Grzegorski, S. J. | bioRxiv Science

bioRxiv2019

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

The 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 prothrombins 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.\n\nKey PointsO_LIDisruption of the kringle 1 domain of prothrombin leads to severe impairment of biosynthesis, activation, and activity\nC_LIO_LIProthrombin deficiency is compatible with normal development in zebrafish but leads to inability to form clots followed by early mortality\nC_LI

genetics↗

bioRxiv2019

Analysis of factor V in zebrafish demonstrates minimal levels needed for hemostasis and risk stratifies human variants

In humans, coagulation factor V (F5) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Complete loss of mouse F5 results in early lethality. Zebrafish possess many distinct advantages including high fecundity, optical clarity, external development, and homology with the mammalian hemostatic system, features that make it ideal for genetic studies. Our aim was to study the role of F5 in zebrafish through targeted mutagenesis, and apply the model to the study of human F5 variants. CRISPR-mediated genome editing of the zebrafish f5 locus was performed, generating mutants homozygous for a 49 base pair deletion in exon 4. Thrombus formation secondary to vascular endothelial injury was absent in f5-/- mutant embryos and larvae. Despite this severe hemostatic defect, homozygous mutants survived before succumbing to severe hemorrhage in adulthood. Human F5 variants of uncertain significance from patients with F5 deficiency were evaluated, and the causative mutations identified and stratified by their ability to restore thrombus formation in larvae. Analysis of these novel mutations demonstrates variable residual F5 function, with minimal activity being required to restore hemostasis. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology. Furthermore, homozygous mutant embryos tolerate what is a severe and lethal defect in mammals, suggesting the possibility of species-specific factors enabling survival, and allowing further study not possible in the mouse. Identification of these factors or other genetic modifiers could lead to novel therapeutic modalities.\n\nKey PointsO_LIF5 mutant fish embryos tolerate symptoms lethal in mammals but succumb to bleeding in adulthood\nC_LIO_LIAnalysis of human variants demonstrate that all have some residual function and that minimal F5 activity is required to restore hemostasis\nC_LI

genetics↗