In humans, coagulation factor V (FV) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. 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 FV 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 FV 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 FV function, with minimal activity being required to restore hemostasis in response to laser-induced endothelial injury. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology, although limitations exist. 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.

The ability to prevent blood loss in response to injury is a conserved function of all vertebrates. Complete deficiency of the central clotting enzyme prothrombin has never been observed in humans and is incompatible with postnatal life in mice, thus limiting the ability to study its role in vivo. Zebrafish are able to tolerate severe hemostatic deficiencies that are lethal in mammals. We have generated a targeted genetic deletion in the kringle 1 domain of zebrafish prothrombin. Homozygous mutant embryos develop normally into the mid-juvenile stage but demonstrate complete mortality by 2 months of age primarily due to internal hemorrhage. Mutants are unable to form occlusive venous and arterial thrombi in response to endothelial injury, a defect that was phenocopied using direct oral anticoagulants. Human prothrombin engineered with the equivalent mutation exhibits a severe reduction in secretion, thrombin generation, and fibrinogen cleavage. Together, these data demonstrate the conserved function of thrombin in zebrafish and provide insight into the role of kringle 1 in prothrombin maturation and activity. Understanding how zebrafish are able to develop normally and survive into early adulthood without thrombin activity will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.Maintaining blood flow in a closed circulatory system requires a delicate balance between pro-and anticoagulant factors. Disequilibrium of these factors in either direction can lead to pathology. In response to vascular injury, the balance shifts towards coagulation in an effort to stabilize blood clots and prevent exsanguination. Critical to this clot stabilization is the activation of prothrombin, a vitamin K-dependent clotting factor, to form the central clotting enzyme, thrombin 1 . Thrombin cleaves soluble fibrinogen into fibrin monomers, which then polymerize to form the insoluble fibrin clot 2 . Additionally, thrombin interacts with protease activated receptors (PARs) on the surface of various cells, including platelets, which makes it the most potent endogenous agonist of primary hemostasis 3 . Human prothrombin (F2) variants have been linked to both thrombophilia and bleeding diatheses. The most common variant (~2% in European populations) 4 is a guanine to adenine transition in the 3' untranslated region at position 20210 that leads to increased plasma prothrombin levels and a 2-3 fold elevated risk of deep vein thrombosis 5,6 . Congenital prothrombin deficiencies result in a bleeding diathesis, but are rare. Acquired deficiencies due to liver failure or vitamin K deficiency are more common 7,8 .Structurally, prothrombin consists of six domains: signal peptide, propeptide, Gla, kringle 1, kringle 2, and serine protease; a domain composed of a light and heavy chain 1 . Following translation, the propeptide targets the protein for post-translational gamma-carboxylation of the glutamic acid residues within the Gla domain. This vitamin K-dependent process is necessary...

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.

Over the past two decades, the teleost vertebrate Danio rerio (zebrafish) has emerged as a model for hemostasis and thrombosis. At genomic and functional levels, there is a high degree of conservation of the hemostatic system with that of mammals. Numerous features of the fish model offer unique advantages for investigating hemostasis and thrombosis. These include high fecundity, rapid and external development, optical transparency, and extensive functional homology with mammalian hemostasis and thrombosis. Zebrafish are particularly suited to genome‐wide mutagenesis experiments for the study of modifier genes. They are also amenable to whole‐organism small‐molecule screens, a feature that is exceptionally relevant to hemostasis and thrombosis. Zebrafish coagulation factor knockouts that are in utero or neonatal lethal in mammals survive into adulthood before succumbing to hemorrhage or thrombosis, enabling studies not possible in mammals. In this illustrated review, we outline how zebrafish have been employed for the study of hemostasis and thrombosis using modern genome editing techniques, coagulation assays in larvae, and in vivo evaluation of patient‐specific variants to infer causality and demonstrate pathogenicity. Zebrafish hemostasis and thrombosis models will continue to serve as a clinically directed basic research tool and powerful alternative to mammals for the development of new diagnostic markers and novel therapeutics for coagulation disorders through high‐throughput genetic and small‐molecule studies.

Congenital erythropoietic porphyria (CEP) is a rare genetic disorder leading to accumulation of uro/coproporphyrin-I in tissues due to inhibition of uroporphyrinogen-III synthase. Clinical manifestations of CEP include bone fragility, severe photosensitivity and photomutilation. Currently there is no specific treatment for CEP, except bone marrow transplantation, and there is an unmet need for treating this orphan disease. Fluorescent porphyrins cause protein aggregation, which led us to hypothesize that uroporphyrin-I accumulation leads to protein aggregation and CEP-related bone phenotype. We developed a zebrafish model that phenocopies features of CEP. As in human patients, uroporphyrin-I accumulated in the bones of zebrafish, leading to impaired bone development. Furthermore, in an osteoblast-like cell line, uroporphyrin-I decreased mineralization, aggregated bone matrix proteins, activated endoplasmic reticulum stress and disrupted autophagy. Using high-throughput drug screening, we identified acitretin, a second-generation retinoid, and showed that it reduced uroporphyrin-I accumulation and its deleterious effects on bones. Our findings provide a new CEP experimental model and a potential repurposed therapeutic.