Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.
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.
Coorssen
J R
JR
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Davidson
M M
MM
Haslam
R J
RJ
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010539:Permeability; D010703:Phorbol Esters / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D001798:Blood Proteins; D006150:Guanine Nucleotides; D010703:Phorbol Esters; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D011506:Proteins; C039077:platelet protein P47; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D013917:Thrombin; D009218:Myosins; D002118:Calcium