Background: HD1-22 is a bivalent aptamer that binds to thrombin with high affinity (K-d = 0.65 nm) and occupies both anion binding exosites without blocking the active centre of the enzyme. HD1-22 has been developed by connecting the exosite 1 binding aptamer HD1 and the exosite 2 binding aptamer HD22 through a poly-dA linker. Objectives: To characterize the anticoagulant profile of HD1-22 in comparison to the clinically established direct acting thrombin inhibitors bivalirudin and argatroban, and to test the efficacy of antidote-oligodeoxynucleotides. Methods and Results: HD1-22 prolongs clotting times of the thrombin time, activated partial thromboplastin time, ecarin clotting time, and lag-time of the tissue factor triggered thrombin generation assay in a dose-dependent manner. On a molar basis, its anticoagulant activity was nearly identical to bivalirudin and superior to argatroban. Thrombin-induced platelet aggregation was more effectively inhibited by HD1-22 than by bivalirudin. The HD1-22 aptamer retains the ability of the HD1-moiety to bind to (pro)exosite 1 of prothrombin and inhibits the prothrombinase activity nearly 2-fold better than HD1. The anticoagulant activities of HD1-22 are fully reversed by addition of antidote-oligodeoxynucleotides. Conclusions: The strong thrombin-inhibiting activity, together with the availability of a rapid acting antidote strategy, makes HD1-22 an interesting anticoagulant candidate, especially for use in clinical situations where effective anticoagulation and rapid reversal of the anticoagulant effect are required. The data obtained warrant further clinical studies.