The goals of this research are to define the portion of the human blood coagulation factor IX that binds to endothelial cells, platelets, phospholipid vesicles and its protein co-factor, factor VIII; to isolate and clone the factor IX binding protein from endothelial cells or platelets; to identify the amino acids of factor VII responsible for its specific interaction with tissue factor and the region of tissue factor that recognizes factor VIIa. This information should add to our understanding of how these proteins and cells interact in normal hemostasis. A variety of molecular biological techniques will be used to construct and express proteins designed to examine these issues. For the endothelial cell and platelet binding studies, factor VII with various amino terminal residues of factor IX substituted for those of factor VII will be used. For cloning of the endothelial cell receptor two approaches will be used. First we will attempt to clone the gene directly by expression cloning. The second approach will be to make monoclonal anti-idiotype antibodies designed to bind to the receptor. For the study of factor IX and factor VIII interactions a variety of chimeric factor IX-factor VII and factor IX-factor X molecules will be used. Kinetic analysis of the stimulation by the co-factor, factor VIIIa on the generation of factor Xa in a purified system will be examined. For the interaction of factor VII and tissue factor we will sue the assay already developed in our laboratory for examining the interaction on immobilon membranes. Again chimeric proteins, point mutations and combination of mutations will be used to study the interaction. These studies should help to understand the protein structure-function relationships in blood coagulation and, thus the mechanisms involved in the pathological manifestations of hemostasis.
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