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.

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.

[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.

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.

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.

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.

[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.

[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.

[Increased myelopepoxidase activity is a risk factor for ishemic heart disease in patients with diabetes mellitus].

Using previously developed spectro-photonmetrical method (Bioorg. Khim. 2009. V. 35. pp. 629-639), a significant increase of myeloperoxidase (MPO) activity was found in blood plasma of patients with type 2 diabetes mellitus (DM2) without of cardiovascular complications, as well as with ischemic heart disease (IHD). Plasma MPO concentration measured by an enzyme-linked immunosorbent assay was significantly higher only in blood plasma of patient with DM2 and IHD. A direct and significant correlation between MPO activity and MPO concentration was observed only in blood plasma samples from healthy donors. Increased MPO activity did not correlate with MPO concentration in blood plasma of patients with DM2 and DM2 with IHD. Taken together, these results highlight the necessity for studying of the MPO role in the development of pathological processes to determine both the amount of enzyme and its peroxidase activity in the blood. The proposed approach gives comprehensive information about the relationship between MPO activity and MPO concentration in patient blood. Since the high concentration of MPO is a diagnostically significant parameter in the prediction of endothelial dysfunction and cardiovascular disease development, the obtained results evidence the contribution of MPO-dependent reactions in cardiovascular complications associated with diabetes. MPO activity may serve as an additional diagnostic criterion for determination of risk of IHD in DM patients.

Functional activity of neutrophils in diabetes mellitus and coronary heart disease: role of myeloperoxidase in the development of oxidative stress.

We performed a comparative analysis of functional activity of neutrophils in patients with type 2 diabetes mellitus with and without symptoms of CHD. Enhanced H2O2 production by neutrophils in response to N-formyl-Met-Leu-Phe (fMLP) was found in patients with type 2 diabetes mellitus. In patients with type 2 diabetes mellitus associated with CHD, fMLP-induced release of myeloperoxidase from azurophilic granules of neutrophils was reduced and plasma myeloperoxidase level was elevated. Increased peroxidase activity of myeloperoxidase, reduced plasma catalase activity, and increased levels of TBA-reactive lipid peroxidation products and oxidized glutathione were detected in patients of both groups. Since myeloperoxidase is an important neutrophilic mediator of oxidative stress, its increased activity in the blood can be an additional marker of oxidative stress and cardiovascular risk in patients with diabetes mellitus.

[Regulation of the functional and mechanical properties of platelet and red blood cells by nitric oxide donors].

The effect of NO donors (sodium nitroprusside, S-nitrosoglutathione, dinitrosyl-iron complexes) on the functional and mechanical properties of human platelets and red blood cells has been investigated. It has been established by atomic force microscopy that NO donor-induced platelet disaggregation is accompanied by changes in the elastic properties of cells. It has been shown that, in the presence of NO donors, the detergent-induced hemolysis of red blood cells is delayed, and the elasticity modulus of these cells decreases. The results obtained indicate that NO donors regulate the structural and functional properties of platelets and red blood cells.

Effect of dinitrosyl iron complexes on platelet aggregation induced by HeLa cervical carcinoma cells.

We studied the effect of dinitrosyl iron complexes with glutathione ligands on platelet aggregation with HeLa tumor cells. It was shown that dinitrosyl iron complexes not only inhibited cell aggregation, but being added at early stages can also block this process. These findings dictate further studies of dinitrosyl iron complexes as a compound reducing metastasizing and thrombus-formation in tumor patients.

[New approaches to the measurement of the concentration and peroxidase activity of myeloperoxidase in human blood plasma].

A novel method for spectrometrical measurement of myeloperoxidase (MPO) activity in plasma with o-dianisidine (DA) as a substrate is proposed. We have determined the optimal conditions, including the pH and hydrogen peroxide concentration, under which MPO is the main contributor to DA oxidation in plasma. Specific MPO inhibitors, salicylhydroxamic acid or (4-aminobenzoyl)hydrazide, are added to measure the activity of other heme-containing peroxidases (mainly hemoglobin and its derivatives) and subtract their contribution from the total plasma peroxidase activity. Plasma MPO concentrations are quantified by a new enzyme-linked immunosorbent assay (ELISA) developed by us and based on the use of antibodies raised in rats and rabbits. The sensitivity of this ELISA is high: 0.2-250 ng/ml. A direct and significant (P < 0.0001) correlation was observed between the MPO activities measured spectrometrically and by ELISA in blood samples from 38 healthy donors. The proposed approaches to MPO measurement in plasma can be used to evaluate the enzyme activity and concentration, as well as the efficacy of mechanisms by which MPO is regulated under physiological conditions and against the background of various inflammatory diseases.

[The formation of stable aggrregates of human platelets induced by lectin from Solanum tuberosum].

It has been demonstrated for the first time that GlcNAc-specific lectin from Solanum tuberosum induces the formation of haptenic sugar-resistant intercellular contacts (HSR-contacts) in platelet aggregation and does not induce stable neutrophil and lymphocyte aggregation. The formation of HSR-contacts in platelets was significantly impaired by the inhibitors of cAMP phosphodiesterase (papaverine) and arachidonic acid methabolism (indomethacin, aristolochic acid, and MK-886) as well as by the sulfhydryl reagent N-ethylmaleimide. The results obtained indicate that STA can be used to study the mechanisms of stable platelet aggregation, to screen drugs with potential antithrombotic activity, and to develop new cell engineering techniques.

Impact of MK-886 on H2O2 generation by human neutrophils and cell degranulation.

Metabolic inhibitors can clearly affect different aspects of the functional activity of cells. This property was studied in the present work with respect to MK-886, a well-known inhibitor of the 5'-lipoxygenase-activating protein. It was found that this inhibitor in a micromolar concentration range (2-20 microM) induced in a dose-dependent manner H2O2 generation by human neutrophils and the release of lysozyme from the cells. The MK-886-induced activation of neutrophils was accompanied by a significant decrease in N-(1-pyrene)maleimide-accessible SH-groups in the cells. According to its activity, MK-886 can be considered an agonist that causes up-regulation of inherent neutrophil functions. In summary, the results indicate that during the application of MK-886 as a 5;-lipoxygenase inhibitor in neutrophils, the impact of the compound on the functional activity of the cells should be taken into consideration.

Cell-type dependence of stability modulation of lectin-initiated contacts by impairment of multivalent carbohydrate binding and intracellular signaling.

Initial ligand selection and the intermolecular spatial arrangement of glycan-lectin complexes are assumed to be essential to induce formation of stable cell aggregates by a lectin. To distinguish effects of these two processes, the tetrameric mistletoe lectin and its isolated B-chain were used. A reduced impact of multivalency for Ehrlich ascites tumor cells in contrast to rat thymocytes was revealed. Signaling is thus initiated in a cell-type-dependent manner. Using selective metabolic inhibitors to reduce signal transfer for aggregate stability, decrease in cellular SH-group level was shown to be a common effect accompanying suppression of lectin-dependent aggregate stability. The results underscore an intrinsic variability in the relative importance of lectin-dependent glycan aggregation on the cell surface for triggering post-binding lectin effects.

Effect of signaling inhibitors on the release of lysozyme from human neutrophils activated by Sambucus nigra agglutinin.

The effect of alpha-NeuAc(2-->6)Gal/GalNAc-specific lectin from Sambucus nigra (SNA) on the release of lysozyme from human neutrophils was studied in vitro. Interaction of cells with the lectin was accompanied by dose-dependent release of lysozyme, which was increased in the presence of cytochalasin B. The involvement of intracellular signaling pathways in the lectin-induced degranulation of neutrophils was determined using a panel of specific inhibitors tested at concentrations in the range of 10-100 microM. Aristolochic acid (a phospholipase A2 inhibitor), indomethacin (a cyclooxygenase inhibitor), neomycin sulfate (a phospholipase C inhibitor), trifluoperazine (a calmodulin antagonist/protein kinase C inhibitor), N-ethylmaleimide (a sulfhydryl reagent), and guanosine-5;-O-(2-thiodiphosphate) (a G-protein inhibitor) were found to reduce SNA-induced lysozyme release from neutrophils by 20-45%. The treatment of cells with bisindolylmaleimide (a protein kinase C inhibitor), H-8 (an inhibitor of protein kinases A, C, G and of myosin light chain kinase), PD 98059 (a MAP kinase inhibitor), and (+/-)-methoxyverapamil (a Ca2+-channel blocker) failed to affect the release of lysozyme. These results indicate that only selective intracellular pathways associated with activation of G-proteins and phospholipid metabolism as well as the thiol-dependent signaling systems are apparently involved in the realization of the SNA-induced degranulation response of human neutrophils.

Dissection of the impact of various intracellular signaling pathways on stable cell aggregate formation of rat thymocytes after initial lectin-dependent cell association of using a plant lectin as model and target-selective inhibitors.

Bivalent lectins as bridging molecules between cells or cell surface lectins as docking points are involved in mediation of cell adhesion by specific recognition of suitable glycoconjugates on an opposing surface. The initial contact formation by a lectin can lead to intracellular post-binding events which effect stable cell association even in the presence of the haptenic sugar. To delineate the participation of intracellular signaling pathways in the cascade of reactions to establish firm association, reagents with proven inhibitory capacity on certain biochemical targets provide suitable tools. Using this approach with rat thymocytes and the galactoside-binding lectin from mistletoe (Viscum album L. agglutinin, VAA) as a model, a panel of 27 inhibitors with impact on e.g. several types of kinases, tyrosine phosphatases, NO synthases, G proteins, enzymes of arachidonate and cyclic nucleotide metabolism and calmodulin was systematically tested with respect to their capacity to impair the formation of lactose-resistant cell aggregates. In addition to the recently reported effectiveness of N-ethylmaleimide, nordihydroguaiaretic acid, and trifluoperazine the agents diacylglycerol kinase inhibitor II, emodin, D609, DPI, KT5720, KT5926, MK-886, bisindolylmaleimide I, and (+/-)methoxyverapamil were able to reduce aggregate stability in the presence of the haptenic sugar. Thus, various types of kinases including p561lck tyrosine kinase, lipoxygenases, phosphatidylcholine-specific phospholipase C as well as calmodulin and Ca(2+)-currents, but not modulators of the metabolism of cyclic nucleotides, NO synthases, MAP kinases, tyrosine phosphatases and phospholipase A (preferentially group II) and C can play a role in eliciting contact stability. More than one principal signaling pathway appears to be linked to the measurable parameter, since inhibitory substances show additive properties in co-incubation assays and differentially affect two lectin-elicited cellular activities, i.e. intracellular movement of Ca(2+)-ions and H2O2-generation, which can accompany cell adhesion and aggregation. Pronounced differences in the extent of modulation of H2O2-generation in human neutrophils by the same set of substances emphasizes that general conclusions on the post-binding effects for a certain lectin in different cell types are definitely precluded. In aggregate, the approach to employ inhibitors with target selectivity intimates an involvement of protein kinases A, C, Ca2+/calmodulin-dependent protein kinase II, p56lck tyrosine kinase, leukotrienes and/or hydroxyeicosatetraenoic acids, phosphatidylcholine-specific phospholipase C and Ca(2+)-fluxes in events following initial binding of a galactoside-specific plant lectin to rat thymocytes which establish firm cell contacts.