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

Zebrafish were used as a model to study haemostasis, a vertebrate function of paramount importance. A limitation of the zebrafish model is the difficulty in assaying small amounts of blood to detect coagulation mutants. We report the use of a rapid total coagulation activity (TCA) assay to screen for coagulation defects in individual adult zebrafish. We screened the TCA in 1000 gynogenetic half-tetrad diploids derived from 86 clutches. Each clutch was from a single F1 female offspring of males mutagenized with ethylnitrosourea (ENU). We found 30-50% defective zebrafish among six clutches, consistent with a heritable defect. The assay developed here provided a rapid screen to detect overall coagulation defects. However, because of the limited amounts of plasma, we could not detect defects in specific pathways. Therefore, a novel, ultra-sensitive kinetic method was developed to identify specific 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 conclude that it is now possible to identify specific coagulation pathway defects in zebrafish.