Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one-pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against alpha-thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg(-1). The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions, in addition to an active site.