FACTOR VIII INTERACTIONS IN INTRINSIC XASE

血友病A，最常见的严重遗传性出血 由于第VIII因子缺陷所致的紊乱。 激活形式的第VIII因子，即第VIIIa因子，起辅助因子的作用 对于依赖因子IVa的因子X的激活，增加kcat 对这一反应有几个数量级的影响。我们建议 阐明蛋白质间相互作用的精细结构保留 这将定义调节这一关键等离子体的机制 蛋白质和内在因子Xase的活性，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之间的相互作用。我们的目标 是定义有助于提高速率的机制，这是 负责一个新的互动过程，从而实现互惠 辅因子和酶活性的调节。为此，我们将(一) 重组因子VIII和A2亚基的构建及分析 推测因子IXa相互作用表面环中的点突变，(II) 量化绑定参数绑定参数，(Iii)评估 因子IVa对因子VIIIa亚基稳定性的贡献，(Iv) 确定本征因子Xase内的几何形状，(V)评估 C-末端酸性区域在因子IVA催化的酶失活中的作用 辅因子和(Vi)通过裂解决定因子Xase的稳定性。 抗性因子VIII和一个新的因子IXa分子。第二个目标 研究一种新发现的凝血因子VIII和 底物因子X。我们的目标是描述这种相互作用和 确定其功能意义。建议的研究将会(I) 鉴定因子VIIIa中的相互作用区并定量结合 参数和(Ii)确定结果 Xase因子产物生成与调控的相互作用 活动。最终目标是研究活性蛋白C(APC)催化的 VIIIa因子失活。我们的目标是定义机制 有助于底物的选择性和Xase因子的衰变 内皮细胞(EC)表面。研究将(I)量化 APC和因子IXa对因子VIIIa中共同键的攻击，(Ii) 评估因子VIIIa仅由裂解引起的失活 A2位点和(Iii)评估S蛋白在确定 站点选择性。实验将表征因子VIIIa依赖 APC对生理表面EC上Xase的灭活作用 这条途径可能代表了因子的主导模式 XASE减振。对这些问题的定义将产生有价值的见解 进入天然的生物化学以及功能失调的第VIII因子 分子，并为上级设计提供信息 治疗学。

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.