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The present in vitro study was designed to examine the antioxidative activity of red cabbage anthocyanins (ATH) in the protection of blood plasma proteins and lipids against damage induced by oxidative stress. Fresh leaves of red cabbage were extracted with a mixture of methanol/distilled water/0.01% HCl (MeOH/H2O/HCl, 50/50/1, v/v/w). Total ATH concentration [µM] was determined with cyanidin 3-glucoside as a standard. Phenolic profiles in the crude red cabbage extract were determined using the HPLC method. Plasma samples were exposed to 100 µM peroxynitrite (ONOO−) or 2 mM hydrogen peroxide (H2O2) in the presence/absence of ATH extract (5–15 µM); oxidative alterations were then assessed. Pre-incubation of plasma with ATH extract partly reduced oxidative stress in plasma proteins and lipids. Dose-dependent reduction of both ONOO− and H2O2-mediated plasma protein carbonylation was observed. ATH extract partly inhibited the nitrative action of ONOO−, and significantly decreased plasma lipid peroxidation caused by ONOO− or H2O2. Our results demonstrate that anthocyanins present in red cabbage have inhibitory effects on ONOO− and H2O2-induced oxidative stress in blood plasma components. We suggest that red cabbage ATH, as dietary antioxidants, should be considered as potentially usable nutraceuticals in the prevention of oxidative stress-related diseases.

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.

Aim The antioxidative and antinitrative activities of (1→3)-β- d -glucan (1–4 μg/ml) from the yeast cell walls of Saccharomyces cerevisiae in human plasma treated with strong oxidants – peroxynitrite (ONOO − ) (0.1 mM) and hydrogen peroxide (H 2 O 2 ) (2 mM) were studied in vitro. The main purpose of this study was to assess if (1→3)-β- d -glucan, a well known strong immunostimulatory agent, possesses a protective function against dual mechanism of ONOO − stroke associated with nitrative and oxidative damages to human plasma biomolecules. Scope The protein changes were determined in vitro by estimating the level of oxidative stress markers – carbonyl groups, and nitrative products – 3-nitrotyrosine residues. The plasma lipid peroxidation was also investigated. The obtained results show that (1→3)-β- d -glucan inhibits in vitro ONOO − -induced oxidation and nitration of plasma proteins, even by 50% and 30%, respectively. The antioxidative activity of (1→3)-β- d -glucan was confirmed by its inhibitory effect on plasma lipids peroxidation induced by ONOO − or by H 2 O 2 . Conclusions The obtained results demonstrate that (1→3)-β- d -glucan from S. cerevisiae protects plasma components against toxic effects of ONOO − and H 2 O 2 due to its antioxidative and antinitrative activities. Therefore (1→3)-β- d -glucan supplementation during inflammatory may be beneficial not only regard for its ability to stimulate the immune system but also by antioxidative properties.

Oxidative stress (OS) plays an important role in the pathogenesis of multiple sclerosis (MS). In MS patients depression is often observed. Cryotherapy might have an effect on OS. The aim of this study was to compare the effects of whole body cryotherapy (WBCT) on changes in total antioxidative status (TAS) of plasma and activities of antioxidative enzymes in erythrocytes from depressive and non depressive MS patients.Twenty-two MS patients with secondary progressive disease course (12 depressive and 10 non depressive) were treated with 10 exposures in a cryochamber. Before and after WBCT the plasma TAS and the activities of superoxide dismutase (SOD) and catalase (CAT) in the erythrocytes were measured.The level of TAS in depressive MS group was significantly lower than in non depressive MS (P0.0003). WBCT increased the level of TAS in depressive (P0.002) more than in non depressive MS patients (P0.01). WBCT treatment of MS patients resulted in the significant increase of TAS level in plasma but had no effects on activities of SOD and CAT.Our results indicate that WBCT suppresses OS in MS patients, especially in depressive patients.

Epidemiologic studies suggest that the regular consumption of polyphenols, secondary metabolites of plants, is correlated with a decrease of the risk of cardiovascular disease, diabetes, arthritis and cancer. The most abundant flavonoid constituents of plants are anthocyanins – water-soluble, glycosylated, nonacetylated pigments. The profitable effects of these compounds may be partly attributed to their antioxidative and anti-inflammatory activity. The supplementation of anthocyanins or an anthocyanin-rich diet has been reported to significantly increase serum antioxidant potential.

The oxidative stress induced by acute exertion may interfere with blood platelet activation. The beneficial effect of L-carnitine (gamma-trimethylamino-beta-hydroxybutyric acid) on oxidative stress in blood platelets has not been fully investigated; however, different studies indicate that this compound modulates platelet functions. The aim of our study was to assess the effects of L-carnitine on platelet activation and oxidative/nitrative protein damage (determined by the levels of protein carbonyl groups, thiol groups, and 3-nitrotyrosine residues) in resting blood platelets or platelets treated with peroxynitrite (ONOO(-), a strong physiological oxidant) in vitro. We also investigated the effects of L-carnitine on the level of platelet glutathione and on the formation of superoxide anion radicals O2(-*), lipid peroxidation measured by thiobarbituric acid reactive substances (TBARS) in blood platelets stimulated by thrombin (a strong physiological agonist), and platelet aggregation induced by adenosine diphosphate (a strong physiological stimulator). We have observed that carnitine decreases platelet activation (measured by platelet aggregation, the generation of O2(-*), and TBARS production). Moreover, our results in vitro demonstrate that carnitine may protect against oxidation of thiol groups induced by ONOO(-). Thus, carnitine may have some protectory effects against oxidative changes induced in blood platelets.

The effects of the beta-D-glucan, a polysaccharide derived from the yeast cell walls of species such as Saccharomyces cerevisiae, on blood platelets activation induced by physiological agonists (thrombin, ADP, collagen) in vitro were studied. The aim of our study was to assess in vitro if beta-glucan, a naturally strong biological response modifier, may modify platelet activation, i.e. platelet aggregation and degranulation (release of proteins and adenine nucleotides) induced by thrombin, ADP and collagen. Cytochrome c reduction method was used to test the ability of beta-glucan to change superoxide anion generation in platelets. Moreover, we determined also its effect on enzymatic arachidonic acid cascade. The obtained results indicate that beta-glucan has the inhibitory effects on platelet aggregation and secretion. beta-glucan distinctly reduced the arachidonic acid pathway and superoxide anion radical generation in platelets stimulated by biological agonists. The results of the present study suggest that beta-glucan from Saccharomyces cerevisiae has antiplatelet and antioxidative activities, and therefore may be beneficial in the prevention of the excessive blood platelet activation-related diseases, such as cardiovascular or inflammatory diseases.

The aim of our work was to study the anti-aggregatory and antioxidative effects of natural dietary products, d-glucaro 1,4-lactone (1,4-GL) in combination with phenolic compound resveratrol (trans-3,4′,5-trihydroxystilbene). Our results in vitro showed that 1,4-GL alone slightly inhibits platelet aggregation induced by thrombin. The combination of resveratrol (0.1 μM) with 0.5 mM of 1,4-GL caused a significant decrease of thrombin-induced platelet aggregation; however separately, neither of studied compound at used concentrations was not effective. When platelets were treated with 1,4-GL (at the concentration of 0.1 mM and higher) and resveratrol (0.1 μM), similar synergistic action of both tested compound on markers of oxidative stress formation was observed. We measured the levels of different specific markers of oxidative stress, e.g., superoxide anion radicals $$ {\left( {O^{{ - \bullet }}_{2} } \right)} $$ , thiobarbituric acid-reactive substances and carbonyl group formation. Both tested compounds inhibited also the generation of $$ {\left( {O^{{ - \bullet }}_{2} } \right)} $$ and malondialdehyde that represents enzymatical peroxidation of arachidonic acid leading to thromboxane A2 (TXA2) formation in platelets after thrombin stimulation. The obtained in vitro results demonstrate that anti-platelet and antioxidative properties of resveratrol may be significantly augmented by another dietary agent such as 1,4-GL, but mechanism synergistic action of these compounds is not yet known.

Many selenoorganic compounds play an important role in biochemical processes and act as antioxidants, enzyme inhibitors, or drugs. The effects of five new synthesized selenoorganic compounds (2-(5-chloro-2-pyridyl)-7-azabenzisoselenazol-3(2H)-one; 2-phenyl-7-azabenzisoselenazol-3(2H)-one; 2-(pyridyl)-7-azabenzisoselenazol-3(2H)-one; 7-azabenzisoselenazol-3(2H)-one; bis(2-aminophenyl) diselenide) on oxidative changes in human blood platelets and in plasma were studied in vitro and compared with those of ebselen, a well known antioxidant. Our studies demonstrated that bis(2-aminophenyl) diselenide has distinctly protective effects against oxidative stress in blood platelets and in plasma. It might have greater biological relevance and stronger pharmacological effects than ebselen.

The antioxidative activity of the polysaccharide extract from Conyza canadensis in blood platelets treated with peroxynitrite (ONOO-) was studied. Peroxynitrite as a strong biological oxidant has toxic effects on blood platelets and induces the oxidation of thiols, carbonylation and nitration of platelet proteins and lipid peroxidation. Therefore, the aim of our study was to assess if the natural extract from herbal plant, Conyza Canadensis, may protect platelet proteins against nitrative and oxidative damage induced by ONOO-. In our study we measured oxidative damage of platelet proteins induced by peroxynitrite and protectory effects of this extract by estimation of the level of carbonyl groups and nitrotyrosine (a marker of platelet protein nitration). We also used cytochrome c reduction method to test the ability of this extract to change O2-* generation in platelets. Moreover, we determined the effects of the extract on blood platelet aggregation induced by ADP. We observed that the extract from Conyza canadensis distinctly reduced oxidation and nitration of proteins in blood platelets treated with ONOO-(0.1mM) and O2-* production in these cells. The extract from Conyza canadensis also inhibited platelet aggregation. The ability of the extract to decrease O2-* generation in blood platelets supports the importance of free radicals in platelet functions, including aggregation process. The present study suggests that the natural polysaccharide extract from Conyza canadensis has antiaggregatory and antioxidative activities, and therefore may be beneficial in the prevention of peroxynitrite-related diseases, such as cardiovascular or inflammatory diseases.

Resveratrol (3, 4', 5-trihydroxystilbene), a natural polyphenol, is found in some plants that are used in human nutrition. Grapes are a major source for resveratrol, and a significant amount can also be found in red wine. Several experimental studies have demonstrated biological properties of resveratrol, especially its anti-inflammatory, antioxidant, anti-platelet and antitumor effects. In the present study, we investigated the first step of platelet activation-platelet adhesion stimulated by lipopolysaccharide (LPS) from Proteus mirabilis (weak stimulator) and thrombin (strong activator) in the presence of resveratrol. Our studies show that endotoxin (0.3 microg/10(8) platelets), like thrombin (0.2 U/10(8) platelets), induced the adhesion of platelets (expressed as absorbance of cell attached proteins) to collagen and fibrinogen. Preincubation of washed platelets with resveratrol at physiological plasma concentrations (25-100 microg/ml, 30 min, 37 degrees C) had an inhibitory effect on adhesion of platelets to collagen after activation by LPS alone or LPS with thrombin. The strongest effect on this process was caused by resveratrol at the concentration of 100 microg/ml. Pretreatment of platelets with resveratrol (25-100 microg/ml, 30 min, 37 degrees C) had also inhibitory effects on adhesion of platelets to fibrinogen after stimulation of these cells by LPS alone or by LPS with thrombin at the same concentration. In conclusion, we suggest that resveratrol present in human diet may be an important compound responsible for the reduction of platelet adhesion and changed reactivity of blood platelets in inflammatory process.

Lipopolysaccharide (LPS, endotoxin) is a well-known causative agent of septic shock and disseminated intravascular coagulation. The action of LPS is related to the activation of many types of cells, including blood platelets. We examined the effects of different LPSs from Proteus mirabilis (smooth form LPS S1959 and rough forms LPSs R110 and R45) and fragments of LPS structure (lipid A and polysaccharide part) on platelet adhesion to collagen in the presence or absence of the phoinositide 3-kinase (PI 3-K) inhibitor, wortmannin. We found that all forms of LPS and its fragments caused the stimulation of platelet adhesion to collagen, but the polysaccharide part (PS S1959) was the most important in this action. Wortmannin had no effect on LPS-stimulated platelet adhesion to collagen. We conclude that both lipid A and PS S1959 play important roles in LPS-stimulated platelet adhesion to collagen independent on the PI 3-K action.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria may activate blood platelets. The aim of our study was to evaluate the effects of different forms of Proteus mirabilis LPS and isolated lipid A and polysaccharide part on the production of superoxide radicals in blood platelets and to estimate the role staurosporin, wortmannin and indomethacin on this process. We compared the generation of superoxide radicals in platelets treated with LPS after preincubation with inhibitors of the signal transduction pathways, namely staurosporin (inhibitor of protein kinase C), wortmannin (inhibitor of phosphoinositide 3-kinase), and indomethacin (inhibitor of cycloxygenase). Our results demonstrate that all LPS molecules and their fragments caused a stimulation of O2- generation in platelets (P.5). LPSS1959 had the strongest stimulatory effect. Straurosporin and wortmannin, but not indomethacin inhibited O2- production in LPS-stimulated platelets. Staurosporin (8 nM) and wortmannin (50 nM) caused about 50% inhibition of thrombin-induced O2- generation in platelets, while indomethacin (10 microM) had only a slight inhibitory effect on this process. Our results provide support that in LPS- and thrombin-activated platelets, at least part of O2- generation in platelets, while indomethacin (10 microM) had only a slight inhibitory effect on this process. Our results provide support that in LPS- and thrombin-activated platelets, at least part of O2- is generated due to the activation of the enzymes (protein kinase C and phosphoinositide 3-kinase) involved in signal transduction pathway. Cycloxygenase seems to be not involved in this process.

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.

The present in vitro study was designed to estimate the antioxidative activity of curcumin in the protection of human blood platelets and plasma against peroxynitrite (ONOO(-))-induced oxidative stress. The effects of curcumin (12.5-50 µg/ml) on ONOO(-)-induced damage of proteins and lipids were determined by the estimation of protein carbonyl groups, 3-nitrotyrosine formation, and thiobarbituric acid reactive substance (TBARS) generation. Exposure of blood platelets and plasma to 100 µM ONOO(-) resulted in an increased level of carbonyl groups, nitration of protein tyrosine residues, and enhanced lipid peroxidation. Curcumin inhibited carbonyl group formation in plasma and in platelet proteins. The highest dose of curcumin (50 µg/ml) reduced blood platelet protein carbonylation by approximately 40%. In the protection of blood plasma protein, the lower doses of curcumin (12.5 and 25 µg/ml) were more effective. Curcumin partially prevented 3-nitrotyrosine formation in plasma proteins; the effect of curcumin was only statistically significant in blood platelets at the highest dose (50 µg/ml). The antioxidative action of curcumin in the protection against lipid peroxidation caused by ONOO(-) was also observed. Curcumin suppressed the formation of TBARS both in blood platelets and in plasma samples. The highest concentration of curcumin (50 µg/ml) decreased the TBARS level by approximately 35% in both blood platelets and plasma samples. In conclusion, the present study demonstrates the antioxidative properties of curcumin and its protective effects against oxidative/nitrative changes of blood platelets and plasma components, especially proteins and lipids.

D-glucono-1,4-lactone, sodium D-gluconate and calcium D-glucarate are non-toxic glucose derivatives occurring naturally in fruits and vegetables. Calcium D-glucarate is promoted as an orally bioavailability dietary supplement with potential chemopreventive activity without adverse effects. Despite many commercial applications in pharmaceutical and food industries the potential activity mechanisms of glucarate and gluconate are not clear. The purpose of this study was to investigate and compare the effects of these compounds on blood platelets under oxidative stress conditions and to examine their role in thrombin-induced platelet activation. Platelet activation is essential in haemostasis, tumor progression and allergic and non-allergic inflammation, where reactive oxygen species are involved. The antiplatelet and antioxidative activity was studied in vitro by measuring levels of specific oxidative stress markers: thiobarbituric acid reactive substances, superoxide anion, carbonyl groups, 3-nitrotyrosine, protein and low molecular weight thiols. All tested compounds significantly inhibited thrombin-induced arachidonic peroxidation, O₂⁻ⁱ production and also platelet protein oxidation/nitration induced by peroxynitrite, which is a strong oxidant formed intravascularly in vivo. Carbonyl group generation, thiol oxidation and nitrotyrosine formation were significantly decreased in the presence of glucose derivatives. The obtained results demonstrate that tested compounds may be helpful in the prevention of excessive platelet activation through the antioxidant mechanisms. Comparative studies indicate the predominant preventive activity of sodium D-gluconate. In general, the consumption of apples or apple juice as well as oranges, grapefruit and cruciferous vegetables, sources of large amounts of tested derivatives, have beneficial effects on platelets under oxidative stress.

Lots of plants belonging to Asteraceae family are very popular in folk medicine in Poland. These plants are also known as being rich in acidic polysaccharides, due to the presence of hexuronic acids or its derivatives. Our preliminary experiments have shown that the extract from Conyza canadensis L. possesses various biological activity, including antiplatelet, antiocoagulant and antioxidant properties. The aim of our study was to assess if macromolecular glycoconjugates from selected herbal plants of Asteraceae family: Achillea millefolium L., Arnica montana L., Echinacea purpurea L., Solidago virgaurea L., Chamomilla recutita (L.) Rauschert., and Conyza canadensis L. protect platelet proteins against nitrative and oxidative damage induced by peroxynitrite, which is responsible for oxidative/nitrative modifications of platelet proteins: the formation of 3-nitrotyrosine and carbonyl groups. These modifications may lead to changes of blood platelet functions and can have pathological consequences. The role of these different medicinal plants in the defence against oxidative/nitrative stress in human platelets is still unknown, therefore the oxidative damage to platelet proteins induced by peroxynitrite and protectory effects of tested conjugates by the estimation of carbonyl group level and nitrotyrosine formation (a marker of protein nitration) were studied in vitro. The antioxidative properties of the polyphenolic-polysaccharide conjugates from selected tested medicinal plants were also compared with the action of a well characterized antioxidative commercial polyphenol - resveratrol (3,4',5-trihydroxystilbene). The obtained results demonstrate that the compounds from herbal plants: A. millefolium, A. montana, E. purpurea, C. recutita, S. virgaurea, possess antioxidative properties and protect platelet proteins against peroxynitrite toxicity in vitro, similar to the glycoconjugates from C. canadensis. However, in the comparative studies, the polyphenolic-polysaccharide conjugates from selected tested medicinal plants were not found to be more effective antioxidant, than the solution of pure resveratrol.

Oxidative stress has been implicated in the pathogenesis of variety of diseases. Since the endogenous antioxidant defense may be not adequate to counteract the enhanced generation of oxidants, a growing interest in research for exogenous nutrients has been observed. The present study was designed to assess in vitro the antioxidative properties of the glucose derivatives: calcium d-glucarate, d-gluconic acid lactone, and sodium d-gluconate (0.5–3 mM) in the protection of plasma proteins and lipids, against the damage caused by 0.1 mM peroxynitrite (ONOO−). Exposure of plasma to ONOO− resulted in carbonyl groups increase, 3-nitrotyrosine (3-NT) formation, reduction in thiol groups, and enhanced lipid peroxidation. d-Gluconic acid lactone and sodium d-gluconate effectively decreased 3-NT formation; the antinitrative action of calcium d-glucarate was less effective. In plasma samples incubated with ONOO− and tested compounds, the level of carbonyl groups was decreased in comparison to plasma samples treated only with ONOO−. The level of protein −SH groups and glutathione was significantly higher in the presence of glucose derivatives than in plasma samples treated with ONOO− only. All the tested compounds had the inhibitory effect on the peroxynitrite-induced plasma lipids peroxidation. The results obtained from our work indicate that calcium d-glucarate, d-gluconic acid lactone, and sodium d-gluconate may partly protect plasma proteins and lipids against peroxynitrite-induced damages.

Blood platelets in addition to their haemostatic role can function as inflammatory cells. The aim of our study was to assess if beta-D-glucan, the natural, very strong biological response modifier, may protect platelet proteins and lipids against oxidative/nitrative damages. The antioxidative activity of the beta-D-glucan, a known immunomodulator derived from the yeast cell walls of species such as Saccharomyces cerevisiae, on blood platelets treated with oxidants-peroxynitrite and hydroperoxide-was studied in vitro. The levels of different specific markers of oxidative stress, thiobarbituric acid reactive substances (TBARS), carbonyl groups and 3-nitrotyrosine (3-NT) were measured. Our studies showed that beta-glucan possesses significant defence properties against peroxynitrite or hydroperoxide induced lipid peroxidation. The level of TBARS was decreased by 80% at the highest dose of beta-glucan. In the presence of beta-glucan the distinct reduction of platelet protein oxidation was observed; the level of carbonyl groups was decreased by 50%. The results indicate that beta-glucan may also be effective in the protection against the nitrative action of peroxynitrite on platelet proteins, as in the presence of beta-glucan the level of 3-nitrotyrosine, measured by a competition-ELISA method, was diminished. The obtained in vitro results demonstrate that antiplatelet activity of beta-glucan from Saccharomyces cerevisiae is dependent on its antioxidative properties, and therefore beta-glucan supplementation may be beneficial in the prevention of excessive blood platelet activation-related diseases, such as cardiovascular or inflammatory diseases.

Objective The protective effects of D-glucaro 1,4-lactone (1,4-GL) against oxidative/nitrative protein damage (determined by parameters such as levels of protein carbonyl groups and nitrotyrosine residues) to human plasma treated with peroxynitrite (ONOO−) or hydroperoxide (H2O2) were studied in vitro. We also investigated the effects of 1,4-GL on the level of total free thiol groups and low-molecular-weight thiols (glutathione and homocysteine) in plasma treated with ONOO− (0.1 mM). Methods Levels of carbonyl groups and nitrotyrosine residues in human plasma proteins were measured by ELISA and a competition ELISA, respectively. High-performance liquid chromatography (HPLC) was used to analyze free thiols from plasma. Results Exposure of plasma to ONOO− (0.1 mM) resulted in an increase of the level of carbonyl groups and nitrotyrosine residues in plasma proteins and in a distinct decrease in total thiols and low-molecular-weight thiols (glutathione and homocysteine) measured by high-performance liquid chromatography. In the presence of 1,4-GL (0.4–6.4 mM), a distinct decrease in carbonyl group formation and tyrosine nitration in plasma proteins and changes in plasma thiols caused by 0.1 mM of peroxynitrite were observed. Moreover, 1,4-GL inhibited plasma protein oxidation induced by H2O2 (2 mM). Conclusion The obtained results indicate that in vitro 1,4-GL has inhibitory effects on ONOO−- or hydroperoxide-mediated oxidative stress in human plasma and changes plasma redox thiol status. The mechanism of the antioxidative action of 1,4-GL present in plasma is not known yet.

Background and aim Peroxynitrite (ONOO − ), a highly reactive species, modulates platelet activation and function. Modifications of platelet proteins induced by ONOO − may be an important factor in the pathogenesis of platelet-related diseases. The defence mechanisms against ONOO − are therefore crucial for normal cellular function. Recently, there has been an increased interest in the screening of natural products present in the diet and herbals for possible antioxidative agents (ONOO − scavengers). d-glucaro-1,4-lactone (1,4-GL), formed from d-glucaric acid (GA), a natural compound found in fruits and vegetables, possesses detoxifying and anticancerogenic properties. However, the effect of this compound on platelet activation is unknown. Methods and results We investigated the effects of 1,4-GL on nitrative and oxidative alteration of platelet proteins and lipid peroxidation caused by two strong oxidants: ONOO − and hydroperoxide (H 2 O 2 ). The action of 1,4-GL on platelet aggregation induced by adenosine diphosphate (ADP) was also studied. Exposure of platelets to ONOO − or H 2 O 2 resulted in an increase in the level of carbonyl groups (approximately three-fold and two-fold, respectively). In the presence of 1,4-GL, a significant decrease (about 50% for the highest concentration of 1,4-GL) in carbonyl group formation was observed; however the level of nitrotyrosine residues in platelets treated with ONOO − remained unchanged. Conclusions We demonstrated an inhibitory effect of 1,4-GL on lipid peroxidation in platelets treated with ONOO − (0.1mM) or H 2 O 2 (2mM). 1,4-GL inhibited platelet lipid peroxidation by about 40%. In the presence of 1,4-GL, peroxidation of plasma lipids was also reduced by about 40%. These results demonstrate that 1,4-GL possesses antioxidative properties and reduces the activation of blood platelets.

Nitric oxide ((*)NO) plays an important role in a number of physiologic processes. Evidence exists that (*)NO, which stimulates soluble guanylate cyclase and enhances cyclic guanosine monophosphate (cGMP) levels, may inhibit platelet activation. In contrast, during platelet activation induced by different agonists, synthesis of (*)NO in platelets occurs. In these studies, production of the stable end-products of (*)NO-nitrite and nitrate (NO(x)) in human platelets, stimulated by different doses of lipopolysaccharide from Proteus mirabilis (LPS; endotoxin), has been evaluated. LPS is a weak platelet agonist that may activate various steps of platelet activation with the generation of reactive oxygen species. The mechanism of platelet activation induced by the endotoxin is not known. The aim of the present study was to measure the level of nitrite and NO(x) in blood platelets treated with LPS and to examine the level of nitrotyrosine in platelet proteins caused by LPS. Our results show that LPS at a low concentration (6.8 ng/ml) caused a decrease (approximately 80%) in the NO(x) level, whereas at higher concentrations (13.6 and 25 ng/ml) it induced an increase in the NO(x) level (approximately 210% and 260%, respectively). Our results indicate that LPS, like other agonists (thrombin, platelet-activating factor), can stimulate (*)NO production in platelets. After incubating platelets with LPS, we also observed a distinct increase in platelet protein nitration (3-nitrotyrosine).

Lipopolysaccharide (LPS) is the major component of the outer membranes of the walls of gramnegative bacteria that protects bacterial cells against antibacterial agents. LPS is the heteropolimer consisting of two parts: the hydrophobic lipid A and hydrophilic polisaccharide. After lysis of bacterial cells LPS is released into the circulation and it is able to activate the immunological system by stimulation of monocytes, macrophages, neutrophils, blood platelets and endothelial cells. LPS can bind to plasma proteins and form complexes that increase toxic activity of LPS and affinity of LPS to cell receptors. The activation of immune cells by LPS leads to release of inflammatory mediators: cytokines, chemokines, enzymes, eicosanoids, adhesion agents and free radicals that are responsible for progression of inflammatory reactions and may induce pathophysiological processes including septic shock.

Many selenoorganic compounds play an important role in biochemical processes and act as antioxidants, enzyme inhibitors or drugs. The effects of a new selenocompound — bis(2-aminophenyl)-diselenide on oxidative/nitrative changes in human plasma proteins induced by peroxynitrite (ONOO−) were studied in vitro and compared with the those of ebselen, a well-known antioxidant. We also studied the role of the tested selenocompounds in peroxynitrite-induced plasma lipid peroxidation. Exposure of the plasma to peroxynitrite (0.1 mM) resulted in an increase in the level of carbonyl groups and nitrotyrosine residues in plasma proteins (estimated using the ELISA method and Western blot analysis). In the presence of different concentrations (0.025–0.1 mM) of the tested selenocompounds, 0.1 mM peroxynitrite caused a distinct decrease in the level of carbonyl group formation and tyrosine nitration in plasma proteins. Moreover, these selenocompounds also inhibited plasma lipid peroxidation induced by ONOO−1 (0.1 mM). The obtained results indicate that in vitro bis(2-aminophenyl)-diselenide and ebselen have very similar protective effects against peroxynitrite-induced oxidative/nitrative damage to human plasma proteins and lipids.

Adhesion of blood platelets to collagen may be involved in pathogenesis of septic shock after damage of endothelial cells by LPS or inflammatory cytokines. Therefore, analysis of platelet adhesion in the presence of endotoxin seems to be of great importance. We studied in vitro the effects of LPSs (S1959, R110, R45) from Proteus mirabilis (smooth and rough types differing significantly in their composition) on the adhesion of unstimulated and thrombin-stimulated platelets to collagen. The adhesion was measured by a static method as absorbance of cell attached proteins. In this work we report that adhesion of resting platelets to collagen was stimulated by all tested LPSs. The effects were dependent on the doses of LPSs. In the presence of LPSs the inhibition of adhesion of thrombin-treated platelets to collagen was observed. The results presented in this paper indicate that LPSs from Proteus mirabilis may act directly on blood platelets and stimulate adhesion of resting platelets to collagen. On the other hand, LPSs can have an inhibitory effect on adhesion of thrombin-stimulated platelets to collagen.