AJP - Cell Physiology, Vol 256, Issue 1 C35-C43, Copyright © 1989 by American Physiological Society
Response of aequorin-loaded platelets to activators of protein kinase C
J. A. Ware, M. Saitoh, M. Smith, P. C. Johnson and E. W. Salzman
Department of Medicine, Harvard-Thorndike Laboratory, Boston, Massachusetts.
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.
