Abstract 7: Targeted Mutagenesis of Antithrombin III in Zebrafish Results in Disseminated Intravascular Coagulation and Thrombosis
Pathologic blood clotting (thrombosis) is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Identification of small molecule modifiers using an in vivo thrombosis would facilitate novel and improved therapeutics for patients. The zebrafish is a powerful genetic model in which the hemostatic system is nearly entirely conserved with mammals and its ability to generate thousands of offspring at low cost makes it a powerful tool to screen chemical modifiers of coagulation disorders. We have generated a zebrafish model of complete antithrombin III (at3) deficiency by targeted mutagenesis using zinc finger nucleases. at3 heterozygotes with 22 and 90 base pair deletions (at3[[Unable to Display Character: ∆]]22 and at3[[Unable to Display Character: ∆]]90) were intercrossed, and no homozygotes have survived beyond 7 months of age. Offspring were genotyped at various time points revealing a significant reduction of expected homozygotes compared to the expected Mendelian frequency as early as 1-2 months post fertilization. This lethality was rescued by an at3 cDNA transgene, enabling survival to at least 15 months of age. Analysis of adult homozygous mutants revealed evidence of large intravascular thrombi and externally visible secondary hemorrhage. We induced thrombosis at 3-4 days post fertilization in at3[[Unable to Display Character: ∆]]90 mutant larvae by laser injury and injection of bovine thrombin. Contrary to our expectations, we observed diminished rates of cardinal vein occlusion in homozygous mutants, and this could be rescued by an at3 cDNA transgene. We hypothesized that this was due to disseminated intravascular coagulation (DIC), and observed reduced fibrinogen levels in at3 homozygous mutant plasma. Furthermore, injection of mutant larvae with human fibrinogen prior to laser injury rescued the mutant phenotype. In conclusion, loss of At3 protein results in subclinical DIC in zebrafish larvae secondary to unopposed thrombin activity, but they survive and succumb to lethal thrombosis in adults. Future studies will adapt this model for high throughput screening for genetic and chemical modifiers of DIC and thrombosis. Genes and small molecules identified will be potential novel diagnostic and therapeutic modalities for patients with these disorders.
- © 2013 by American Heart Association, Inc.