Potentiation by thrombin of the secretion of serotonin from permeabilized platelets equilibrated with Ca2+ buffers. Relationship to protein phosphorylation and diacylglycerol formation

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Abstract
After human platelets are rendered permeable to small molecules by high voltage electric discharges, addition of buffered micromolar concentrations of Ca2+ causes an ATP-dependent secretion of dense granule serotonin. Platelets permeabilized by this technique showed an up to 10-fold increase in their sensitivity to Ca2+ after exposure to thrombin. In permeabilized platelets, as in the intact cells, release of serotonin was associated with the Ca2+-dependent phosphorylation of 47,000 and 20,000 Da [dalton] polypeptides (P47 and P20). Thrombin markedly increased the phosphorylation of P47 in the presence of 0.1-1.0 .**GRAPHIC**. but had a much smaller effect on phosphorylation of P20. Thrombin also stimulated the formation of 1,2-diacylglycerol in the presence of 0.1 .**GRAPHIC**. and even more effective with 1.0 .**GRAPHIC**. suggesting that receptor-activated hydrolysis of phosphoinositides to 1,2-diacylglycerol was preserved in permeabilized platelets and was potentiated by low intracellular concentrations of Ca2+. The increase in phosphorylation of P47 on addition of thrombin may therefore be accounted for by the stimulatory action of 1,2-diacylglycerol on Ca2+-activated, phospholipid-dependent protein kinase. However, in both the presence and absence of thrombin, higher Ca2+ concentrations were required for optimal secretion than for maximal phosphorylation of both P47 and P20, indicating that additional actions of Ca2+ and thrombin, perhaps also mediated by 1,2-diacylglycerol formation, may be involved in the release of serotonin.