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

Richard J Fish,Corinne Di Sanza,Cristina Freire,Marguerite Neerman-Arbez

doi:10.3390/ijms22020655

Richard J Fish, Corinne Di Sanza + Show 2 more

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https://doi.org/10.3390/ijms22020655

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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.

Highlights

Mutations in the three fibrinogen genes give rise to congenital fibrinogen disorders [1].Fibrinogen has a crucial role as the soluble fibrin precursor in blood clotting, and its deficiency or dysfunction manifests in both bleeding and thrombotic clinical events in these disorders [2,3]
Laser injury of the posterior cardinal vein in 3-day postfertilization (3 dpf) zebrafish larvae can lead to occlusive venous thrombosis and when monitored gives the time-toocclusion (TTO, Figure 1A)
We aimed to determine whether the experimental venous thrombosis phenotype of afibrinogenemia, a quantitative disorder, differs from that of dysfibrinogenemia—a disorder of fibrinogen quality

Summary

Introduction

Mutations in the three fibrinogen genes give rise to congenital fibrinogen disorders [1]. Fibrinogen has a crucial role as the soluble fibrin precursor in blood clotting, and its deficiency or dysfunction manifests in both bleeding and thrombotic clinical events in these disorders [2,3]. Mutations in any of the genes can lead to a quantitative or qualitative fibrinogen disorder. Quantitative disorders include afibrinogenemia, where plasma fibrinogen antigen is undetectable, and hypofibrinogenemia where fibrinogen is below the typical healthy range (1.5–4 g/L). The qualitative disorders are dysfibrinogenemia, where a normal antigen level is accompanied by a lower functional level of fibrinogen when compared to control plasma in clotting assays, and hypodysfibrinogenemia where an abnormally low antigen level is discordant with an even lower functional activity [4]

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