Factor Vllla interactions in the intrinsic factor Xase

血友病A是最常见的严重遗传性出血性疾病，由第VIII因子缺乏或缺陷引起。第VIII因子的激活形式，第VIIIa因子，作为依赖第IXa因子激活X因子的辅因子，使该反应的kcat增加几个数量级。我们实验室正在进行的研究集中在这种具有临床重要性和生物化学吸引力的蛋白质上。我们建议阐明蛋白质间相互作用的细微结构细节，这些细节将定义催化速率增强和内在因子Xase活性调节的机制。目的研究因子VIIIa(亚基)在与因子IXa相互作用及其活性部位调控中的作用。研究将集中在A2亚基上，这是基于我们的观察，该亚基包含一个扩展的因子IXa相互作用表面，并且分离的A2增强了因子IXa催化的因子X的激活的kCAT。主要工作将集中在实验评估和完善我们的A2因子IXa界面的模型。原辅因子到活性因子Villa的蛋白水解性转换在A2结构域上暴露了一个功能因子IXa相互作用的位点，我们建议确定这个关键区域(S)，从而定义因子VIII“激活”的分子机制。使用天然和突变蛋白质的功能分析将辅之以物理方法，如基于荧光的分析、化学交联、氢/氢交换和质谱分析，以评估参与扩展相互作用表面的重要残基/区域。相关研究将确定因子VIIIa对因子X的因子Xase的Km的贡献的基础，以及新的底物静电转向机制的基础。目的II将研究失活的蛋白酶激活蛋白C(APC)和凝血因子Xa对因子VIIIa的调节，重点研究利用独特的蛋白酶形式的外切体之间的相互作用。这些相互作用导致因子VIIIa失活，从而调节因子Xase的机制仍然知之甚少。我们将使用天然和重组因子VIIIa(以及分离的亚基)来探索这些相互作用，这些亚基在假定的外切体相互作用区以及抗切割形式中具有变化。一个重点是研究A1亚基Arg336的蛋白降解，A1亚基是APC和因子Xa共同切割的一个主要位点，以及该位点的攻击对更多次级位点切割的影响。对这些问题的定义将对天然和功能失调的因子viii分子的生物化学产生有价值的和基本的见解，并为设计更好的治疗方法提供信息。

Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. The activated form of factor VIII, factor VIIIa, functions as a cofactor for the factor IXa-dependent activation of factor X, increasing the kcat for this reaction by several orders of magnitude. On-going research in our laboratory has focused upon this clinically important and biochemically fascinating protein. We propose to elucidate fine point structural details of inter-protein interactions that will define mechanisms for catalytic rate enhancement and the regulation of activity of the intrinsic factor Xase. Aim I will study the role of factor VIIIa (subunits) in the interaction with factor IXa and modulation of its active site. Studies will focus on the A2 subunit based upon our observations that this subunit contains an extended factor IXa-interactive surface and that isolated A2 enhances the kcat for factor IXa-catalyzed activation of factor X. A primary effort will focus on experimentally assessing and refining our model for the A2-factor IXa interface. Proteolytic conversion of procofactor to active factor Villa exposes a functional factor IXa-interactive site on the A2 domain that is otherwise cryptic, and we propose to identify this critical region(s) thereby defining the molecular mechanism for factor VIII "activation." Functional assays using native and mutant proteins will be complemented with physical methods such as fluorescence-based assays, chemical crosslinking, hydrogen/deuterium exchange and mass spectrometry to assess important residues/regions that participate in the extended interactive surface. Related studies will determine the bases for the factor VIIIa-dependent contributions to the Km of factor Xase for factor X and for a novel substrate electrostatic steering mechanism. Aim II will examine regulation of factor VIIIa by the inactivating proteinases activated protein C (APC) and factor Xa, with emphasis on examining interactions of exosites using unique proteinase forms. Mechanisms for these interactions leading to factor VIIIa inactivation, and hence factor Xase regulation remain poorly understood. We will probe these interactions using native and recombinant factor VIIIa (and isolated subunits) possessing alterations in putative exosite-interactive regions as well as cleavage-resistant forms. A focal point is the study of proteolysis at Arg336 in the A1 subunit, a predominant site cleaved by both APC and factor Xa, and the influence of attack at this site on cleavage at more secondary sites. Definition of these issues will yield valuable and fundamental insights into the biochemistry of the native as well as dysfunctional factor VIII molecules, and provide information for the design of superior therapeutics