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L Touqui

Publications and source records attributed to L Touqui.

32 records · Page 2Linked to original sources
PubMed1991

The metabolism of 1-acyl-PAF in rabbit platelets and its possible interaction with PAF.

The metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF), a naturally occurring analogue of platelet activating factor (PAF), was investigated in rabbit platelets. Our studies showed that 1-acyl-[3H]PAF (1-palmitoyl-2-acetyl-sn-glycero-3-phospho[N-methyl-3H]-choline) was converted by platelets into phosphatidyl-[3H]choline [( 3H]PC) in a time-dependent fashion. The formation of [3H]PC occurred at a rate similar to that observed when lyso-[3H]PC (palmitoyl-sn-glycero-3-phospho[N-methyl-3H]choline) was used as substrate. In addition, a time-dependent increase in the level of water-soluble radioactivity was observed during the incubation of platelets with either 1-acyl-[3H]PAF or lyso-[3H]PC. This increase was parallel to the formation of [3H]PC and was not observed in the presence of [14C]PAF (1-octadecyl-2-acetyl-sn-glycero-3-phospho[N methyl-14C]-choline). Analysis by thin-layer chromatography showed that the soluble radioactivity was mainly associated with glycerophosphocholine (GPC). On the other hand, the preincubation of platelets with phenylmethylsulfonyl fluoride, an inhibitor of the acetylhydrolase, reduced the hydrolysis of 1-acyl-[3H]PAF to [3H]GPC with a concomitant accumulation of radioactivity in 1-acyl-PAF. These findings suggest that 1-acyl-PAF is converted into PC through deacetylation-reacylation with lysoPC as an obligatory intermediate. The findings also indicate that the lysoPC resulting from 1-acyl-PAF is either reacylated to phosphatidylcholine (PC) or hydrolyzed to GPC by lysophospholipase. Finally, we showed that the stimulation of platelets with PAF led to a time- and concentration-dependent increase in the conversion of 1-acyl-[3H]PAF to [3H]PC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Gamma-thrombin-induced phospholipase A2 activation in rabbit platelets: comparison with alpha-thrombin.

gamma-Thrombin stimulated release of [3H]arachidonic acid ([3H]AA) accompanied by a significant production of PAF and lyso-PAF by rabbit platelets. These responses, which reflect PLA2 activation, were observed after a prolonged lag and to a lower extent when compared to those induced by alpha-thrombin which evoked a much higher elevation in intracellular calcium. This elevation together with [3H]AA release were markedly reduced by EDTA. However, addition of ionophore A23187 enhanced the release of [3H]AA by gamma-thrombin to the levels similar to those of alpha-thrombin. We conclude that gamma-thrombin is able to activate PLA2 and suggest that calcium influx may be a limiting factor for this activation.

Animals↗
PubMed1989

Selective inhibition of adrenaline-induced human platelet aggregation by the structurally related Paf antagonist Ro 19-3704.

1. Two non-lipid antagonists of platelet-activating factor acether (Paf), BN 52021 and WEB 2086, at concentrations which completely blocked Paf-induced platelet aggregation, failed to interfere with aggregation by adrenaline. In contrast, Ro 19-3704, a structurally related antagonist of Paf, inhibited concentration-dependently aggregation induced by adrenaline or by the simultaneous addition of submaximal concentrations of adrenaline and Paf. Reversal of aggregation was obtained when Ro 19-3704 was added to the platelet suspension after adrenaline. 2. Ro 19-3704 was selective for Paf and adrenaline since it failed to interfere with platelet aggregation induced by arachidonic acid or ADP. CV-3988, an antagonist of Paf structurally similar to Ro 19-3704, also inhibited adrenaline-induced aggregation. However, a morpholine analogue (MA) of Paf, which has no anti-Paf activity, failed to interfere with the aggregation induced by adrenaline. This suggests that the effect of Ro 19-3704 and CV-3988 on adrenaline is not simply due to their lipid structure. 3. Experiments on plasma membrane preparations showed that Ro 19-3704 inhibited [3H]-yohimbine binding with an inhibition constant (Ki) of 7 +/- 3 microM. In contrast, BN 52021 and MA did not interfere with [3H]-yohimbine binding. Equilibrium binding experiments showed that Ro 19-3704 increased the apparent KD of [3H]-yohimbine binding from 2.02 +/- 0.15 to 7.3 +/- 0.4 nM. The Paf antagonist Ro 19-3704 interacts specifically with the alpha 2-adrenoceptor and may thus prevent the early steps involved in the mechanism of adrenaline-induced platelet activation.

5-Hydroxytryptophan↗
PubMed1987

The role of Ca2+ in regulating the catabolism of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in rabbit platelets.

In the present study we have investigated the effect of changes in the concentration of cytosolic free Ca2+ ([Ca2+]i) on the deacetylation-reacylation of PAF-acether (alkylacetylglycerophosphocholine, alkylacetyl-GPC) by rabbit platelets. Washed platelets were incubated with alkyl[3H]acetyl-GPC ([3H]acetyl-PAF) or [3H]alkylacetyl-GPC ([3H]alkyl-PAF) and [Ca2+]i was subsequently elevated by the addition of the ionophore A23187 or thrombin. The catabolism of PAF-acether was studied by measuring the release of [3H]acetate or the formation of [3H]alkylacyl-GPC. The ionophore inhibited the release of [3H]acetate and the formation of [3H]alkylacyl-GPC with no accumulation of lyso-[3H]PAF, indicating that the deacetylation of PAF-acether was blocked. The effect of ionophore on the deacetylation of PAF-acether was parallel with the increase of [Ca2+]i and could be reversed by the addition of EGTA. In contrast with the prolonged inhibition evoked by ionophore, thrombin, which induced a transient elevation of [Ca2+]i, merely delayed the deacetylation of PAF-acether. Since intact platelets failed to convert exogenous lyso-PAF, the effect of Ca2+ on its acylation was investigated by using platelet homogenates. These experiments showed that the acylation of lyso-PAF was inhibited by the exogenously added Ca2+, with a maximum effect at 1 mM. When the formation of endogenous lyso-PAF from the labelled pool of alkylacyl-GPC was examined, a prolonged increase in the concentration of lyso-PAF with a parallel and equally prolonged decrease in the cellular level of alkylacyl-GPC were observed after the addition of ionophore to intact platelets. The addition of EGTA reversed the effect of ionophore, thus permitting reacylation of lyso-PAF. In contrast, only a transient change in the level of lyso-PAF and alkylacyl-GPC was evoked by the addition of thrombin. Therefore we conclude that the inhibitory effect of Ca2+ on the deacetylation-reacylation of PAF-acether may have an important role in the regulation of its biosynthesis.

Acetates↗
PubMed1985

Effect of intracellular oxygen-free radicals on the formation of lipid derived mediators in rat renomedullary interstitial cells.

An intracellular generation of oxygen free radicals was induced by phenazine methosulfate (PMS) in rat renomedullary interstitial cells (RMIC) in culture. This response was associated with an increase in PGE2 and 15 HETE production. The synthesis of cyclooxygenase and lipoxygenase derivatives in PMS-treated cells was inhibited by indomethacin and NDGA respectively. Inhibitors of PLA2 such as mepacrine and dexamethasone were able to inhibit partially the PGE2 synthesis induced by PMS. The formation of lyso-platelet activating factor, a product of membrane-bound phospholipid, by a PLA2 catalyzed reaction was also stimulated in PMS-treated cells. Superoxide dismutase added to the incubation medium enhanced the PMS-dependent PGE2 synthesis whereas catalase decreased it, suggesting the involvement of H2O2 in this process. In addition, a depletion of soluble thiol groups was observed in PMS-treated cells. Treatment of RMIC by the thiol oxidative agent, diamide, mimicked the effect of PMS on PGE2 synthesis, whereas diamide did not increase the formation of lyso-PAF indicating its inability to stimulate PLA2. These results suggest that cyclooxygenase may be involved in this process, indeed added arachidonate, bypassing PLA2, enhanced PGE2 synthesis in PMS-treated cells further supporting the involvement of cyclooxygenase. In conclusion, generation of oxygen free radicals by PMS in RMIC enhanced the synthesis of lipid derived mediators. A decrease in the cellular thiol content is partially involved in cyclooxygenase activation but does not appear to be involved in PLA2 activation.

Animals↗
PubMed1985

Human platelets stimulated by thrombin produce platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when the degrading enzyme acetyl hydrolase is blocked.

It has been shown [Touqui, Jacquemin & Vargaftig (1983) Thromb. Haemostasis 50, 163; Touqui, Jacquemin & Vargaftig (1983) Biochem. Biophys. Res. Commun. 110, 890-893; Alam, Smith & Melvin (1983) Lipids 18, 534-538; Pieroni & Hanahan (1983) Arch. Biochem. Biophys. 224, 485-493] that rabbit platelets inactivate exogenous PAF (platelet-activating factor, PAF-acether) by a deacetylation-reacylation mechanism. The deacetylation step is catalysed by an acetyl hydrolase sensitive to the serine-hydrolase inhibitor PMSF (phenylmethanesulphonyl fluoride) [Touqui, Jacquemin, Dumarey & Vargaftig (1985) Biochim. Biophys. Acta 833, 111-118]. We report here that human platelets can produce PAF on thrombin stimulation. This production is marginal and transient, reaching a maximum at 10 min and decreasing thereafter. In contrast, 10-12 times more PAF is produced when platelets are treated with PMSF and stimulated with thrombin. Under these conditions, the maximum formation is observed at 30 min and no decline occurs for up to 60 min after stimulation. In addition, these platelets (treated with PMSF and stimulated with thrombin) incorporate exogenous labelled acetate in the 2-position of PAF, probably by an acetyltransferase-dependent mechanism. Production of PAF by human platelets during physiological stimulation can be demonstrated when PAF degradation is suppressed by the acetyl-hydrolase inhibitor PMSF.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1985

PAF-acether may not mediate the third pathway of platelet aggregation since self-desensitization reduces the effects of low thrombin but enhances those of convulxin.

PAF-acether (platelet-activating factor) was hypothesized as the mediator of the ADP and thromboxane-independent activation of platelets induced by thrombin (Thr) and by the snake venom glycoprotein convulxin (Cx). Aspirinized rabbit platelets self-desensitized to PAF-acether were less responsive to low amounts of Thr, as expected if PAF-acether would be formed, but were hyper-reactive to Cx, in contradiction with its hypothesized mediating role. Aggregation by higher concentrations of Thr overcame inhibition. Experiments with ADP-depleted platelets showed that secretion is neither involved with desensitization to PAF-acether nor with hyper-reactivity to Cx. Those effects required the presence of PAF-acether in the platelet suspension and persisted when transformation of PAF-acether into its recognized metabolite alkyl-acyl-glycerophosphorylcholine was inhibited. The ADP and thromboxane-independent activation of rabbit platelets by low and medium concentrations of Thr may be accounted for by platelet formation of PAF-acether, but overall the contrasting effects of platelet desensitization to PAF-acether on responsiveness to Thr and to Cx suggest that the third pathway of aggregation requires other explanations.

Animals↗
PubMed1985

1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine is the precursor of platelet-activating factor in stimulated rabbit platelets. Evidence for an alkylacetyl-glycerophosphorylcholine cycle.

The metabolism of [3H]PAF-acether ([1',2'-3H]alkyl-2-acetyl-sn-glycero-3-phosphorylcholine ([3H]alkylacetyl-GPC)) by rabbit platelets was investigated using thin-layer chromatography and high-performance liquid chromatography followed by radioactivity detection. After 2 h of incubation at 37 degrees C, 90 +/- 5.3% of [3H]PAF-acether taken up by the platelets were converted into a product identified as sn-2 long-chain acyl analogue ([3H]alkylacyl-GPC) which was incorporated in the membranes. This conversion was independent from extracellular calcium and was completely inhibited by platelet pre-exposure to 2 mM phenylmethylsulfonyl fluoride, a serine hydrolase inhibitor, which failed to inhibit the uptake of [3H]PAF-acether by the cells. The 2-deacetylated derivative, lyso-[3H]PAF-acether was found to be an intermediate of the conversion of [3H]PAF-acether into [3H]alkylacyl-GPC in platelet homogenates. Platelet stimulation with 2.5 U/ml of thrombin induced a reduction (16.5 +/- 2.2%) of its content of [3H]alkylacyl-GPC, accompanied by the release of [3H]PAF-acether and lyso-[3H]PAF-acether to the medium. These effects were suppressed by the phospholipase A2 inhibitor, p-bromophenacyl bromide. Our results demonstrate that intact platelets convert exogenous PAF-acether into alkylacyl-GPC, which can serve as the precursor of PAF-acether released during stimulation. The existence of a metabolic cycle for the uptake, the release and the inactivation of PAF-acether by platelets is suggested.

Acylation↗
PubMed1985

The effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether) on the arterial wall.

The effect of a topical paf-acether superfusion over an injured arterial segment was assessed in the guinea-pig, using an opto-electronic in vivo thrombosis model allowing on-line quantification of small platelet thrombus dynamics. As compared to control, ADP-induced, thromboformation and behaviour, exogenous paf-acether causes a large, dense platelet thrombus, invaded and surrounded by numerous leukocytes, spreading widely over the adjoining, vacuolized, endothelium. Its embolization has to be forced with prostanoids, mepacrine, EDTA, or with a specific paf-acether antagonist (BN 52021). A few minutes after such forced embolization, a new thrombus starts growing at the same site, without renewal of the paf-acether superfusion. This phenomenon of spontaneous reappearance after forced embolization can be followed during several hours. Experiments with labelled paf-acether and the paf-acether antagonist indicate a possible endogenous paf-acether (or paf-acether-like) production triggered by superfusion with exogenous paf-acether.

Adenosine Diphosphate↗
PubMed1984

Dissociation between inhibition of phospholipid methylation and production of PAF-acether by rabbit platelets.

Platelet-activating-factor (PAF-acether, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is formed by and released from rabbit platelets stimulated with thrombin, with the ionophore A23187, with collagen and with the platelet-stimulating glycoprotein convulxin. We here show that 3-deazaadenosine (C3ado) and L-homocysteine (HCy), two inhibitors of platelet methylation, reduced the formation of PAF-acether and of its deacetylated product lyso-PAF-acether by rabbit platelets challenged with thrombin, under conditions where the accompanying aggregation was not significantly modified. In contrast, platelet aggregation induced by convulxin was completely and irreversibly blocked when C3ado and HCy were associated. Aggregation by thrombin was not affected by the methylation inhibitors even when ADP was scavenged and thromboxane formation was suppressed. Our results indicate that phospholipid methylation, thrombin-induced platelet aggregation and formation of PAF-acether can be dissociated. The formation of PAF-acether by rabbit platelets can be blocked by mechanisms other than inhibition of phospholipase A2, since the latter is not affected by C3ado and/or HCy.

Animals↗
PubMed1983

Conversion of 3H-PAF acether by rabbit platelets is independent from aggregation: evidences for a novel metabolite.

3H-PAF-acether (Alkyl-[1', 2'-3H]- 2-acetyl-sn-glyceryl-3-phosphorylcholine) was time-dependently transformed by plasma-free rabbit platelets into an unknown product, "PX", which was distinct from lyso-PAF-acether. This effect was specific for platelets since plasma only converted 3H-PAF-acether into lyso-3H-PAF-acether and platelets were not effective in metabolizing lyso-3H-PAF-acether. Platelet aggregation did not interfere with the formation of "PX". The latter is a novel platelet metabolite of PAF-acether with as yet unknown biological properties.

Animals↗
PubMedDate not supplied

Platelet activation--a role for a 40K anti-phospholipase A2 protein indistinguishable from lipocortin.

Stimulus-response (S-R) coupling in platelets requires an intermediary other than an elevation in cytosolic free calcium ([Ca2+]i). While an increase in [Ca2+]i is essential in S-R coupling, effecting phosphorylation of myosin of relative molecular mass (Mr) 20,000 (20 K), platelet activation is also associated with phosphorylation of a 40K protein, which can occur in the absence of changes in [Ca2+]i. The 40K protein is the substrate for protein kinase C (PKC). Mounting evidence suggests that activation of PKC by diacylglycerol is the other signal involved in S-R coupling. Although phosphorylation of the 40K protein is associated with certain platelet functional responses, no precise role has been accredited to it. Recently, we and others have described several proteins (collectively known as lipocortin) which inhibit phospholipase A2 (PLA2). One of the most conspicuous proteins of this group is a 40K peptide whose inhibitory activity can be suppressed by prior phosphorylation. We hypothesized that the 40K protein described in platelets may possess anti-PLA2 activity and that phosphorylation by PKC, suppressing its inhibitory activity, may represent the mechanism underlying mobilization of arachidonic acid, the precursor of prostaglandins. The results of the present study strongly support this hypothesis.

Alkaline Phosphatase↗