No 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...

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.

SummaryPyridoxal 5’-phosphate (PALP) inhibited ADP, thrombin, adrenaline, PAF and AA induced aggregation and 14C-5HT release. Thromboxane B2 (TxB2) generation induced by all the above agents except AA was also inhibited indicating that PALP may be inhibiting AA release via phospholipase A2 activation rather than AA metabolism. PALP inhibited ristocetin induced aggegation in PRP and agglutination in formaldehyde-treated washed platelets (FWP). Inhibition of ADP, adrenaline, PAF and AA-induced aggregation and 14C-5HT release by PALP was found in resuspended platelets pretreated with PALP and sodium borohydride suggesting that inhibition was mediated by Schiff base formation with platelet surface amino groups.Irreversible fixation of PALP to the platelet membrane by borohydride reduction also inhibited thrombin induced 14C-5HT release and TxB2 generation but not thrombin induced primary aggregation or ristocetin induced agglutination in FWP. This suggests that PALP may interact with specific glycoproteins on the platelet membrane involved in ADP, adrenaline and PAF induced primary aggregation and that PALP could be inhibiting ristocetin induced agglutination by direct interaction with ristocetin or FVIII RCoF.