title: Thrombin and activators of protein kinase C modulate secretory responses of permeabilised human platelets induced by Ca2+.
abstract: Addition of thrombin enhances secretion of both [14C]serotonin and beta-N-acetylglucosaminidase induced by Ca2+ in human platelets rendered permeable by exposure to intense electric fields. Enhancement of beta-N-acetylglucosaminidase secretion by thrombin results from an increase in the maximal extent of the response with no significant change in the median effective concentration EC50 for Ca2+. In contrast, thrombin shifts the dose/response curve for Ca2+-induced [14C]serotonin secretion to the left and has little effect on the maximal extent of this response even when this extent is reduced by use of a non-saturating concentration of MgATP2-. The relationship between extent of response and [MgATP2-] is similar for secretion of [14C]serotonin and of beta-N-acetylglucosaminidase in the presence or absence of thrombin. Similar nucleotide specificities are also observed. Activators of protein kinase C have previously been shown to mimic quantitatively the effect of thrombin on [14C]serotonin secretion induced by Ca2+ [D. E. Knight & M. C. Scrutton (1984) Nature (Lond.) 309, 66-68]. Such activators have the same qualitative effect as thrombin on the properties of beta-N-acetylglucosaminidase secretion induced by Ca2+ but are less effective. The EC50 for thrombin observed for enhancement of [14C]serotonin and beta-N-acetylglucosaminidase secretion is in the same range as that obtained for intact platelets under comparable conditions [D. E. Knight, T. J. Hallam & M. C. Scrutton (1982) Nature (Lond.) 296, 256-257]. The EC50, and the specificity of response, observed for activators of protein kinase C in these systems are consistent with those reported previously for the purified enzyme. Addition of 1-10 microM Ca2+ to permeabilised platelets in the presence of [gamma-32P] ATP causes marked enhancement of 32P incorporation into polypeptides of molecular mass 20 kDA, 45 kDA and 66 kDA. No additional polypeptides become phosphorylated in this system when thrombin is added together with 10 microM Ca2+, but some increase is observed in the extent of phosphorylation of the 45-kDa polypeptide. Addition of 1-oleyl-2-acetylglycerol + 1 - 2 microM Ca2+ causes enhanced phosphorylation of the 45-kDa polypeptide and to a lesser extent of the 20-kDa polypeptide. The dose/response curves for Ca2+-dependent phosphorylation of the 45-kDa polypeptide in the presence and absence of 1-oleyl-2-acetylglycerol are similar to those observed for Ca2+-dependent [14C]serotonin secretion under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D004075:Diglycerides / Q000494:pharmacology; D004558:Electric Stimulation; D004789:Enzyme Activation / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000097:blood*; D012701:Serotonin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D004075:Diglycerides; D012701:Serotonin; C038237:1-oleoyl-2-acetylglycerol; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011493:Protein Kinase C; D000118:Acetylglucosaminidase; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium