YS02 Screening and Characterisation of Novel Platelet Candidate Genes Derived from Genome‐Wide Association Studies for Function in Relation to Haemostasis and Thrombosis in the Model Organism Danio Rerio

Abstract

Complementary approaches of microarray, proteomics, genome wide association and platelet functional studies have identified novel platelet proteins which are candidates for playing a role in thrombosis or conferring risk of myocardial infarction. The function of many of the proteins encoded by these candidate genes is unknown, and will need to be determined. To address the need for high‐throughput screening of candidate genes in a model organism, we have investigated the suitability of the zebrafish (ZF; Danio rerio), a genetically tractable vertebrate with nucleated thrombocytes. It is known that mouse knockouts of critical pro‐thrombotic genes [e.g. coagulation factor (F) VIII, integrin αIIb (ITGA2B), or glycoprotein VI] have subtle phenotypes and do not bleed spontaneously. We hypothesised that the sensitivity of the ZF model to screen for loss of platelet function could be enhanced by concurrent knock down of the FVIII gene and a pro‐thrombotic platelet gene. To test this, the genes for FVIII, ITGA2B or both were silenced by antisense oligonucleotides (morpholinos). Embryos were then assessed for body plan and bleeding at 3 days post‐fertilization (dpf). In parallel, a thrombosis model using laser injury to initiate thrombus formation was developed. This permits time to adhesion (TTA), time to occlusion (TTO) and time to thrombolysis (TTL) to be determined in morpholino‐injected and wild type ZF. As expected, individual knockdown of the FVIII or ITGA2B genes did not lead to spontaneous bleeding. Knockdown of the ITGA2B gene did, however, result in morphological malformations. Simultaneous knockdown of both genes resulted in a clear and reproducible bleeding phenotype visible in ∼20% of embryo's. Using the laser injury model, venous thrombosis could be reproducibly induced in 3 and 6 dpf wild‐type ZF larvae with partial but not complete thrombolysis occurring within 5–10 min. In conclusion, ZF are a fast and informative way to screen for platelet function modifying genes.