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

Abstract

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.

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

“…We used this technique in fibrinogen mutants to test whether the absence of fibrinogen alters the TTO. As we reported previously [ 35 ] and in similar mutants [ 27 ], injury in fga −/− larvae did not lead to occlusion ( Figure 1 B,C). Of the larvae supporting occlusion the mean TTO was 72 s in fga +/+ and 114 s in fga +/− animals, respectively ( Figure 1 B).…”

“…Afibrinogenemic larvae ( fga −/− ) failed to support venous thrombosis, as described previously [ 27 , 35 ], and had markedly reduced thrombocyte binding and accumulation at sites of injury, with aggregates showing a tendency to embolize. In preparing a zebrafish line designed to model a dysfibrinogenemia-causing missense mutation, we detected fga transcripts with exon 2 skipping and an mRNA encoding an Aα chain resembling a fibrinogen found in hypodysfibrinogenemia [ 9 ].…”

“…This lower level may explain our inability to detect the AαE chain in fga Δ 19–56/ Δ 19–56 plasma. In contrast to the situation in larvae [ 35 ], the Aα chain is detected more abundantly than the AαE chain in adult zebrafish plasma (RJF, MNA, unpublished). As this immunoblotting was intended to determine the presence or absence of mutated fibrinogen, sampling was not repeated, and relative band quantifications are not reported with n = 1.…”

“…Two minutes after laser injury, fga −/− larvae typically had few bound thrombocytes at the injury site and showed a clear tendency for embolism of clumps of fluorescent cells after initial adhesion at the injury site ( Figure 2 B). Thrombocyte aggregation data were reported previously for Tg(itga2b:EGFP) fga +/+ and fga −/− genotypes [ 35 ], but not compared to fga +/− . We made this replication in order to present the fga +/− larvae, the embolic phenotype seen in fga −/− , and to provide images ( Figure 2 B) and videos of the result that were not used previously.…”

“…In contrast to fga +/+ larvae from the AB background ( Figure 1 B) used for generating fga −/− , we recorded a relatively short TTO for all fga +/+ fish with the TU background (mean 23 s, Figure 5 A). This difference has been investigated in other work and relates to the ability to express the predominant larval fibrinogen alpha chain isoform (AαE) in larvae [ 35 ]. All fga +/ Δ 19–56 larvae also supported vessel occlusion after injury with a mean TTO of 29 s. A two-tailed unpaired t -test between TTOs measured for fga +/+ ( n = 17) versus fga +/Δ19–56 ( n = 42) gave a p -value of 0.361.…”