Thrombin generated by the prothrombinase complex contributes significantly to the pathogenesis of common clinical disorders such as myocardial infarction, deep vein thrombosis, pulmonary embolism, and stroke. The prothrombinase complex consists of the enzyme factor Xa, the cofactor factor Va and a phospholipid membrane surface. The interaction of factor Va with platelet membranes requires expression of phosphatidylserine (PS) on the surface of activated platelets or endothelial cells. The long-term goal of this project is to use integrated molecular, structural and biophysical approaches to understand the interaction of factor Va with biological membranes. A membrane binding site was localized to the factor V C2 domain and the structures of two crystal forms of this domain have been elucidated. These structures have suggested a working model for the interaction of factor Va with phospholipid membranes that includes:(1) immersion of exposed hydrophobic residues in the apolar membrane core; (2) stereospecific interactions with PS head groups and (3) favorable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups. Several aspects of this model have been validated. The indole side chains of two tryptophans located on a mobile solvent exposed loop within the C2 domain insert into the membrane bilayer and are required for high affinity membrane binding. Tyrosine and leucine residues located in an analogous location within the homologous factor V C1 domain play a role in binding to membranes containing low concentrations of PS. The goals of the present study are to validate and refine our working model for the association of factor Va with biological membranes.
The specific aims of the present proposal are to define the PS specificity pocket(s) and membrane binding sites within the factor V C1 and C2 domains and to determine their contribution to prothrombinase assembly on both phospholipid vesicles and cellular membranes. Binding sites will be defined using recombinant factor Va mutants, recombinant light chain domains, scFv antibodies and soluble phospholipid analogues. Binding interactions will be characterized using quantitative fluorescence binding assays and functional prothrombinase assays. We hypothesize that complex interactions between factor Va and PS containing membranes allow for the fine regulation of the prothrombinase complex. The proposed experiments could lead to novel targets for anti-thrombotic therapy. ? ? ?
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