Proteinase Allostery and the Regulation of Blood Coagulation

描述（由申请方提供）：凝血酶是凝血级联反应的末端丝氨酸蛋白酶，在血栓形成及其调节中起关键作用。凝血酶在凝血中的这些多重且有时相反的作用受到酶的相对面上的两个外部位点（ABE1和ABE2）以及与这些或其他位点结合以变构调节蛋白酶的辅因子或配体的影响。主要的差距和不一致性仍然存在于凝血酶变构如何实现和转化为调节功能的机制理解。我们提出了这个问题，意想不到的发现，凝血酶可以容易地和可逆地相互转换沿着一个连续的酶原样和蛋白酶样状态取决于互补的配体结合到酶。我们认为，这些相互转换的核心在于凝血酶变构。我们的研究集中在发现片段1.2（F12），ABE 2的真实蛋白配体，在生物学上有利于酶原样形式。我们现在将采用滴定量热法来建立热力学基础，通过该热力学基础，与ABE 1结合的血栓调节蛋白（TM）选择性地稳定并有利于蛋白酶样形式以对抗F12的作用。我们还发现，meizothrombin（mIIa），作为凝血酶形成过程中的中间体，特别是酶原样，因为F12和蛋白酶结构域之间的共价键。我们将采用停流动力学的方法来研究mIIa酶原样和蛋白酶样形式之间的分布。虽然F12内的F2区预计接触ABE2，我们现在提出了一个新的功能，钙离子稳定的结构的puperminate遥远的F1在加强F12的能力，有利于酶原样形式。基于这些机制的研究，我们将研究凝血酶功能的调节ABE1结合不同的配体和F12结合ABE2的相反效果，着眼于解释归因于mIIa和各种抗凝凝血酶的特异性的明显差异。我们推测，它们改变的特异性在于它们显著的酶原样特征，可以通过配体和底物的结合而被拯救。这些概念还为开发新型适体探针提供了适当的框架，所述适体探针可以调节凝血酶在酶原样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竞技场，我们提出了解决一系列新结构的长期目标，以建立F12的酶促功能的结构基础。我们的策略为凝血酶变构的重要问题带来了新的统一概念。我们期望我们的研究结果能为在正常止血和疾病状态下调节凝血酶功能的机制提供新的线索。我们的研究结果有可能揭示这种酶在血栓和血管疾病中的治疗靶向的新策略。 公共卫生相关性：凝血酶是血液中产生的关键酶，可以加速和减缓血凝块的形成。我们的研究带来了新的概念和方法来解决凝血酶如何参与多种反应并产生相反的后果。我们的研究结果将揭示选择性干扰凝血酶不同功能的新策略，用于治疗许多人类疾病中危及生命的血栓。

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. PUBLIC HEALTH RELEVANCE: Thrombin is the pivotal enzyme produced in blood that both accelerates and slows down the formation of the blood clot. Our research brings new concepts and methodologies to bear on solving how thrombin participates in multiple reactions with opposing consequences. Our findings will reveal novel strategies for selectively interfering with the different functions of thrombin for the treatment of life-threatening blood clots in a number of human diseases.