Dr. Darrel W. Stafford requests funding for a five year period. The title of the project is Co-factor & Receptor Interaction of Factors IX, X and VII. Goals of the project include definition of the portion of coagulation factor Ixa that binds to factor VIIIa-its co-factor. Energy contributions of individual residues of factor VII responsible for binding to its co-factor-tissue factor (TF) will also be determined. In addition, the structural aspects of factor VII responsible for the increase in activity which it demonstrates upon binding to TF will be discerned. Thus, we have a project on protein-protein interactions which seeks to determine how co-factors undergo an increase in enzyme activity upon binding. Taken together, the approach will seek to determine how coagulation proteins interact with each other in normal hemostasis. The proteins to be studied will be produced via molecular biology approaches and tissue culture. Various chimeras will be constructed from domains of homologous factors (factors IX and X and factors IX and VII). (Chimeras are composed of at least two genetically different cell types). Mutations will also be created from individual or multiple residues derived from the surface. These will be the modalities for investigating structure-function relationships. The x-ray structure of factor IX and the factor VIII-tissue factor complex are now known and therefore, the studies can be facilitated. Kinetic analysis will be used to characterize the factor Ixa-VIIIa interaction. The rate of factor X activation is a measurement which will be made. Factor Ixa-VIIIa binding will be measured directly with the use of surface plasmon resonance. Direct binding assays with radiolabeled factor VII will be used to measure the interaction between factor VII and tissue factor. An indirect measurement will be made with a functional assay which measures cleavage of a peptide substrate or activation of factor X.
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