A Zebrafish Model Of Antithrombin III Deficiency Displays Bleeding and Thrombosis Secondary To Disseminated Intravascular Coagulation

Abstract

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 inhibitors of thrombosis in an in vivo model 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 humans. Its external development, ability to generate thousands of offspring at low cost, and optical transparency all make it a powerful tool to screen for genetic and chemical modifiers of coagulation disorders. We generated a zebrafish model of antithrombin III (AT3) deficiency by targeted mutagenesis using zinc finger nucleases. Homozygous at3 mutants displayed a lethal phenotype due to intracardiac thrombosis between 2 and 7 months of age, yet embryos and larvae appeared grossly normal with no overt evidence of pathologic clotting. Induction of thrombosis at 3-4 days post fertilization (dpf) in homozygous mutant larvae by laser-mediated endothelial ablation resulted in diminished rates of posterior cardinal vein (PCV) occlusion, a bleeding phenotype. To prove functional conservation with mammals, we expressed recombinant zebrafish At3 and demonstrated that it binds human thrombin in vitro. Furthermore, while injection of wild type zebrafish and human cDNAs rescued the laser injury phenotype, zebrafish at3 with a mutation in the putative reactive site failed to do so. We hypothesized that the discrepant larval bleeding and adult thrombotic phenotypes could be accounted for by disseminated intravascular coagulation (DIC). Consistent with this, we observed reduced fibrinogen levels in at3 homozygous mutant plasma, and were able to rescue mutant larvae by injection of human fibrinogen prior to laser injury. To identify the location of consumed fibrinogen, we tagged human fibrinogen with FITC (fluorescein isothiocyanate), followed by infusion into larvae from heterozygous intercrosses at 3 dpf. at3 homozygous mutants displayed extensive PCV fluorescence, which was absent in wild type and heterozygous siblings. Pre-incubation with warfarin completely prevented this phenotype, co-injection of tissue plasminogen activator (TPA) partially prevented fluorescence accumulation, and post-injection of TPA reduced the signal, consistent with our hypothesis of DIC. Our data have uncovered the mechanism underlying the discrepant at3 mutant phenotypes and demonstrated conservation of At3 function in zebrafish. Loss of At3 protein results in DIC in zebrafish larvae secondary to unopposed thrombin activity. Mutants survive, only to succumb to lethal thrombosis as adults. Further study as to why larvae are able to tolerate excessive clot formation, as well as small molecule screens for novel anticoagulants using this model, could potentially lead to innovative therapeutic modalities for affected patients. Disclosures: No relevant conflicts of interest to declare.