Activation of neutrophil membrane receptors initiates intracellular signal transduction cascades that orchestrate the cell's effector functions,including phagocytosis,production of reactive oxygen and halogen species,degranulation,and NETosis(formation of neutrophil extracellular traps[NETs]).NETs,which contain antimicrobial compounds such as myeloperoxidase(MPO),represent a strategy to combat infection.However,excessive production of NETs promotes thrombosis,diabetes mellitus,and other diseases.Therefore,investigations into the mechanisms of NETosis and the identification of modulators of this process are critical for developing strategies to address NETosis-related disorders.Here,we identified a novel NETosis inducer,human serum albumin(HSA)modified by the MPO product hypochlorous acid(HSAHOCl),whose accumulation in vivo was correlated with inflammatory processes.Using human blood neutrophils,we investigated HSAHOCl-induced NETosis and detected NET formation by flow cytometry.The results showed that the mechanism of HSAHOCl-induced NETosis involved MPO,NADPH oxidase,and phosphatidylinositol 3-kinases(PI3Ks),and that HSAHOCl activated a reactive oxygen species-dependent suicidal type of NETosis.Moreover,HSAHOCl-induced NETosis was inhibited by an anti-HSAHOCl monoclonal antibody.Thus,our findings may facilitate the development of strategies to modulate NETosis in inflammation associated with elevated MPO activity.

Introduction. Complement is a key humoral participant of immunity involved in defensive and pathological processes. Human lysozyme (hLZ), hen egg white lysozyme (HEWL) and lactoferrin (LF) are classical constituents of innate immunity. Complement and proteins of innate immunity are often co-localized but their interaction is poorly understood. The aim of the work was to characterize functional interaction of LF and hLZ/HEWL with complement. Materials and methods. Human blood serum was used as a source of complement. The complement classical and alternative pathway models contained antibody-sensitized sheep erythrocytes and rabbit erythrocytes respectively. The level of complement activation was estimated as serum hemolytic activity and anaphylatoxins production measured by ELISA. The bactericidal effect was estimated in colony-count assay. Permeabilization of outer and inner membranes of Escherichia coli ML-35p was evaluated with the use of chromogenic substrates of periplasmic and cytoplasmic enzymes. Enzymatic activity of lysozymes was measured in turbidimetric assay. Results. We confirmed the data that LF selectively inhibits the classical pathway (IC50 ~ 160 μg/mL; ~ 2 μM). We demonstrated for the first time that hLZ at high concentrations (40—160 μg/mL) moderately enhances the activity of the both complement pathways (C3 and C5 conversion as well as lytic activity) while HEWL at concentrations up to 160 μg/mL does not influence on complement activation. LF and hLZ modulated the classical pathway in an independent manner. LF did not prevent E. coli killing by serum but instead slightly increased bacterial viability in diluted serum. Lysozymes cooperated with complement in bacterial killing but LF partially prevented this in 5% serum. hLZ and HEWL accelerated bacterial inner membrane disruption regardless the presence of LF. The two enzymes accelerated the bactericidal effect of 50% serum, and hLZ did it slightly better than HEWL. Conclusion. LF and hLZ produce opposing and independent effects on complement. We refine the idea of the interaction between complement and lysozyme and propose a new model in which hLZ cooperates with complement to accelerate bacterial killing via promotion of the classical and alternative pathways activation as well as synergism with membrane-attack complex. LF does not inhibit bacterial killing by serum but can oppose hLZ and complement synergism in diluted serum.

Objective: Phagocytes activation results in the production of reactive oxygen metabolites exerting antimicrobial and host-damaging activity. Although the main pool of papers shows their potentiating action on a key humoral nexus of innate immunity, complement system, the data are controversial. Combined action of reac­tive oxygen metabolites with antimicrobial peptides of phagocytes also remains poorly characterized. Methods: We have investigated the influence of oxidative burst products on complement activation in various in vitro models and assessed the combined bactericidal action of hypochlorous acid with antimicrobial peptides. Results and Discussion: Hydrogen peroxide, including that in medium with Fe-EDTA did not affect parameters of complement activity in human blood serum. HOCl in millimolar concentrations stimulated production of C3a and C5a anaphylatoxins in 80

BACKGROUND: Coronary heart disease is one of the leading causes of death and disability worldwide. Myeloperoxidase plays the key role in its pathogenesis. Oxidative modification of high-density lipoprotein particles by myeloperoxidase followed by impaired reverse cholesterol transport and the decrease of high-density lipoprotein cholesterol level results in atherosclerosis progression. We studied the effect of myeloperoxidase on reverse cholesterol transport among patients with arterial hypertension and different clinical forms of chronic coronary heart disease, judging by findings in blood plasma. AIM: The ultimate goal was to establish whether that effect is associated with the total amount of myeloperoxidase or its activity. METHODS: 93 patients were recruited (65.4 ± 10.1 years old in average; men — 30 (32%)) with arterial hypertension and different clinical forms of chronic coronary heart disease. Depending on the diagnosis established, all participants were divided into 3 groups. Group I (control) contained patients with arterial hypertension, but without chronic coronary heart disease (n = 46). Group II (n = 26) included patients with initially stable coronary syndromes of chronic coronary heart disease (stable angina and/or scheduled surgical interventions for stable coronary heart disease), who never experienced acute adverse cardiac events. Group III (n = 21) contained patients with acute coronary syndrome (acute myocardial infarction) in the past 6 months or earlier. The total myeloperoxidase content (MPO-T) was assayed by enzyme-linked immunosorbent assay (ELISA). Home-modified specific immune-extraction followed by enzymatic detection (SIEFED) test was used to measure the active myeloperoxidase (MPO-A). Then, the coefficient of myeloperoxidase activity (MPO-CA) and the ratio of coefficient of myeloperoxidase activity to high-density lipoprotein cholesterol (MPO-CA/HDL-C) were calculated. RESULTS: The level of MPO-A was higher in patients from group III with complicated form of chronic coronary heart disease, as compared with group II (p 0.05). MPO-CA in patients of group III also was higher in comparison with group II (p = 0.001). Weak positive correlation was found between MPO-T and MPO-A in the whole cohort under investigation (r = 0.26; p 0.05), and the relationship was stronger in the group III (r = 0.59; p 0.05). In addition, negative correlation between MPO-A and HDL-C was found in group III (r = –0.46; p 0.05). The MPO-CA/HDL-C ratio was higher in patients with anamnestic acute coronary syndrome, as compared with patients manifesting non-complicated stable coronary heart disease (p 0.001) and with patients of group I who had no coronary heart disease (p 0.001). To determine diagnostic value of the MPO-CA/HDL-C the receiver operating characteristic curve (ROC-curve) was plotted. The calculated area under curve (AUC) was 0.8 which indicates a high predictive value of the MPO-CA/HDL-C ratio for different forms of chronic coronary heart disease. CONCLUSION: The results of our study demonstrate that in patients with preceding history of acute coronary syndrome, as compared with those having a stable course of chronic coronary heart disease, the effect of myeloperoxidase on reverse cholesterol transport depends on its activity rather than concentration. MPO-CA/HDL-C ratio mirrors the complicated chronic coronary heart disease and might serve as an additional indicator of residual risk.

A study was carried out on the spectralluminescent properties of fl uorescein after its reaction with various reactive oxygen and halogen species ( O_2^∙-, H2O2, HOCl, HOBr, HOSCN, N-chloramine, taurine N-chloramine, and taurine N-bromamine) as well as in the myeloperoxidase (MPO)–H2O2–Cl–/Br–/SCN– system. Reaction with only HOBr or with the MPO–H2O2–Br system turns fluorescein into a compound with an absorption maximum at 518 nm. The fluorescence maximum is recorded at 540 nm when excited at 520 nm, corresponding to eosin Y (brominated fluorescein). Conditions with phosphatebuffered saline (PBS) at pH 7.4 containing 137 mM NaCl, 5 mM fluorescein, 15–30 mM NaBr, and 25–50 mM H2O2 were found to be optimal for detecting HOBr in solution. A qualitative method for determining the brominating activity of MPO in vitro has been proposed. This method was used to study the effect of physiological and synthetic inhibitors as well as reactive oxygen and halogen species scavengers on the brominating activity of MPO. Our results indicate that fluorescein holds promise for use in a fluorescent method for detecting the brominating activity of mammalian hemecontaining peroxidases.

Type 2 diabetes mellitus (T2DM) is accompanied by halogenative stress resulting from the excessive activation of neutrophils and neutrophilic myeloperoxidase (MPO) generating highly reactive hypochlorous acid (HOCl). HOCl in blood plasma modifies serum albumin (Cl-HSA). We studied the formation of neutrophil extracellular traps (NETs) in the whole blood and by isolated neutrophils under the action of Cl-HSA. It was found that Cl-HSA induces neutrophil priming and NETosis. MPO-containing as well as MPO-free NETs were found. These NETs with different composition can be a product of NETosis of one and the same neutrophil. NET formation in neutrophils with vacuolated cytoplasm was detected. In the presence of Cl-HSA, acceleration of NET degradation was observed. Accelerated NET degradation and neutrophil priming can be the factors contributing to the development of complications in T2DM.

BACKGROUND: Leukocyte myeloperoxidase catalyzes the formation of HOCl, which, by oxidizing and chlorinating biomolecules, contributes to the development of oxidative/halogenative stress. The latter in hyperglycemia may interfere with wound healing in patients with diabetes mellitus complications. AIM: Evaluation of the concentration of markers of oxidative/halogenative stress and NETosis in the blood of experimental rats with hyperglycemia, its correction with lactoferrin, as well as elucidation of the effect of this multifunctional protein on skin wound healing. MATERIALS AND METHODS: For the experimental modeling of hyperglycemia, the animals were injected once with streptozotocin at a dosage of 43 mg/kg body weight. Blood samples were collected from the tail vein of anesthetized animals. Glucose was measured by the electrochemical method. Markers of oxidative/halogenative stress were detected by the immunoenzymatic and spectrophotometric methods. RESULTS: It was shown that in rats with streptozotocin-induced hyperglycemia, a “prophylactic+therapeutic” supplementation of lactoferrin (at a dose of 250 mg/kg on days 5, 3, and 1 before and days 2, 4, 6, and 8 after streptozotocin injection) significantly decreased blood concentration of glucose (fasting), myeloperoxidase, chlorinated ceruloplasmin, complexes of myeloperoxidase/DNA, and also prevented the decrease in thiols (SH-groups) and the activity of erythrocyte glutathione peroxidase. Moreover, lactoferrin administered according to the above regimen to rats with experimental hyperglycemia promoted wound healing, which was manifested by a 28% decrease in the wound area compared to the control animals. CONCLUSIONS: The results obtained indicate that lactoferrin has an ability in a hyperglycemia model in animals to reduce not only the hyperglycemia level, but also to prevent the development of oxidative/halogenative stress and NETosis, which leads to improved wound healing.

COVID-19 cases caused by new variants of highly mutable SARS-CoV-2 continue to be identified worldwide. Effective control of the spread of new variants can be achieved through targeting of conserved viral epitopes. In this regard, the SARS-CoV-2 nucleocapsid (N) protein, which is much more conserved than the evolutionarily influenced spike protein (S), is a suitable antigen. The recombinant N protein can be considered not only as a screening antigen but also as a basis for the development of next-generation COVID-19 vaccines, but little is known about induction of antibodies against the N protein via different SARS-CoV-2 variants. In addition, it is important to understand how antibodies produced against the antigen of one variant can react with the N proteins of other variants. Here, we used recombinant N proteins from five SARS-CoV-2 strains to investigate their immunogenicity and antigenicity in a mouse model and to obtain and characterize a panel of hybridoma-derived monoclonal anti-N antibodies. We also analyzed the variable epitopes of the N protein that are potentially involved in differential recognition of antiviral antibodies. These results will further deepen our knowledge of the cross-reactivity of the humoral immune response in COVID-19.

BACKGROUND: The modern market of medical devices in Belarus and Russia is represented by a wide range of hemostatic agents, of which the most popular are local hemostatics of plant origin possessing the significant technological potential for renewal and improvement. A promising reserve for this may be biologically active compounds of mosses, which are characterized by anti-inflammatory, antibacterial and antifungal effects. However, their hemostatic effect is almost not studied, which determines the relevance of this work. AIM: The aim of this work is to study the effect of lectin-containing substances from mosses of three species collected in East Antarctica and Belarus on the parameters of human blood hemostasis in vitro. MATERIALS AND METHODS: We studied mosses of the genera Bryum, Ceratodon, and Coscinodon, collected in the area of the Belarusian Antarctic station Gora Vechernyaya in East Antarctica and in Belarus. Lectin-containing substances of mosses were obtained by extracting shoots in 0.05 M tris-HCl buffer (pH 8.0), centrifugation, filtration. The assessment of the biological activity of lectin-containing substances in mosses was carried out by the agglutination reaction of rabbit erythrocytes, as well as the effect on human platelet aggregation and in the test for activated partial thromboplastin time. RESULTS: It was established that lectin-containing substances of the studied moss species had agglutinating activity against erythrocytes in the range from 11708.28 (Belarusian samples) to 1333979.59 U/mg of protein (Antarctic samples) depending on the species and localization; initiated the aggregation of human platelets (25–80% of the effect of thrombin) regardless of blood group, Rh and gender of donors; influenced the plasma link of hemostasis, reducing activated partial thromboplastin time (by 15–18%). CONCLUSIONS: It was found that some species of mosses of the genera Bryum, Ceratodon and Coscinodon of Antarctica and Belarus had an agglutinating and hemostatic effect on erythrocytes and platelets, with the greatest activity noted for Antarctic species. A hypothesis has been put forward that the observed phenomenon is due to the structural features of proteins, including lectins. It is assumed that lectins are possible inducers of erythrocyte agglutination and platelet aggregation in mosses. It is shown that the moss species Bryum pseudotriquetrum and Ceratodon purpureus have a certain resource potential in Belarus for their annual harvest. The results obtained expand the list of moss species with hemostatic activity, and can be used to develop new hemostatics of plant origin for local use from Belarusian plant materials.

The widespread resistance to antibiotics in pathogenic bacteria makes the development of a new generation of antimicrobials an urgent task. The development of new antibiotics must be accompanied by a comprehensive study of all of their biological activities in order to avoid adverse side-effects from their application. Some promising antibiotic prototypes derived from the structures of arenicins, antimicrobial peptides from the lugworm Arenicola marina, have been developed. Previously, we described the ability of natural arenicins -1 and -2 to modulate the human complement system activation in vitro. In this regard, it seems important to evaluate the effect of therapeutically promising arenicin analogues on complement activation. Here, we describe the complement-modulating activity of three such analogues, Ar-1[V8R], ALP1, and AA139. We found that the mode of action of Ar-1[V8R] and ALP1 on the complement was similar to that of natural arenicins, which can both activate and inhibit the complement, depending on the concentration. However, Ar-1[V8R] behaved predominantly as an inhibitor, showing only a moderate increase in C3a production in the alternative pathway model and no enhancement at all of the classical pathway of complement activation. In contrast, the action of ALP1 was characterized by a marked increase in the complement activation through the classical pathway in the concentration range of 2.5–20 μg/mL. At the same time, at higher concentrations (80–160 μg/mL), this peptide exhibited a complement inhibitory effect characteristic of the other arenicins. Peptide AA139, like other arenicins, exhibited an inhibitory effect on complement at a concentration of 160 μg/mL, but was much less pronounced. Overall, our results suggest that the effect on the complement system should be taken into account in the development of antibiotics based on arenicins.

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.

The protective effects of recombinant human lactoferrin rhLF (branded “CAPRABEL™”) on the cognitive functions of rat offspring subjected to prenatal hypoxia (7% O2, 3 h, 14th day of gestation) have been analyzed. About 90% of rhLF in CAPRABEL was iron-free (apo-LF). Rat dams received several injections of 10 mg of CAPRABEL during either gestation (before and after the hypoxic attack) or lactation. Western blotting revealed the appearance of erythropoietin (EPO) alongside the hypoxia-inducible factors (HIFs) in organ homogenates of apo-rhLF-treated pregnant females, their embryos (but not placentas), and in suckling pups from the dams treated with apo-rhLF during lactation. Apo-rhLF injected to rat dams either during pregnancy or nurturing the pups was able to rescue cognitive deficits caused by prenatal hypoxia and improve various types of memory both in young and adult offspring when tested in the radial maze and by the Novel Object Recognition (NOR) test. The data obtained suggested that the apo-form of human LF injected to female rats during gestation or lactation protects the cognitive functions of their offspring impaired by prenatal hypoxia.

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.

Cationic antimicrobial peptides (CAMPs) have gained attention as promising antimicrobial therapeutics causing lower or no bacterial resistance. Considerable achievements have been made in designing new CAMPs that are highly active as antimicrobials. However, there is a lack of research on their interaction with biologically important proteins. This study focused on CAMPs’ effects on myeloperoxidase (MPO), an enzyme which is microbicidal and concomitantly damaging to host biomolecules and cells due to its ability to produce reactive oxygen and halogen species (ROS/RHS). Four CAMPs designed by us were employed. MPO catalytic activity was assessed by an absorbance spectra analysis and by measuring enzymatic activity using Amplex Red- and Celestine Blue B-based assays. The peptide Hm-AMP2 accelerated MPO turnover. Pept_1545 and Hm-AMP8 inhibited both the MPO chlorinating and peroxidase activities, with components of different inhibition types. Hm-AMP8 was a stronger inhibitor. Its Ki towards H2O2 and Cl– was 0.3–0.4 μM vs. 11–20 μM for pept_1545. Peptide tyrosine and cysteine residues were involved in the mechanisms of the observed effects. The results propose a possible dual role of CAMPs as both antimicrobial agents and agents that downregulate MPO activation, and suggest CAMPs as prototypes for the development of antioxidant compounds to prevent MPO-mediated ROS/RHS overproduction.

The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves dysregulations of iron metabolism, and although the mechanism of this pathology is not yet fully understood, correction of iron metabolism pathways seems a promising pharmacological target. The previously observed effect of inhibiting SARS-CoV-2 infection by ferristatin II, an inducer of transferrin receptor 1 (TfR1) degradation, prompted the study of competition between Spike protein and TfR1 ligands, especially lactoferrin (Lf) and transferrin (Tf). We hypothesized molecular mimicry of Spike protein as cross-reactivity of Spike-specific antibodies with Tf and Lf. Thus, strong positive correlations (R2 > 0.95) were found between the level of Spike-specific IgG antibodies present in serum samples of COVID-19-recovered and Sputnik V-vaccinated individuals and their Tf-binding activity assayed with peroxidase-labeled anti-Tf. In addition, we observed cross-reactivity of Lf-specific murine monoclonal antibody (mAb) towards the SARS-CoV-2 Spike protein. On the other hand, the interaction of mAbs produced to the receptor-binding domain (RBD) of the Spike protein with recombinant RBD protein was disrupted by Tf, Lf, soluble TfR1, anti-TfR1 aptamer, as well as by peptides RGD and GHAIYPRH. Furthermore, direct interaction of RBD protein with Lf, but not Tf, was observed, with affinity of binding estimated by KD to be 23 nM and 16 nM for apo-Lf and holo-Lf, respectively. Treatment of Vero E6 cells with apo-Lf and holo-Lf (1–4 mg/mL) significantly inhibited SARS-CoV-2 replication of both Wuhan and Delta lineages. Protective effects of Lf on different arms of SARS-CoV-2-induced pathogenesis and possible consequences of cross-reactivity of Spike-specific antibodies are discussed.

The work is devoted to the study of the structural characteristics of the myeloperoxidase-ceruloplasmin-thrombin complex using small-angle neutron scattering methods in combination with computer modeling, as well as surface plasmon resonance and solid-phase enzyme assay. We have previously shown that the functioning of active myeloperoxidase during inflammation, despite the presence in the blood of an excess of ceruloplasmin which inhibits its activity, is possible due to the partial proteolysis of ceruloplasmin by thrombin. In this study, the myeloperoxidase-ceruloplasmin-thrombin heterohexamer was obtained in vitro. The building of a heterohexamer full-atomic model in silico, considering the glycosylation of the constituent proteins, confirmed the absence of steric barriers for the formation of protein-protein contacts. It was shown that the partial proteolysis of ceruloplasmin does not affect its ability to bind to myeloperoxidase, and a structural model of the heterohexamer was obtained using the small-angle neutron scattering method.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to have a significant impact on global public health. Multiple mechanisms for SARS-CoV-2 cell entry have been described; however, the role of transferrin receptor 1 (TfR1) in SARS-CoV-2 infection has received little attention. We used ferristatin II to induce the degradation of TfR1 on the surface of Vero cells and to study the consequences of such treatment on the viability of the cells and the replication of SARS-CoV-2. We demonstrated that ferristatin II is non-toxic for Vero cells in concentrations up to 400 µM. According to confocal microscopy data, the distribution of the labeled transferrin and receptor-binding domain (RBD) of Spike protein is significantly affected by the 18h pretreatment with 100 µM ferristatin II in culture medium. The uptake of RBD protein is nearly fully inhibited by ferristatin II treatment, although this protein remains bound on the cell surface. The findings were well confirmed by the significant inhibition of the SARS-CoV-2 infection of Vero cells by ferristatin II with IC50 values of 27 µM (for Wuhan D614G virus) and 40 µM (for Delta virus). A significant reduction in the infectious titer of the Omicron SARS-CoV-2 variant was noted at a ferristatin II concentration as low as 6.25 µM. We hypothesize that ferristatin II blocks the TfR1-mediated SARS-CoV-2 host cell entry; however, further studies are needed to elucidate the full mechanisms of this virus inhibition, including the effect of ferristatin II on other SARS-CoV-2 receptors, such as ACE2, Neuropilin-1 and CD147. The inhibition of viral entry by targeting the receptor on the host cells, rather than the viral mutation-prone protein, is a promising COVID-19 therapeutic strategy.

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.