Dual roles of proton pump inhibitors in estrogen-induced venous thromboembolism Free

Venous thromboembolism (VTE), including deep vein thrombosis and pulmonary embolism, is the third leading cause of cardiovascular mortality in the U.S., affecting approximately 900,000 individuals and causing up to 100,000 deaths annually. VTE arises from both inherited (e.g., protein C (PROC) deficiency) and acquired (e.g., surgery, cancer, elevated estrogen from hormone therapy or contraceptives) factors. Current therapies such as warfarin and direct oral anticoagulants (DOACs) carry significant bleeding risks (over 7 per 100 patient-years) and can be fatal. DOACs now account for nearly 40% of oral anticoagulant-related emergency visits, highlighting that there is still a need for safer, more targeted interventions. In previous zebrafish FDA-repurposing drug screens for mitigation of estrogen-induced thrombosis, we have found that proton pump inhibitors (PPIs) were antithrombotic at 5 micromolar (μM) concentrations. However, our examination of two distinct electronic health record (EHR) databases revealed increased thrombotic events in patients co-prescribed PPIs and hormonal contraceptives. To address this, we leveraged zebrafish, which shares highly conserved hemostatic pathways with humans. Zebrafish larvae are well-suited for drug repurposing studies due to rapid external development, optical transparency, and high fecundity, enabling real-time, in vivo analysis of thrombosis. Zebrafish also recapitulate both inherited (e.g., PROC-deficiency) and acquired (e.g., estrogen-induced) thrombosis, allowing for direct comparison of mechanistically distinct modalities. To test PPI-associated thrombotic risk, transgenic zebrafish expressing GFP-tagged fibrinogen under control of a liver-specific promoter were treated at 3 days post-fertilization (dpf) with a range of PPI concentrations, from clinically relevant nanomolar (nM) levels to higher μM doses. Estrogen treatments were performed at 4 dpf to induce thrombosis in wild-type larvae. Additionally, PPIs were examined in the proc mutant background, which develops spontaneous thrombosis. Thrombosis phenotypes were assessed at 5 dpf in the venous system by visualization of fluorescent thrombi, and all analyses were blinded and evaluated using ordinal logistic regression with correction for multiple testing. PPIs showed biphasic, dose-dependent effects in the acquired model, with clinically relevant concentrations increasing thrombosis levels on average 25%, while higher levels (>5 μM) reduced thrombosis by ~15%, suggesting involvement of off-target or indirect mechanisms. This explains our paradoxical EHR findings. In contrast, the prothrombotic effect of PPIs was absent in the inherited model of PROC deficiency, highlighting differential drug responses and suggesting drug-gene interactions. To test whether PPIs act via the V-type ATPase proton pump, we studied zebrafish orthologs of ATP6V1A, a catalytic subunit of the complex. Inhibitors (bafilomycin A1 and concanamycin A) enhanced estrogen-induced thrombosis by ~55%, with no effect in proc mutants, mirroring the response seen with nM-to-μM PPI concentrations. CRISPR-mediated knockdown of atp6v1a similarly increased thrombosis by ~70% from baseline, identifying ATP6V1A as a key modifier and likely mediator of PPI-induced prothrombotic effects. In summary, these studies serve to reconcile conflicting preclinical and clinical data, showing that PPIs have biphasic, concentration-dependent effects on thrombosis and at standard dosing may increase risk in patients on estrogen. These findings highlight the importance of accounting for both drug concentration and genetic context in thrombosis risk. ATP6V1A is a mechanistic candidate for future study and potential risk stratification in estrogen-induced thrombosis. Further study of higher antithrombotic PPI concentrations is needed to determine if they can be a safe therapeutic and preventive modality for VTE.