Analysis of blood coagulation in the zebrafish

The zebrafish (Danio rerio) is a unique animal model in which saturation mu tagenesis has been used to identify genes involved in vertebrate developmen t. The relevance of the zebrafish as a genetic model for hemostasis depends , in large part, on the degree of similarity between the zebrafish and mamm alian systems. The diminutive size of the zebrafish poses technical problem s for analysis of coagulation. This study describes methods to obtain citra ted whole blood and plasma fi om the zebrafish, analyze in vitro coagulatio n in small plasma volumes, obtain uniform dosing of zebrafish with oral ant icoagulants, and demonstrate specific factor activities via chromogenic ass ays. Analysis of the zebrafish system demonstrates the presence of both the intrinsic and extrinsic pathways of coagulation, evidence for prothrombin, factor X, protein C, antithrombin, and heparin cofactor IJ activity, and a requirement for vitamin K dependent gamma-carboxylation of zebrafish hemos tatic proteins. Induction of a morphologically recognizable bleeding phenot ype by warfarin treatment is also demonstrated. Characterization of zebrafi sh coagulation provides evidence that major hemostatic pathways are conserv ed between zebrafish and man. These similarities indicate that the zebrafis h is a relevant genetic model for identification of novel genes involved in hemostasis and thrombosis.