Estradiol-Responsive miR-365a-3p Interacts with Tissue Factor 3′UTR to Modulate Tissue Factor-Initiated Thrombin Generation

Background High estradiol (E (2) ) levels are linked to an increased risk of venous thromboembolism; however, the underlying molecular mechanism(s) remain poorly understood. We previously identified an E (2) -responsive microRNA (miR), miR-494-3p, that downregulates protein S expression, and posited additional coagulation factors, such as tissue factor, may be regulated in a similar manner via miRs. Objectives To evaluate the coagulation capacity of cohorts with high physiological E (2) , and to further characterize novel E (2) -responsive miR and miR regulation on tissue factor in E (2) -related hypercoagulability. Methods Ceveron Alpha thrombin generation assay (TGA) was used to assess plasma coagulation profile of three cohorts. The effect of physiological levels of E (2) , 10nM, on miR expression in HuH-7 cells was compared using NanoString nCounter and validated with independent assays. The effect of tissue factor-interacting miR was confirmed by dual-luciferase reporter assays, immunoblotting, flow cytometry, biochemistry assays, and TGA. Results Plasma samples from pregnant women and women on the contraceptive pill were confirmed to be hypercoagulable (compared with sex-matched controls). At equivalent and high physiological levels of E (2) , miR-365a-3p displayed concordant E (2) downregulation in two independent miR quantification platforms, and tissue factor protein was upregulated by E (2) treatment. Direct interaction between miR-365a-3p and F3- 3UTR was confirmed and overexpression of miR-365a-3p led to a decrease of (1) tissue factor mRNA transcripts, (2) protein levels, (3) activity, and (4) tissue factor-initiated thrombin generation. Conclusion miR-365a-3p is a novel tissue factor regulator. High E (2) concentrations induce a hypercoagulable state via a miR network specific for coagulation factors.