Thrombin and activators of protein kinase C modulate secretory responses of permeabilised human platelets induced by Ca2+

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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 [median effective concentration] for Ca2+. 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 were previously shown to mimic quantitatively the effect of thrombin on [14C]serotonin secretion induced by Ca2+. 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. 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 .mu.M Ca2+ to permeabilized platelets in the presence of [.gamma. 32P] ATP causes marked enhancement of 32P incorporation into polypeptides of molecular mass 20 kDA [kilodalton], 45 kDA and 66 kDA. No additional polypeptides become phosphorylated in this system when thrombin is added together with 10 .mu.M Ca2+, but some increase is observed in the extent of phosphorylation of the 45-kDa polypeptide. Addition of 1-oleyl-2-acetylglycerol+1-2 .mu.M 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. At 0.01 .mu.M Ca2+ addition of either thrombin or 1-oleyl-2-acetylglycerol in the presence of [.gamma.-32P]ATP causes enhanced phosphorylation of the 45-kDa polypeptide but not of the 20-kDa or 66-kDa polypeptides without significant release of [14C]serotonin. The results are consistent with the postulate that thrombin enhances [14C]serotonin secretion induced by Ca2+ in permeabilized platelets by increasing the level of 1,2-diacylglycerol and hence activating protein kinase C, but that this mechanism is not adequate to explain the enhancement of .beta.-N-acetylglucosaminidase secretion caused by addition of thrombin in this preparation.