The initiation of coagulation via the extrinsic pathway results in the activation of factors X and IX. The enzyme complex that catalyses these reactions (extrinsic Xase) is composed of the serine protease factor VIIa, and the integral membrane protein, tissue factor (TF) that associate tightly on a cellular or phospholipid surface in the presence of calcium ions. Thus, exposure of TF upon vascular damage or the action of stimulants on the appropriate cells leads to the initiation of coagulation. The approaches outlined in this proposal involve the use of purified proteins and synthetic phospholipid vesicles to examine selected aspects of extrinsic Xase function reconstituted under defined conditions. Kinetic studies of factor X activation are proposed to investigate the contributions of the interaction of factor X with the membrane surface to the catalytic cycle.
The aim will be accomplished by the application of the fluorescent probe p-aminobenzamidine in continuous measurements of factor Xa generation in steady state and stopped-flow measurements. These studies will evaluate the hypothesis that the delivery of factor X to the assembled Xase complex occurs via an initial interaction with available sites on the membrane surface. In collaborative studies, we will also prepare and evaluate specific, fluorescent transition state inhibitors of factor Xa. The successful development of these inhibitors will provide additional tools for the dissection of factor X activation with the complicating effects of side reactions catalyzed by factor Xa. Studies are also proposed to examine the molecular mechanisms of the activation of factor VII by the action of factor Xa. The dissection of this activation reaction is feasible by quenched-flow techniques and can provide information that is relevant of an understanding of the initiation of factor X activation. The equilibrium and dynamic aspects of Xase assembly will be examined using fluorescent peptidyl chloromethyl ketones and by resonance energy transfer between appropriately modified VIIa and TF. These studies will provide insight into the thermodynamics and the kinetics of Xase assembly. Collectively, these studies will provide a kinetic and thermodynamic foundation for the molecular details of Xase function which is relevant to an understanding of the initiation of coagulation and thrombotic complications in atherosclerotic vascular disease.
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