ATP IS REQUIRED IN PLATELET SEROTONIN EXOCYTOSIS FOR PROTEIN-PHOSPHORYLATION AND PRIMING OF SECRETORY VESICLES DOCKED ON THE PLASMA-MEMBRANE

Calcium-evoked secretion generally requires the presence of millimolar concentrations of Mg-ATP. We investigated the role of Mg-ATP in the s ecretion of serotonin from electropermeabilized bovine platelets, The secretion of serotonin was lost within 5 minutes when the Mg-ATP conce ntration was diluted to less than 0.1 mM, but was maintained when ATP- gamma S (adenosine 5'-O-3-thiotriphosphate) was used instead of ATP, O kadaic acid, a potent inhibitor of protein phosphatase, could also mai ntain the exocytotic activity even when ATP was diluted, Decrease in t he secretory activity was paralleled by a decrease in phosphorylation level of four proteins after dilution of ATP, but the activity was mai ntained when the thiophosphorylation level of these proteins was maint ained, Two of these proteins were digested by a protease, calpain, whi ch has been shown to lead to a loss in the exocytotic activity, Electr on microscopic studies showed that calcium did not induce the formatio n of distinct bridge-like structures between the granule membrane and the plasma membrane in Mg-ATP-diluted cells, previously shown as the s tructure transiently formed prior to fusion of the two membranes. Anch orage of the secretory dense granules to the plasma membrane and the p resence of the amorphous structures between the granules and the plasm a membrane were unchanged by dilution of ATP, These results indicate t hat ATP is not required for the anchorage itself, but is required to p rime anchored granules for calcium-triggered secretion, Maintenance of the phosphorylated state of proteins by ATP enables the calcium trigg er to form the bridge-like structures preceding membrane fusion events .