Thrombin-induced platelet activation involves cleavage of protease-activated receptors (PARs) 1 and 4, and interaction, via glycoprotein (Gp)Ibalpha, with the platelet GpIb/IX/V complex. This study investigated inhibition of platelet activation by thrombin inhibitors with different modes of action: two reversible direct thrombin inhibitors, melagatran and inogatran; hirudin, a tightly binding direct thrombin inhibitor; and two indirect thrombin inhibitors, heparin and dalteparin. Up-regulation of P-selectin (CD62P) and PAR-1 cleavage was measured in human whole blood, by flow cytometry. The thrombin concentration that induced 50% of maximum (EC50 ) PAR-1 cleavage was 0.028 nmol/l, while that of platelet activation (CD62P) was over two-fold higher (0.64 nmol/l). The EC50 of a PAR-1-independent component, defined as a further activating effect of thrombin on top of the maximum PAR-1-activating peptide (AP) effect, was 3.2 nmol/l. All anticoagulants were concentration-dependent inhibitors of thrombin-induced platelet activation and PAR-1 cleavage, but none inhibited PAR-1-AP or PAR-4-AP induced activation. Melagatran and inogatran were more potent inhibitors of CD62P up-regulation than of PAR-1 cleavage; conversely, hirudin, heparin and dalteparin were more potent inhibitors of PAR-1 cleavage.Thus, reversible direct thrombin inhibitors, such as melagatran, are potent inhibitors of thrombin-induced platelet activation, acting mainly by inhibition of a PAR-1-independent component.