Grazyna Rajtar

Effect of trans‐resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function

Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. The aim of the present study was to investigate the effects of trans‐resveratrol (3,4′,5‐trihydroxy‐trans‐stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. trans‐Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 μM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3±0.13 μM, mean±s.e.mean), as evaluated by luminol‐amplified chemiluminescence. trans‐Resveratrol prevented the release of elastase and β‐glucuronidase by PMN stimulated with the receptor agonists fMLP (1 μM, IC50 18.4±1.8 and 31±1.8 μM), and C5a (0.1 μM, IC50 41.6±3.5 and 42±8.3 μM), and also inhibited elastase and β‐glucuronidase secretion (IC50 37.7±7 and 25.4±2.2 μM) and production of 5‐lipoxygenase metabolites leukotriene B4 (LTB4), 6‐trans‐LTB4 and 12‐trans‐epi‐LTB4 (IC50 48±7 μM) by PMN stimulated with the calcium ionophore A23187 (5 μM). trans‐Resveratrol significantly reduced the expression and activation of the β2 integrin MAC‐1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti‐MAC‐1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation‐dependent epitope on MAC‐1. Consistently, PMN homotypic aggregation and formation of mixed cell‐conjugates between PMN and thrombin‐stimulated fixed platelets in a dynamic system were also prevented by trans‐resveratrol. These results, indicating that trans‐resveratrol interferes with the release of inflammatory mediators by activated PMN and down‐regulates adhesion‐dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.

Salicylate reverses in vitro aspirin inhibition of rat platelet and vascular prostaglandin generation.

Abstract It is generally accepted that aspirin inhibits platelet function by irreversible acetylation of prostaglandin cyclo-oxygenase. The salicylate moiety seems not to be causally involved in the inhibitory effect of aspirin, a concept supported by the virtual inactivity of sodium salicylate. However, prostaglandin synthesis is also inhibited by numerous compounds which have no acetylating properties. Recent evidence indicates that salicylate may prevent the inhibitory effect of aspirin on rabbit platelet cyclo-oxygenase, suggesting that interaction of the salicylate moiety of aspirin with this enzyme is important. This study was aimed at evaluating whether the inhibitory effect of aspirin on platelet prostaglandin generation could be reversed by sodium salicylate. We therefore measured spectrophotometrically malondialdehyde (MDA) generated by arachidonate in rat platelet-rich plasma and evaluated the effect of short-term incubation with either aspirin or salicylate or both. In the experimental conditions used, salicylate not only prevented but also reversed aspirin-inhibition of MDA formation. This interaction was not peculiar for platelets, since salicylate also reversed the in vitro inhibitory effect of aspirin on vascular prostacyclin generation (measured by a bioassay). These findings suggest that irreversible acetylation of cyclo-oxygenase does not account for the early in vitro inhibitory effect of aspirin on prostaglandin synthesis.

Effects of glycosaminoglycans on platelet and leucocyte function: Role of N-sulfation

The effect of glycosaminoglycans (GAGs) such as sulodexide, low molecular mass dermatan sulfate, heparin and some derivatives with different degrees and types of sulfation was studied on cathepsin G- or thrombin-stimulated platelets and n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated polymorphonuclear leucocytes (PMNs). All GAGs (0.01-20 micrograms/mL) inhibited both platelet aggregation induced by cathepsin G and its catalytic activity. Thrombin-induced platelet aggregation in contrast was only prevented by heparin, sulodexide and dermatan (2-100 micrograms/mL). All GAGs, except 2-O,N-desulfated heparin, inhibited beta-glucuronidase and lysozyme release, as well as beta-glucuronidase activity and PMN superoxide production by the peptide fMLP. The efficacy of GAGs was clearly dependent on the degree and type of sulfation since dermatan and N-desulfated heparins were comparatively less effective. The observation that heparin and other GAGs inhibit platelet activation induced by the PMN protease cathepsin G may help determine whether mechanisms of action other than anticoagulation are critical in the antithrombotic activity of heparin and related compounds.

Comparison of radioimmunoassay and high-resolution gas chromatography mass spectrometry for the quantitative determination of serum thromboxane B2 and 6-keto-PGF1α after pharmacological blockade of thromboxane synthetase

Radioimmunoassay (RIA) and high-resolution gas chromatography-mass spectrometry (HRGC-MS) were compared for the determination of serum 6-keto-PGF-1 alpha and TXB2 in a situation of drug-altered arachidonate metabolism. Results were comparable for TXB2 in both conditions. 6-keto-PGF1 alpha RIA determinations (with two different antisera) revealed increased levels in dazoxiben-treated samples, which were not confirmed by HRGC-MS. The assay were repeatedly checked in controlled conditions to investigate these discrepancies. Interference was found with both antisera, due to the drug-induced change in metabolism.

Prostaglandins and human platelet aggregation: Implications for the anti-aggregating activity of thromboxane-synthase inhibitors

Selective pharmacological blockade of thromboxane-synthase in human platelets by dazoxiben resulted in the reorientation of cyclic-endoperoxides towards PGE2, PGD2 and PGF2 alpha. At concentrations which can be reached when thromboxane-synthase is inhibited, PGE2 (100-500 nM) exerted a marked, concentration-dependent pro-aggregatory effect. This required the formation of endogenous or the addition of exogenous endoperoxides and was prevented by PGD2 or 13-aza-prostanoic acid, a selective antagonist of PGH2/TxA2 receptors. The anti-aggregating effect of PGD2 was evident at concentrations lower than those obtained in dazoxiben-treated platelets. It is proposed that in the absence of TxA2 generation, a combination of endoperoxides and PGE2 may result in normal aggregation. The latter may be inhibited by PGD2. No interference of PGF2 alpha on platelet function could be shown.

Sulphinpyrazone prevents in vivo the inhibitory effect of aspirin on rat platelet cyclo-oxygenase activity

Abstract Sulphinpyrazone reportedly inhibits in vitro platelet cyclo-oxygenase activity. This study shows that Sulphinpyrazone administration (200 mg/kg) to rats was followed by long lasting inhibition of platelet cyclo-oxygenase, as measured by malondialdehyde generation by sodium arachidonate. The inhibition was apparently competitive and could in fact be ascribed to supposed metabolites of the drug. When given 30min–6 hours before aspirin (5–25mg/kg), Sulphinpyrazone and to a considerable extent its metabolites significantly prevented permanent inhibition of platelet cyclo-oxygenase activity normally produced in vivo by aspirin. Sulphinpyrazone at 100 mg/kg was unable by itself to modify platelet malondialdehyde or thromboxane B2 generation, yet if effectively interfered with aspirin activity. This suggests that Sulphinpyrazone and its metabolites may interact with a binding site on cyclo-oxygenase not directly involved with the enzyme activity. Interaction of aspirin with this binding site would be a prerequisite for its inhibitory effect on the enzyme active site. The clinical implications of this study include a reappraisal of the pharmacological basis for the association of Sulphinpyrazone and aspirin in thrombosis prevention trials.

Arachidonic acid-induced malondialdehyde formation in rat platelets - Kinetic aspects and inhibition by acetylsalicylic acid and indomethacin

SummaryMalondialdehyde (MDA) is a stable product of arachidonic acid metabolism, catalyzed by the enzyme cyclo-oxygenase. The experimental conditions for measuring the kinetic of MDA formation in rat platelet-rich plasma were defined. In platelets stimulated with arachidonic acid MDA formation was almost linear for a limited period of time (between 0 and 2 min) and was concentration-dependent with saturation kinetics. The hyperbolic curves obtained were recast in a linear plot (according to transformation S/V versus S) and straight lines fitting all experimental points were obtained. The apparent Km value of arachidonic acid was 0.49±0.09 mM and Vmax 1.44±0.06 nmoles MDA/1.4×109 platelets/min.The apparent type of inhibition and the relative potency of acetylsalicylic acid and indomethacin on MDA formation were also investigated. Inhibition by both drugs was concentration-dependent, and was much stronger when either drug was preincubated with platelets for 10 min than for 1 min. Analysis of the data by Dixon plots (1/V versus inhibitor concentrations) revealed an apparently competitive type of inhibition.It is suggested that both acetylsalicylic acid and indomethacin inhibit cyclo-oxygenase in platelet-rich plasma by a similar mechanism, not involving covalent binding of either drug to the enzyme.