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

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Abstract
1 The effect of the membrane‐permeable diacylglycerol analogues, 1,2‐dioctanoylglycerol (Oco2Gro) 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 Oco2Gro (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 Ca2+ ([Ca2+]i) mobilisation and arachidonate/thromboxane B2 release. OleAcGro (62–125 μM) had no effect on thrombin‐induced [Ca2+]i elevations but had a slight (15%) inhibitory effect on thrombin‐induced arachidonate release with a 5‐min pre‐incubation. Addition of Oco2Gro, PMA or OleAcGro on their own caused no rise in [Ca2+]i levels or arachidonate release. 3 Collagen (20 μg/ml) induced substantial arachidonate release without a detectable rise in [Ca2+]i. Pretreatment (10–300 s) with Oco2Gro (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 B2 formation in response to collagen was inhibited in the presence of Oco2Gro or PMA. 4 Both Oco2Gro 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 Oco2Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 μM), suggesting that inhibition of thromboxane B2 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 32Plabelled platelets showed that while Oco2Gro (60 μM) and PMA (16 nM) caused a 4–5‐fold increase in 32P‐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, Oco2Gro 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.