CALPAIN-INDUCED DOWN-REGULATION OF PROTEIN-KINASE-C INHIBITS DENSE-GRANULE SECRETION IN HUMAN PLATELETS - INHIBITION OF PLATELET-AGGREGATION OR CALPAIN ACTIVITY PRESERVES PROTEIN-KINASE-C AND RESTORES FULL SECRETION

The relationship between platelet aggregation, calpain activation, PKC activities and the secretory response have been examined in PMA-and i onomycin-stimulated platelets. Go-addition of PMA and ionomycin result ed in a maximal synergistic secretion of [C-14]S-hydroxytryptamine ([C -14]5-HT) from platelet dense granules. However, prior addition of PMA for 5 or 10 min resulted in a reduction of this secretory response. I nclusion of either RGDS (to inhibit platelet aggregation) or E64-d (to inhibit calpain activity) resulted in full restoration of the secreto ry response. In experiments to determine the activity status of PKC, P MA was found to induce a loss in cytosolic and total PKC activity with out an increase in membrane-associated activities during this time per iod. Inhibition of either platelet aggregation or calpain activity res ulted in preservation of total and cytosolic activities with a measura ble increase in membrane translocated activity. PMA-induced phosphoryl ation of a number of PKC substrates was measured in P-32-labelled plat elets. PMA induced potent phosphorylation of the 45 and 20 kDa species and also proteins of the molecular masses 66, 80, 97 and 119 kDa. Pho sphorylation was maximal at either 1 or 2 min after which dephosphoryl ation occurred. Inclusion of either RGDS or E64-d resulted in a reduct ion of the dephosphorylation rates, and sustained phosphorylation of t he 66, 80, 97 and 119 kDa proteins. These studies suggest that the act ivity status of PKC is an important factor in the level of secretion o btained and that platelet aggregation is involved in calpain-initiated down-regulation of PKC.