Venous thromboembolism (VTE) is the third leading cause of cardiovascular mortality in the U.S., affecting ~900,000 individuals and causing up to 100,000 deaths annually. VTE arises from both inherited and acquired risk factors, such as surgery, cancer, trauma, and exogenous hormone exposure. Among these, estrogen-based therapies (e.g., combined oral contraceptives and hormone replacement therapy) are well-established contributors. Globally, over 100 million premenopausal women use combined oral contraceptives, leading to tens to hundreds of thousands of thrombotic events annually. Estrogens are thought to drive this risk, but the mechanisms remain poorly defined. Clarifying this link is key to improving risk stratification and developing safer, targeted interventions for hormone-associated VTE. Zebrafish offer a uniquely powerful in vivo model for studying thrombosis. Their hemostatic and coagulation systems are highly conserved with mammals, and their optical transparency, external development, and high fecundity enable real-time visualization and high-throughput thrombus analysis. Notably, zebrafish are the only robust in vivo system for modeling estrogen-induced thrombosis, as no similar mammalian model exists, and most findings are based on clinical studies. Previously, we have shown that estrogen exposure triggers rapid fibrin(ogen)-rich thrombosis in the zebrafish venous system when administered at 4 days post-fertilization (dpf) in a transgenic line visualizing GFP-tagged fibrinogen expressed under control of a liver-specific promoter. Fluorescent thrombi are evaluated at 5 dpf and all phenotypic assessments performed blinded to genotype and treatment, and statistical analysis conducted using ordinal logistic regression with a correction for multiple testing. To identify the relevant estrogen receptor, we knocked out all known estrogen-responsive nuclear hormone and G-protein-coupled receptors individually and in combination. The levels of thrombosis were unaffected, suggesting the existence of a novel receptor. We next determined the involvement of the coagulation cascade, by examining prothrombin (f2), factor X (f10), and factor VIII (f8) knockouts. Thrombosis persisted in all mutants despite that we have previously proven their involvement in induced thrombosis in response to endothelial injury. To rule out any residual thrombin activity, f2-/- larvae were pretreated with warfarin (10 μg/mL) beginning at 2 dpf, a regimen which we have previously shown to completely block induced thrombosis in wild-type larvae. Warfarin reduced thrombosis by ~50% in f2-/- larvae but also had the same effect on wild-type controls. Notably, in this model, thrombi appeared as widely dispersed speckles throughout the caudal vein, reminiscent of tissue factor (TF, f3 gene) distribution along the subendothelium, prompting us to assess the role of TF in estrogen-induced thrombosis. f3 is duplicated in zebrafish, and using our established knockout lines, we found that f3b-/- larvae exhibited ~45% reduction in fluorescent thrombus formation, whereas f3a-/- mutants showed no significant effect, identifying f3b as the predominant modulator in this context. However, double f3a/f3b mutants demonstrated a synergistic effect, with >80% reduction in thrombosis. Given that TF classically initiates coagulation through the formation of a complex with factor VII (FVII, f7 gene), and the warfarin effect above, we next tested whether its activity was required. Zebrafish possess three paralogs: f7, f7l, and f7i. We have previously shown that all procoagulant FVII activity is from the f7 and f7l loci, while others have shown that f7i is not procoagulant. Surprisingly, homozygous f7/f7l mutants displayed no detectable changes in estrogen-induced thrombosis at 5 dpf. In summary, we have found that estrogen-induced thrombosis is mediated through a novel estrogen receptor and via a noncanonical, FVII- and thrombin-independent mechanism that requires TF, bypassing both the extrinsic and common coagulation pathways. The lack of a thrombin effect suggests that estrogen-induced thrombosis may be due to fibrinogen rather than fibrin crosslinking, and points to a novel paradigm in this acquired thrombotic disease. Identification of the novel estrogen receptor, downstream pathways, and role of TF may discover new methods for prevention and treatment of affected patients.

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2025
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