Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes inman

Zebrafish were used as a model to study haemostasis, a vertebrate function of paramount importance. A limitation of the zebrafish model is the difficu lty in assaying small amounts of blood to detect coagulation mutants. We re port the use of a rapid total coagulation activity (TCA) assay to screen fo r coagulation defects in individual adult zebrafish. We screened the TCA in 1000 gynogenetic half-tetrad diploids derived from 86 clutches. Each clutc h was from a single F1 female offspring of males mutagenized with ethylnitr osourea (ENU). We found 30-50% defective zebrafish among six clutches, cons istent with a heritable defect. The assay developed here provided a rapid s creen to detect overall coagulation defects. However, because of the limite d amounts of plasma, we could not detect defects in specific pathways. Ther efore, a novel, ultra-sensitive kinetic method was developed to identify sp ecific pathway defects. To test whether the kinetic assay could be used as a screening tool, 1500 Florida wild-type zebrafish pairs were analysed for naturally occurring coagulation defects. We detected 30 fish with extrinsic pathway defects, but with intact common and intrinsic pathways. We conclud e that it is now possible to identify specific coagulation pathway defects in zebrafish.