SATURATION MUTAGENESIS OF ZEBRAFISH COAGULATION PATHWAY

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DESCRIPTION: (Investigator's abstract) The initiation of blood coagulation through the extrinsic pathway is a threshold-mediated event influenced by relative levels of factor VIIa, tissue factor and TFPI activity. Despite intensive study, important questions remain regarding initiation and regulation of the coagulation response. The applicant hypothesizes that novel genes, involved in initiation and regulation of the extrinsic pathway of coagulation, remain to be identified. The current proposal employs the zebrafish as a genetic tool to identify novel genes in this pathway. The novel genes will be identified by chemical mutagenesis of the zebrafish genome followed by a comprehensive screen for mutants with defective thrombin generation by the extrinsic pathway. Screening for zebrafish phenotypes with defective thrombin generation will yield a significant number of mutations in both known and unknown genes involved in the extrinsic pathway of coagulation. Zebrafish that display defective thrombin generation will be rapidly sorted by both biochemical and linkage analyses to eliminate mutations in known genes, and focus will be placed on novel defects affecting the expression, activation or activity of factor VIIa. Identification of novel zebrafish genes will ultimately allow identification of human homologues involved in hemostasis and thrombosis. Identification of human homologues will further our understanding of how the coagulation cascade functions in vivo, suggest novel targets for antithrombotic therapy and provide candidate genetic markers for thrombotic disease.