Dabigatran attenuates the binding of thrombin to platelets - a novel mechanism of action.

Abstract

Thrombin is a multifunctional regulatory enzyme of the hemostasis and has both pro- and anticoagulant roles. It has, therefore, been a main target for drug discovery over many decades. Thrombin is a serine protease and possesses two positively charged regions called exosites, through which it is known to bind to many substrates. Dabigatran is a thrombin inhibitor and is widely used as an oral anticoagulant for the antithrombotic treatment of atrial fibrillation and venous thromboembolism. The mechanism by which dabigatran inhibits thrombin is the blockage of the active site, however, its effect on thrombin binding to its substrates has not been studied thoroughly and is thus poorly understood. We have discovered that apart from blocking the active site, dabigatran also inhibits thrombin binding to platelets, which is an important step in the hemostasis. This was achieved by evaluating the binding of fluorescently labelled thrombin to platelets by flow cytometry. Further, to confirm the results we utilized modern techniques for biomolecular binding studies, microscale thermophoresis combined with SPR, which validated the results. The inhibition of thrombin binding to its substrates in flow cytometry was dose dependent with IC50 of 118 nM which was slightly lower than for inhibition of platelet activation and is close to the clinically relevant plasma concentration of dabigatran. Since thrombin has numerous functions beyond the cardiovascular system, this finding may have important implications.