Venous thromboembolism (VTE) in women taking oral contraceptives (OC) is a problem of broad clinical significance that is mediated, at least in part, by estrogen effects on the expression of blood coagulation proteins by the liver or vasculature. Our overall goal is to identify and characterize the underlying coagulation pathways leading to OC-induced hypercoagulability and to identify genetic factors prevalent in individuals at highest risk for VTE. OC use decreases the plasma concentration of two endogenous anticoagulant proteins, tissue factor pathway inhibitor (TFPI) and protein S (PS) that modulate plasma FIXa production and activity. We have developed a highly sensitive and specific (<10 pmol/L) technique for measurement of plasma FIXa activity using an enhanced thrombin generation assay, which amplifies the FIXa signal from subject plasma via thrombin generation in FIX-deficient plasma. Using this assay in plasma samples from blood donors we found that OC-induced changes in TFPI and PS are associated with elevated plasma factor IXa (FIXa) activity in ~30% of women taking OC. Our central hypothesis is that OC use alters the inhibition of procoagulant responses by the natural anticoagulants TFPI and PS, producing large increases in plasma factor IXa (FIXa) activity and a systemic hypercoagulable state. This hypothesis will be probed to identify biochemical and genetic correlates of OC-induced hypercoagulability. Biochemical studies using recombinant FIX variants resistant to antithrombin or PS binding will be used to define how TFPI and PS modulate FIXa generation in human plasma-based systems. The clinical impact of the biochemical findings will be examined by measurement of plasma levels of TFPI, PS, and FIXa, GWAS analysis, and a thrombosis history questionnaire in two cohorts of pre-menopausal women: 1) a cross-sectional cohort of ~1000 pre-menopausal blood donors; and 2) a longitudinal cohort of ~45 adolescent girls to be followed for one year after initiation of OC-therapy. The data collected in the cross-sectional cohort will be modeled to identify non-genetic and genetic factors that correlate with OC-associated differences in plasma TFPI, PS and FIXa and thrombosis history. The data collected in the longitudinal cohort will be used to examine the time course and stability of the OC-induced plasma hypercoagulable state within individuals using repeated measures analysis. These findings will be used to validate plasma protein changes and the effect of SNPs identified by GWAS in the cross-sectional cohorts. The proposed experiments will define the biochemical and genetic underpinnings of the OC-induced hyper- coaguable state focused on our finding of elevated amounts of an activated clotting factor, FIXa, in the plasma of about 30% of women using OC. These results will also establish rationale for further clinical studies to define the predictive value of plasma FIXa and associated genetic markers for OC-induced thrombosis and facilita...