PB0560 - The vkorc1 paralog vkorc1l1 and vitamin K reductase fsp1 regulate coagulation in vivo in zebrafish

Background: Vitamin K-dependent clotting factor deficiency (VKCFD) is a rare inherited disorder presenting with neonatal hemorrhage. Vitamin K (VK) epoxide reductase (VKOR) and VKR (Vitamin K reductase) are essential enzymes for coagulation. Genes for the former (VKORC1 and VKORC1L1) have been known for two decades, while VKR was only recently identified in vitro as FSP1. VKORC1 is thought to be the primary VKOR in mammals, while the role of VKORC1L1 is less clear.

Aims: To determine whether VKORC1L1 and FSP1 regulate coagulation in vivo using zebrafish, which has a high degree of homology with mammalian hemostasis.

Methods: Human and zebrafish VKORC1/VKORC1L1/FSP1 functions were assessed in double (VKORC1/VKORC1L1) or triple (VKORC1/VKORC1L1/FSP1) knockout HEK293 cells transfected with cDNAs. Venous clotting was evaluated in zebrafish larvae using time-to-occlusion (TTO) after laser-induced endothelial injury. CRISPR/Cas9 genome editing in zebrafish produced null alleles for vkorc1 and vkorc1l1, and knockdowns of fsp1.

Results: Human and zebrafish VKORC1/VKORC1L1/FSP1 demonstrated equivalent carboxylation activity between each ortholog (Figure 1). Homozygous zebrafish mutants with large deletions in vkorc1 or vkorc1l1 had a normal TTO in 5-day larvae. A significantly increased TTO was only observed in vkorc1-/-;vkorc1l1+/- or vkorc1-/-;vkorc1l1-/- larval genotypes and with a maternal genotype that included vkorc1l1-/-, highlighting the importance of maternal VK stores (Figure 2). Pretreatment with VK rescued the defects in 43% of vkorc1-/-;vkorc1l1+/-, but not vkorc1-/-;vkorc1l1-/- larvae. Knockdown of fsp1 in the vkorc1/vkorc1l1 mutant background exacerbated the failure to occlude in vkorc1-/-;vkorc1l1+/- larvae from 42% to 93% (Figure 2).

Conclusion(s): Like humans, we find evidence that maternal VK is critical for hemostasis in the embryonic/larval period. We also confirmed that fsp1 functions in the VK pathway in vivo. The role of vkorc1l1 in coagulation has been questionable, but here we show a key role in fish, suggesting that strategies increasing VKORC1L1 could be novel therapies for patients with VKCFD.