Factor VIIIa interactions in the intrinsic factor Xase

描述(申请人提供)：血友病A是最常见的严重遗传性出血性疾病，由因子缺乏或缺陷引起。我们建议阐明蛋白质间相互作用的细微结构细节，这些细节将确定因子VIIIa依赖的催化速率增强因子IXa及其活性下调导致固有因子Xase抑制的机制。目的研究凝血因子VIIIa对凝血因子Xase催化效率的调节机制。我们继续研究因子VIIIaA2亚基的作用，因为这个亚基直接影响因子IXa活性部位的事件。新型重组A2试剂的使用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了因子VIIIa A3C1C2亚单位在因子Xase形成中的作用。这一目的是重要的，因为这个亚基为因子VIIIa与因子IXa的相互作用提供了大部分结合能。我们开发的杆状病毒表达系统产生了功能完全的A3C1C2亚基，这将有助于实现这一目标。凝血因子VIIIa增强Xase中底物凝血因子X的结合，我们将研究A3C1C2亚基对凝血因子VIIIa A1亚基中凝血因子X相互作用位点的稳定性。A2亚基中的正电荷势在底物周转和产物释放中的作用将使用重组试剂进行评估。目的II主要研究因子VIIIa的蛋白水解性失活，基于我们最近的研究，表明激活蛋白C(APC)和因子Xa对辅因子失活具有外源依赖性，以及P1 Arg残基两侧的序列在APC催化的因子VIIIa失活中的重要贡献。导致因子VIIIa失活和随后抑制因子Xase的这些酶的反应的分子相互作用仍然知之甚少。我们将使用天然和突变因子VIIIa变异体来探索这些相互作用，这些变异体在假定的胞外相互作用区以及抗切割形式中具有变化。一个焦点是A1亚基Arg336的蛋白降解，这是APC和因子Xa共同切割的主要位置，并阐明了这一催化事件的机制。这些目的将通过使用新的试剂来促进，包括重组蛋白酶形式，由高度纯化的因子VIII(A)链/亚单位组成的底物，以及在感兴趣的残留处具有点突变的变体。我们将使用高分辨率技术，包括荧光能量转移和各向异性，表面等离子体共振和MALDI-TOF质谱学来完成这些研究。我们期望我们的结果将为辅因子介导的催化及其调控提供重要的见解，并为设计治疗血友病A的高级疗法提供有用的信息。公共卫生相关性：血友病A是最常见的严重遗传性出血疾病，由凝血因子VIII的缺陷或缺陷引起。在这一应用中，我们将阐明蛋白质间相互作用的细微结构细节，这些细节将定义因子VIIIa辅因子的功能及其在凝血因子Xase中活性的调节机制。

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.