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M Hatmi

Publications and source records attributed to M Hatmi.

31 records · Page 2Linked to original sources
PubMed1993

Carrageenan-induced activation of human platelets is dependent on the phospholipase C pathway.

Stimulation of washed human platelets by the pro-inflammatory polysaccharide carrageenan is accompanied by shape change, aggregation and release of granule contents and unaccompanied by thromboxane A2 synthesis. Carrageenan triggers platelet activation through a prostaglandin synthetase-independent mechanism. The phospholipase A2 (PLA2) inhibitor, p-bromophenacyl bromide suppresses platelet responses to carrageenan (Vargaftig et al, 1980) probably by mechanism(s) other than those which involve PLA2 activity. Exposure of platelets to carrageenan (2-25 micrograms/ml) induced inositol phosphate formation in a time- and concentration-dependent manner, the level of inositol phosphate formation correlating with the intensity of aggregation. Neomycin, an aminoglycoside antibiotic which inhibits the phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate breakdown, suppressed both platelet activation and inositol phosphate formation. Inhibition was concentration-dependent with an IC50 value of about 180 microM. Platelet-activating factor (PAF) is not responsible for carrageenan-induced platelet activation and inositol phosphate formation, since exposure of platelets to carrageenan (25 micrograms/ml) in the presence of compound WEB 2086 (100 microM), a PAF antagonist, failed to inhibit carrageenan responses. However, compound Ro 19-3704, a structurally related antagonist of PAF reported to be also an inhibitor of phospholipases A2 and C, inhibited concentration-dependently (0.1-10 microM) aggregation and ATP release induced by carrageenan (25 micrograms/ml). These findings indicate that carrageenan activates human platelets through a phospholipase C-dependent mechanism and show that neomycin, at low concentrations, can be a selective inhibitor of phospholipase C-mediated PIP2-breakdown.

Blood Platelets↗
PubMed1993

Competitive inhibition of phospholipase A2 activity by the platelet-activating factor antagonist Ro 19-3704 and evidence for a novel suppressive effect on platelet activation.

The compound Ro 19-3704 [3-4(R)-2-(methoxycarbonyl) oxy-3-(octadecylcarbamoyl)oxy-propoxy butylthiazolium iodide], initially described as an antagonist of platelet-activating factor, is reported here to directly inhibit rabbit platelet phospholipase (PL) A2 activity, with an IC50 value of 4 to 7 microM. Classical Michaelis-Menten analysis showed that inhibition was reversible and competitive, inasmuch as apparent Km values increased in the presence of Ro 19-3704 (from 0.2-0.4 to 2 microM), whereas Vmax values remained constant (200 +/- 20 nmol/min/10(9) cells). Ro 19-3704 inhibited platelet aggregation, PLA2 release and thromboxane B2 formation induced by thrombin (0.25 U/ml), with IC50 values of 8, 15 and below 5 microM, respectively. Aggregation and PLA2 release by arachidonic acid (100 microM) were also inhibited, but thromboxane B2 formation was unaffected, indicating that Ro 19-3704 does not inhibit cyclooxygenase. Platelet activation by collagen (5 micrograms/ml), the thromboxane mimetic U46619 ([15(S)-hydroxy-11,9(epoxymethano)-prosta-5Z,13E-dienoic acid] 1 microM) and low concentrations of thrombin (0.05-0.1 U/ml) was also inhibited by Ro 19-3704. Inhibition of platelet activation was reversible, suggesting that its suppressive effect was not due to cytotoxicity. Finally, Ro 19-3704 did not stimulate cyclic AMP formation or inhibit phosphodiesterase activity. Ro 19-3704 is a competitive inhibitor of PLA2 activity, and is also endowed with a potent suppressive effect on platelet activation induced by different agonists.

Animals↗
PubMed1992

Azelastine potentiates the prostaglandin-induced increase of cyclic AMP content in human platelets and in guinea-pig alveolar macrophages.

The effect of azelastine on intracellular cyclic AMP concentration and on various indexes of cell activation was evaluated in guinea-pig alveolar macrophages and in human platelets. The effect of azelastine was further investigated on adenylate cyclase activity using membranes and homogenates from guinea-pig alveolar macrophages. Pretreatment of alveolar macrophages with azelastine prevented the activation induced by PAF-acether and by the chemotactic peptide fMLP as estimated by the reduced liberation of arachidonic acid metabolites formed by the cyclooxygenase and the lipoxygenase pathways. The effect of azelastine was concentration-dependent (50 to 500 microM) and reversible. Similarly, a short pretreatment with azelastine (100 microM) prevented arachidonic acid-induced platelet aggregation. This effect was also reversible after washing the platelets. In guinea-pig alveolar macrophages, azelastine induced a concentration-dependent (10 to 500 microM) increase in intracellular cyclic AMP and markedly potentiated the increase induced by PGE2. In human platelets, azelastine alone increased intracellular cyclic AMP concentration marginally only but, as in the case of macrophages, synergized with PGI2. Azelastine did not activate significantly adenylate cyclase unless a cytosolic factor was included within the membrane fraction. This effect of azelastine was not due to Ca2+ movements and was not modified by GTP. Our findings show that azelastine interferes with cell activation through a mechanism related to an increase in intracellular cyclic AMP concentration. The increase in cyclic AMP was induced by azelastine in intact cells and in homogenates but not in a crude membrane fraction. Those results indicate that azelastine modifies a cytosolic factor that may be phosphodiesterase. In addition, similarities between the effects of azelastine and those of reference phosphodiesterase inhibitors (theophylline, isobutyl-methyl-xanthine) are shown in this study, suggesting that azelastine might behave as a phosphodiesterase inhibitor.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Alkaline phosphatase prevents platelet stimulation by thromboxane-mimetics.

1. The effects of alkaline phosphatase on platelet aggregation, secretion and thromboxane B2 (TxB2) generation induced by the full dose-range of common platelet agonists were studied in human platelet-rich plasma and washed platelets. 2. Platelet aggregation and adenosine 5'-triphosphate (ATP) secretion induced by threshold and supramaximal concentrations of arachidonate and stable TxA2 and prostaglandin endoperoxide-mimetics (compounds U46619 and EP171) were abolished in the presence of alkaline phosphatase (0.5-1 u ml-1), even though the synthesis of TxB2 persisted. In contrast, platelet aggregation by PAF-acether and by supramaximal concentrations of thrombin as well as the primary wave of aggregation by adenosine diphosphate (ADP) and adrenaline were unaffected by alkaline phosphatase under conditions where the secondary wave of aggregation by ADP was blocked. 3. Alkaline phosphatase, unlike prostacyclin, failed to raise the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of the platelets. Also, the pretreatment of platelets by inorganic phosphate or by ATP plus creatine phosphate/creatine phosphokinase reversed the inhibitory effect of alkaline phosphatase. 4. Experiments performed in the guinea-pig in vivo showed that alkaline phosphatase was effective on thrombocytopenia induced by arachidonate. 5. Our results provide the first direct evidence for a specific inhibitory effect of alkaline phosphatase at a site sensitive to TxA2 and prostaglandin endoperoxides and suggest that its phosphorylation/dephosphorylation state may play an important role in modulating platelet activation. These results also suggest the presence of ecto-protein kinases on membrane platelets.

Alkaline Phosphatase↗
PubMed1988

Interference of eicosapentaenoic and docosahexaenoic acids with arachidonate-and U46619-induced platelet activation and desensitization.

In previous studies we showed that arachidonate (AA) and the cyclic endoperoxide/thromboxane (Tx) A2 mimic U46619-induced auto- and cross-desensitization of human platelets to either agonist. The desensitizing effect of U46619 is direct, whereas that of AA is mediated by a cyclooxygenase-dependent metabolite. Desensitization by AA and U46619 is suppressed by antagonists of the endoperoxide/Tx receptor sites. In the present investigation we demonstrated that eicosapentaenoic (EPA) and docosahexaenoic acid (DCHA) the major polyunsaturated fatty acids of fish oil suppressed TxB2 formation and prevented platelet activation by AA and U46619. This inhibition required the presence of EPA or DCHA, since platelets pre-treated with these fatty acids and washed before testing responded as controls to the stimulating agents. At 0.1 and 0.3 mM respectively, DCHA and EPA behaved as reversible inhibitors of cyclooxygenase or Tx synthetase (inhibition of the effects of AA) and as endoperoxides/TxA2 receptor antagonist (inhibition of the effects of U46619). Co-exposure of DCHA (0.1 mM) with AA or U46619 prevents auto- and cross-desensitization to AA and U46619. Platelets exposed to 0.3 mM DCHA and washed became refractory to stimulation by AA, but responded as controls to U46619. EPA (0.3 mM) was fully removed from platelets, which responded to AA and to U46619. EPA and DCHA antagonize endoperoxide/TxA2 directly, and thus prevent the stimulation-dependent desensitization, and additionally, inhibit the cyclooxygenase activity required for desensitization.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1987

Interference of the thromboxane antagonist SK&F 88046 with platelet activation and subsequent desensitization by arachidonic acid and the thromboxane mimetics U46619 and EP171.

We have investigated the effects of the thromboxane antagonist SK&F 88046 on human platelet activation and desensitization by arachidonic acid (AA) and by the thromboxane A2 mimetics U46619 or EP171. SK&F 88046 inhibited platelet aggregation and secretion induced by AA, U46619, EP171 and thrombin at low (0.05 U/ml) but not at a high (1 U/ml) concentrations. Platelet inhibition was reversed by washing the cells. Platelets pre-exposed to AA, U46619 or EP171 and then disaggregated with prostacyclin, washed and resuspended, failed to respond with aggregation or secretion to a second challenge by either agonist. In the presence of the endoperoxide/thromboxane receptor antagonist L636499 or of SK&F 88046, pre-exposure to AA, U46619 or EP171 failed to prevent subsequent responses to the related agonists. Our results suggest that SK&F 88046 is a selective antagonist of platelet activation and desensitization induced by TxA2 or prostaglandin endoperoxides and that it may have utility as an anti-platelet drug.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1987

Effects of sulfinpyrazone and its metabolite G25671 on platelet activation and desensitization and on bronchoconstriction induced by the prostaglandin endoperoxide analog U46619.

Sulfinpyrazone (100 microM) and its thioether metabolite G25671 (50 microM) suppressed arachidonic acid-induced platelet aggregation, thromboxane (Tx) B2 formation and ATP release. Platelet activation by the endoperoxide analog U46619 also was prevented by sulfinpyrazone or G25671 (0.3-1 mM). Previous studies have shown that human platelets pre-exposed to arachidonic acid or to U46619 and then washed and resuspended failed to respond to a second challenge by both arachidonic acid and U46619; desensitization by arachidonic acid and U46619 occurred at a site sensitive to endoperoxides/Tx receptor antagonists; and the desensitizing effects of U46619 were direct, whereas those of arachidonic acid were mediated by a cyclooxygenase-dependent metabolite. We now demonstrate that the presence of sulfinpyrazone or G25671 during platelet exposure to arachidonic acid or U46619 prevented desensitization. We also studied the threshold aggregating concentration of arachidonic acid and U46619 in healthy subjects before and after treatment with sulfinpyrazone and we found a good correlation between ex vivo and in vitro findings. We finally examined the actions of sulfinpyrazone and G25671 on the bronchoconstriction in vivo and parenchymal lung strip contraction in vitro induced by U46619. Neither drug had any preventive effect. Our results demonstrate that sulfinpyrazone and its metabolite G25671 are not only cyclooxygenase inhibitors but can also act as endoperoxide/Tx antagonists and indicate clearly that antagonism of U46619 by both drugs is selective for platelets.

Arachidonic Acid↗
PubMed1986

Modulation by cyclic AMP of arachidonic acid-induced platelet desensitization.

We have previously demonstrated that arachidonic acid (AA) and the stable cyclic endoperoxide analogue (U46619) desensitize human platelets at a common site, which is sensitive to endoperoxides/thromboxane receptor antagonists. We now report on the influence of agents which evaluate intracellular levels of platelet adenosine 3',5'-cyclic monophosphate (cAMP) on AA- and U46619-induced platelet desensitization. Prostaglandin E1, prostacyclin, carbacyclin, forskolin or dibutyryl cAMP prevented platelet activation by and desensitization to AA and to U46619 under conditions where the formation of thromboxane B2 was not significantly modified. Inhibition of platelet activation (aggregation and secretion) required a lower increase of the cAMP content than was needed to inhibit desensitization, confirming previous findings that desensitization to and by AA or U46619 are independent from the platelet release reaction. Together, these results indicate that AA-induced desensitization can be modulated by the adenylate cyclase/cAMP system acting at a site distinct from the known mechanisms of Ca2+ sequestration. This site is shared by the AA metabolite responsible for desensitization and by U46619 and is related to their common platelet membrane receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
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

Platelet desensitization induced by arachidonic acid is not due to cyclo-oxygenase inactivation and involves the endoperoxide receptor.

Human platelets pre-exposed to arachidonic acid (AA) (0.1-1 mM) or to the endoperoxide analogue U46619 (1-3 microM) and then washed and resuspended, failed to respond with aggregation or secretion to a second challenge by either agonist. The response to thrombin at low (0.04-0.1 u ml-1) but not at high (2.5 u ml-1) concentrations was also inhibited by pre-exposure to AA and U46619. The ability of platelets to synthesize thromboxane (Tx) B2 from AA or upon challenge with thrombin persisted despite platelet desensitization. In the presence of the reversible cyclo-oxygenase (CO) inhibitors methyl salicylate (MS) or L8027, pre-exposure to AA had no effect on subsequent challenge by the same agonist or by U46619, whereas platelet desensitization by pre-exposure to U46619 persisted. However, platelet activation by, and desensitization to AA and U46619, was prevented by trimetoquinol and compound L636499, two thromboxane/endoperoxide receptor antagonists. In contrast to the CO inhibitors, the thromboxane synthetase inhibitor dazoxiben, which in 3 'responders' out of 5 subjects suppressed aggregation, secretion, and Tx formation induced by AA, failed to prevent AA-induced desensitization. Compared to quiescent cells the distances between platelets desensitized after re-exposure to AA were reduced in electron microscopy, but the tight connections associated with aggregated cells were not observed. Degranulation was also not observed and cell morphology resembled that of normal quiescent platelets. In conclusion, (a) AA and U46619 desensitize human platelets at a similar site sensitive to prostaglandin/thromboxane receptor antagonists, and show cross-desensitization; (b) desensitization by AA appears to be mediated by a CO-dependent metabolite, as CO inhibitors prevent desensitization by AA but not to U46619; (c) the failure of dazoxiben to prevent desensitization by AA suggests that a metabolite other than TxA2, possibly the endoperoxides, mediates the phenomenon; (d) desensitization does not involve inactivation of CO or thromboxane synthetase enzymes.

Adenosine Triphosphate↗
PubMed1984

Structural requirements for preventing the aspirin- and the arachidonate-induced inactivation of platelet cyclo-oxygenase: additional evidence for distinct enzymatic sites.

2-Hydroxybenzoic acid (salicylic acid) prevents the inhibition by aspirin (ASA) of platelet aggregation and of the generation of thromboxane A2 from arachidonic acid (AA). We studied the ability of 2-hydroxybenzoic acid analogues to block ASA and to prevent the platelet desensitization due to a first exposure to AA. Inactivation was prevented when exposure to AA was done in the presence of reversible inhibitors of cyclo-oxygenase. Phenol, methyl salicylate and L8027 were thus strong inhibitors of AA-induced platelet activation and desensitization. The minimal structural requirement for inhibition of thromboxane A2 generation from AA was a phenol group as benzoic acid was fully inactive. 2-Hydroxybenzoic acid, and to some extent 2,6-dihydroxybenzoic acid were effective against ASA, the most active substances being methyl salicylate and L8027. The minimal structural requirement for blocking ASA was that 2-hydroxybenzoic acid, 2-methoxybenzoic acid should be devoid of activity, which highlights the fact that the hydroxyl group must be available. Our work favours the hypothesis that non-steroidal anti-inflammatory drugs react with two sites of cyclo-oxygenase, which were named the supplementary and the catalytic sites. The interaction of 2-hydroxybenzoic acid and of its analogues with the supplementary site is necessary but not sufficient for the efficacy of these compounds as cyclo-oxygenase inhibitors. The intensity of interaction with the supplementary site and the modifications of the catalytic site determine the potency of these compounds as cyclo-oxygenase inhibitors. For preventing ASA inactivation, an interaction with the supplementary sites is always necessary, but furthermore an appropriate group, preferentially in the position ortho to the hydroxyl, is needed.

Animals↗