P2Y12 component of the calcium response is mediated through PI 3-kinase and inhibition of cAMP. (A) Platelets were preincubated with LY294002 (LY; 10 μM) or vehicle as control for 25 minutes and stimulated with ADP (10 μM) or thrombin (0.1 U/mL) for 3 minutes, as indicated. Reactions were stopped by addition of an equal volume of Laemmli sample solvent, and proteins from whole-cell lysates (WCLs) were separated by SDS-PAGE and blotted using the phospho-peptide-specific anti-pThr308 PKB antibody. (B-C) Fura 2-loaded platelets were incubated for 25 minutes with or without LY294002 (10 μM), followed by incubation for 5 minutes with a submaximal concentration of A3P5P (200 μM; B-C) or AR-C69931MX (1 μM; B), as indicated. Platelets were then stimulated with ADP (10 μM), and 340:380-nm fluorescence ratios were plotted (B) or the peak rise in cytosolic calcium was calculated and represented as a bar graph (C). For panel B, data are representative of 3 separate experiments and for panel C, data shown are mean ± SEM (n = 3). (D) Platelets were pretreated for 5 minutes with AR-C69931MX and various concentrations of the adenylate cyclase inhibitor SQ22536 for 25 minutes. The peak rise in cytosolic calcium in response to ADP (10 μM) is plotted against log concentration of SQ22536. Data shown are mean ± SEM (n = 3).
