Abstract Number: PB0732
Meeting: ISTH 2020 Congress
Background: A fibrinogen isoform with C-terminally-extended Aα chains (AαE) represents ≈1% of circulating fibrinogen molecules. Larval zebrafish produce predominantly AαE-containing fibrinogen, with unknown functional significance. Distinct zebrafish strains differ in their ability to produce fibrinogen AαE, which correlates with inter-strain variability in experimental venous thrombosis. A causal role for altered AαE expression in thrombosis has not been demonstrated.
Aims: To determine if fibrinogen AαE expression is the basis of inter-strain variability in larval zebrafish venous thrombosis.
Methods: Time-to-occlusion and thrombocyte activity were monitored in larval zebrafish after venous laser injury. Aα or AαE chain overexpression and fibrinogen knockdowns were made using transgenesis and an antisense morpholino, respectively. Inter-strain sequence variation was assessed by Sanger and whole-genome sequencing.
Results: We measured the time-to-occlusion (TTO) of laser-induced venous thrombosis in AB and TU zebrafish strains. The AB strain gave a longer mean TTO than TU, and mating AB with TU produced larvae with the TU phenotype. AB larvae failed to produce fibrinogen AαE due to a mutation in the AαE-specific fga exon 6: c.1428delG (p.Ser477LeufsX4). The link between delayed TTO and the mutation was confirmed in larvae through mapping crosses of AB with the TL strain, followed by whole genome sequencing. We investigated whether the lack of AαE explained the delayed AB strain TTO. Transgenic expression of AαE shortened the AB TTO to that of TU and rescued venous occlusion in fibrinogen null mutants or larvae with morpholino-directed fibrinogen knockdown, but Aα expression was less effective. In 5-day-old larvae, when thrombocytes begin to contribute to hemostasis, laser-induced thrombocyte adhesion and aggregation was reduced in fibrinogen mutants. Expression of Aα or AαE restored thrombocyte accumulation similarly.
Conclusions: We define a genetic modifier of venous thrombosis, a role for fibrinogen AαE in early hemostasis, and suggest a link between differentially expressed fibrinogen isoforms and cell types available for clotting.
To cite this abstract in AMA style:Fish RJ, Freire C, Vilar R, Di Sanza C, Grzegorski SJ, Richter CE, Shavit JA, Neerman-Arbez M. A Genetic Modifier of Venous Thrombosis in Zebrafish Reveals a Functional Role for Fibrinogen AαE in Early Hemostasis [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-genetic-modifier-of-venous-thrombosis-in-zebrafish-reveals-a-functional-role-for-fibrinogen-a%ce%b1e-in-early-hemostasis/. Accessed May 6, 2021.
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