Factor VIIIa interactions in the intrinsic factor Xase

因子 VIIIa 在内因子 Xase 中的相互作用

• 批准号: 7459299
• 负责人: PHILIP J. FAY
• 金额: $ 39.66万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-09 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7459299
• 关键词: Active Sites; Address; Anisotropy; Baculovirus Expression System; Binding; Biological Assay; Catalysis; Charge; Cleaved cell; Complement; Complex; Defect; Dependence; Development; Disease; Down-Regulation; Endopeptidases; Energy Transfer; Enzymes; Event; Factor IXa; Factor VIII; Factor VIIIa; Factor X; Factor Xa; Fluorescence; Hemophilia A; Hemorrhage; Inherited; Intrinsic factor; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Mediating; Membrane; Mutation; Peptide Hydrolases; Point Mutation; Proteins; Proteolysis; Public Health; Rate; Reaction; Reagent; Recombinants; Regulation; Relative (related person); Resistance; Resolution; Role; Serine Protease; Site; Surface Plasmon Resonance; Techniques; Therapeutic; Variant; activated Protein C; base; cancer procoagulant; cofactor; design; insight; interest; mutant; novel

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.

描述（由申请人提供）：血友病A是最常见的严重遗传性出血性疾病，由因子VIII缺乏或缺陷引起。我们建议阐明蛋白质间相互作用的精细点结构细节，这些细节将定义因子IXA依赖于因子VIII a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。目的研究凝血因子VIIIa调节凝血因子Xase催化效率的机制。我们继续研究因子VIIIa A2亚基的作用，因为该亚基直接影响因子IXa活性位点的事件。使用新型重组A2试剂将有助于鉴定关键因子IXa相互作用残基。这些研究的一个新的重点是研究因子VIIIa A3 C1 C2亚基在因子Xase形成中的作用。这一目的是重要的，因为该亚基提供了因子VIIIa与因子IXa相互作用的大部分结合能。我们开发的杆状病毒表达系统产生全功能A3 C1 C2亚基将有助于解决这一目标。因子VIIIa增强Xase中的底物因子X结合，我们将研究A3 C1 C2亚基对因子VIIIa A1亚基中因子X相互作用位点的稳定性。将使用重组试剂评价A2亚基中正电荷电位在促进底物转换和产物释放中的作用。目的II的重点是因子Ⅷ a的蛋白水解失活，是基于我们最近的研究表明外切位点依赖的辅因子失活的活化蛋白C（APC）和因子Xa，以及一个重要的贡献侧翼序列的P1精氨酸残基在APC催化失活因子Ⅷ a。导致这些负责因子VIIIa失活的酶的反应和随后的因子Xase抑制的分子相互作用仍然知之甚少。我们将探测这些相互作用，使用本机和突变体因子VIIIa的变体拥有在假定的外来位点相互作用的区域，以及与抗裂解形式的改变。一个焦点是A1亚基Arg 336处的蛋白水解，这是APC和Xa因子裂解的主要位点，并阐明了这种催化事件的机制。这些目的将通过使用新型试剂来促进，所述新型试剂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质谱完成这些研究。我们预计我们的结果将定义机制，为辅因子介导的催化及其调控提供重要见解，并为设计治疗血友病A的上级疗法提供有用信息。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，由因子VIII缺乏或缺陷引起。在这个应用程序中，我们将阐明蛋白质间相互作用的精细点结构的细节，这将定义因子VIII a辅因子的功能和调节其在因子Xase的活性的机制。