In the United States, cardiovascular disease results in one death every 30 seconds. Clinical disorders such as myocardial infarction, deep vein thrombosis and pulmonary embolism, and stroke are usually precipitated by thrombotic events. Although basic research in thrombosis has lead to significant advances in the diagnosis and treatment of thrombotic disorders current approaches remain sub optimal. Generation of thrombin by the prothrombinase complex plays a particularly important role in the pathogenesis venous thrombosis. The prothrombinase complex consists of the enzyme factor Xa, the cofactor factor Va and a phospholipid membrane surface. The interaction of factor Xa with the factor Va requires cofactor activation for expression of factor Xa binding sites. The interaction of factor Va with platelet membranes requires expression of phosphatidylserine on the surface of activated platelets or endothelial cells. The binding sites for factor Xa and phospholipid membranes are discontinuous and are located in several different domains. The complexity of these binding sites may allow for the fine regulation of the prothrombinase complex. The molecular bases for these interactions remain poorly understood. 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. During the previous funding period the factor C2 domain was expressed using insect cells and the structures of two crystal forms were elucidated. Expression of factor Va mutants in mammalian cells demonstrated that glycosylation of the C2 domain modulates membrane binding and that two tryptophans located in a mobile solvent exposed loop play a critical role in high affinity binding of factor V to phospholipid membranes containing low concentrations of phosphatidylserine.
The specific aims of the present proposal are to further define the binding sites in the factor Va light chain for phospholipid membranes and cellular membranes. Binding sites will be localized using recombinant factor Va mutants, recombinant light chain domains, domain specific and monoclonal antibodies. Experiments will be designed using available crystal structures or molecular models for individual domains. Binding interactions will be characterized using surface plasmon resonance and fluorescence binding assays. This information will provide important new insights into regulation of the prothrombinase complex and may identify sites that could be exploited as novel targets for anti-thrombotic therapy.
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