P2Y₁, but not P2Y₁₂, activates Src kinase. (A) Platelets were prepared with or without indomethacin pretreatment for 10 minutes as indicated. Platelets were then preincubated for 1 minute with or without EGTA (1 mM) and stimulated for a further 3 minutes with 10 μM ADP. Proteins were separated by SDS-PAGE and blotted with phospho-peptide-specific anti-Tyr416 Src antibody. IP indicates immunoprecipitate; IB, immunoblot; and coll, collagen. (B) Src was immunoprecipitated from basal platelets or platelets incubated for 1 minute with EGTA (1 mM) and stimulated for a further 3 minutes with 10 μM ADP. Some platelets had been preincubated for 5 minutes with A3P5P (1 mM), AR-C69931MX (1 μM), or both inhibitors, as indicated. Immunoprecipitates were incubated with the exogenous tyrosine kinase substrate, Raytide, and incorporation of ³²P into Raytide was measured by liquid scintillation counting. Data shown are mean ± SEM (n = 3). (C) This diagram summarizes the data presented. Although P2Y₁ is absolutely required for induction of a calcium response, P2Y₁₂ is able to contribute to this response through 2 mechanisms: activation of PI3K and lowering of cellular cAMP levels. P2Y₁₂ thus feeds forward on the P2Y₁-mediated calcium response. On the other hand, P2Y₁ activates Src kinase, which negatively regulates the PI3K component of the P2Y₁₂-mediated calcium response. P2Y₁ thus negatively feeds back on this part of the P2Y₁₂-mediated response, although not upon the cAMP component of the P2Y₁₂ signaling pathway. This demonstrates an intricate level of feedback cross-talk between these 2 receptors at the level of calcium signaling in platelets.