors or ligands that bind to these or other sites to allosterically regulate the proteinase. Major gaps and inconsistencies remain in the mechanistic understanding of how thrombin allostery is achieved and translates into regulated function. We broach the problem with the unexpected finding that thrombin can readily and reversibly interconvert along a continuum of zymogen-like and proteinase-like states depending on the complement of ligands bound to the enzyme. We propose that these interconversions lie at the heart of thrombin allostery. Our studies center on the finding that fragment 1.2 (F12), the authentic protein ligand for ABE2, thermodynamically favors zymogen- like forms. We will now employ titration calorimetry to establish the thermodynamic basis by which thrombomodulin (TM), which binds to ABE1, selectively stabilizes and favors proteinase-like forms to oppose the effects of F12. We also find that meizothrombin (mIIa), produced as an intermediate during thrombin formation, is particularly zymogen-like because of covalent linkage between F12 and the proteinase domain. We will employ a stopped-flow kinetic approach to examine the distribution of mIIa between zymogen-like and proteinase-like forms. Although the F2 region within F12 is expected to contact ABE2, we now propose a novel function for the Ca2+ stabilized structure of the putatively distant F1 in enforcing the ability of F12 to favor zymogen-like forms. Based on these mechanistic studies, we will study the regulation of thrombin function by opposing effects of ABE1 binding by different ligands and F12 binding to ABE2, with an eye to explaining the apparent differences in specificity ascribed to mIIa and various anticoagulant thrombins. We hypothesize that their altered specificity lies in their significantly zymogen-like character that can be variably rescued by the binding of ligands and substrates. These concepts also provide the appropriate framework for the development of novel aptamer probes that can modulate the distribution of thrombin between zymogen-like and proteinase- like states and thereby regulate its specificity with therapeutic potential. Finally, based on our recent successful entry into the structural biology arena, we propose the long term goal of solving a series of novel structures to establish the structural basis for the zymogenizing function of F12. Our strategies bring fresh and unifying concepts to the important problem of thrombin allostery. We anticipate our findings to shed new light on the mechanisms at play in regulating thrombin function in normal hemostasis and in disease states. Our findings have the potential to reveal new strategies for therapeutic targeting of this enzyme in thrombotic and vascular disease.