Signalling components underlying platelet aggregation to a Ca2+ ionophore and a phorbol ester

Although it is well established that G(q)- and G(i)-coupled receptors can c ombine to mediate platelet aggregation, the signalling events underlying th e synergy are not fully characterised. This study has used the calcium iono phore, A23187, and phorbol ester, PMA, to investigate this question. We sho w that aggregation to submaximal but not maximal concentrations of ionophor e is partially inhibited by antagonism of the P2Y(12) ADP receptor or PKC b lockade. However, a full aggregation response can be restored under these c onditions by addition of PMA or ADP. Studies using PI 3-kinase inhibitors d emonstrate that this is the second messenger pathway that restores aggregat ion by the G(i)-coupled receptor in the presence of PKC blockade. However, under normal circumstances, PI 3-kinase activity is not a major requirement for aggregation to the ionophore. PMA stimulates a weak aggregation which takes several minutes to reach a maximum. Threshold concentrations of PMA a nd a G(i)-coupled receptor agonist when added alone show no effect on aggre gation, but when combined induce aggregation responses. This study demonstr ates that calcium and PKC interact synergistically with a G(i) coupled rece ptor agonist to mediate aggregation, and also with each other. Activation o f G(i) supports aggregation in part through the PI 3-kinase pathway. High c oncentration of ionophore on their own can induce aggregation independent o f PKC and activation of G(i). Multiple signalling pathways mediate platelet aggregation and their relative importance depends on experimental conditio ns.