Incorporation of Pectin into Vaterite Microparticles Prevented Effects of Adsorbed Mucin on Neutrophil Activation.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP-luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

pH-Dependent HEWL-AuNPs Interactions: Optical Study.

Optical methods (spectroscopy, spectrofluorometry, dynamic light scattering, and refractometry) were used to study the change in the state of hen egg-white lysozyme (HEWL), protein molecules, and gold nanoparticles (AuNPs) in aqueous colloids with changes in pH, and the interaction of protein molecules with nanoparticles was also studied. It was shown that changing pH may be the easiest way to control the protein corona on gold nanoparticles. In a colloid of nanoparticles, both in the presence and absence of protein, aggregation-deaggregation, and in a protein colloid, monomerization-dimerization-aggregation are the main processes when pH is changed. A specific point at pH 7.5, where a transition of the colloidal system from one state to another is observed, has been found using all the optical methods mentioned. It has been shown that gold nanoparticles can stabilize HEWL protein molecules at alkaline pH while maintaining enzymatic activity, which can be used in practice. The data obtained in this manuscript allow for the state of HEWL colloids and gold nanoparticles to be monitored using one or two simple and accessible optical methods.

Methylglyoxal-Modified Human Serum Albumin Binds to Leukocyte Myeloperoxidase and Inhibits its Enzymatic Activity.

Hyperglycemia in diabetes mellitus induces modification of proteins by glucose and its derivative methylglyoxal (MG). Neutrophils perform their bactericidal activity mainly via reactive halogen (RHS) and oxygen (ROS) species generation catalyzed by myeloperoxidase (MPO) stored in neutrophil azurophilic granules (AGs) and membrane NADPH oxidase, respectively. Herein, we study the binding of human serum albumin (HSA) modified with MG (HSA-MG) to MPO and its effects on MPO activity and release by neutrophils. Peroxidase activity of MPO was registered by oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and chlorinating activity by decolorization of Celestine blue B dye. Binding of HSA-MG to MPO was studied by affinity chromatography, disc-electrophoresis, ligand Western blotting and enzyme-linked solid phase immunoassay using monoclonal antibodies (mAbs) to MPO. ROS and RHS generation were detected by lucigenin (Luc) and luminol (Lum) chemiluminescence (CL), respectively. Neutrophil degranulation was assessed by flow cytometry using fluorescent labeled antibodies to the marker proteins CD63 from AGs and CD11b from peroxidase-negative granules (PNGs). NETosis was assayed by quantifying DNA network-like structures (NET-like structures) in blood smears stained by Romanowsky. HSA-MG bound to MPO, giving a stable complex (Kd = 1.5 nM) and competing with mAbs, and non-competitively inhibited peroxidase and chlorinating MPO activity and induced degranulation of PNGs but not of AGs. HSA-MG enhanced Luc-CL per se or following PMA, unlike Lum-CL, and did not affect spontaneous or PMA-stimulated NETosis. Thus, HSA modified under hyperglycemia-like conditions stimulated NADPH oxidase of neutrophils but dampened their functions dependent on activity of MPO, with no effect on its release via degranulation or NETosis. This phenomenon could underlie the downregulation of bactericidal activity of MPO and neutrophils, and hence of innate immunity, giving rise to wound healing impairment and susceptibility to infection in patients with hyperglycemia.

Preparation and Investigation of Spherical Powder Made from Corrosion-Resistant 316L Steel with the Addition of 0.2% and 0.5% Ag.

The paper describes the production and study of spherical powder made from corrosion-resistant 316L steel with the addition of 0.2% and 0.5% Ag. The study of granulometric composition, morphology, fluidity and bulk density, phase composition, microhardness and impurity composition of the spherical powders was carried out. The study showed compliance of the spherical powders with the requirements for powders used for additive manufacturing. The fluidity of the powders was 17.9 s, and the bulk density was 3.76 g/cm3. The particles have a spherical shape with a minimum number of defects and an austenitic-ferritic structure. The study of the phase composition of ingots, wires and powders showed that the ingot structure of all samples consists of austenite. According to the results of studies of the phase composition of the wire, there is a decrease in γ-Fe and an increase in α-Fe and σ-NiCr in going from wire No. 1 to wire No. 3. According to the results of studies of the phase composition of the powder particles, there are three phases, γ-Fe, α-Fe, and Fe3O4. The study of microhardness showed a decrease in HV depending on the increase in silver. The hardness of the powder is lower than that of the ingot by 16-24% due to the presence of a ferritic phase in the powder. As a result of plasma spraying, an increase in residual oxygen is observed, which is associated with the oxidation of the melt during plasma dispersion. The amount of nitrogen and sulfur does not change, while the amount of carbon and hydrogen decreases, and the impurities content corresponds to the standards for corrosion-resistant steel. Qualitative and quantitative analysis of the silver content in the samples indicates that it was not affected by the stages involved in obtaining the spherical powder.

New Application of the Commercially Available Dye Celestine Blue B as a Sensitive and Selective Fluorescent "Turn-On" Probe for Endogenous Detection of HOCl and Reactive Halogenated Species.

Hypochlorous acid (HOCl) derived from hydrogen peroxide and chloride anion by myeloperoxidase (MPO) plays a significant role in physiological and pathological processes. Herein we report a phenoxazine-based fluorescent probe Celestine Blue B (CB) that is applicable for HOCl detection in living cells and for assaying the chlorinating activity of MPO. A remarkable selectivity and sensitivity (limit of detection is 32 nM), along with a rapid "turn-on" response of CB to HOCl was demonstrated. Furthermore, the probe was able to detect endogenous HOCl and reactive halogenated species by fluorescence spectroscopy, confocal microscopy, and flow cytometry techniques. Hence, CB is a promising tool for investigating the role of HOCl in health and disease and for screening the drugs capable of regulating MPO activity.

Neutrophil Activation by Mineral Microparticles Coated with Methylglyoxal-Glycated Albumin.

Hyperglycemia-induced protein glycation and formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of diabetic complications and pathological biomineralization. Receptors for AGEs (RAGEs) mediate the generation of reactive oxygen species (ROS) via activation of NADPH-oxidase. It is conceivable that binding of glycated proteins with biomineral particles composed mainly of calcium carbonate and/or phosphate enhances their neutrophil-activating capacity and hence their proinflammatory properties. Our research managed to confirm this hypothesis. Human serum albumin (HSA) was glycated with methylglyoxal (MG), and HSA-MG was adsorbed onto mineral microparticles composed of calcium carbonate nanocrystals (vaterite polymorph, CC) or hydroxyapatite nanowires (CP). As scopoletin fluorescence has shown, H2O2 generation by neutrophils stimulated with HSA-MG was inhibited with diphenyleneiodonium chloride, wortmannin, genistein and EDTA, indicating a key role for NADPH-oxidase, protein tyrosine kinase, phosphatidylinositol 3-kinase and divalent ions (presumably Ca2+) in HSA-MG-induced neutrophil respiratory burst. Superoxide anion generation assessed by lucigenin-enhanced chemiluminescence (Luc-CL) was significantly enhanced by free HSA-MG and by both CC-HSA-MG and CP-HSA-MG microparticles. Comparing the concentrations of CC-bound and free HSA-MG, one could see that adsorption enhanced the neutrophil-activating capacity of HSA-MG.

Ex vivo observation of granulocyte activity during thrombus formation.

BACKGROUND: The process of thrombus formation is thought to involve interactions between platelets and leukocytes. Leukocyte incorporation into growing thrombi has been well established in vivo, and a number of properties of platelet-leukocyte interactions critical for thrombus formation have been characterized in vitro in thromboinflammatory settings and have clinical relevance. Leukocyte activity can be impaired in distinct hereditary and acquired disorders of immunological nature, among which is Wiskott-Aldrich Syndrome (WAS). However, a more quantitative characterization of leukocyte behavior in thromboinflammatory conditions has been hampered by lack of approaches for its study ex vivo. Here, we aimed to develop an ex vivo model of thromboinflammation, and compared granulocyte behavior of WAS patients and healthy donors. RESULTS: Thrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips. Moving granulocytes were observed in hirudinated or sodium citrate-recalcified blood under low wall shear rate conditions (100 s-1). These cells crawled around thrombi in a step-wise manner with an average velocity of 90-120 nm/s. Pre-incubation of blood with granulocyte priming agents lead to a significant decrease in mean-velocity of the cells and increase in the number of adherent cells. The leukocytes from patients with WAS demonstrated a 1.5-fold lower mean velocity, in line with their impaired actin polymerization. It is noteworthy that in an experimental setting where patients' platelets were replaced with healthy donor's platelets the granulocytes' crawling velocity did not change, thus proving that WASP (WAS protein) deficiency causes disruption of granulocytes' behavior. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. As most of the crawling granulocytes carried procoagulant platelets teared from thrombi, we propose that the role of granulocytes in thrombus formation is that of platelet scavengers. CONCLUSIONS: We have developed an ex vivo experimental model applicable for observation of granulocyte activity in thrombus formation. Using the proposed setting, we observed a reduction of motility of granulocytes of patients with WAS. We suggest that our ex vivo approach should be useful both for basic and for clinical research.

Lactoferrin modified by hypohalous acids: Partial loss in activation of human neutrophils.

Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are produced at high concentrations during inflammation and oxidative/halogenative stress by myeloperoxidase, an enzyme of azurophilic neutrophilic granules. Here we compared the effects of recombinant human lactoferrin (rhLTF) and its halogenated derivatives (rhLTF-Cl and rhLTF-Br) on functional responses of neutrophils. Our results demonstrated that after halogenative modification, rhLTF lost its ability to induce mobilization of intracellular calcium, actin cytoskeleton reorganization, and morphological changes in human neutrophils. Moreover, both forms of the halogenated rhLTF prevented binding of N-acetylglucosamine-specific plant lectin Triticum vulgaris agglutinin (WGA) to neutrophils and, in contrast to native rhLTF, inhibited respiratory burst of neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine and by two plant lectins (WGA and PHA-L). However, we observed no differences between the effects of rhLTF, rhLTF-Cl, and rhLTF-Br on respiratory burst of neutrophils induced by phorbol 12-myristate 13-acetate (PMA), digitonin, and number of plant lectins with different glycan-binding specificity. Furthermore, all rhLTF forms interfered with PMA- and ionomycin-induced formation of neutrophil extracellular traps. Thus, halogenative modification of LTF is one of the mechanisms involved in modulating a variety of signaling pathways in neutrophils to control their pro-inflammatory activity.

Investigation of Antibacterial Properties of Corrosion-Resistant 316L Steel Alloyed with 0.2 wt.% and 0.5 wt.% Ag.

The article is devoted to the study of melted ingots, plates rolled from them, and the resulting spherical powder made of corrosion-resistant 316L steel with the addition of 0.2 wt.% and 0.5 wt.% Ag. The study of antibacterial properties, microstructure, and distribution of silver concentrations, as well as qualitative analysis of silver content was carried out. The optimal mode of homogenization annealing of the ingot was 1050 °C for 9 h, which leads to the formation of an austenitic structure. It is shown that the addition of a small amount of silver does not affect the formation of the austenitic structure and silver is distributed evenly throughout the volume of the ingot. The austenitic structure also prevails in the plates after rolling. Silver is distributed evenly throughout the entire volume of the plate. It is noted that the addition of 0.2 wt.% Ag does not affect the strength, elongation, and microhardness of steel, and the addition of 0.5 wt.% Ag does not significantly reduce the strength of steel, however, all samples meet the mechanical characteristics according to the ASTM A240 standard. The qualitative chemical composition of samples made of corrosion-resistant steels was confirmed by X-ray fluorescence analysis methods. By the method of energy-dispersion analysis, the presence of a uniform distribution of silver over the entire volume of the powder particle was determined. The particles have a spherical shape with a minimum number of defects. The study of the antibacterial activity of plates and powder shows the presence of a clear antibacterial effect (bacteria of the genus Xanthomonas campestris, Erwinia carotovora, Pseudomonas marginalis, Clavibacter michiganensis) in samples No. 2 and No. 3 with the addition of 0.2 wt.% and 0.5 wt.% Ag.

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells.

Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to influence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulfide bond but in inflammatory foci as a result of disulfide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct effects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band 3 protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, significant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the first time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer leaflet of RBC membrane. However, the effects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These findings suggest that the ability of MPO protein to influence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inflammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.

Effects of recombinant human lactoferrin on calcium signaling and functional responses of human neutrophils.

Lactoferrin is a non-heme iron-binding glycoprotein with multiple health-beneficial functions including antimicrobial, antioxidant, anticarcinogenic, and immunomodulatory effects. There is emerging evidence that neutrophils may serve as targets of lactoferrin in vivo, and here we show how recombinant human lactoferrin (rhLf) can contribute to this regulation. Indeed, our results demonstrate that rhLf binds efficiently to human neutrophils and induces a variety of early cellular responses such as mobilization of intracellular Ca2+, remodeling of actin cytoskeleton, and degranulation (release of lysozyme and myeloperoxidase). In addition, rhLf facilitates lectin-induced H2O2 production and stabilization of lectin-induced cellular aggregates. The role of calcium signaling seems to be essential for rhLf-induced activation of neutrophils, as Ca2+-chelators inhibit degranulation response while lectin-induced H2O2 production correlates significantly with cytoplasmic Ca2+ elevation. Taken together, our findings justify that rhLf can activate neutrophil functions in a calcium-dependent manner and hence, can potentiate innate immune responses.

Protective Effect of Dinitrosyl Iron Complexes with Glutathione in Red Blood Cell Lysis Induced by Hypochlorous Acid.

Hypochlorous acid (HOCl), one of the major precursors of free radicals in body cells and tissues, is endowed with strong prooxidant activity. In living systems, dinitrosyl iron complexes (DNIC) with glutathione ligands play the role of nitric oxide donors and possess a broad range of biological activities. At micromolar concentrations, DNIC effectively inhibit HOCl-induced lysis of red blood cells (RBCs) and manifest an ability to scavenge alkoxyl and alkylperoxyl radicals generated in the reaction of HOCl with tert-butyl hydroperoxide. DNIC proved to be more effective cytoprotective agents and organic free radical scavengers in comparison with reduced glutathione (GSH). At the same time, the kinetics of HOCl-induced oxidation of glutathione ligands in DNIC is slower than in the case of GSH. HOCl-induced oxidative conversions of thiolate ligands cause modification of DNIC, which manifests itself in inclusion of other ligands. It is suggested that the strong inhibiting effect of DNIC with glutathione on HOCl-induced lysis of RBCs is determined by their antioxidant and regulatory properties.

Neutrophil activation in response to monomeric myeloperoxidase.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also regulate cellular functions via its nonenzymatic effects. Mature active MPO isolated from normal human neutrophils is a 145 kDa homodimer, which consists of 2 identical protomers, connected by a single disulfide bond. By binding to CD11b/CD18 integrin, dimeric MPO induces neutrophil activation and adhesion augmenting leukocyte accumulation at sites of inflammation. This study was performed to compare the potency of dimeric and monomeric MPO to elicit selected neutrophil responses. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. Analysis of the crucial signal transducer, intracellular Ca2+, showed that dimeric MPO induces Ca2+ mobilization from the intracellular calcium stores of neutrophils and influx of extracellular Ca2+ whereas the effect of monomeric MPO on Ca2+ increase in neutrophils was less. It was also shown that monomeric MPO was less efficient than dimeric MPO at inducing actin cytoskeleton reorganization, cell survival, and neutrophil degranulation. Furthermore, we have detected monomeric MPO in the blood plasma of patients with acute inflammation. Our data suggest that the decomposition of dimeric MPO into monomers can serve as a regulatory mechanism that controls MPO-dependent activation of neutrophils and reduces the proinflammatory effects of MPO.

Enzymatic and bactericidal activity of myeloperoxidase in conditions of halogenative stress.

Myeloperoxidase (MPO), found mainly in neutrophils, is released in inflammation. MPO produces reactive halogen species (RHS), which are bactericidal agents. However, RHS overproduction, i.e., halogenative stress, can also damage host biomolecules, and MPO itself may be targeted by RHS. Therefore, we examined the susceptibility of MPO to inactivation by its primary products (HOCl, HOBr, HOSCN) and secondary products such as taurine monochloramine (TauCl) and taurine monobromamine (TauBr). MPO was dose-dependently inhibited up to complete inactivity by treatment with HOCl or HOBr. TauBr diminished the activity but did not eliminate it. TauCl had no effect. MPO became inactivated when producing HOCl or HOBr but not HOSCN. Taurine protected MPO against inactivation when MPO was catalyzing oxidation of Cl- to HOCl, whereas taurine failed to prevent inactivation when MPO was working with Br-, either alone or in combination with Cl-. SCN- interfered with HOCl-mediated MPO inhibition. UV-vis spectra showed that heme degradation is involved in HOCl- and HOBr-mediated MPO inactivation. A negative linear correlation between the remaining chlorinating activity of HOCl- or HOBr-modified MPO and Escherichia coli survival upon incubation with MPO/H2O2/Cl- was found. This study elucidated the possibility of MPO downregulation by MPO-derived RHS, which could counteract halogenative stress.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca(2+), and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

Proatherogenic modification of LDL by surface-bound myeloperoxidase.

One of the factors promoting oxidative/halogenating modification of low-density lipoproteins (LDL) is myeloperoxidase (MPO). We have shown previously that MPO binds to the LDL surfaces. The LDL-MPO complex is uncoupled in the presence of peptide EQIQDDCTGDED that corresponds to a fragment of apoB-100 (445-456). In this paper we studied how this peptide, as well as inhibitors and modulators of halogenating activity of MPO such as ceruloplasmin (CP), 4-aminobenzoic acid hydrazide (ABAH) and thiocyanate (SCN(-)) affect the accumulation of cholesterol and its esters in monocytes/macrophages after incubation with LDL subjected to different kinds of MPO-dependent oxidative/halogenating modification. In the presence of H2O2 and halides MPO causes stronger proatherogenic modification of LDL than exogenous reactive halogen species (HOCl and HOBr). Both monocytes, which differentiate into macrophages, and neutrophils secrete MPO in response to the presence of damaged LDL. The peptide EQIQDDCTGDED preventing interaction between MPO and LDL reduces the uptake of modified LDL and MPO by monocytes/macrophages and thus precludes the accumulation of intracellular cholesterol. Our results indicate that binding to MPO is important for LDL to become modified and acquire proatherogenic properties. The peptide EQIQDDCTGDED, CP, ABAH, and SCN(-) can play the role of anti-atherogenic factors reducing the deleterious effect of catalytically active MPO on LDL and accumulation of cholesterol in macrophages.

Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation, degranulation, and shape change.

Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the "inflammatory loop" under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the ß subunit of ß2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators.

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry.

Myeloperoxidase (MPO) is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist). In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca(2+) through enhancement of store-operated Ca(2+) entry (SOCE). Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils.

The gp91phox subunit of flavocytochrome b(558) is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b(558). gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin-gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H(2)O(2) generation by human neutrophils treated with the lipid raft disrupting agent methyl-ß-cyclodextrin (MßCD). MßCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MßCD treatment either stimulated or inhibited H(2)O(2) production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.

Redox regulation of morphology, cell stiffness, and lectin-induced aggregation of human platelets.

Redox regulation and carbohydrate recognition are potent molecular mechanisms which can contribute to platelet aggregation in response to various stimuli. The purpose of this study is to investigate the relationship between these mechanisms and to examine whether cell surface glycocalyx and cell stiffness of human platelets are sensitive to the redox potential formed by glutathione. To this end, human platelets were treated with different concentrations (0.05 µM to 6 mM) and ratios of reduced or oxidized glutathione (GSH or GSSG), and platelet morphological, mechanical, and functional properties were determined using conventional light microscopy, atomic force microscopy, and lectin-induced cell aggregation analysis. It was found that lowering the glutathione redox potential changed platelet morphology and increased platelet stiffness as well as modulated nonuniformly platelet aggregation in response to plant lectins with different carbohydrate-binding specificity including wheat germ agglutinin, Sambucus nigra agglutinin, and Canavalia ensiformis agglutinin. Extracellular redox potential and redox buffering capacity of the GSSG/2GSH couple were shown to control the availability of specific lectin-binding glycoligands on the cell surface, while the intracellular glutathione redox state affected the general functional ability of platelets to be aggregated independently of the type of lectins. Our data provide the first experimental evidence that glutathione as a redox molecule can affect the mechanical stiffness of human platelets and induce changes of the cell surface glycocalyx, which may represent a new mechanism of redox regulation of intercellular contacts.