Background: P2Y(12) plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity.
Objective: To characterize the receptor biology of the first reversibly binding oral P2Y(12) antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development.
Methods: Ticagrelor displayed apparent non-competitive or insurmountable antagonism of ADP-induced aggregation in human washed platelets. This was investigated using competition binding against [(3)H]ADP, [(33)P]2MeS-ADP and the investigational CPTP compound [(125)I]AZ11931285 at recombinant human P2Y(12). Functional receptor inhibition studies were performed using a GTPgammaS-binding assay, and further binding studies were performed using membranes prepared from washed human platelets.
Results: Radioligand-binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y(12) with K(on) and K(off) of (1.1 +/- 0.2) x 10(-4) nm(-1) s(-1) and (8.7 +/- 1.4) x 10(-4) s(-1), respectively. Ticagrelor does not displace [(3)H]ADP from the receptor (K(i) > 10 mum) but binds competitively with [(33)P]2MeS-ADP (K(i) = 4.3 +/- 1.3 nm) and [(125)I]AZ11931285 (K(i) = 0.33 +/- 0.04 nm), and shows apparent non-competitive inhibition of ADP-induced signaling but competitive inhibition of 2MeS-ADP-induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non-competitive binding for ADP and ticagrelor.
Conclusions: These data indicate that P2Y(12) is targeted by ticagrelor via a mechanism that is non-competitive with ADP, suggesting the existence of an independent receptor-binding site for CPTPs.