FACTOR VIII INTERACTIONS IN INTRINSIC XASE

点击翻译按钮获取中文摘要

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.