Hormone-induced thrombosis is mediated through non-canonical fibrin(ogen) aggregation and a novel estrogen target in zebrafish.

Venous thrombosis is a well-known complication of sex hormone therapy, with onset typically within weeks to months after initiation. Worldwide, more than 100 million pre-menopausal women use combined oral contraceptives, with tens to hundreds of thousands developing thrombosis annually, resulting in significant morbidity and mortality. Although it is known that estrogens can alter expression of coagulation factors, the pathways and mechanisms that connect the two systems, as well as the proteins involved in progression to thrombosis, are poorly understood. Identification of these mediators are central to any comprehensive understanding of hormone-induced pathophysiology, could help ascertain patients at higher risk for thrombosis, and may also pinpoint future therapeutic targets. The zebrafish is a powerful genetic model in which the hemostatic system is almost entirely conserved with humans. Its external development, ability to generate thousands of offspring at low cost, and optical transparency all make it a powerful tool to study the genetics of coagulation disorders. We previously produced a transgenic line () that generates GFP-tagged fibrinogen that labels induced and spontaneous fibrin-rich thrombi. Here we show rapid onset of thrombosis after exposure to various estrogens, but not progestins or testosterone. Thrombi are localized to the venous system, develop broadly along the posterior cardinal vein, and show evidence for clot contraction. Thrombosis is only partially impeded by anticoagulants, occurs in the absence of factor X and prothrombin, but is completely blocked in the absence of fibrinogen. Furthermore, although an estrogen receptor antagonist is partially inhibitory, targeted knockout of all known estrogen receptors does not eliminate thrombosis. These data suggest that zebrafish can be used to model human estrogen-induced thrombosis, although the lack of dependence on the canonical coagulation cascade is surprising. The inability to completely inhibit thrombosis through genetic/pharmacologic anticoagulation or estrogen receptor disruption suggests that the mechanisms may be multifactorial. We hypothesize that thrombi are composed of fibrin(ogen) aggregates rather than purely fibrin. Results of further studies could lead to novel therapeutic targets and ascertain patients at higher risk for thrombosis.