Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders

Abstract

Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga−/− larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga+/− larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding AαΔ19–56. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα+/Δ19–56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga−/− mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα+/Δ19–56. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.

High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies

“…Although the sequence alignment analyses indicate a lower homology in the case of the zebrafish, it must be noted that that is one of the teleost species where a greater number of orthologous genes are involved in hemostasis [ 79 , 80 ]. That is the reason why zebrafish has been postulated as a useful model for in vivo coagulation studies [ 81 , 82 ].…”

Role of Neutrophils and NETs in Animal Models of Thrombosis

“…As shown in Table 1, the main species used to study thrombosis are zebrafish [25][26][27], mice [8,[28][29][30][31][32][33], rats [34,35], rabbit [36][37][38] hamsters [39,40], guinea pigs [41,42], pigs [43][44][45], dogs [46][47][48], and baboons [9,49,50]. Of course, the mouse remains the reference animal model in the study of cardiovascular disease for several reasons: (1) it is a mammalian species with many physiological similarities to humans, (2) it is economical to house, (3) it is easy to manipulate, and (4) its genome is easily modifiable, allowing the creation of numerous transgenic lines [25].…”

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

“…fga mutant larval fish exhibit a hemostatic defect As in mammals, targeted mutation of fga results in loss of fibrinogen, and absence of thrombus formation following endothelial injury. 35,67 This can be rescued by the expression of zebrafish fga cDNA or human fibrinogen infusion. Surprisingly, incubation with ε-aminocaproic acid also partially reverses this hemostatic defect.…”