Prevention of fouling from proteins in blood plasma attracts significant efforts, and great progress is made in identifying surface coatings that display antifouling properties. In particular, poly(ethylene glycol) (PEG) is widely used and dense PEG-like cylindrical brushes of poly[oligo(ethylene glycol) methacrylate] (poly(OEGMA)) can drastically reduce blood plasma fouling. Herein, a comprehensive study of the variation of blood plasma fouling on this surface, including the analysis of the composition of protein deposits on poly(OEGMA) coatings after contact with blood plasma from many different donors, is reported. Correlation between the plasma fouling behavior and protein deposit composition points to the activation of the complement system as the main culprit of dramatically increased and accelerated deposition of blood plasma proteins on this type of antifouling coating, specifically through the classical pathway. These findings are consistent with observations on PEGylated drug carriers and highlight the importance of understanding the potential interactions between antifouling coatings and their environment.

• Keywords
• antifouling coatings, donor variability, mass spectrometry, polyethylene glycol, polymer brushes, protein adsorption, protein identification, surface plasmon resonance,
• MeSH
• Coated Materials, Biocompatible chemistry   MeSH
• Biofouling prevention & control   MeSH
• Blood Proteins chemistry   analysis   MeSH
• Humans MeSH
• Methacrylates chemistry   MeSH
• Polyethylene Glycols * chemistry   MeSH
• Surface Properties MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Coated Materials, Biocompatible MeSH
• Blood Proteins MeSH
• Methacrylates MeSH
• Polyethylene Glycols * MeSH

• Keywords
• Myelodysplastic syndrome, Plasma proteome, Proteomics,
• MeSH
• Humans MeSH
• Myelodysplastic Syndromes * diagnosis   MeSH
• Proteomics * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Whenever an artificial surface comes into contact with blood, proteins are rapidly adsorbed onto its surface. This phenomenon, termed fouling, is then followed by a series of undesired reactions involving activation of complement or the coagulation cascade and adhesion of leukocytes and platelets leading to thrombus formation. Thus, considerable efforts are directed towards the preparation of fouling-resistant surfaces with the best possible hemocompatibility. Herein, a comprehensive hemocompatibility study after heparinized blood contact with seven polymer brushes prepared by surface-initiated atom transfer radical polymerization is reported. The resistance to fouling is quantified and thrombus formation and deposition of blood cellular components on the coatings are analyzed. Moreover, identification of the remaining adsorbed proteins is performed via mass spectroscopy to elucidate their influence on the surface hemocompatibility. Compared with an unmodified glass surface, the grafting of polymer brushes minimizes the adhesion of platelets and leukocytes and prevents the thrombus formation. The fouling from undiluted blood plasma is reduced by up to 99%. Most of the identified proteins are connected with the initial events of foreign body reaction towards biomaterial (coagulation cascade proteins, complement component, and inflammatory proteins). In addition, several proteins that are not previously linked with blood-biomaterial interaction are presented and discussed.

• Keywords
• MS identification, antifouling surfaces, hemocompatibility, polymer brushes, protein adsorption,
• MeSH
• Adsorption MeSH
• Biocompatible Materials pharmacology   chemistry   MeSH
• Biofouling * prevention & control   MeSH
• Humans MeSH
• Polymers chemistry   MeSH
• Surface Properties MeSH
• Proteins MeSH
• Thrombosis * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biocompatible Materials MeSH
• Polymers MeSH
• Proteins MeSH

BACKGROUND: Leucine-rich alpha-2-glycoprotein (LRG) has been repeatedly proposed as a potential plasma biomarker for myelodysplastic syndrome (MDS). OBJECTIVE: The goal of our work was to establish the total LRG plasma level and LRG posttranslational modifications (PTMs) as a suitable MDS biomarker. METHODS: The total plasma LRG concentration was determined with ELISA, whilst the LRG-specific PTMs and their locations, were established using mass spectrometry and public mass spectrometry data re-analysis. Homology modelling and sequence analysis were used to establish the potential impact of PTMs on LRG functions via their impact on the LRG structure. RESULTS: While the results showed that the total LRG plasma concentration is not a suitable MDS marker, alterations within two LRG sites correlated with MDS diagnosis (p= 0.0011). Sequence analysis and the homology model suggest the influence of PTMs within the two LRG sites on the function of this protein. CONCLUSIONS: We report the presence of LRG proteoforms that correlate with diagnosis in the plasma of MDS patients. The combination of mass spectrometry, re-analysis of publicly available data, and homology modelling, represents an approach that can be used for any protein to predict clinically relevant protein sites for biomarker research despite the character of the PTMs being unknown.

• Keywords
• LRG, MDS, Myelodysplastic syndrome, leucine-rich alpha-2-glycoprotein, proteomics,
• MeSH
• Biomarkers MeSH
• Glycoproteins * genetics   metabolism   MeSH
• Mass Spectrometry MeSH
• Leucine metabolism   MeSH
• Humans MeSH
• Myelodysplastic Syndromes * diagnosis   MeSH
• Protein Processing, Post-Translational MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• Glycoproteins * MeSH
• Leucine MeSH

Non-specific protein adsorption (fouling) triggers a number of deleterious events in the application of biomaterials. Antifouling polymer brushes successfully suppress fouling, however for some coatings an extremely high variability of fouling for different donors remains unexplained. The authors report that in the case of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) this variability is due to the complement system activation that causes massive acceleration in the fouling kinetics of blood plasma. Using plasma from various donors, the fouling kinetics on poly(HEMA) is analyzed and correlated with proteins identified in the deposits on the surface and with the biochemical compositions of the plasma. The presence of complement components in fouling deposits and concentrations of C3a in different plasmas indicate that the alternative complement pathway plays a significant role in the fouling on poly(HEMA) through the "tick-over" mechanism of spontaneous C3 activation. The generated C3b binds to the poly(HEMA) surface and amplifies complement activation locally. Heat-inactivated plasma prevents accelerated fouling kinetics, confirming the central role of complement activation. The results highlight the need to take into account the variability between individuals when assessing interactions between biomaterials and blood plasma, as well as the importance of the mechanistic insight that can be gained from protein identification.

• Keywords
• antifouling coatings, complement C3, polymer brushes, protein adsorption, protein identification, surface plasmon resonance,
• MeSH
• Complement Activation MeSH
• Biocompatible Materials pharmacology   MeSH
• Biofouling * prevention & control   MeSH
• Plasma MeSH
• Humans MeSH
• Methacrylates MeSH
• Surface Properties MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biocompatible Materials MeSH
• hydroxyethyl methacrylate MeSH Browser
• Methacrylates MeSH

BACKGROUND: Extracellular vesicles are released into body fluids from the majority of, if not all, cell types. Because their secretion and specific cargo (e.g., proteins) varies according to pathology, extracellular vesicles may prove a rich source of biomarkers. However, their biological and pathophysiological functions are poorly understood in hematological malignancies. OBJECTIVE: Here, we investigated proteome changes in the exosome-rich fraction of the plasma of myelodysplastic syndrome patients and healthy donors. METHODS: Exosome-rich fraction of the plasma was isolated using ExoQuick™: proteomes were compared and statistically processed; proteins were identified by nanoLC-MS/MS and verified using the ExoCarta and QuickGO databases. Mann-Whitney and Spearman analyses were used to statistically analyze the data. 2D western blot was used to monitor clusterin proteoforms. RESULTS: Statistical analyses of the data highlighted clusterin alterations as the most significant. 2D western blot showed that the clusterin changes were caused by posttranslational modifications. Moreover, there was a notable increase in the clusterin proteoform in the exosome-rich fraction of plasma of patients with more severe myelodysplastic syndrome; this corresponded with a simultaneous decrease in their plasma. CONCLUSIONS: This specific clusterin proteoform seems to be a promising biomarker for myelodysplastic syndrome progression.

• MeSH
• Biomarkers blood   MeSH
• Chromatography, Liquid MeSH
• Extracellular Vesicles metabolism   MeSH
• Humans MeSH
• Myelodysplastic Syndromes metabolism   pathology   MeSH
• Proteome analysis   metabolism   MeSH
• Proteomics methods   MeSH
• Aged MeSH
• Case-Control Studies MeSH
• Tandem Mass Spectrometry MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• Proteome MeSH

Fibrinogen, an abundant plasma glycoprotein, is involved in the final stage of blood coagulation. Decreased fibrinogen levels, which may be caused by mutations, are manifested mainly in bleeding and thrombotic disorders. Clinically relevant mutations of fibrinogen are listed in the Human Fibrinogen Database. For the αC-connector (amino acids Aα240-410, nascent chain numbering), we have extended this database, with detailed descriptions of the clinical manifestations among members of reported families. This includes the specification of bleeding and thrombotic events and results of coagulation assays. Where available, the impact of a mutation on clotting and fibrinolysis is reported. The collected data show that the Human Fibrinogen Database reports considerably fewer missense and synonymous mutations than the general COSMIC and dbSNP databases. Homozygous nonsense or frameshift mutations in the αC-connector are responsible for most clinically relevant symptoms, while heterozygous mutations are often asymptomatic. Symptomatic subjects suffer from bleeding and, less frequently, from thrombotic events. Miscarriages within the first trimester and prolonged wound healing were reported in a few subjects. All mutations inducing thrombotic phenotypes are located at the identical positions within the consensus sequence of the tandem repeats.

• Keywords
• Human Fibrinogen Database, afibrinogenemia, dysfibrinogenemia, fibrinogen, hypodysfibrinogenemia, hypofibrinogenemia, mutations, αC-connector,
• MeSH
• Fibrinogen genetics   MeSH
• Blood Coagulation genetics   MeSH
• Hemorrhage genetics   MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Thrombosis genetics   MeSH
• Blood Coagulation Tests methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Fibrinogen MeSH

Early and late thrombosis remain the most frequent reasons for the failure of synthetic cardiovascular grafts. Long-term hemocompatibility of implanted synthetic grafts can be achieved if a natural living endothelium is formed over its blood-contacting surface. Here we present a modification of a standard expanded polytetrafluorethylene (ePTFE) vessel prosthesis by a controlled preparation of a fibrin mesh enriched with covalently bound heparin and noncovalently bound vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Compared to a bare prosthesis, the coated prosthesis showed excellent antithrombogenic properties after contact with heparinized fresh human blood. Human umbilical vein endothelial cells seeded on the inner surface of the coated prosthesis formed a confluent layer in 5 days, whereas only small colonies of cells were scattered on the bare prosthesis. Viability of the cells was promoted mainly by FGF immobilized on the coating. These findings suggest that the coating may prevent acute thrombus formation and support the self-endothelialization of an implanted ePTFE vascular graft in vivo.

• Publication type
• Journal Article MeSH

Oxidative stress in humans is related to various pathophysiological processes, which can manifest in numerous diseases including cancer, cardiovascular diseases, and Alzheimer's disease. On the atomistic level, oxidative stress causes posttranslational modifications, thus inducing structural and functional changes into the proteins structure. This study focuses on fibrinogen, a blood plasma protein that is frequently targeted by reagents causing posttranslational modifications in proteins. Fibrinogen was in vitro modified by three reagents, namely sodium hypochlorite, malondialdehyde, and 3-morpholinosydnonimine that mimic the oxidative stress in diseases. Newly induced posttranslational modifications were detected via mass spectrometry. Electron microscopy was used to visualize changes in the fibrin networks, which highlight the extent of disturbances in fibrinogen behavior after exposure to reagents. We used molecular dynamics simulations to observe the impact of selected posttranslational modifications on the fibrinogen structure at the atomistic level. In total, 154 posttranslational modifications were identified, 84 of them were in fibrinogen treated with hypochlorite, 51 resulted from a reaction of fibrinogen with malondialdehyde, and 19 were caused by 3-morpholinosydnonimine. Our data reveal that the stronger reagents induce more posttranslational modifications in the fibrinogen structure than the weaker ones, and they extensively alter the architecture of the fibrin network. Molecular dynamics simulations revealed that the effect of posttranslational modifications on fibrinogen secondary structure varies from negligible alternations to serious disruptions. Among the serious disruptions is the oxidation of γR375 resulting in the release of Ca2+ ion that is necessary for appropriate fibrin fiber formation. Folding of amino acids γE72-γN77 into a short α-helix is a result of oxidation of γP76 to glutamic acid. The study describes behaviour of fibrinogen coiled-coil connecter in the vicinity of plasmin and hementin cleavage sites.

• MeSH
• Fibrinogen chemistry   metabolism   MeSH
• Humans MeSH
• Protein Processing, Post-Translational * MeSH
• Protein Structure, Secondary MeSH
• Molecular Dynamics Simulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fibrinogen MeSH

Myelodysplastic syndromes (MDS) represent a heterogeneous group of pre-leukemic disorders, characterized by ineffective hematopoiesis and the abnormal blood cell development of one or more lineages. Oxidative stress, as an important factor in the carcinogenesis of onco-hematological diseases, is also one of the known factors involved in the pathogenesis of MDS. An increase of reactive oxygen species (ROS) may lead to the oxidation of DNA, lipids, and proteins, thereby causing cell damage. Protein carbonylation caused by ROS is defined as an irreversible post-translational oxidative modification of amino acid side chains, and could play an important role in signaling processes. The detection of protein carbonyl groups is a specific useful marker of oxidative stress. In this study, we examined 32 patients divided into three different subtypes of MDS according to the World Health Organization (WHO) classification criteria as refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blasts-1,2 (RAEB-1,2). We found significant differences in protein carbonylation between the group of all MDS patients and healthy controls (P=0.0078). Furthermore, carbonylated protein levels were significantly elevated in RARS patients compared to healthy donors (P=0.0013) and to RCMD patients (P=0.0277). We also found a significant difference in the total iron binding capacity (TIBC) between individual subgroups of MDS patients (P=0.0263). Moreover, TIBC was decreased in RARS patients compared to RCMD patients (P=0.0203). TIBC moderately negatively correlated with carbonyl levels (r=-0.5978, P=0.0054) in the MDS patients as a whole. Additionally we observed changes in the carbonylated proteins of RARS patients in comparison with healthy controls and their negative controls. Using tandem mass spectrometry (LC-MS/MS) we identified 27 uniquely carbonylated proteins of RARS patients, which were generated by ROS and could influence the pathophysiology of low-risk MDS. These data indicate that increased protein carbonylation is related with RARS as low-risk MDS subgroup. We suggest that this type of post-translational modification in MDS disease is not "only" a consequence of oxidative stress, but also plays an active role in the pathophysiology and iron metabolism within the RARS subgroup of MDS. Measurement of plasma carbonyl levels and the isolation of carbonylated plasma proteins, followed by their identification, could serve as a potential diagnostic and prognostic tool in MDS.

• Keywords
• Carbonyl levels, Carbonylated proteins, Myelodysplastic syndromes, Oxidative stress,
• MeSH
• Adult MeSH
• Protein Carbonylation MeSH
• Blood Proteins metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Myelodysplastic Syndromes diagnosis   metabolism   MeSH
• Oxidative Stress MeSH
• Prognosis MeSH
• Reactive Oxygen Species metabolism   MeSH
• Anemia, Refractory MeSH
• Aged MeSH
• Tandem Mass Spectrometry MeSH
• Protein Binding MeSH
• Iron metabolism   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Blood Proteins MeSH
• Reactive Oxygen Species MeSH
• Iron MeSH