Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Hu, Zhilian; Lavik, Kari; Liu, Yang; Vo, Andy H.; Richter, Catherine; Paola, Jorge Di; Shavit, Jordan A.

doi:10.1111/jth.14391

Cited by 14 publications

(27 citation statements)

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“…In contrast to mammals, in which a number of coagulation factors are necessary for embryonic and/or neonatal viability, zebrafish are able to survive the loss of many aspects of the canonical cascade at least until early adulthood. Loss of antithrombin, fibrinogen, FV, and FX are all compatible with development to adulthood 22,[26][27][28]37 . Targeting of f10 in fish results in the absence of larval hemostasis, but is accompanied by extended survival 28 .…”

Section: Discussionmentioning

confidence: 99%

“…Embryonic and adult studies have demonstrated the benefits of zebrafish for the study of hemostatic and other human diseases 19,20 . Conservation of coagulation factors in zebrafish is well characterized with a variety of techniques and genetic models [21][22][23][24][25][26][27] . FX knockout (f10 −/− ) zebrafish survive several months into adulthood before succumbing to lethal hemorrhage, suggesting that the model is more resistant to extreme disturbances in hemostasis than mammals 28 .…”

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

Grzegorski

Liu

et al. 2020

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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...

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Section: Discussionmentioning

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

Grzegorski

Liu

et al. 2020

Sci Rep

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“…The zebrafish has emerged as a model for thrombosis and hemostasis studies 13 due to the conservation of coagulation proteins 14 and the successful adaptation of in vivo hemostasis tests using transparent zebrafish larvae. [15][16][17][18][19] In addition, targeted mutations in a number of coagulation-related proteins, including fibrinogen, 20,21 have demonstrated the zebrafish's utility in modeling factor deficiencies and for assessing the impact of patient-derived polymorphisms. [22][23][24][25] Zebrafish have nucleated thrombocytes, 26 which are considered cellular equivalents to platelets in hemostasis.…”

Section: Introductionmentioning

confidence: 99%

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

et al. 2020

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Plasma fibrinogen molecules comprise 2 copies of Aα, Bβ, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.

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“…In contrast to mammals, in which a number of coagulation factors are necessary for embryonic and/or neonatal viability, zebrafish are able to survive the loss of many aspects of the canonical cascade at least until early adulthood. Loss of antithrombin, fibrinogen, FV, and FX are all compatible with development to adulthood 22,[26][27][28]45 . Targeting of f10 in fish results in the absence of larval hemostasis, but is accompanied by extended survival 28 .…”

Section: Discussionmentioning

confidence: 99%

“…Embryonic and adult studies have demonstrated the benefits of zebrafish for the study of hemostatic and other human diseases 19,20 . Conservation of the coagulation cascade in zebrafish is well characterized with a variety of techniques and genetic models [21][22][23][24][25][26][27] . FX knockout (f10 -/-) in zebrafish demonstrated that the model is more resistant to extreme disturbances in hemostasis than mammals 28 .…”

Section: Introductionmentioning

confidence: 99%

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

Grzegorski

Liu

et al. 2019

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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.

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Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Cited by 14 publications

References 70 publications

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

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

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

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

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