OC 33.4 - Loss of Factor VIII in Zebrafish Does Not Result in Severe Hemophilia A Due to an Altered Balance with Natural Anticoagulants

Background: Hemophilia A (factor VIII [FVIII] deficiency), is a severe bleeding disorder; novel therapies include modulation of hemostatic balance through targeting natural anticoagulants. The zebrafish has a high degree of homology with the mammalian hemostatic system. However, common pathway factor knockouts have shown differences in hemostatic balance when compared to mammals.

Aims: To study hemostatic balance in a zebrafish model of hemophilia A.

Methods: We used CRISPR/Cas9 genome editing to create null alleles for FVIII and von Willebrand Factor (VWF). Two assays were used to assess venous thrombus formation in larvae: grading spontaneous fibrin clots tagged with fluorescence and measuring time to occlusion after laser-induced endothelial injury.

Results: Homozygous mutants with a 117 base pair deletion in exon 4 of the zebrafish FVIII gene (f8) survived into adulthood with no signs of hemorrhage, and surprisingly formed normal thrombi when subjected to laser-mediated endothelial injury. We hypothesized that this might be due to altered hemostatic balance in the fish as compared to humans. We have previously shown that antithrombin mutant (at3-/-) larvae develop disseminated intravascular coagulation (DIC), with spontaneous thrombosis and fibrinogen consumption, resulting in a secondary lack of induced thrombus formation upon endothelial injury. We found that with loss of FVIII (f8-/-; at3-/-), larvae no longer developed spontaneous fibrin thrombi (Figure 1) and did produce clots in response to endothelial injury (Figure 2). Both outcomes are reversals of the at3 mutant (DIC) phenotype. VWF loss also mitigated the at3 mutant DIC phenotype, but to a lesser degree.

Conclusion(s): We found that an altered balance of natural anticoagulants mitigates FVIII deficiency in zebrafish, similar to human clinical pipeline products. However, in zebrafish the baseline state of AT3 activity prevents a bleeding phenotype in f8 mutants, without development of spontaneous thrombosis. Further study of this unique balance could provide new insights for management of hemophilia A.