Response of aequorin-loaded platelets to activators of protein kinase C

The protein kinase C activators phorbol ester 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (DAG) cause platelet aggregation, secretion, and a rise in aequorin-indicated cytoplasmic Ca2+ ([Ca2+]i), but the importance of this action to platelet activation by these agonists has not been established. We found that the previous addition of PMA or DAG either enhanced or inhibited the platelet response if thrombin was subsequently added, depending on the latter's concentration. The effects of PMA or DAG on the response to thrombin were obtained only if the agonists were added in concentrations sufficient to elevate [Ca2+]i themselves. A [Ca2+]i rise also occurred after the second agonist (thrombin), but its magnitude did not necessarily correlate with subsequent aggregation, secretion, or the activation of protein kinase C as reported by the phosphorylation of a 47-kDa protein (p47). The protein kinase C inhibitor sphingosine inhibited aggregation and p47 phosphorylation caused by PMA or DAG alone or with thrombin, but the [Ca2+]i rise in response to the first agonist was not affected. PMA-induced aggregation and p47 phosphorylation were inhibited by quin2, which also inhibited protein kinase C activity in a cell-free system. We conclude that a rise in aequorin-indicated [Ca2+]i is necessary for PMA or DAG to activate platelets or to alter the subsequent platelet response to thrombin; this [Ca2+]i rise may be a prerequisite for activation of protein kinase C.