Antithrombin III and C1 inhibitor are homologous plasma protease inhibitors which play important regulatory roles in the coagulation, complement, kinin-generating and fibrinolytic pathways. Genetic deficiency of antithrombin III causes hereditary thrombosis, while familial C1 inhibitor deficiency causes hereditary angioneurotic edema. The work proposed in this application focuses on structure-function and gene expression aspects of antithrombin III and C1 inhibitor biology. We will identify the structural elements responsible for conferring heparin activation on antithrombin III, and target protease specificity on antithrombin III and C1 inhibitor. Initial work in this area will involve characterization of dysfunctional inhibitor molecules and genes from families with hereditary thrombosis and hereditary angioneurotic edema. We will also identify elements which are highly conserved in the primary structures of ATIII, C1 inhibitor and other serine protease family members, in order to gain some idea about what residues are required for maintaining inhibitor function. Finally, we will test and extend our knowledge of the structure-function principles underlying the design of serine protease inhibitors by determining the functional properties of abnormal ATIII and C1 inhibitor molecules we have generated by expression of in vitro mutagenized genes. The goal of another set of studies will be to discover how a drug cures the symptoms of a genetic disease. The attenuated androgen danazol prevents attacks of hereditary angioneurotic edema by raising plasma levels of C1 inhibitor. In order to determine how the hormone regulates gene expression, we will develop an in vitro model of the androgen - C1 inhibitor response, and characterize it at the protein, RNA and DNA levels.
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