Identifying Thrombosis Modifier Genes and Novel Anticoagulants in Zebrafish

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Identifying Thrombosis Modifier Genes and Novel Anticoagulants in Zebrafish This project will take advantage of the powerful genetic tools available in zebrafish to conduct a large scale genomic screen for hemostasis regulatory genes that could modify the severity of human hemorrhagic and thrombotic disorders. This system will also facilitate a novel approach for high-throughput screening of chemical compound libraries in an effort to identify new candidate anticoagulant and hemostatic therapeutics. In preliminary studies, we have positionally cloned a novel ENU-induced mutation in the zebrafish pak2a gene that results in intracerebral hemorrhage, likely due to a defect in endothelial cell function and vascular integrity. We have also engineered a specific mutation (M385L) associated with human protein C deficiency into the zebrafish protein C gene. Additional preliminary studies include a high throughput chemical screen that successfully identified compounds that modulate streptokinase gene expression in pathogenic group A streptococci. In Specific Aim I we will characterize M385L and additional engineered zebrafish protein C mutants to develop in vivo thrombosis models that can be used as the basis for both positive and negative genetic and pharmacologic screens. Specific Aim II will perform large scale chemical library screens to identify "enhancer" compounds that induce thrombosis in partially protein C deficient fish and "suppressor" compounds that "rescue" a lethal thrombosis model. Aim III will use the same zebrafish models to perform whole genome END mutagenesis screens to identify both prothrombotic and anticoagulant modifier genes. Taken together, these studies should identify multiple candidate genes for modifiers of human hemorrhagic and thrombotic disorders, provide new insight into the regulation of hemostasis in vivo, and suggest novel pharmaceutical agents for the treatment of bleeding and thrombosis in humans.