Vascular injury occurring in response to hemostatic and thrombotic events is a major cause of morbidity and mortality. The initiation of these reactions may involve both soluble and cellular components of the vasculature. Indeed, cellular disruption resulting from trauma and cellular stimulation from the introduction of bacterial products such as endotoxin can initiate the expression of tissue factor, the obligatory cofactor for Factor VII activity, which can contribute to the overall reactions of thrombosis and the Shwartzman reaction leading to intravascular coagulation, respectively. The purpose of this proposal is to investigate the detailed molecular interactions of Factor VII with its cofactor and substrates which should provide a basic understanding of the initiation and regulation of the coagulation system by defining the loci on Factor VII which mediate these molecular associations. Identification of specific regions on Factor VII will primarily involve an immunochemical study using previously generated monoclonal antibodies directed to native Factor VII; some of which neutralize coagulant activity. The possible mechanisms for this neutralization will be investigated. Another aspect of this application will be to identify the specific peptides of Factor VII which are recognized by the monoclonal antibodies. Peptides will be generated from chemical and enzymatic cleavage of Factor VII or the chains of Factor VIIa and bound to immobilized antibodies. The bound peptides will be eluted, identified by amino acid sequence analysis and synthetic peptides made. These epitopes and potential association sites will be investigated in binding and inhibition studies using monoclonal antibodies and the synthetic reagents. New hybridoma antibodies will be generated to novel epitopes to further define the regions on Factor VII mediating these interactions. The second area in the proposal focuses on the isolation and characterization of specific molecular variants of human Factor VII. These studies will include an immunochemical analysis of the epitopes expressed on these abnormal molecules. The binding of normal and variant Factor VII to tissue factors of different species origin will be studied under conditions of limiting cofactor and correlated with biological activity. The ability of the variants to cleave and activate Factors IX and X under these conditions, and to be cleared by Factor Xa will be investigated. Finally, differences in the primary structure will be addressed using peptide mapping and amino acid sequencing approaches.
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