Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

Abstract

Running title: Loss of prothrombin in zebrafish Key Points• Disruption of the kringle 1 domain of prothrombin leads to severe impairment of biosynthesis, activation, and activity • Prothrombin deficiency is compatible with normal development in zebrafish but leads to inability to form clots followed by early mortality AbstractThe ability to prevent blood loss in response to injury is a critical, evolutionarily conserved function of all vertebrates. Prothrombin (F2) contributes to both primary and secondary hemostasis through the activation of platelets and the conversion of soluble fibrinogen to insoluble fibrin, respectively. Complete prothrombin deficiency has never been observed in humans and is incompatible with life in mice, limiting the ability to understand the entirety of prothrombin's in vivo functions. We have previously demonstrated the ability of zebrafish to tolerate loss of both pro-and anticoagulant factors that are embryonic lethal in mammals, making them an ideal model for the study of prothrombin deficiency. Using genome editing with TALENs, we have generated a null allele in zebrafish f2. Homozygous mutant embryos develop normally into early adulthood, but demonstrate eventual complete mortality with the majority of fish succumbing to internal hemorrhage by 2 months of age. We show that despite the extended survival, the mutants are unable to form occlusive thrombi in both the venous and arterial systems as early as 3-5 days of life, and we were able to phenocopy this early hemostatic defect using direct oral anticoagulants. When the equivalent mutation was engineered into the homologous residues of human prothrombin, there were severe reductions in secretion and activation, suggesting a possible role for kringle 1 in thrombin maturation, and the possibility that the F1.2 fragment has a functional role in exerting the procoagulant effects of thrombin. Together, our data demonstrate the conserved function of thrombin in zebrafish, as well as the requirement for kringle 1 for biosynthesis and activation by prothrombinase. Understanding how zebrafish are able to develop normally and survive into early adulthood without prothrombin will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.

Nuclear Progestin Receptor–mediated Linkage of Blood Coagulation and Ovulation

“…Each ovary was immediately cut into three equal pieces and then transferred into medium containing vehicle, 2 mM EDTA, or an anticoagulant (100 µg /mL heparin, 50 µg /mL warfarin, 50 µM dabigatran etexilate, or 250 µM rivaroxaban), to examine the inhibitory effect of anticoagulants on ovulation. The dose of each drug was chosen based on previous reports (37)(38)(39). Follicles were immediately dispersed by pipetting using a glass pipette (~ 1 mm in diameter).…”

Fishing for answers to hemostatic and thrombotic disease: Genome editing in zebrafish

“…They develop normally into the mid-juvenile stage but exhibit spontaneous hemorrhage beginning around 2 months of age, with complete lethality by 4 months. 31,34,60 Intrinsic In zebrafish, common pathway knockouts survive into early adulthood before succumbing to lethal hemorrhage.…”

“…Complete loss of common pathway factors is embryonic lethal in mouse models and presumed lethal in humans. 31,34,60 Hypotheses for the differences between mammals and zebrafish include: absence of birth trauma differential hemostatic challenges in an aquatic environment lower blood pressures in zebrafish species-specific genetic differences resulting in a differential baseline hemostatic balance 60,72 Since hemostasis must occur within a closed circulatory system, consisting of flowing blood, proteins, cells, and endothelium, the zebrafish is superior to cell culture systems for assessing variants.…”

Multi-ancestry GWAS for venous thromboembolism identifies novel loci followed by experimental validation in zebrafish

“…Our previous studies validated several known coagulation factors using the genome edited zebrafish models of hemostasis and thrombosis: F2 (Grzegorski et al, 2020) F5 (Weyand et al, 2019), F10 (Hu et al, 2017), PROS1 (Ku et al, 2020), and PROC (Ku et al, 2020). Using this model, we evaluated 6 prioritized genes (F7, RASIP1, TC2N, STAB2, TSPAN15, and PLCG2) from regions that demonstrate conservation of synteny with the zebrafish genome.…”