Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa

Abstract

AbstractEP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (kon > 107M−1s−1), a high affinity (KI = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (KD = 30nM) and activated the serpin to rapidly (kass ∼ 106M−1s−1) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (∼ 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone.