Electropermeabilized human platelets containing 5-hydroxy[C-14]tryptamine ([C-14]5-HT)1 were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [C-14]5-HT and beta-thromboglobulin (beta-TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log ]Ca2+ free[ (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta-TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta-TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca2+ - dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP-gamma-S had distinct effects in the absences of Ca2+ (pCa > 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta-TG, whereas 100-mu-M GTP-gamma-S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP-gamma-S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-P-32]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100-mu-M GTP-gamma-S was approximately 50% as effective as PMA; neither GTP-gamma-S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP-gamma-S exerted additional stimulatory effects on secretion from both denses and alpha-granules that were not mediated by PKC. Measurement of ]H-3[inositol phosphate formation in permeabilized platelets containing ]H-3[phosphoinositides showed that GTP-gamma-S did not stimulate phosphoinositide-specific phospholipase C in the absence of CA2+. It follows that in permeabilized platelets, GTP-gamma-S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.
