Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Abstract

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) 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 [14C]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 Ca(2+)-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 absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM 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-32P]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 microM 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 dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]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.

Protein kinase C-dependent and Ca2+-dependent mechanisms of secretion from streptolysin O-permeabilized platelets: effects of leakage of cytosolic proteins

“…The final concentration of SLO was 0.4 unit\ml and that of ATP was 4 mM. Additional EGTA was included to obtain a pCa value 9 (final concentration 12.5 mM) [5]. The compositions of Ca# + buffers and pCa values were calculated using the binding constants given in [22].…”

“…These studies have shown that the sensitivity of secretion to Ca# + is greatly enhanced by activation of PKC [4,5]. In addition, use of this experimental system has led to the recognition of at least two distinct roles for GTP-binding proteins in secretion from platelets [5], as found in experiments with other permeabilized cells [6]. Thus GTP or its more stable analogue, guanosine 5h-[γ-thio]-triphosphate (GTP [S]) is required for the G-protein-and Ca# + -dependent activation of phospholipase C in permeabilized platelets [7], which results in the stimulation of PKC by 1,2-diacylglycerol [8].…”

“…The mechanisms by which this is achieved have been analysed in some detail using electropermeabilized platelets in which the intracellular concentrations of Ca# + and other small molecules can be precisely controlled without disruption of the physical process of secretion [3]. These studies have shown that the sensitivity of secretion to Ca# + is greatly enhanced by activation of PKC [4,5]. In addition, use of this experimental system has led to the recognition of at least two distinct roles for GTP-binding proteins in secretion from platelets [5], as found in experiments with other permeabilized cells [6].…”

“…Thus GTP or its more stable analogue, guanosine 5h-[γ-thio]-triphosphate (GTP [S]) is required for the G-protein-and Ca# + -dependent activation of phospholipase C in permeabilized platelets [7], which results in the stimulation of PKC by 1,2-diacylglycerol [8]. Secondly, experiments with electropermeabilized platelets have shown that GTP[S] acts synergistically with PKC when this enzyme is stimulated by phorbol 12-myristate 13-acetate (PMA), to induce a Ca# + -independent secretion of granule constituents [5] ; this effect is associated with the activation of phospholipase D, but not of phospholipase C [8,9]. GTP[S] also potentiated Ca# + -induced secretion from permeabilized platelets after protein kinase activities had been Abbreviations used : PKC, protein kinase C ; SLO, streptolysin O ; 5-HT, 5-hydroxytryptamine ; PMA, phorbol 12-myristate 13-acetate ; GTP [S], guanosine 5h-[γ-thio]triphosphate ; ECL, enhanced chemiluminescence.…”

“…Thus results with electropermeabilized platelets have indicated that an optimal stimulation of secretion of 5-hydroxytryptamine (5-HT) from platelet dense granules or of β-thromboglobulin from α-granules can be achieved with any two of GTP [S], PMA and Ca# + [8]. In the latter work the role of PKC was readily defined by monitoring the phosphorylation of its major platelet substrate, pleckstrin [5,8].…”