Hemophilia A and B are inherited hemorrhagic disorders that result from alterations in the coagulation cascade. Aside from spontaneous bleeding, the main complication of hemophilia is hemarthrosis. Progress over the last three decades, specifically prophylaxis using recombinant factor, has prevented hemarthrosis and lengthened patient life expectancies. However, many treatments require frequent dosing up to three times a week, and alloantibodies (inhibitors) against replacement factor continues to be an issue. These problems call for novel treatments for patients with hemophilia. Although there has been progress in extended half‐life factors and mimetics of factor VIII, an alternative treatment methodology is to rebalance the activities of pro‐ and anticoagulant factors through inhibition of the natural anticoagulants: antithrombin, tissue factor pathway inhibitor, protein C, and protein S. This review will explore the efficacy of targeting these inhibitory pathways from preclinical development through clinical trials, and delve into concerns of thrombotic risk.

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.

Ovulation is a remodeling process including blood capillary rupture and coagulation. Until now, there is no regulation and functional studies of coagulation factors in ovulation. Here, we report dramatic increases of coagulation factors (f5, f3a) in zebrafish preovulatory follicles. This upregulation was induced by progestin (DHP: 17α, 20β-dihydroxy-4-pregnen-3-one), a native ligand for nuclear progestin receptor (Pgr) that is essential for ovulation in zebrafish; but was abolished in pgr-/-. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin via zebrafish Pgr. Similarly, we found promoter activities of human F5 were significantly stimulated by progesterone (P4) via human PGRB. Moreover, a dramatic increase of erythrocyte numbers in capillaries on ovarian follicles was associated with ovulation. Importantly, heparin, an anticoagulant, inhibited ovulation. Furthermore, reduced fecundity and impaired ovulation were observed in f5+/- female zebrafish. Together, our results provide plausible evidence for an exceptional function of coagulation factors in ovulation.Significance StatementPrecise activation of metalloprotease is essential for ovulation. This activation is controlled by serum-derived protease inhibitors. However, the association between coagulation factors and ovulation is unclear. We demonstrated that nuclear progestin receptor (Pgr), a well-established initiator for ovulation, was a major regulator for a dramatic increase of coagulation factors (f5 & f3a) in preovulatory follicles of zebrafish. Blood coagulation in the capillaries of preovulatory follicles occurred prior to follicular rupture in wildtype zebrafish. Interestingly, this blood coagulation was blocked in Pgr knockout fish, which can’t ovulate. Inhibition of blood coagulation significantly reduced ovulation. Decreased fertility was also found in F5 heterozygous zebrafish. Our findings reveal critical roles of steroid receptor-regulated coagulation factors in blood coagulation that controls ovulation.

Tissue factor (TF) is an evolutionarily conserved protein necessary for initiation of hemostasis. Zebrafish have two copies of the tissue factor gene (f3a and f3b) as the result of an ancestral teleost fish duplication event (so called ohnologs). In vivo physiologic studies of TF function have been difficult given early lethality of TF knockout in the mouse. We used genome editing to produce knockouts of both f3a and f3b in zebrafish. Since ohnologs arose through sub- or neofunctionalization, they can unmask unknown functions of non-teleost genes and could reveal whether mammalian TF has developmental functions distinct from coagulation. Here we show that a single copy of either f3a or f3b is necessary and sufficient for normal lifespan. Complete loss of TF results in lethal hemorrhage by 2–4 months despite normal embryonic and vascular development. Larval vascular endothelial injury reveals predominant roles for TFa in venous circulation and TFb in arterial circulation. Finally, we demonstrate that loss of TF predisposes to a stress-induced cardiac tamponade independent of its role in fibrin formation. Overall, our data suggest partial subfunctionalization of TFa and TFb. This multigenic zebrafish model has the potential to facilitate study of the role of TF in different vascular beds.