The role of protein kinase C in the initial events of platelet activation by thrombin assessed with a selective inhibitor.

The role of protein kinase C (PKC) in platelet activation by thrombin was assessed using a PKC inhibitor Ro 31-7549/001 (R2) which, in vitro, shows more selectivity for PKC than other kinase inhibitors. During early (1.5 s) thrombin-induced platelet activation, when phosphorylation of 47 kDa protein (pleckstrin) and myosin light chain by PKC and myosin light chain kinase, respectively, are most readily differentiated, R2 suppressed phosphorylation of pleckstrin more effectively than myosin light chain. R2-inhibited dense granule secretion (measured 0-10 s using quenched-flow techniques) with a dose dependency similar to that for inhibition of pleckstrin phosphorylation, supporting a role for PKC in this process. R2, at 0.5 microM inhibited 47 kDa protein phosphorylation by more than 60%, but had only minimal effects on the kinetics (0-3s) of ADP-induced primary aggregation. At this same concentration, R2 potentiated the thrombin-induced rise in cytosolic calcium during early (0-15 s) activation as measured in the presence or absence of external calcium. These data support the hypothesis that activation of PKC during early platelet function helps regulate cytosolic calcium levels by limiting calcium release into the cytosol.