P2Y12 receptor blockade potentiates the anti-platelet effects of prostacyclin and nitric oxide

Nicholas S. Kirkby1,2, Rachit Singhal1, Jane A. Mitchell2, Timothy D. Warner1. 1William Harvey Research Institute, Barts & the London School of Medicine, London EC1M 6BQ, United Kingdom, 2National Heart & Lung Institute, Imperial College, London SW3 6LY, United Kingdom.

ADP is an important mediator of secondary platelet aggregation, which acts via P2Y1 and P2Y12 receptors, the target of the thienopyridine anti-platelet drugs (clopidorel). P2Y12 activation inhibits adenylate cyclase and facilitates aggregation by reducing inhibitory cAMP. In vivo, prostacyclin (PGI2) increases cAMP, which, both directly, and in synergy with NO-stimulated cGMP, serves to supress platelet reactivity. We hypothesised that blockade of P2Y12 may exert its anti-platelet effect in part by sensitising platelets to the inhibitory effects of PGI2 and NO.

Blood was collected from healthy human volunteers (n = 5) and platelets isolated by centrifugation. Platelets were washed with modified Tyrodes buffer and treated prasugrel-active metabolite (PAM; 3uM; P2Y12 antagonist) or vehicle. Light transmission aggregometry was used to determine reponses to thrombin (0.01-1U/ml) and the ability of PGI2 (0.3-100nM) and DEA/NONOate (0.1nM-10uM) to inhibit thrombin (1U/ml)-induced aggregation. In parallel, washed platelets treated with PAM or vehicle, were incubated with PGI2 or DEA/NONOate with or without thrombin, and [cAMP] and [cGMP] measured. Aggregation data were analysed by t-test and [cAMP] and [cGMP] data by 1-way ANOVA.

P2Y12 blockade reduced sensitivity but not maximal responses to thrombin (Emax vehicle: 59.2±1.5%, PAM: 61.3±4.0%; p = 0.635). PGI2 caused concentration-dependent inhibition of thrombin-induced aggregation (-logEC50: 8.07±0.06), which was potentiated by PAM (-logEC50: 8.96±0.05; p<0.0001). PGI2 increased platelet cAMP content (Emax: 5.09±0.39pmoles) and this was reduced by thrombin (Emax: 3.03±0.29pmoles; p<0.05). In the presence of PAM, the effects of PGI2 on cAMP were augmented (Emax: 7.40±0.75pmoles) and were not altered by thrombin (Emax: 7.63±0.66pmoles; p<0.05). DEA/NONOate also inhibited platelet aggregation (-logEC50: 7.00±0.29) and this was enhanced in the presence of PAM (-logEC50: 8.31±0.17; p = 0.0052). [cGMP] was increased by DEA/NONOate, but this was not altered by thrombin or PAM. Similar aggregation data were obtained in platelet-rich plasma using 96 well plate aggregometry.

P2Y12 blockade potentiates the inhibitory effects of PGI2 on platelet aggregation, probably by preventing agnoist-induced reductions in [cAMP]. P2Y12 blockade enhances the anti-platelet effects of DEA/NONOate to a similar extent but this is not accompanied by changes in [cGMP]. As such, this may reflect synergy between NO-induced cGMP and the increased levels of cAMP that follow P2Y12 blockade.