Exposure Of Blood Platelets Research Articles

Comparison of biological activity of phenolic fraction from roots of Alhagi maurorum with properties of commercial phenolic extracts and resveratrol

Phenolic compounds have different biological properties, including antioxidative activities, but they may also be prooxidants. The effect of phenolic fraction from roots of Alhagi maurorum on oxidative protein/lipid damages (determined by such parameters as levels of protein thiol groups and the concentration of thiobarbituric acid reactive species – TBARS) in human blood platelets and human plasma after treatment with hydrogen peroxide – H2O2 (which is the strong biologic oxidant and inflammatory mediator) was studied in vitro. We also studied the effect of A. maurorum extract on blood platelet activation corresponding to thrombin-induced arachidonic acid pathway. Moreover, the present work was designed to study the effect of A. maurorum extract on selected physiological function of blood platelets – adhesion of blood platelets to collagen in vitro. The action of phenolic fraction from A. maurorum was compared with the selected commercial phenolic extracts: extract from berries of Aronia melanocarpa (Aronox®), extract from bark of Yucca schidigera and monomeric polyphenol–resveratrol (3,4′,5-trihydroxystilbene). Exposure of blood platelets or plasma to H2O2 resulted in a decrease of the level of thiol groups in proteins, and an increase of TBARS. In the presence of phenolic fraction from A. maurorum (0.5–50 µg/ml), a reduction of thiol groups oxidation together with the decrease of autoperoxidation of lipids and lipid peroxidation caused by H2O2 or thrombin was observed. The inhibitory, concentration-dependent effects of A. maurorum extract on adhesion of thrombin-activated platelets to collagen were also found. The phenolic fraction from A. maurorum acts as an antioxidant and can be useful as the natural factor protecting against diseases associated with oxidative stress. Tested fraction from A. maurorum has more effective antioxidative activity and antiplatelet properties than aronia extract or other commercial extract, however differences between their actions are not statistically significant.

Antioxidative properties of curcumin in the protection of blood platelets against oxidative stress in vitro

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.

Effects of garcinol and guttiferone K isolated from Garcinia cambogia on oxidative/nitrative modifications in blood platelets and plasma

The effects of garcinol and guttiferone K, two polyisoprenylated benzophenones occurring a food plant called Garcinia cambogia, on oxidative/nitrative protein damage (determined by parameters such as levels of protein carbonyl groups and nitrotyrosine residues) in human blood platelets and plasma after treatment with peroxynitrite (ONOO−) were studied in vitro. We also investigated the effects of garcinol and guttiferone K on lipid peroxidation in blood platelets and plasma induced by ONOO− (100 µM). Exposure of blood platelets or plasma to peroxynitrite (100 µM) resulted in an increased level of carbonyl groups and nitrotyrosine residues in proteins, and an increase of lipid peroxidation measured by the level of thiobarbituric acid reactive species (TBARS). In the presence of garcinol and guttiferone K (0.1–25 µg/ml), a distinct reduction in the formation of carbonyl groups in plasma and platelet proteins together with the decrease of TBARS caused by 100 µM peroxynitrite, was observed. However, garcinol and guttiferone K did not inhibit plasma and platelet protein nitration induced by peroxynitrite. Polyisoprenylated benzophenones present in human diet such as garcinol or guttiferone K in vitro have protective effects against lipid and protein oxidation and may have some promising effects in vivo because they are good antioxidants in the tested models in vitro. Garcinol and guttiferone K can be also useful as protecting factors against diseases associated with oxidative stress.

Studies on pig blood platelet responses to peroxynitrite action

The action of peroxynitrite (ONOO - ) on pig blood platelet function was studied. Incubation of washed blood platelets with ONOO - (0.062-2 mM) caused strong, dose- and time-dependent inhibitory effects on adhesion of resting and thrombin-stimulated platelets to collagen and on platelet aggregation induced by thrombin. The platelet responses were measured 10 s after addition of peroxynitrite to platelets ('zero time') and after 60 min incubation of platelets with ONOO - . Exposure of blood platelets to peroxynitrite (60 min) brought about platelet lipid peroxidation assessed by the thiobarbituric acid method but did not cause any changes in blood platelet proteins as determined by SDS-polyacrylamide gel electrophoresis.

Modulation of cisplatin toxicity in blood platelets by glutathione depletion.

The effect of a specific inhibitor of gamma-glutamylcysteine synthetase, L-buthionine sulfoximine (BSO), on the action of cisplatin in pig blood platelets was investigated. We show that the effect of BSO, blocking glutathione (GSH) synthesis, on depletion of GSH level in platelets was time and dose dependent (p<0.05). Exposure of blood platelets to BSO reduced not only the cellular GSH content, but diminished also cytotoxicity effects of cisplatin on platelets. Cytotoxic effects of cisplatin were determined by the extent of oxidative stress in these cells, i.e. lipid peroxidation expressed as thiobarbituric acid reactive substances and oxygen free radicals (superoxide anion). The obtained results confirm that the intracellular level of GSH plays an important role in the action of cisplatin on blood platelets.

Antiaggregatory Properties of Beta-adrenoceptor Blocking Drugs are Related to their Physico-chemical Properties

The effects of the beta-adrenoceptor blockers (BAB) alprenolol, atenolol, metipranolol, oxprenolol, practolol and propranolol on platelet aggregation stimulated by ADP, thrombin and collagen were investigated. The BAB were divided into three groups according to their liposolubility and antiaggregatory activity. The first group consists of the highly active drugs alprenolol and propranolol; metipranolol and oxprenolol are in the second group; the least active atenolol and practolol are in the third group. Collagen induced platelet aggregation was inhibited by BAB to the highest extent, followed by inhibition of thrombin stimulated aggregation. ADP-induced aggregation was inhibited to the lowest extent. Consistent with the rank order of their partition coefficients, BAB inhibited aggregation with the following order of potency: propranolol > alprenolol > metipranolol > oxprenolol > practolol > atenolol. A positive correlation was found between inhibition of thrombin and collagen stimulated platelet aggregation and the partition coefficients of the individual drugs. A significant time-dependent inhibition of platelet aggregation developed within 30 s of exposure of blood platelets to BAB.

Evidence for Two Populations of Disulphide Bonds on Blood Platelets

The aggregation of human blood platelets is inhibited when membrane disulphide bonds are reduced (Born, 1967). Exposure of blood platelets to U.V. light induces platelet aggregation (Dickson et al., 1971) possibly also by the reduction of disulphide bonds (Doery et al., 1973). By using dithiothreitol as a reducing agent, we have confirmed that low concentrations inhibit platelet aggregation but at higher concentrations dithiothreitol itself induces aggregation. Platelet-rich plasma was prepared from human and rat blood by differential centrifugation, and platelet aggregation was measured photometrically in 0.1 ml samples (Gordon & Drummond, 1974). Suspensions of washed human platelets were prepared from 2ml volumes of platelet-rich plasma by using an albumin density gradient (Walsh, 1972). Total adenine nucleotides and 5-hydroxytryptamine in platelets and in plasma or suspending medium were measured fluorimetrically (Gordon & Drummond, 1974; Drummond & Gordon, 1974) before and after platelet aggregation to determine the extent of the platelet-release reaction. Lactate dehydrogenase was measured by the method of Wroblewski & La Due (1955) to check for possible lysis of platelets. Platelets aggregate when collagen or ADP is added to stirred platelet-rich plasma. The response to collagen is characterized by a lag of several seconds duration, followed by irreversible aggregation caused by release of amines and nucleotides from the platelets. In contrast, the aggregation response to low concentrations of ADP is rapid, reversible and not associated with the release of platelet constituents. The addition of dithiothreitol to platelet-rich plasma samples inhibited aggregation induced by both ADP and collagen, but dithiothreitol was less effective against ADP. The I.C.50 value (concentration of dithiothreitol inhibiting aggregation by 50%) was 0 .45m~ for collagen and 2 .15m~ for ADP. In each case, a half-maximal aggregating stimulus was used and dithiothreitol was preincubated in platelet-rich plasma for 2min at 37°C before adding the aggregating agent. The addition of 1 m-dithiothreitol abolished collagen-induced aggregation in human platelet-rich plasma and also completely inhibited the accompanying plateletrelease reaction : collagen released 34 % of platelet 5-hydroxytryptamine in control samples, but caused no release in samples containing 1 m-dithiothreitol. The inhibitory potency of dithiothreitol was similar in human and in rat platelet-rich plasma. When samples of platelet-rich plasma were preincubated with dithiothreitol at 37°C before addition of the aggregating agent, inhibition increased with time, reaching a maximum after 10-2Omin. When dithiothreitol was added to human platelet-rich plasma in concentrations of 3 m ~ and above, it caused platelet aggregation after a prolonged lag phase (lamin). The duration of the lag decreased and the rate of aggregation increased with increasing dithiothreitol concentrations. In suspensions of washed human platelets dithiothreitol did not produce aggregation unless fibrinogen was also added. Even when fibrinogen was present there was always a lag of about 2min between the addition of dithiothreitol and the start of aggregation. When dithiothreitol had been incubated with the platelet suspension for 2min or longer in the absence of fibrinogen, the subsequent addition of fibrinogen caused immediate aggregation. The lag before dithiothreitol-induced aggregation was due to an action of dithiothreitol on the platelets and not on the fibrinogen, since preincubation of dithiothreitol with fibrinogen did not alter the duration of the lag (Fig. 1). Aggregation of human platelets by dithiothreitol was associated with the release of 10-15 % of platelet 5-hydroxytryptamine and adenine nucleotides. No release of lactate dehydrogenase from the platelets could be detected during aggregation by dithiothreitol, suggesting that the release of amines and nucleotides was selective and not caused by platelet lysis. When disulphide bonds are reduced by dithiothreitol, there is concomitant formation