1,2-DIOCTANOYLGLYCERIOL (diC8) BUT NOT 1-OLEOYL 2-ACETYLGLYCEROL (OAG) INHIBITS AGONIST-INDUCED PLATELET RESPONSES

Abstract

Inhibition of agonist-induced platelet responses by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PrkC), has been reported. We have examined the effects of the diacylglycerol (DG) analogues OAG and diCg, both PrkC activators, as well as PMA on intracellular Ca2+ ([Ca2+]i) mobilisation and 5-hydroxytryptamine (5HT) release induced by thrombin (T), collagen (Coll.) and the thromboxane (Tx) mimetic U46619. All studies were performed using washed human platelets pre-labelled with either quin-2 or [14C]-5HT and maximal concentrations of agonists. Neither diCg or PMA elevated [Ca2+]i above resting levels though both agents induced a small (10-15%) secretory response. In response to 0.2U/ml T or 0.6uM U46619, but not 20μg/ml Coll., [Ca2+]i increased from resting levels of lOOnM to 758±108nM and 712±58nM respectively. Addition of diCg (60μM) or PMA (16nM) 10 sec before or after T or U46619 reduced the control responses by 10-15% and 30-80% respectively. In contrast, [14C]-5HT secretion in response to T and Coll. was unaffected by diCg or PMA and in the case of U46619 was potentiated 1.4-1.6 fold over control levels. With longer pre-incubation times (5 min) [Ca2+]i mobilisation was further reduced and an inhibitory effect (10-40%) on agonist-induced secretion was evident. Unlike diCg or PMA, OAG (63μM) had no significant inhibitory effect on agonist-induced [Ca2+]i mobilisation and [14C]-5HT secretion even with long pre-incubation times (5 min). However, like diCg and PMA, OAG potentiated U46619-induced secretion with a 10 sec incubation though it induced no secretion itself. The inability of OAG to inhibit may be related to its lesser potency as a PrkC activator. Over a 10 sec-5 min period OAG caused significantly less 40Kd protein phosphorylation ( < 2-fold increase in [32P]-labelling), compared to diCg and PMA (4-6-fold increase). Our results suggest that diCg may be a better tool as an activator of PrkC and DG mimic than OAG. Further, the time course of inhibition of agonist-induced [Ca2+]i mobilisation by diCg suggests that this effect may constitute a physiologically relevant phenomenon mediated by DG within a single cycle of agonist-induced events.