FACTOR VIII INTERACTIONS IN INTRINSIC XASE

内在 XASE 中因子 VIII 的相互作用

• 批准号: 6302186
• 负责人: PHILIP J. FAY
• 金额: $ 25.58万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6302186
• 关键词: binding sites; blood coagulation; cell line; coagulation factor IX; coagulation factor VIII; coagulation factor X; enzyme activity; fluorescent dye /probe; protein C; protein protein interaction; protein structure function; proteolysis; prothrombin; site directed mutagenesis; thrombin; transfection

Hemophilia A, the most common of the severe, inherited bleeding disorder, resulting from a deficiency of defect in factor VIII. The activated form of factor VIII, factor VIIIa, functions as a co-factor for the factor IVa-dependent activation of factor X, increasing the kcat for this reaction by several orders of magnitude. We propose to elucidate fine point structural detains of inter-protein interactions that will define mechanisms for the regulation of this critical plasma protein and activity of the intrinsic factor Xase, applying physical and biochemical approaches and utilizing molecular biological methods. The first aim will study interactions of factors VIIIa and IXa. Our goal is to define mechanisms that contribute to rate enhancement and that are responsible for a novel interactive process resulting in reciprocal regulation of cofactor and enzyme activities. To this end we will (i) construct and analyze recombinant factor VIII and A2 subunit containing point mutations in a putative factor IXa-interactive surface loop, (ii) quantitate binding parameters binding parameters, (iii) assess contribution of factor IVa to factor VIIIa subunit stability, (iv) determine geometry within the intrinsic factor Xase, (v) evaluate the role of C-terminal acidic region in factor IVa-catalyzed inactivation of the co-factor and (vi) determine factor Xase stability using a cleavage- resistant factor VIII and a novel factor IXa molecule. The second aim studies a newly identified interactions between factor VIII and substrate factor X. Our goal is to characterize this interaction and determine its functional significance. Proposed studies will (i) identify the interactive region in factor VIIIa and quantitate binding parameters and (ii) determine the consequence the consequence of this interaction in terms of product generation and regulation of factor Xase activity. A final aim will study activated protein C (APC) catalyzed inactivation of factor VIIIa. Our goal is to define mechanisms contributing to substrate site selectivity and decay of factor Xase on the endothelial cell (EC) surface. Studies will (i) quantitate rates of attack by APC and factor IXa at a common bond in factor VIIIa, (ii) evaluate factor VIIIa inactivation resulting solely from cleavage at the A2 site and (iii) assess the basis for the role of protein S in defining site selectivity. Experiments will characterize factor VIIIa-dependent inactivation of factor Xase by APC on the EC, a physiologic surface where this pathway potentially represents the dominant mode for factor Xase damping. Definition of these issues will yield valuable insights into the biochemistry of the native as well as dysfunctional factor VIII molecules, and provide information for the design of superior therapeutics.

血友病A是最常见的严重遗传性出血 一种疾病，由于缺乏缺陷因子VIII。的 因子VIII的活化形式，因子VIIIa，作为辅因子发挥作用 对于因子X的因子IVa依赖性活化，增加kcat 对这个反应的影响要大上几个数量级。我们建议 阐明蛋白质间相互作用的精细点结构 它将定义这种关键等离子体的调节机制， 蛋白质和活性的内在因子Xase，应用物理和 生物化学方法和利用分子生物学方法。 第一个目标是研究因子VIIIa和IXa的相互作用。我们的目标 是定义有助于提高速率的机制， 负责一个新的互动过程， 辅因子和酶活性的调节。为此，我们将（一） 构建并分析了含有因子VIII和A2亚基的重组体 推定的因子IXa相互作用表面环中的点突变，（ii） 定量结合参数结合参数，（iii）评估 因子IVa对因子VIIIa亚单位稳定性的贡献，（iv） 确定内在因子Xase内的几何结构，（v）评估 C-末端酸性区在因子IVa催化失活中的作用 辅因子和（vi）使用裂解酶确定因子X酶稳定性。 耐药因子VIII和新型因子IXA分子。第二个目的 研究了一种新发现的因子VIII和 底物因子X。我们的目标是描述这种相互作用， 确定其功能意义。拟议的研究将（i） 鉴定因子VIIIa中的相互作用区域并定量结合 参数和（ii）确定的后果，这一后果 产物生成和因子Xase调节方面的相互作用 活动最后一个目标是研究活化蛋白C（APC）催化的 因子VIIIa失活。我们的目标是定义机制 有助于底物位点的选择性和Xase因子的衰减， 内皮细胞（EC）表面。研究将（i）定量 APC和因子IXa攻击因子VIIIa中的共同键，（ii） 评价仅由在以下位点的切割引起的因子VIIIa失活： A2位点和（iii）评估蛋白S在定义 位点选择性实验将表征VIII因子依赖性 APC在EC（一种生理表面）上灭活因子Xase 其中该途径可能代表因子的主导模式 Xase阻尼。对这些问题的界定将产生有价值的见解 研究了先天性和功能失调的凝血因子VIII的生物化学， 分子，并为设计上级 治疗学