Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor

Ticagrelor is a potent antagonist of the P2Y(12) receptor (P2Y(12)R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5'-diphosphate (ADP)-induced Ca2+ release in washed platelets vs other P2Y(12)R antagonists. This additional effect of ticagrelor beyond P2Y(12)R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of G(s)-coupled adenosine A(2A) receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y(12)R signaling, and this was validated in 1321N1 cells stably transfected with humanP2Y12R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of theP2Y(12)R, limiting basalGi-coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca2+ release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of plateletENT1 and inverse agonism at the P2Y(12)Rthat contribute to its effective inhibition of platelet activation.