Structural Correlates of Protease and Cofactor Function

项目主任/首席调查员(最后、第一、中间)：KrJShnaSWamy，Sriram 项目总结(见说明)： 丝氨酸蛋白酶因子Xa(FXA)和辅因子Va(FVA)是 酶复合凝血酶原酶，已知的唯一的凝血酶原的生理激活剂。一个主要因素是 凝血酶原酶通过胞外酶之间的相互作用实现其显著的特异性， 底物和辅因子。这些结合位点中的许多在这些蛋白质的前体上不起作用 它们被构象激活的机制还不是很清楚。此外，还有 目前对这些位点如何进行变构调节以及对什么进行调节的理解仍然存在重大差距 这些相互作用在多大程度上有助于增强酶复合体的功能。宽泛的长期 这项工作的目标是破译这些分子过程，并提供详细的机制洞察 FXA和FVA在凝血酶原酶中发挥作用。在第一个目标中，我们将研究FX酶原的作用 将蛋白水解酶过渡途径中的大分子结合部位表达在催化结构域内。我们 假设在蛋白酶构象激活后FX的分子内激活起到了 在底物、辅因子和抑制剂结合部位的暴露中起主要作用。在第二个目标中，我们将 利用独特的FVA变异体来检查凝血酶原结合，并检查这种相互作用在 凝血酶原酶功能。我们假设FVA重链中的特定酸性序列是 对凝血酶结合很重要，但在凝血酶原识别中不起主要作用。在最终目标上，我们 将使用能被FVA热力学挽救的FXA“类酵素”变种来检查 他们使用血友病的体外和体内模型提供有效的止血。我们假设这些 变异体将对蛋白酶抑制剂产生抗药性，并在循环中存活较长时间，但会促进凝血酶 在FVA存在的情况下生成。我们将测试这些衍生品是否具有治疗作用 血友病动物中的促凝剂旁路制剂。理解分子事件导致的 FXA和FVA上结构决定因素的表达以及这些位点如何相互影响可能 在开发用于调节凝血酶生成的药理制剂方面证明是有用的。 相关性(请参阅说明)： 形成血块的蛋白质和细胞必须保持静止状态，只有激活。 在受伤之后。这项建议旨在更好地了解其中一种蛋白质，因子X是如何转化为 活动表单FXA。这一过程背后的机制与更好地理解和 可能治疗血友病和血栓形成的病理状态。 项目/

Program Director/Principal Investigator (Last, First, Middle): KrJShnaSWamy, Sriram PROJECT SUMMARY(See instructions): The serine proteinase factor Xa (FXa) and the cofactor factor Va (FVa) are essential components of the enzymatic complex prothrombinase, the only known physiological activator of prothrombin. A major factor by which prothrombinase achieves its remarkable specificity is through exosites interactions between enzyme, substrate and cofactor. Many of these binding sites are not functional on the precursor of these proteins and the mechanism by which they are conformationally activated is not well understood. Additionally, there are still major gaps in the current understanding of how these sites are allosterically modulated and to what extent these interactions contribute to the enhanced function of the enzyme complex. The broad long-term objective of this work is to decipher these molecular processes and provide detailed mechanistic insight into FXa and FVa function within prothrombinase. In the first aim, we will investigate the role of the FX zymogen to protease transition pathway in macromolecular binding site expression within the catalytic domain. We hypothesize that intramolecular activation of FX following conformational activation of the protease plays a major role in the exposure of substrate, cofactor, and inhibitor binding sites. In the second aim, we will exploit unique FVa variants to examine prothrombin binding and examine the role this interaction plays in prothrombinase function. We hypothesize that specific acidic sequences within the heavy chain of FVa are important for thrombin binding but do not play a major role in prothrombin recognition. In the final aim, we will use FXa "zymogen-like" variants which can be thermodynamicallyrescued by FVa to examine whether they provide effective hemostasis using in vitro and in vivo models of hemophilia. We hypothesize that these variants will be resistant to protease inhibitors and be long lived in the circulation but promote thrombin generation in the presence of FVa. We will test whether these derivatives will be useful therapeutic procoagulant bypass agents in hemophilic animals. Understanding the molecular events leading to the expression of structural determinants on FXa and FVa as well as how these sites influence each other may prove useful in developing pharmacological agents directed at regulating thrombin generation. RELEVANCE (See instructions): The proteins and cells that form a blood clot must remain in a quiescent state and only become activated following injury. This proposal seeks to better understand how one of these proteins, factor X is converted to the active form FXa. The mechanism underlying this process has relevance for better understanding and possibly treating the pathological states of hemophilia and thrombosis. PROJECT/