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

Abstract

1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycero1 (OcozGro) and 1 -oleoyl-2-acetyl-glycerol (OleAcGro) on agonist-induced platelet activation processes were compared with those of the phorbol ester, phorbol12-myristate 13-acetate (PMA), using appropriately labelled washed human platelets.2. Pre-treatment (10 -300 s) with OcozGro (1 5 -60 pM) 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 CaZ ' ([Ca2'Ii) mobilisation and arachidonate/thromboxane B2 release.OleAcGro (62-125 pM) had no effect on thrombin-induced [Ca2+Ii elevations but had a slight (15%) inhibitory effect on thrombin-induced arachidonate release with a 5-min pre-incubation. Addition of OcozGro, PMA orOleAcGro on their own caused no rise in [Ca2 'Ii levels or arachidonate release.3. Collagen (20 pg/ml) induced substantial arachidonate release without a detectable rise in [Ca2+Ii. Pretreatment (10-300 s) with OcozGro (15-60 pM), PMA (16 nM) or OleAcGro (62 pM) 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 OcozGro 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 pM), 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 32P-labelled platelets showed that while OcozGro (60 pM) 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 pM) 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, OcozGro 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 arachidonat...

Na+/H+ exchange is not necessary for protein kinase C‐mediated effects in platelets

“…), such that we regard them to be unsuitable reagents in this respect. Furthermore, there is no evidence implicating PKC as a positive modulator of Ca*+ mobilisation in platelets; rather, some studies [17,26,27] have convincingly shown PKC to be an inhibitor of receptor-mediated Ca*+ mobilisation in platelets. It is difficult therefore to see how PKC can enhance Ca*+ mobilisation via Na+/H+ exchange, and it is our view, as judged from the present results, that none of the effects actually mediated by PKC activation in platelets are dependent on Na+/H+ exchange.…”

“…Effect of [Na+je removal on potentiatorylinhibitory effects of PMA/diG on receptormediated responses Our previous studies [17] have shown that PMA and diCs have potentiatory effects on collageninduced arachidonate release with incubation times l-5 min. Substitution of [Na+le with choline+ reduced the extent of collagen (20 pg/ml)-induced arachidonate release but did not affect its enhancement by PMA (increase from 6.11 to 8.7% in Na+ buffer; increase from 4.04 to 7.04% in choline+ buffer, with a 5 min pre-incubation of 16 nM PMA before addition of collagen; p < 0.01 in both cases).…”

“…Washed platelet suspensions resuspended in a pH 7.4 buffer (buffer B) composed of 10 mM Hepes, 5 mM KCI, 1 mM MgS04, 1 mM CaC12, 0.35% BSA, 0.09% glucose, 0.05 U/ml hirudin and 145 mM NaC or choline+ as the chloride salt were prepared from titrated platelet-rich plasma foilowing a centrifugation-wash step in a pH 6.5 buffer (buffer A), comprising 35 mM citric acid, 5 mM KCl, 0.5 mM CaCla, 0.35% bovine serum albumin (BSA), 0.09% glucose, 0.05 U/ml hirudin and 103 mM Na+ or choline as described [17]. Loading of platelets with quin-2 and [t4C]5HT was carried out by incubating 20pM quin-2 ester or 1 &i/ml [r4C]5HT with PRP for 30 min at 37°C.…”

Activation of human platelets by mastoparan

“…Platelet aggregation, 5-hydroxytryptamine (5-HT) secretion, liberation of arachidonic acid (AA), beta-thromboglobulin (BTG) release and protein phosphorylation were measured in washed human platelets prepared from citrate anticoagulated blood using previously described procedures [6]. Intracellular CAMP levels ([CAMP],) were monitored using a modification of the procedure described by Gilman [7].…”

Protein kinase C is not involved in the desensitization of platelet activating factor receptor in rabbit platelets

“…Abbreviations: Bmax, msximal binding site density; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol b/s(aminoethyl ether}N,N,N',hr-tetraacetic acid; GTP, guanine nucleotide triphosphate; H7, 1-(5-isoquinoline sulfonyl}-2methylpiperazine; HPLC, high performance liquid chromatography; IP 1, inositolmonophosphate; IP2, inositolbisphosphate; IP 3, inositoltrisphosphate; Kd, dissociation constant; PAF, platelet activating factor; PMA, phorbol 12-myristate-13-acetate; PMSF, phenylmethylsulfonyl fluoride. protein kinase C activator, such as PMA, the inositol phospholipid breakdown and other responses induced by PAF, thrombin and collagen were attenuated markedly (17)(18)(19)(20)(21)(22), we asked whether protein kinase C could serve as feed-back regulator in the PAF-induced desensitization of platelets.…”