Factor VIIIa interactions in the intrinsic factor Xase

描述（由申请人提供）：血友病A，最常见的严重遗传性出血性疾病，由因子VIII的缺乏或缺陷引起。我们建议阐明蛋白间相互作用的精细点结构细节，这将定义因子viia依赖性因子IXa的催化速率增强及其活性下调导致内在因子Xase抑制的机制。目的1研究因子viia调节因子Xase催化效率的机制。我们继续研究因子IXa A2亚基的作用，因为该亚基直接影响因子IXa活性部位的事件。使用新的重组A2试剂将有助于鉴定关键因子ixa相互作用残基。这些研究的一个新焦点现在检查因子viii A3C1C2亚基在因子Xase形成中的作用。这个目标很重要，因为这个亚基为因子viia与因子IXa的相互作用提供了大部分结合能。我们开发的具有全功能A3C1C2亚基的杆状病毒表达系统将有助于解决这一问题。因子viia增强了底物因子X在Xase中的结合，我们将研究因子viia A1亚基中因子X相互作用位点被A3C1C2亚基所稳定。A2亚基中正电荷电位在促进底物周转和产物释放中的作用将使用重组试剂进行评估。Aim II关注的是VIIIa因子的蛋白水解失活，并基于我们最近的研究表明，活化蛋白C （APC）和因子Xa对辅因子失活的外源依赖性，以及APC催化VIIIa因子失活中P1精氨酸残基侧翼序列的重要贡献。导致这些酶的反应的分子相互作用负责因子viia失活和随后的因子Xase抑制，但人们对这些酶的反应知之甚少。我们将使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原生和突变因子viii - ia变体来探索这些相互作用。重点是A1亚基Arg336的蛋白水解，这是APC和Xa因子共同切割的主要位点，并阐明了这一催化事件的机制。这些目标将通过使用新的试剂来实现，包括重组蛋白酶形式，由高度纯化的因子VIII(a)链/亚基组成的底物，以及在感兴趣的残基上具有点突变的变体。我们将使用高分辨率技术完成这些研究，包括荧光能量转移和各向异性，表面等离子体共振和MALDI-TOF质谱。我们预计我们的研究结果将定义机制，为辅助因子介导的催化及其调控提供重要的见解，并为设计更好的血友病A治疗方法提供有用的信息。公共卫生相关性：血友病A是最常见的严重遗传性出血性疾病，是由因子VIII的缺乏或缺陷引起的。在本应用中，我们将阐明蛋白间相互作用的精细点结构细节，这些相互作用将定义因子viia辅助因子功能及其在因子Xase中的活性调节机制。

DESCRIPTION (provided by applicant): Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. We propose to elucidate fine point structural details of inter-protein interactions that will define mechanisms for factor VIIIa-dependent catalytic rate enhancement of factor IXa and the down- regulation of its activity leading to dampening of the intrinsic factor Xase. Aim I studies mechanisms by which factor VIIIa modulates catalytic efficiency within factor Xase. We continue to study the role of the factor VIIIa A2 subunit as this subunit directly influences events at the active site of factor IXa. Use of novel recombinant A2 reagents will facilitate identification of critical factor IXa-interactive residues. A new focus of these studies now examines the role of the factor VIIIa A3C1C2 subunit in factor Xase formation. This aim is significant since this subunit provides the majority of the binding energy for the interaction of factor VIIIa with factor IXa. Our development of a baculovirus expression system yielding fully functional A3C1C2 subunit will be instrumental in addressing this aim. Factor VIIIa enhances substrate factor X binding in Xase and we will investigate the stabilization of a factor X-interactive site in the factor VIIIa A1 subunit by the A3C1C2 subunit. The role of positive charge potential in the A2 subunit in contributing to substrate turnover and product release will be evaluated using recombinant reagents. Aim II focuses on proteolytic inactivation of factor VIIIa and is based upon our recent studies showing exosite-dependence for cofactor inactivation by activated protein C (APC) and factor Xa, as well as an important contribution of sequences flanking the P1 Arg residues in APC-catalyzed inactivation of factor VIIIa. The molecular interactions leading to reactions of these enzymes responsible for factor VIIIa inactivation and consequent dampening of factor Xase remain poorly understood. We will probe these interactions using native and mutant factor VIIIa variants possessing alterations in putative exosite-interactive regions as well as with cleavage-resistant forms. A focal point is proteolysis at Arg336 in the A1 subunit, the predominant site cleaved by both APC and factor Xa, and elucidation of mechanisms for this catalytic event. These aims will be facilitated by use of novel reagents including recombinant protease forms, substrate comprised of highly purified factor VIII(a) chains/subunits, as well as variants possessing point mutations at residues of interest. We will accomplish these studies using high resolution techniques including fluorescence energy transfer and anisotropy, surface plasmon resonance and MALDI-TOF mass spectrometry. We anticipate our results will define mechanisms providing significant insights into cofactor-mediated catalysis and its regulation, as well as provide useful information for the design of superior therapeutics for the treatment of hemophilia A. PUBLIC HEALTH RELEVANCE: Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. In this application we will elucidate fine point structural details of inter-protein interactions that will define mechanisms for factor VIIIa cofactor function and the regulation of its activity in factor Xase.