EFFECT OF TUMOR-PROMOTING PHORBOL ESTER, THROMBIN AND VASOPRESSIN ON TRANSLOCATION OF 3 DISTINCT PROTEIN-KINASE-C ISOFORMS IN HUMAN PLATELETS AND REGULATION BY CALCIUM

Protein kinase C (PKC) acts in synergy with Ca2+ mobilization for the activation of platelets. Three different PKC subtypes that specifically react with antibodies to alpha- beta- and zeta-PKC have been detected in human platelets. We have compared the subcellular redistribution of these isoforms in platelets after exposure to the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and to two physiological agonists, thrombin and vasopressin. In the presence of PMA, beta-PKC is most rapidly translocated to membranes, followed by zeta-PKC and alpha-PKC [membrane contents of 39+/-6, 31+/-4 and 24+/-4% (means+/-S.E.M.) respectively after 2 min incubation]. In contrast, both thrombin and vasopressin induced a biphasic translocation of PKC isoforms. For both agonists, the first phase of translocation occurred within 1 min and was identical for the three isoforms. However, during the second phase, the translocation of zeta-PKC by thrombin and vasopressin differed [membrane contents (mean+/-S.E.M.) of 24+/-3 and 46+/-4 % respectively after 10 min]. These results suggest a differential activation of zeta-PKC by vasopressin and thrombin. PMA-induced translocation of alpha-PKC was decreased from 278+/-27 to 198+/-24 (mean+/-S.E.M., P = 0.02; percentage increase over control value) in the presence of 1 mM-EDTA, whereas chelation of intracellular Ca2+ by Quin2-AM does not influence this response. These results suggest that the PMA-induced translocation of alpha-PKC depends on the presence of 1 mm concentration of extracellular Ca2+. In addition, the chelation of either extracellular or intracellular Ca2+ inhibited both vasopressin- and thrombin-induced translocation of all three isoforms, suggesting that Ca2+ is an important requirement for the translocation of alpha-, beta- and zeta-PKC by physiological agonists. In conclusion, the translocation of PKC varies between different isoforms and between different agonists.