Barbara Wachowicz

Generation of reactive oxygen species in blood platelets.

The generation of superoxide anion radicals (O . - 2 ) and the other reactive oxygen species (ROS) was estimated by means of cytochrome c reduction and chemiluminescence, as well in resting blood platelets and in platelets stimulated by thrombin in the presence or absence of some inhibitors of pathways involved in platelet activation. We used allopurinol (xanthine oxidase inhibitor), wortmannin (PI 3-kinase inhibitor) and staurosporine (protein kinase C inhibitor). To determine the involvement of the glutathione in ROS generation, we used L-buthionine sulfoximine (BSO) which blocks GSH synthesis. Our results confirmed that thrombin stimulates the production of ROS concomitant with metabolism of arachidonate and production of malonyldialdehyde (MDA) in blood platelets ( P < 0.05) and showed that, in the presence of inhibitors, the generation of ROS in platelets (resting and stimulated) was reduced. This indicates that xanthine oxidase, PI 3-kinase or protein kinase C take part in the formation of ROS in blood platelets. Moreover, adhesion of platelets to fibrinogen and secretion of adenine nucleotides from platelets after wortmannin and staurosporine action was also inhibited. BSO not only decreased GSH level, but also reduced the amount of ROS; a correlation between the depletion of GSH and the decrease of ROS was observed ( R = -0.987; P < 0.02). It is concluded that in blood platelets, ROS are produced in the receptor-mediated signaling pathways and platelet activation (arachidonic acid metabolism, the glutathione cycle, metabolism of phosphoinositoides and due to xanthine oxidase). Our results support the importance of ROS in platelet function.

Resveratrol, a phenolic antioxidant with effects on blood platelet functions

The main purpose of this article is to provide an overview of the currently available evidence of antiplatelet properties of resveratrol (3,4′,5-trihydroxystilbene). Resveratrol, a phenolic compound found naturally in fruits, nuts, flowers, seeds and bark of different plants is integral part of human diet. It exhibits a wide range of biological effects, including antiplatelet, anti-inflammatory, anticancer, antimutagenic and antifungal properties. It is also a potent antioxidant, reactive oxygen species scavenger and metal chelators. Resveratrol reduces lipid peroxidation, oxidation and nitration of platelet and plasma proteins. This review article describes the chemical structure of resveratrol, its biological activity, the effects on blood platelet functions and the mechanisms involved in its action on blood platelets, the cells which play an important role not only in the haemostatic process, but also in pathogenesis of cardiovascular diseases.

Comparative anti-platelet and antioxidant properties of polyphenol-rich extracts from: berries of Aronia melanocarpa, seeds of grape and bark of Yucca schidigera in vitro

The aim of the present study was to investigate and compare the anti-platelet action of extracts from three different plants: bark of Yucca schidigera, seeds of grape and berries of Aronia melanocarpa (chokeberry). Anti-platelet action of tested extracts was compared with action of well characterized antioxidative and anti-platelet commercial monomeric polyphenol–resveratrol. The effects of extracts on platelet adhesion to collagen, collagen–induced platelet aggregation and on the production of in resting platelets and platelets stimulated by a strong platelet agonist–thrombin were studied. The in vitro experiments have shown that all three tested extracts (5–50 µg/ml) rich in polyphenols reduce platelet adhesion, aggregation and generation of in blood platelets. Comparative studies indicate that all three plant extracts were found to be more reactive in reduction of platelet processes than the solution of pure resveratrol. The tested extracts due to their anti-platelet effects may play an important role as components of human diet in prevention of cardiovascular or inflammatory diseases, where blood platelets are involved.

Role of reactive nitrogen species in blood platelet functions.

Blood platelets, in analogy to other circulating blood cells, can generate reactive oxygen/nitrogen species (ROS/RNS) that may behave as second messengers and may regulate platelet functions. Accumulating evidence suggest a role of ROS/RNS in platelet activation. On the other hand, an increased production of ROS/RNS causes oxidative stress, and thus, may contribute to the development of different diseases, including vascular complications, inflammatory and psychiatric illnesses. Oxidative stress in platelets leads to chemical changes in a wide range of their components, and platelet proteins may be initial targets of ROS/RNS action. It has been demonstrated that reaction of proteins with ROS/RNS results in the oxidation and nitration of some amino acid residues, formation of aggregates or fragmentation of proteins. In oxidized proteins new carbonyl groups and protein hydroperoxides are also formed. In platelets, low molecular weight thiols such as glutathione (GSH), cysteine and cysteinylglycine and protein thiols may be also target for ROS/RNS action. This review describes the chemical structure and biological activities of reactive nitrogen species, mainly nitric oxide (•NO) and peroxynitrite (ONOO−) and their effects on blood platelet functions, and the mechanisms involved in their action on platelets.

Antioxidant activity of resveratrol in endotoxin-stimulated blood platelets.

Resveratrol (3,4′,5-trihydroxystilbene) is a natural molecule with antioxidant action. It is also considered to be a molecule with antiplatelet, anticancer and anti-inflammatory action. The effects of trans-resveratrol on the reactive oxygen species (ROS) generation and thiobarbituric acid-reactive substances (TBARS) in blood platelets induced by endotoxin (lipopolysaccharide, LPS) or thrombin were studiedin vitro. The production of superoxide radicals (O2.–) and other reactive oxygen species (H2O2, singlet oxygen, and organic radicals) in the presence of resveratrol was measured by a chemiluminescence method in resting blood platelets and platelets stimulated by LPS (0.3 μg/108 platelets) or thrombin (2.5 U/108 platelets). We have shown that resveratrol (6.25–100 μg/ml) inhibits chemiluminescence and generation of O2.– in blood platelets. It has an inhibitory effect on the production of ROS and TBARS in platelets caused by LPS or thrombin.

Peroxynitrite-mediated modification of fibrinogen affects platelet aggregation and adhesion.

The reaction of peroxynitrite with fibrinogen resulted in both structural modifications and altered biological properties of this glycoprotein. SDS-PAGE analysis of peroxynitrite-treated fibrinogen, performed under non-reducing conditions, showed some aggregated material on the top of the gel (5-10% of total staining bands) and the presence of nitrotyrosine. The amount of nitrotyrosine, detected by immunoassay with anti-nitrotyrosine antibodies, was dependent on peroxynitrite concentration. In comparison with native molecule, peroxynitrite-treated fibrinogen subjected to SDS-PAGE under reducing conditions revealed not only three bands corresponding to A f , B g and n chains, but the existence of additional high molecular bands probably due to the formation of dityrosine crosslinking between fibrinogen subunits. The different susceptibility in tyrosine nitration of fibrinogen subunits was also observed. The A f chain was the most intensely nitrated, while B g and n chains were nitrated much less? Peroxynitrite-treated fibrinogen in comparison with native molecule had a distinct capability to mediate platelet adhesion and aggregation. Both unstimulated and ADP-activated platelets showed a reduced ability to adhere to peroxynitrite-modified fibrinogen. The percentage of ADP-induced platelet aggregation decreased as a function of peroxynitrite-mediated modification of fibrinogen molecule.

Resveratrol may reduce oxidative stress induced by platinum compounds in human plasma, blood platelets and lymphocytes.

Resveratrol (trans-3,4′,5-trihydroxystilbene), a polyphenolic compound found in grapes and wine, has been shown to have anti-inflammatory, anti-oxidant, anti-tumor and anti-platelet activities. Using different methods, we show that resveratrol reduces oxidative stress induced by cisplatin (cis-diamminedichloroplatinum II) and selenium–cisplatin conjugate ([NH3]2Pt(SeO3), Se-Pt) in human blood platelets, lymphocytes and plasma. Resveratrol decreased the production of 8-epi-prostaglandin F2 (a biomarker of lipid peroxidation) in control blood platelets and platelets treated with platinum compounds (10 μg/ml), and markedly reduced activities of different anti-oxidative enzymes (glutathione peroxidase, superoxide dismutase and catalase) in these cells. A combined action of resveratrol and Se-Pt evoked a significant decrease of DNA damage (measured by comet assay) in lymphocytes compared with cells treated with Se-Pt only. Resveratrol also caused a distinct reduction of total anti-oxidant level in plasma after incubation with platinum compounds. Therefore, anti-oxidative activity of resveratrol may diminish oxidative stress and damage to cellular biomolecules (lipids, proteins and DNA) induced by platinum compounds.

Peroxynitrite-induced changes of thiol groups in human blood platelets

In this study we investigated sulfhydryl and disulfide metabolism in blood platelets treated with peroxynitrite (ONOO−) at concentrations of 10, 100, and 1000 µM. In platelets incubated with peroxynitrite, a decrease of both protein and low molecular weight thiols (glutathione, cysteine and cysteinylglycine) was observed. The reduction of platelet free thiols caused by ONOO− was dose dependent. We found that peroxynitrite induces the oxidation of platelet free SH-groups. Platelets contain a large fraction of glutathione in reduced form (GSH). We showed that after ONOO− treatment, GSH/GSSG ratio in platelets was distinctly decreased. The presented results suggest that oxidation of thiols and depletion of GSH in platelets caused by peroxynitrite may be responsible for cytotoxic effects of this compound.

An Extract from Berries of Aronia melanocarpa Modulates the Generation of Superoxide Anion Radicals in Blood Platelets from Breast Cancer Patients

Plant antioxidants protect cells against oxidative stress. Because oxidative stress (measured by different biomarkers) is observed in breast cancer patients, the aim of this study was to establish the effects of a polyphenol-rich extract of Aronia melanocarpa (final concentration of 50 microg/mL, 5 min, 37 degrees C) on superoxide anion radicals (O(2)(-*)) and glutathione (GSH) in platelets from patients with breast cancer and in a healthy group in vitro. Generation of O(2)(-*) in platelets before and after incubation with the extract was measured by cytochrome C reduction. Using HPLC, we determined the level of glutathione in blood platelets. We observed a statistically significant increase of biomarkers of oxidative stress such as O(2)(-*) and a decrease in GSH in platelets from patients with breast cancer compared with the healthy group. We showed that the extract from A. melanocarpa added to blood platelets significantly reduced the production of O(2)(-*) in platelets not only from the healthy group but also from patients with breast cancer. Considering the data presented in this study, we have demonstrated the protective role of the extract from A. melanocarpa in patients with breast cancer in vitro.

L-Carnitine protects plasma components against oxidative alterations.

OBJECTIVE L-Carnitine as a dietary supplement has been reported to have a beneficial effect on several cardiovascular risk parameters and exercise capacity, but the biological relevance of its activity is poorly understood. Dietary supplements (including L-carnitine) are often used to foster exercise performance; however, these may affect some pathways of human body metabolism. The aim of this study in vitro was to determine antioxidative properties of L-carnitine (0.1-100 μM) added to plasma and to assess if L-carnitine might protect plasma proteins and lipids against oxidative/nitrative damage (determined by levels of protein carbonyl groups, thiols, 3-nitrotyrosine formation and thiobarbituric-acid reactive substances generation) caused by 100 μM peroxynitrite (ONOO(-)), a strong physiologic oxidative/nitrative agent. METHODS The level of carbonyl group generation was measured by a colorimetric method. For the estimation of 3-nitrotyrosine formation, a competition enzyme-linked immunosorbent assay was performed. Plasma lipid peroxidation was measured spectrophotometrically as the production of thiobarbituric-acid reactive substances. High-performance liquid chromatography was used to analyze total free thiol groups of plasma proteins and low-molecular-weight thiols (glutathione, cysteine, and homocysteine) in plasma. RESULTS The L-carnitine added to plasma inhibited in vitro ONOO(-)-induced oxidation and nitration of blood plasma proteins. Incubation of plasma with peroxynitrite resulted in the decrease of protein thiols. L-Carnitine had a protective effect on peroxynitrite-induced decreased -SH level in plasma proteins. The presence of L-carnitine also prevented the decrease of low-molecular-weight thiols (glutathione, cysteine, and homocysteine) in plasma caused by peroxynitrite and protected plasma lipids against peroxidation induced by peroxynitrite. CONCLUSIONS These results demonstrated that L-carnitine possesses antioxidative activity.