1,2‐Dioctanoylglycerol but not 1‐oleoyl‐2‐acetylglycerol inhibits agonist‐induced platelet responses

1 The effect of the membrane‐permeable diacylglycerol analogues, 1,2‐dioctanoylglycerol (Oco 2 Gro) and 1‐oleoyl‐2‐acetyl‐glycerol (OleAcGro) on agonist‐induced platelet activation processes were compared with those of the phorbol ester, phorbol 12‐myristate 13‐acetate (PMA), using appropriately labelled washed human platelets. 2 Pre‐treatment (10–300 s) with Oco 2 Gro (15–60 μM) or PMA (16 nM) before addition of thrombin (0.2 U/ml) or, addition of these agents 10–20 s after thrombin, resulted in a significant reduction (20–80%) in the extent of thrombin‐induced intracellular Ca 2+ ([Ca 2+ ] i ) mobilisation and arachidonate/thromboxane B 2 release. OleAcGro (62–125 μM) had no effect on thrombin‐induced [Ca 2+ ] i elevations but had a slight (15%) inhibitory effect on thrombin‐induced arachidonate release with a 5‐min pre‐incubation. Addition of Oco 2 Gro, PMA or OleAcGro on their own caused no rise in [Ca 2+ ] i levels or arachidonate release. 3 Collagen (20 μg/ml) induced substantial arachidonate release without a detectable rise in [Ca 2+ ] i . Pretreatment (10–300 s) with Oco 2 Gro (15–60 μM), PMA (16 nM) or OleAcGro (62 μM) before collagen addition or addition of these agents 30–60 s after collagen addition resulted in a significant potentiation of arachidonate release (1.2–2‐fold over control), even though thromboxane B 2 formation in response to collagen was inhibited in the presence of Oco 2 Gro or PMA. 4 Both Oco 2 Gro and PMA had dual effects on 5‐hydroxytryptamine secretion induced by thrombin or collagen. Short pre‐incubations (< 2 min) with these agents caused a potentiation of sub‐maximal agonist‐induced secretion, while not affecting secretion induced by maximal agonist concentrations. With longer pre‐incubation times (5–15 min) however, a significant reduction in the level of agonist‐induced secretion in the presence of Oco 2 Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 μM), suggesting that inhibition of thromboxane B 2 formation alone does not account for inhibition of 5‐hydroxytryptamine secretion. OleAcGro had no inhibitory effects on agonist‐induced secretion even though it potentiated it (with < 2‐min incubations) at sub‐maximal agonist concentrations. 5 Time courses of phosphorylation of a 45‐kDa protein, a marker of protein kinase C activation, in 32 Plabelled platelets showed that while Oco 2 Gro (60 μM) and PMA (16 nM) caused a 4–5‐fold increase in 32 P‐labelling of this protein over a 5‐min incubation period, OleAcGro (62–125 μM) caused a 1.5‐fold increase in labelling which was only maintained for a 10–30‐s period. The inability of OleAcGro to exert any significant inhibitory effects on agonist‐induced platelet responses may therefore be due to insufficient activation of protein kinase C and/or phosphorylation of the 45‐kDa protein. Hence, Oco 2 Gro may be a better tool as a diacylglycerol analogue. However, the potentiatory effects of OleAcGro with short pre‐incubations (agonist‐induced 5‐hydroxytryptamine secretion and collagen‐induced arachidonate release) is indicative of a diversity in the effects of protein kinase C activators, determined not only by the type of activator and time of exposure to it, but also by the agonist and activation process involved.