Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Human Fibrinogen Disorders
ISTH Academy. Freire Sanz C. Jul 10, 2019; 274062; OC 74.4 Topic: Fibrinogen & Factor XIII
Cristina Freire Sanz
Cristina Freire Sanz
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OC 74.4

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

C. Freire Sanz, R. Fish, M. Neerman-Arbez
University of Geneva, Genetic Medicine and Development, Geneva, Switzerland

Main Topic: Fibrinolysis and Proteolysis
Category: Fibrinogen & Factor XIII

Background: Afibrinogenemia and dysfibrinogenemia are caused by mutations in the fibrinogen genes and typically lead to a bleeding tendency and the possibility of thromboembolic complications. Bleeding can be explained by deficiency in the major blood clotting protein substrate, but the pathophysiology of thromboembolic events in fibrinogen deficiency has not been extensively studied.
Aims: Using zebrafish with a deletion in fga (fga-/-), and a mutant designed to mimic a human dysfibrinogenemia mutation (Aα R35C) lacking a functional Aα chain N-terminal thrombin cleavage site, our study aimed to demonstrate the impact of afibrinogenemia and dysfibrinogenemia on hemostasis upon vascular challenge.
Methods: Zebrafish mutants were prepared using genome-editing of fga exon 2. Fibrinogen susceptibility to thrombin cleavage was assessed in plasma. Embryonic vascular injuries were made using a laser targeting the caudal vein. Time-to-occlusion (TTO) was measured as a global assessment of hemostasis and thrombosis. Thrombocyte adhesion and aggregation were recorded by monitoring fluorescent thrombocytes at the injury site.
Results: 3-day-old fga-/- zebrafish failed to support venous thrombosis (n=26) after laser injury whereas >90% of wild-type (n=29) and heterozygous (n=43) siblings showed vessel occlusion. Thrombocyte adhesion and aggregation were reduced in 5-day-old afibrinogenemic fish, compared to controls, and embolic cell aggregates were observed. The R28C mutation in fga lead to in-frame exon 2 skipping of its mRNA. The encoded Aα chain lacks a thrombin cleavage site, resembling the Aα chain in hypodysfibrinogenemia patients with an FGA splicing mutation. Fibrinogen Aα from fgaR28C/R28C plasma was insensitive to thrombin cleavage. Prolonged TTO and reduced thrombocyte adhesion and aggregation were measured in fga+/R28C (n=42) and fgaR28C/R28C (n=16) fish, compared to wild-types (n=17).
Conclusions: Zebrafish with afibrinogenemia cannot support venous thrombosis or sustained thrombocyte aggregation, whereas those with a mutated fibrinogen Aα chain thrombin cleavage site show prolonged thrombosis times and reduced thrombocyte aggregation, even in heterozygosity.

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