Synergy between Gq- and Gi-coupled receptors to activate PKD.
(A) Washed platelets were resuspended to a concentration of 1 × 109/mL. Those activated with ADP or ADP receptor antagonists were treated with the cyclooxygenase (COX) inhibitor indomethacin (10 μM) and those stimulated with U46619 or epinephrine were treated with the ADP scavenging enzyme apyrase (2 U/mL). Platelets were stimulated with threshold concentrations of (i) U46619 (0.1 μM), epinephrine (10 μM), and both agonists together, or maximum concentrations of (ii) ADP (20 μM)+ 1 μM AR-C67085 (ARC), ADP (20 μM)+ 1 mM A3P5P (A3P) and ADP (20 μM) alone for 1 minute. Western blot analysis was performed using pS916 following lysis of cells with 2 × sample buffer. Lysates from cells stimulated for 1 minute with 30 nM PMA were used as a positive control. Results show the mean ± SEM of results from 3 different donors. (B) Platelets were lysed and PKD activity was assayed by in vitro kinase assay (as described in “Materials and methods”). Results are the mean ± SEM of 3 separate experiments, and each sample was in duplicate.
