Background: The G-protein-coupled P2Y12 -receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y12 -receptor antagonist.
Objective: The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y12 -receptors.
Methods: Recombinant wild-type or mutant human P2Y12 -receptors were stably expressed in Chinese hamster ovary Flp-In cells. Receptor function was assessed by quantification of ADP- and 2-methylthio-ADP-mediated inhibition of forskolin-induced cellular cAMP production either using a [(3) H]cAMP-radioaffinity assay or a cAMP response element-driven luciferase reporter gene assay.
Results: The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y12 -receptor with a lower potency (EC50 209 nm) than the synthetic agonist 2-methylthio-ADP (EC50 1.0 nm). Ticagrelor shifted the concentration-response curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA2 values of 8.85 for ticagrelor against ADP, and 8.69 against 2-methylthio-ADP, and slopes of the regression lines not different from unity. In cells expressing a recombinant C194A(5.43) -mutant P2Y12 -receptor construct, ticagrelor lost antagonistic potency when tested against ADP or 2-methylthio-ADP.
Conclusions: The experiments reveal a surmountable and competitive mode of antagonism of ticagrelor at P2Y12 -receptors activated by either the natural agonist ADP or the synthetic agonist 2-methylthio-ADP. Cys194(5.43) is likely to be involved in the interaction of ticagrelor with ADP and 2-methylthio-ADP. The data give new insights into the site and mode of action of ticagrelor at the human P2Y12 -receptor.
Keywords: binding site; mode of action; platelets; receptors, purinergic P2Y12.
© 2014 International Society on Thrombosis and Haemostasis.