Factor Vllla interactions in the intrinsic factor Xase

A型血友病是最常见的严重遗传性出血性疾病，是由于因子VIII缺乏或缺陷造成的。因子VIII的活化形式，因子VIIIa，作为因子ixa依赖的因子X活化的辅助因子，将该反应的kcat增加了几个数量级。我们实验室正在进行的研究主要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精细点结构细节，这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iiia（亚基）在与因子IXa相互作用及其活性位点调控中的作用。基于我们的观察，研究将集中在A2亚基上，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催化因子x活化的kcat。主要的工作将集中在实验评估和完善我们的A2-因子IXa界面模型。原因子向活性因子Villa的蛋白水解转化暴露了A2结构域上的一个功能因子ixa相互作用位点，否则该位点是隐藏的，我们建议确定这个关键区域，从而定义因子VIII“激活”的分子机制。使用天然和突变蛋白的功能分析将与基于荧光的分析、化学交联、氢/氘交换和质谱等物理方法相辅相成，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viia对因子Xase对因子X的Km的依赖贡献的基础，以及一种新的衬底静电转向机制。Aim II将通过失活蛋白酶活化蛋白C （APC）和因子Xa来检查因子viii ia的调节，重点是使用独特的蛋白酶形式检查外来物的相互作用。这些相互作用导致因子viia失活和因子Xase调控的机制仍然知之甚少。我们将利用在假定的外源相互作用区域以及抗切割形式中具有改变的天然和重组因子viii（和分离亚基）来探测这些相互作用。一个重点是研究A1亚基Arg336的蛋白水解，以及该位点的攻击对更多次级位点裂解的影响。A1亚基是APC和Xa因子均可切割的主要位点。这些问题的定义将产生对天然因子VIII分子和功能失调因子VIII分子的生物化学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