Potentiation by thrombin of the secretion of serotonin from permeabilized platelets equilibrated with Ca2+ buffers. Relationship to protein phosphorylation and diacylglycerol formation.
After human platelets have been 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 [Knight & Scrutton (1980) Thromb. Res. 20, 437-446]. In the present study, platelets permeabilized by this technique were found to show 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 polypeptides (P47 and P20). Thrombin markedly increased the phosphorylation of P47 in the presence of 0.1-1.0 microM-Ca2+ free 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 microM-Ca2+ free and was even more effective with 1.0 microM-Ca2+ free, 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.
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002021:Buffers; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D004075:Diglycerides / Q000096:biosynthesis / Q000097:blood; D004558:Electric Stimulation; D006801:Humans; D066298:In Vitro Techniques; D010766:Phosphorylation; D012701:Serotonin / Q000097:blood*; D013917:Thrombin / Q000494:pharmacology*
C011439:1,2-diacylglycerol; D001798:Blood Proteins; D002021:Buffers; D004075:Diglycerides; D012701:Serotonin; D013917:Thrombin; D002118:Calcium