Chiara Cerletti

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.

Prevention of thrombosis and vascular inflammation: benefits and limitations of selective or combined COX-1, COX-2 and 5-LOX inhibitors

Anti-thrombotic therapy with aspirin, which at low doses acts as a selective inhibitor of platelet cyclooxygenase 1 (COX-1) activity, is well established. However, a major limitation of aspirin treatment is its gastrointestinal toxicity, which is thought to be linked to the suppression of COX-1-mediated production of cytoprotective prostaglandins. Selective COX-2 inhibitors are effective anti-inflammatory agents with lower gastrointestinal toxicity than aspirin. These inhibitors might also downregulate vascular and leukocyte inflammatory components that play a major part in atherothrombotic disease. However, some selective COX-2 inhibitors appear to increase cardiovascular risk. Newly developed dual COX-5-lipoxygenase (5-LOX) inhibitors share the anti-inflammatory effect and gastric safety of COX-2 inhibitors, but also inhibit COX-1-mediated platelet function and 5-LOX-mediated synthesis of gastrotoxic leukotrienes. Dual inhibitors might thus be beneficial in the treatment of atherosclerosis, where platelet-leukocyte interaction dominates the underlying inflammatory process.

Recent Advances in Platelet-Polymorphonuclear Leukocyte Interaction

Epidemiological evidence suggests a positive correlation between the number of PMN and the risk of ischemic vascular disease. The observation that activated PMN induce platelet activation my provide some biological plausibility to the role of PMN in thrombogenesis. Between other PMN products, cathepsin G, a protease released during PMN activation, is a potent platelet agonist. However, the antiproteinases present in plasma could virtually abolish its activity. Indeed it was shown that, when PMN were stimulated after interaction with platelets in mixed cell population, P-selectin-mediated platelet-PMN adhesion may result in the formation of a sequestered microenvironment in which cathepsin G activity is protected by antiproteases. P-selectin-mediated adhesion was also shown to facilitate the transcellular metabolism of arachidonic acid, resulting in increased production of both thromboxane B2 and leukotriene C4. PMN adhesion to activated platelets in mixed cell suspensions subjected to high shear rate can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step followed by an adhesion-strengthening interaction mediated by the β2-integrin Mac-1. Moreover, an intermediate tyrosine-kinase-dependent signal regulating β2-integrin adhesiveness is required. Indeeed activated platelets express not only P-selectin but also different β2-integrin ligands including fibrinogen and ICAM-2. Some of the functional responses elicited by P-selectin on PMN could be prevented by specific antibody to the P-selectin glycoprotein ligand-1, indicating that this adhesive receptor is able to transduce an ‘outside-in’ signal when engaged by the ligand. By using activated platelets, P-selectin-expressing CHO cells and soluble recombinant P-selectin, P-selectin was shown to trigger protein tyrosine phosphorylation in PMN and the tyrosine kinase-dependent function of Mac-1. In conclusion, adherence of activated platelets to PMN may be a key event in the sequence of thrombus formation. The recognition of the essential contribution of PMN β2-integrins in addition to P-selectin in platelet-PMN adhesion provides an additional evidence to the broad range of function and mechanisms in which PMN integrins are involved and may be potential targets for pharmacological intervention.

Pharmacokinetic and Pharmacodynamic Differences Between Two Low Dosages of Aspirin May Affect Therapeutic Outcomes

AbstractBackground: Meta-analyses of the prevention of major vascular events by aspirin suggest therapeutic equivalence of all dosages. However, the optimal dosage still remains problematic, and a recent trial found aspirin 160 mg/day to be more effective than 80 mg/day for secondary prevention of ischaemic stroke. Objective: To evaluate two low dosages of aspirin in terms of pharmacokinetics and pharmacodynamics (inhibition of platelet thromboxane generation and urinary excretion of thromboxane and prostacyclin metabolites). Design and Participants: A randomised cross-over study was performed in 16 healthy volunteers (9 women and 7 men, 33.8 ± 5.1 years old) given enteric-coated aspirin 80 or 160 mg/day for 7 days. Methods: Plasma concentrations of salicylate and aspirin were measured by high-performance liquid chromatography (HPLC) after both the first and the last dose (days 1 and 7). The usual pharmacokinetic parameters were then derived. Serum thromboxane B2 (TxB2) was measured by radioimmunoassay. The urinary excretion of 11-dehydro-TxB2 and 2,3-dinor-6-keto-prostaglandin F1α were measured on 8-hour urine samples by immunoassay after extraction and HPLC separation, both before and after 7 days of drug administration. Results: With the 160mg dosage, but not with the 80mg dosage, higher concentrations of aspirin were found at day 7 compared with day 1. For aspirin 80 mg/day, 24-hour area under the concentration-time curve (AUC24) was similar on days 1 and 7 (569 ± 339 vs 605 ± 377 γ · h/L), but increased from 904 ± 356 ⧎g · h/L on day 1 to 1355 ± 883 ⧎g · h/L on day 7 with the higher dosage. Similarly, the AUC24 for salicylate was similar on days 1 and 7 with the lower dosage, but significantly increased from day 1 to day 7 after the higher dosage. This paralleled inhibition of serum TxB2 levels (99% vs 95% average inhibition by 160 and 80 mg/day) and of urinary excretion of thromboxane metabolite (77% vs 61% average inhibition by 160 and 80 mg/day), without altering the excretion of prostacyclin metabolite. Conclusions: Inhibition of serum TxB2 generation and of thromboxane metabolite urinary excretion by the lower dosage of aspirin, although substantial, still appeared incomplete. The small but significant further increase of serum TxB2 inhibition by the higher dosage was accompanied by an even greater inhibition of urinary excretion. We suggest that in some instances this difference would translate into a greater clinical benefit with the higher aspirin dosage. Our findings may also contribute to better definition of the recent concept of ‘aspirin resistance’.

PROSTACYCLIN OVERPRODUCTION IN BARTTER'S SYNDROME

Urinary excretion of 6-keto-prostaglandin F1alpha and thromboxane B2, the major metabolites of prostacyclin and of thromboxane A2, respectively, was measured by specific radioimmunoassays in five female patients with Bartters syndrome and in five normal female controls. The patients with Bartters syndrome excreted about four times as much 6-keto-PGF1alpha as the controls; their excretion of thromboxane B2 was no different from that of the controls. These data suggest that overproduction of prostacyclin mediates both the hyper-reninaemia and the hyporesponsiveness of blood-pressure to pressor agents in Bartters syndrome.

Non-steroidal anti-inflammatory drugs react with two sites on platelet cyclo-oxygenase evidence from ‘in vivo’ drug interaction studies in rats

Non-steroidal anti-inflammatory drugs inhibit platelet cyclo-oxygenase activity. This study shows that salicylate, diflunisal, sulphinpyrazone and indomethacin prevention vivo aspirin inhibitory effect on cyclo-oxygenase activity as measured by the formation of malondialdehyde and thromboxane B2, two products of platelet arachidonic acid metabolism. Salicylate also prevents the inhibitory effect of indomethacin. All these drugs therefore appear to interact with the same site on platelet cyclo-oxygenase. Since salicylate is inactive by itself on this platelet enzyme and diflunisal and sulphinpyrazone were used at ineffective doses, it is suggested that their interaction with aspirin (or indomethacin) occurs at the level of a supplementary binding site, rather than directly on the substrate active site. Interaction with this putative supplementary site is necessary but not sufficient for the efficacy of these drugs on cyclo-oxygenase inhibitors. Acetylation by aspirin of the active site appears to be a phenomenon secondary to the binding of salicylate moiety to the supplementary site.

Licofelone, a dual lipoxygenase–cyclooxygenase inhibitor, downregulates polymorphonuclear leukocyte and platelet function

Polymorphonuclear leukocytes are strongly implicated in the pathogenesis of inflammatory disease. Polymorphonuclear leukocyte recruitment at sites of inflammation, mainly sustained by the beta2-integrins, is followed by the synthesis and release of inflammatory mediators, such as leukotrienes, proteolytic enzymes and reactive oxygen species. Functional and metabolic interactions between polymorphonuclear leukocytes and platelets can contribute to and exacerbate the process. The effects of the dual 5-lipoxygenase and cyclooxygenase inhibitor licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) were studied on arachidonic acid transcellular metabolism occurring between polymorphonuclear leukocytes and platelets. The formation of leukotriene C(4), a leukotriene A(4)-derived metabolite, by mixed polymorphonuclear leukocyte/platelet suspensions stimulated with 10 microM A23187 was inhibited by licofelone with an IC(50) of 3.8 +/- 0.07 microM. The formation of 5,12-di-hydroxy-eicosatetraenoic acid (HETE) was abolished at concentrations > or = 10 microM. Licofelone also inhibited the generation of reactive oxygen species by polymorphonuclear leukocytes stimulated with 1 microM n-formyl-methionyl-leucyl-phenylalanine (fMLP), 10 nM complement fraction 5a (C5a) and 1 microM platelet activating factor (PAF) with IC(50)s of 24.4 +/- 0.6, 11.0 +/- 1.5 and 11.7 +/-1.2 microM; elastase release induced by the three agonists was inhibited with IC(50)s of 12.2 +/- 2.2, 23.5 +/- 8 and 2.6 +/- 1 microM, respectively. Homotypic polymorphonuclear leukocyte aggregation induced by fMLP, C5A and PAF was inhibited by licofelone with IC(50)s of 23.7 +/- 4.8, 15.6 +/- 3.4 and 15.4 +/- 4 microM, respectively. The present study extends the anti-lipoxygenase and anti-cyclooxygenase activities of licofelone to the production of arachidonic acid metabolites generated as a consequence of polymorphonuclear leukocyte-platelet transcellular metabolism and to polymorphonuclear leukocyte responses relevant to the pathogenesis of inflammation. The coexistence within the same molecule of a wide spectrum of anti-inflammatory properties is of interest.

Inhibition by heparin of platelet activation induced by neutrophil-derived cathepsin G

Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombotic activity of this drug. The present study shows that different heparin preparations (50 nM) completely prevent human platelet aggregation, serotonin release and thromboxane B2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an inactivated active for antithrombin III was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade of protease activity. Heparins were also shown to reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used to optimize its antithrombotic efficacy.

Salicylate fails to prevent the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on human platelet cyclo-oxygenase and lipoxygenase activities

Sodium salicylate is inactive both on cyclo-oxygenase and lipoxygenase prepared from human platelets. It prevents the inhibition of cyclo-oxygenase induced by aspirin, but does not counteract the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on both enzymes. It also fails to interfere with the inhibitory activity of nordihydroguaiaretic acid on lipoxygenase. These data indicate that, unlike eicosatetraynoic acid, non-steroidal anti-inflammatory drugs interact with a site on cyclo-oxygenase distinct from the catalytic site, although related to it. Such a supplementary binding site is lacking on lipoxygenase.

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.