Synergy between GPVI and Gi leading to PKD activation.
Indomethacin (10 μM)–treated platelets were resuspended to a density of 1 × 109/mL. These were then split into 2, and half were pretreated with apyrase (2 U/mL). (A) Platelets minus apyrase were then stimulated with threshold concentrations of convulxin (cvx; 2 nM) or ADP (1 μM) in the presence of the P2Y1 receptor antagonist (1 mM A3P5P) and the agonist combination. Similarly, platelets treated with apyrase were activated by threshold concentrations of convulxin (Cvx; 2 nM) or epinephrine (Epin; 10 μM) and both together. Aliquots were removed from each sample after 1 minute and subjected to Western blot analysis using pS916 antisera to determine PKD activity. One experiment representative of 3 is shown. (B) In parallel, PKD was immunoprecipitated from the remaining sample for an in vitro kinase assay to observe syntide-2 phosphorylation. Results are the mean ± SEM of 3 independent experiments, carried out in duplicate.
