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，由因子VIII缺乏或缺陷引起。因子VIII的活化形式，即因子VIIIa，作为因子IX a依赖性活化因子X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主要集中在这种临床上重要且生物化学上迷人的蛋白质上。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。目的研究凝血因子VIIIa（亚基）在与凝血因子IXa相互作用中的作用及其活性位点的调节。研究将集中在A2亚基的基础上，我们的观察，该亚基包含一个扩展的因子IXa相互作用的表面，并隔离A2增强因子IXa催化的因子X的活化的kcat。一个主要的努力将集中在实验评估和完善我们的模型A2因子IXa接口。前辅因子向活性因子VIIIa的蛋白水解转化暴露了A2结构域上的功能性因子IXa相互作用位点，否则该位点是隐蔽的，我们建议鉴定该关键区域，从而确定因子VIII“活化的分子机制。“使用天然和突变蛋白质的功能测定将与物理方法（如基于荧光的测定、化学交联、氢/氘交换和质谱法）相补充，以评估参与扩展的相互作用表面的重要残基/区域。相关的研究将确定的基础因子VIII依赖的贡献Km的因子Xase的因子X和一种新的基板静电转向机制。目的II将检查调节因子Ⅷ a的失活蛋白酶活化蛋白C（APC）和因子Xa，重点检查使用独特的蛋白酶形式的相互作用的exosites。这些相互作用导致因子VIIIa失活的机制，因此因子Xase调节仍然知之甚少。我们将使用天然和重组因子VIIIa（和分离的亚基）探测这些相互作用，这些因子VIIIa（和分离的亚基）在假定的外切位点相互作用区域以及抗切割形式中具有改变。一个重点是在A1亚基，一个主要的网站切割APC和因子Xa，在这个网站上的攻击切割在更多的次要网站的影响Arg 336蛋白水解的研究。这些问题的定义将产生有价值的和基本的见解的生物化学的天然以及功能失调的因子VIII分子，并提供信息的设计上级治疗

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