Platelets become activated by multiple stimuli, one of these being ADP. ADP acts on two platelet GPCRs, P2Y₁ which is coupled to Gq, and P2Y₁₂ which is coupled to Gi. It has been generally accepted that P2Y₁ is solely responsible for the rise in cytosolic calcium elicited by ADP. However, recently controversy has appeared surrounding the role of P2Y₁₂ in the previously accepted P2Y₁-mediated rise in cytosolic calcium. We have clarified the role of P2Y₁₂ in the rise in cytosolic calcium induced by ADP.

Human platelets were isolated from drug-free volunteers on the day of experiment and prepared as previously described (Crosby & Poole, 2003). For measurements of cytosolic calcium, platelets were loaded with Fura 2-AM. For all experiments stimulations were made at 30° C in the presence of 1 mM EGTA to block platelet aggregation, other than measurements of intracellular calcium where stimulations were made at room temperature in the absence of EGTA. For measurements of cAMP accumulation, stimulations were made using 10 µM ADP for 5 minutes in the presence of 10 µM Forskolin and 100 µM IBMX.

The peak calcium response elicited by 10 µM ADP is inhibited by the structurally unrelated specific P2Y₁₂ antagonists AR-C69931MX (1 µM; ARC) and 2MeSAMP (10 µM) by 54 ± 4.9% and 39 ± 2.9% respectively, with no significant increase in the EC₅₀ of ADP (the EC₅₀ for ADP is 2.5 ± 1x10^-7 M versus 6.1 ± 2x10^-7 M in the presence of ARC). Additionally, we were able to reveal a calcium response dependent upon P2Y₁₂ potentiation when P2Y₁ was submaximally inhibited. 200 µM A3P5P, a concentration of the specific P2Y₁ antagonist which caused a 21 +/- 2.6% reduction in the peak calcium response to ADP, caused complete ablation of the calcium response when combined with maximal P2Y₁₂ antagonism (1 µM ARC). This indicates that submaximal inhibition of P2Y₁ with 200 µM A3P5P yields a calcium response dependent upon P2Y₁₂ stimulation and provides a useful means of studying the P2Y₁₂-mediated potentiatory pathway. Using this method, we showed that this pathway is mediated by PI3-Kinase and the inhibition of adenylate cyclase.

Interestingly, the selective Src kinase inhibitor PP1 (20 µM) potentiated the P2Y₁₂-mediated calcium response (an increase of 46 ± 6 %, p<0.01), but had no effect on the P2Y₁-mediated calcium response (p=0.99) or the maximum calcium response (p>0.1). In addition, PP1 also had no effect on the P2Y₁₂-mediated cAMP response (p=0.93). Using an in vitro kinase assay, we demonstrated that P2Y₁ selectively activates Src kinase (an increase of 57 ± 4.5% above basal, p<0.01).

Taken together, these data indicate that there is a reciprocal interplay between the two ADP receptors at the level of calcium signalling which we hypothesise may help contribute to the delicate balance between rest and activation of circulating platelets.

Crosby, D & Poole, AW (2003) J Biol Chem 278, 24533-24541