The Effect of Vitamin D3 on Neutrophil Extracellular Trap Formation in High-Glucose Conditions.

NETosis, i.e., the formation of neutrophil extracellular traps (NET), and neutrophil autophagy are important elements in the pathogenesis and the development of complications of type 2 diabetes mellitus (T2DM). Therefore, the search of drugs that can regulate the level of NETosis and autophagy in T2DM is relevant. Here we studied an ex vivo NET formation and neutrophil death in whole blood from healthy subjects upon the addition of glucose up to a high concentration of 15 mM or/and the phorbol ester PMA (phorbol-12-myristate-13-acetate). Their individual and combined action caused neutrophil death and an increase in NET content. It can be hypothesized that this resulted from activation of NETosis and autophagy. It was also shown that this activation of NETosis and autophagy is completely prevented by daily intake of 1000 IU vitamin D3 for 14 days. Therefore, vitamin D3 supplementation can be considered as a preventive measure against the development of T2DM complications.

Structure-biological activity relationships of myeloperoxidase to effect on platelet activation.

Myeloperoxidase (MPO), an oxidant-producing enzyme of neutrophils, has been shown to prime platelet activity promoting immunothrombosis. Native MPO is a homodimer, consisting of two identical protomers (monomer) connected by a single disulfide bond. But in inflammatory foci, MPO can be found both in the form of a monomer and in the form of a dimer. Beside MPO can also be in complexes with other molecules and be modified by oxidants, which ultimately affect its physicochemical properties and functions. Here we compared the effects of various forms of MPO as well as MPO in complex with ceruloplasmin (CP), a physiological inhibitor of MPO, on the platelet activity. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. MPO was modified with HOCl in a molar ratio of 1:100 (MPO-HOCl). Using surface-enhanced Raman scattering (SERS) spectroscopy we showed that peaks at about 510 and 526 cm-1 corresponded to disulfide bond was recognizable in the SERS-spectra of dimeric MPO, absent in the spectrum of hemi-MPO and less intense in the spectra of MPO-HOCl, which indicates the partial decomposition of dimeric MPO with a disulfide bond cleavage under the HOCl modification. It was shown hemi-MPO to a lesser extent than dimeric MPO bound to platelets and enhanced their agonist-induced aggregation and platelet-neutrophil aggregate formation. MPO modified by HOCl and MPO in complex with CP did not bind to platelets and have no effect on platelet activity. Thus, the modification of MPO by HOCl, its presence in monomeric form as well as in complex with CP reduces MPO effect on platelet function and consequently decreases the risk of thrombosis in inflammatory foci.

Hyperglycemia and Some Aspects of Leukocyte Activation In Vitro.

We analyzed functional status of blood leukocytes in diabetes mellitus and after addition of glucose in vitro. To this end, generation of ROS and reactive halogen species by monocytes and neutrophils from patients with diabetes mellitus and healthy donors was assayed using lucigenin- and luminol-dependent chemiluminescence after stimulation with phorbol 12-myristate 13-acetate or opsonized zymosan in vitro. Formation of neutrophil extracellular traps was evaluated in the blood after addition of glucose. In comparison with donors, leukocytes from patients with diabetes mellitus were primed and this effect can be modeled by addition of glucose to the blood in vitro. Addition of glucose to donor blood also triggered the formation of neutrophil extracellular traps.

Binding of lactoferrin to the surface of low-density lipoproteins modified by myeloperoxidase prevents intracellular cholesterol accumulation by human blood monocytes.

Myeloperoxidase (MPO) is a unique heme-containing peroxidase that can catalyze the formation of hypochlorous acid (HOCl). The strong interaction of MPO with low-density lipoproteins (LDL) promotes proatherogenic modification of LDL by HOCl. The MPO-modified LDL (Mox-LDL) accumulate in macrophages, resulting in the formation of foam cells, which is the pathognomonic symptom of atherosclerosis. A promising approach to prophylaxis and atherosclerosis therapy is searching for remedies that prevent the modification or accumulation of LDL in macrophages. Lactoferrin (LF) has several application points in obesity pathogenesis. We aimed to study LF binding to Mox-LDL and their accumulation in monocytes transformed into macrophages. Using surface plasmon resonance and ELISA techniques, we observed no LF interaction with intact LDL, whereas Mox-LDL strongly interacted with LF. The affinity of Mox-LDL to LF increased with the degree of oxidative modification of LDL. Moreover, an excess of MPO did not prevent interaction of Mox-LDL with LF. LF inhibits accumulation of cholesterol in macrophages exposed to Mox-LDL. The results obtained reinforce the notion of LF potency as a remedy against atherosclerosis.

[Biomarkers of system inflammation in local and diffuse peritonitis]. / Markery sistemnogo vospaleniia pri mestnom i rasprostranennom peritonite.

In cases of any acute surgical abdominal disease the progression of purulent inflammation can lead to local or diffuse peritonitis. The indicators of the degree and specificity of the inflammatory response in blood such as cytokine concentration, neutrophil activity, plasma antioxidant capacity (thiols concentration) could be considered as potential predictors of complications. The luminol-dependent chemiluminescence (CL) response of blood activated by the phorbol ester (PMA), and the concentration of cytokines IL-6, IL-8, IL-10, myeloperoxidase (MPO) and thiols in plasma were measured in patients with uncomplicated condition (group 1, n=8), local peritonitis (group 2, n=9) or diffuse peritonitis (group 3, n=9) at admission to surgery (before surgical operation, b/o), immediately after surgical operation (a/o) and a day after surgery (1 day) as well as in healthy volunteers (norm, n=12). In all time-points the cytokines and MPO concentrations measured by ELISA, in group 3 were higher than in healthy volunteers and in patients in groups 1 and 2. Blood CL demonstrated a more than 5-fold increase above the normal values in all patients, and was also higher in group 2 as compared to group 1 (b/o and a/o). Patients in group 3 had shown both maximum and minimum of CL values, which could be a consequence of neutrophil priming or exhaustion ("immune paralysis"), respectively. The same patients' plasma exhibited low thiol concentration (≤30% vs normal values). In patients with fatal outcomes (group 3, n=2) within a day after surgery, either a decrease of the CL to zero values concurrently with elevated IL-8 and IL-6 concentrations and low thiol levels was observed, or CL exceeded normal values more than 20 times with concurrent complete exhaustion of the plasma thiol pool. No clear dependency between the plasma parameters and neutrophil activity was found. Hence a parameter set for prognosis and/or early diagnosis of infectious complications in acute abdominal pathology should include different biomarkers of the inflammatory response: cytokine profile (IL-6, IL-8, IL-10), MPO and neutrophil activity, antioxidant plasma capacity (e.g., total thiols concentration).

The Role of Halogenative Stress in Atherogenic Modification of Low-Density Lipoproteins.

This review discusses formation of reactive halogen species (RHS) catalyzed by myeloperoxidase (MPO), an enzyme mostly present in leukocytes. An imbalance between the RHS production and body's ability to remove or neutralize them leads to the development of halogenative stress. RHS reactions with proteins, lipids, carbohydrates, and antioxidants in the content of low-density lipoproteins (LDLs) of the human blood are described. MPO binds site-specifically to the LDL surface and modifies LDL properties and structural organization, which leads to the LDL conversion into proatherogenic forms captured by monocytes/macrophages, which causes accumulation of cholesterol and its esters in these cells and their transformation into foam cells, the basis of atherosclerotic plaques. The review describes the biomarkers of MPO enzymatic activity and halogenative stress, as well as the involvement of the latter in the development of atherosclerosis.

[Neutrophils as a source of factors that increase the length of the inflammatory phase of wound healing in patients with type 2 diabetes mellitus]. / Neitrofily kak istochnik faktorov, uvelichivaiushchikh prodolzhitel'nost' fazy vospaleniia ranevogo protsessa u bol'nykh sakharnym diabetom 2-go tipa.

Oxidative stress and neutrophil activation leading to an increase in myeloperoxidase (MPO), elastase and neutrophil extracellular trap (NET) levels in blood are considered as pathogenic mechanisms responsible for the development of extremity damage in people with type 2 diabetes mellitus (T2DM). The aim of this study was to analyze the relationship between factors, associated with neutrophil activation, and the length of the initial phase of wound healing (the inflammatory phase) in T2DM patients. Patients were divided retrospectively into three groups depending on the damage extent: group 1 (wound on toe) < group 2 (wound on foot) < group 3 (wound on lower leg). Compared to the control group (healthy volunteers), T2DM patients at admission to hospital had significantly (p<0.05) increased levels of blood glucose and glycated hemoglobin (groups 1-3), ESR (groups 1 and 3), blood neutrophil count (groups 2 and 3), plasma MPO concentration (groups 1-3) and blood NET concentration (group 3) and decreased levels of plasma thiols (groups 1-3) and erythrocyte glutathione peroxidase activity (groups 2 and 3). The length of hospital stay after surgical procedures corresponded to the length of the inflammatory phase of the wound healing process and correlated with the number of blood neutrophils in patients before surgery (r=0.72, p<0.05). Leukocytic intoxication index depended on wound area (r=0.59, p<0.05), and it was significantly higher for groups 2 and 3 compared to the control group and group 1. The neutrophil count before surgery in T2DM patients with damage in the lower extremities correlated with the length of the inflammatory phase of wound healing. The correlation found can be attributed to an increase in extracellular MPO and NETs, which, in its turn, results from the activation and degranulation of neutrophils and netosis. Thus, the duration of the inflammatory phase of wound healing depends on specific aspects of systemic inflammation increasing oxidative/halogenative stress and intoxication.

[Enzymatic and bactericidal activity of monomeric and dimeric forms of myeloperoxidase]. / Fermentativnaia i bakteritsidnaia aktivnost' monomernoi i dimernoi form mieloperoksidazy.

This study was carried out to compare the enzymatic and bactericidal activity of mature, dimeric myeloperoxidase (MPO) and its monomeric form. Dimeric MPO was isolated from HL-60 cells. Hemi-MPO obtained from dimeric MPO by reductive cleavage of a disulfide bond between protomeric subunits was used as the monomeric form. Both peroxidase and halogenating (chlorinating) activities of MPO were assayed, each of them by two methods. Bactericidal activity of the MPO/Н2О2/Cl- system was tested using the Escherichia coli laboratory strain DH5a. No difference in the enzymatic and bactericidal activity between dimeric MPO and hemi-MPO was found. Both forms of the enzyme also did not differ in the resistance to HOCl, the main product of MPO. HOCl caused a dose-dependent decrease in peroxidase and chlorinating activity, and the pattern of this decrease was identical for dimeric MPO and hemi-MPO. At equal heme concentration, a somewhat higher bactericidal effect was observed for the hemi-MPO/Н2О2/Cl- system compared with the dimeric MPO/Н2О2/Cl- system. However, this is most likely not related to some specific property of hemi-MPO and can be accounted for by the higher probability of contacting between bacterial surface and hemi-MPO molecules due to their two-fold greater number relative to that of dimeric MPO molecules at the same heme concentration. By using Western-blotting with antibodies to MPO, we showed, for the first time, that the dimeric molecule of MPO could be cleaved into two monomeric subunits by HOCl, most probably due to oxidation of the disulfide bond between these subunits. This finding suggests that appearance in blood of MPO corresponding in mass to its monomer may result from the damage of dimeric MPO by reactive halogen species, especially upon their overproduction underlying oxidative/halogenative stress in inflammatory diseases.

[Exocytosis of myeloperoxidase from activated neutrophils in the presence of heparin]. / Ékzotsitoz mieloperoksidazy pri aktivatsii neitrofilov v prisutstvii geparina.

Exocytosis of myeloperoxidase (MPO) from activated neutrophils in the presence of the anionic polysaccharide heparin was studied. It was determined that the optimal concentration of heparin (0.1 u/ml), at which there is no additional activation of cells (absence of amplification of exocytosis of lysozyme contained in specific and azurophilic granules). It was found that after preincubation of cells with heparin (0.1 u/ml) the exocytosis of MPO from neutrophils activated by various stimulants (fMLP, PMA, plant lectins CABA and PHA-L) increased compared to that under the action of activators alone. In addition, it was shown that heparin in the range of concentrations 0.1-50 u/ml did not affect on the peroxidase activity of the MPO isolated from leukocytes. Thus, the use of heparin at a concentration of 0.1 u/ml avoids the artifact caused by the "loss" of MPO in a result of its binding to neutrophils, and increases the accuracy of the method of registration the degranulation of azurophilic granules of neutrophils based on determination of the concentration or peroxidase activity of MPO in cell supernatants.

Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase.

CP is a copper-containing ferroxidase of blood plasma, which acts as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by MPO, such as HOCl, HOBr, and HOSCN, on its spectral, enzymatic, and anti-inflammatory properties was studied. We monitored the chemiluminescence of lucigenin and luminol along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by PMA or fMLP. Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr, and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced, along with a drop in the ferroxidase and amino oxidase activities of CP. Likewise, its inhibitory effect on the halogenating activity of MPO was diminished. Sera of either healthy donors or patients with Wilson disease were co-incubated with neutrophils from healthy volunteers. In these experiments, we observed an inverse relationship between the content of CP in sera and the rate of H2O2 production by activated neutrophils. In conclusion, CP is likely to play a role of an anti-inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO-mediated oxidative modifications.

[The role of neutrophil myeloperoxidase in the development of inflammation after thermal skin burns]. / Rol' mieloperoksidazy neitrofilov v razvitii vospaleniia pri termicheskikh ozhogakh kozhi.

In the blood of children (n=16) with large thermal skin burns (> 20% of total body surface), luminol-dependent chemiluminescence (CL) of neutrophils stimulated with phorbol-12-myristate-13-acetate (PMA) and myeloperoxidase (MPO) activity in neutrophils and plasma were assayed in the early period (1-7 post-burn days). PMA-stimulated neutrophils in thermally injured patients produced higher CL than those in a reference group of healthy children (n=24), p<0.01. MPO activity was elevated in neutrophils and plasma in 40% and 57% of patients' blood samples, respectively. The albumin fraction isolated from plasma of burned patients enhanced the PMA-stimulated CL response of blood samples from healthy volunteer. Our results suggest that the acute inflammatory response induced by thermal injury involves activation of neutrophils and is accompanied by MPO release into the plasma. MPO-mediated modification of serum albumin induces its capacity to prime neutrophils and thus to enhance further inflammatory reaction.

Myeloperoxidase Stimulates Neutrophil Degranulation.

Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation.

[Myeloperoxidase activity in blood plasma as a criterion of therapy for patients with cardiovascular disease]. / Aktivnost' mieloperoksidazy v plazme krovi kak kriterii éffektivnosti lecheniia patsientov s serdechno-sosudistymi zabolevaniiami.

A significant increase in the myeloperoxidase (MPO) activity has been found in plasma of patients with stable angina and with acute coronary syndrome (ACS) in comparison with the control group. MPO concentration was significantly increased in plasma of ACS patients. Reduced MPO activity in the treated ACS patients correlated with a favorable outcome of the disease. Generally, changes in plasma MPO concentration coincided with changes in lactoferrin concentration thus confirming the role of neutrophil degranulation in the increase of plasma concentrations of these proteins. The increase in MPO activity was obviously determined by modification of the MPO protein caused by reactive oxygen species and halogen in the molar ratio of 1 : 25 and 1 : 50. The decrease in plasma MPO activity may be associated with increased plasma concentrations of the physiological inhibitor of its activity, ceruloplasmin, and also with modification of the MPO protein with reactive oxygen species and halogen at their molar ratio of 1 : 100 and higher. Thus, MPO activity may be used for evaluation of effectiveness of the treatment of cardiovascular diseases.

Kinetic method for assaying the halogenating activity of myeloperoxidase based on reaction of celestine blue B with taurine halogenamines.

Myeloperoxidase (MPO) is a challenging molecular target which, if put under control, may allow regulating the development of inflammatory reactions associated with oxidative/halogenative stress. In this paper, a new kinetic method for assaying the halogenating activity of MPO is described. The method is based on measuring the rate of iodide-catalyzed oxidation of celestine blue B (CB) by oxygen and taurine N-chloramine (bromamine). The latter is produced in a reaction of taurine with HOCl (HOBr). CB is not a substrate for the peroxidase activity of MPO and does not react with hydrogen peroxide and superoxide anion radical. Taurine N-chloramine (bromamine) reacts with CB in molar ratio of 1:2. Using the new method, we studied the dependence of MPO activity on concentration of substrates and inhibitors. The specificity of MPO inhibition by non-proteolyzed ceruloplasmin is characterized. The inhibition of taurine N-chloramine production by neutrophils and HL-60 cells in the presence of MPO-affecting substances is demonstrated. The new method allows determining the kinetic parameters of MPO halogenating activity and studying its inhibition by various substances, as well as screening for potential inhibitors of the enzyme.

Interaction of ceruloplasmin with eosinophil peroxidase as compared to its interplay with myeloperoxidase: Reciprocal effect on enzymatic properties.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex between MPO and ceruloplasmin (CP). Considering the high structural homology between MPO and EPO, we studied the latter's interaction with CP and checked whether EPO becomes inhibited in a complex with CP. Disc-electrophoresis and gel filtration showed that CP and EPO form a complex with the stoichiometry 1:1. Affinity chromatography of EPO on CP-agarose (150 mM NaCl, 10 mM Na-phosphate buffer, of pH 7.4) resulted in retention of EPO. EPO protects ceruloplasmin from limited proteolysis by plasmin. Only intact CP shifted the Soret band typical of EPO from 413 to 408 nm. The contact with CP likely causes changes in the heme pocket of EPO. Peroxidase activity of EPO with substrates such as guaiacol, orcinol, o-dianisidine, 4-chloro-1-naphtol, 3,3',5,5'-tetramethylbenzidine, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) is inhibited by CP in a dose-dependent manner. Similar to the interaction with MPO, the larger a substrate molecule, the stronger the inhibitory effect of CP upon EPO. The limited proteolysis of CP abrogates its capacity to inhibit the peroxidase activity of EPO. The peptide RPYLKVFNPR (corresponding to amino acids 883-892 in CP) inhibits the peroxidase and chlorinating activity of EPO. Only the chlorinating activity of EPO is efficiently inhibited by CP, while the capacity of EPO to oxidize bromide and thiocyanate practically does not depend on the presence of CP. EPO enhances the p-phenylenediamine-oxidase activity of CP. The structural homology between the sites in the MPO and EPO molecules enabling them to contact CP is discussed.

Pluronics suppress association of low-density lipoproteins inducing atherogenesis.

We studied the effect of pluronics P85, L61, and F68 with different hydrophilic-lipophilic characteristics on association of LDL. It was found that pluronics with pronounced hydrophobic properties (P85 and L61) in concentrations close to or surpassing the critical concentration of micelle formation inhibited LDL association, while hydrophilic pluronic F68 in all concentrations had no effect on LDL association.

Measurement of plasma hemoglobin peroxidase activity.

We described a spectrophotometric method for measuring hemoglobin peroxidase activity in human plasma using o-dianisidine (o-DA) as the substrate and myeloperoxidase specific inhibitor 4-aminobensoic acid hydrazide (ruling out the probable contribution of myeloperoxidase to the measured parameter value). The optimal conditions (pH 5.5; 2 mM H2O2) have been determined, at which hemoglobin makes the main contribution to plasma oxidation of o-DA. A significant positive correlation between hemoglobin peroxidase activity measured by the spectrophotometric method and hemoglobin level measured by the pyridine hemochromogenic method has been detected (r=0.624; p<0.01) in plasma specimens from 16 donors. Plasma hemoglobin peroxidase activities were measured in healthy individuals and patients with type 2 diabetes mellitus and coronary heart disease. High plasma hemoglobin peroxidase activities in both groups of patients indicates disorders in the mechanisms of clearance of hemoglobin and its highly reactive derivatives and can serve as specific markers of diseases associated with oxidative stress.

[Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils].

It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase--4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC50 = 20 microM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a "vicious circle" of neutrophil activation at the inflammatory site.

Hypochlorous acid as a precursor of free radicals in living systems.

Hypochlorous acid (HOCl) is produced in the human body by the family of mammalian heme peroxidases, mainly by myeloperoxidase, which is secreted by neutrophils and monocytes at sites of inflammation. This review discusses the reactions that occur between HOCl and the major classes of biologically important molecules (amino acids, proteins, nucleotides, nucleic acids, carbohydrates, lipids, and inorganic substances) to form free radicals. The generation of such free radical intermediates by HOCl and other reactive halogen species is accompanied by the development of halogenative stress, which causes a number of socially important diseases, such as cardiovascular, neurodegenerative, infectious, and other diseases usually associated with inflammatory response and characterized by the appearance of biomarkers of myeloperoxidase and halogenative stress. Investigations aimed at elucidating the mechanisms regulating the activity of enzyme systems that are responsible for the production of reactive halogen species are a crucial step in opening possibilities for control of the development of the body's inflammatory response.

[Glutathione-dependent regulation of platelet aggregation with neutrophils and tumor cells].

It is shown that in the presence of reduced glutathione at low concentrations (1-5 microM) the extent of platelet aggregation with neutrophils increases and the lag period of platelet aggregation induced by tumor cells decreases. At the same time in the presence of reduced glutathione at high concentration (3 mM) the extent of platelet aggregation with neutrophils decreases, and the lag period of platelet aggregation induced by tumor cells increases. It is established that glutathione-dependent regulation of the intercellular contact formation between platelets and neutrophils depends on the ratio of glutathione oxidized and reduced forms: at fixed total glutathione concentration of 5 microM, increase of glutathione redox potential from -175 mV to 0 mV led to reduction in platelet aggregation with neutrophils. Thus, it is shown for the first time, that GSH has priming effect on the platelet aggregation with neutrophils and tumor cells, which may contribute to the regulation of inflammatory diseases and cancer.