ABSTRACT
Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/-OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/-OCl, 10 µM HOCl/-OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BßMet367, γMet264, and γMet94, identified in 10 µM HOCl/-OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.
Subject(s)
Fibrinogen , Methionine , Oxidation-Reduction , Oxidative Stress , Fibrinogen/chemistry , Fibrinogen/metabolism , Humans , Methionine/metabolism , Methionine/chemistry , Protein Processing, Post-Translational , Reactive Oxygen Species/metabolism , Hypochlorous Acid/chemistry , Hypochlorous Acid/metabolism , Fibrin/metabolism , Fibrin/chemistry , Tandem Mass SpectrometryABSTRACT
Plasminogen is a zymogenic form of plasmin, an enzyme that plays a fundamental role in the dissolution of fibrin clots as well as in many other physiological processes. For the first time, by the method of gas chromatography-mass spectrometry, post-translational modifications in the primary structure of plasminogen treated with physiologically relevant amounts of hydrogen peroxide were identified. It was found that methionine and tryptophan residues located in different structural regions of plasminogen served as targets of the oxidant. Plasminogen oxidation caused a dose-dependent effect in decreasing the fibrinogenolytic activity of plasmin evidenced by the formation of fibrinogen degradation products. The possible antioxidant role of methionines in the oxidative modification of plasminogen is discussed.
Subject(s)
Peroxides , Plasminogen , Fibrin , Fibrinogen , Fibrinolysin , Fibrinolysis , OxidantsABSTRACT
Using the methods of dynamic and elastic light scattering and confocal laser scanning microscopy, the damage in the spatial fibrin structure during peroxide- and hypochlorite-induced oxidation of fibrinogen was studied. Peroxide had a weak effect on the structural organization of fibrin, whereas hypochlorite caused the formation of abnormal fibrin with reduced individual fiber diameter and decreased porosity. Measurements of the size distributions of the native and oxidized fibrinogen revealed a decrease in the hydrodynamic size of the oxidized fibrinogen molecule with an increase in the concentration of oxidizers. These results indicate that the hydrophobicity of fibrinogen surface increased and its colloidal stability decreased. The possible role of oxidative sites in the assembly of structurally abnormal fibrin is analyzed.
Subject(s)
Fibrin/chemistry , Fibrinogen/metabolism , Hypochlorous Acid/pharmacology , Peroxides/pharmacology , Fibrin/metabolism , Oxidation-Reduction/drug effectsABSTRACT
The damage to blood coagulation factor XIII (FXIII) at different stages of its enzymatic activation under the action of various physiological amounts of hypochlorite ion was studied. The results obtained by HPLC-MS/MS, SDS-PAGE, and colorimetry showed that, during the conversion of FXIII to FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased. FXIII oxidized with 150 µM hypochlorite completely retained its enzymatic activity inherent to the intact protein, whereas FXIIIa treated with 50 µM hypochlorite showed sharply reduced enzymatic activity. It was shown that a number of methionine and cysteine residues on the catalytic subunit can perform antioxidant function; additionally, the regulatory subunits of FXIII-B contribute to the antioxidant protection of the catalytic center of the FXIII-A subunit, which, together with the tight packing of the tetrameric structure of the FXIII proenzyme, are the three factors that provide high protein resistance to the oxidizing agent.
Subject(s)
Factor XIII/chemistry , Hypochlorous Acid/pharmacology , Oxidants/pharmacology , Catalytic Domain , Enzyme Activation , Humans , Oxidation-Reduction , Tandem Mass Spectrometry/methodsABSTRACT
The effect of peroxide-induced oxidation of fibrinogen on modification of its primary structure and functional properties was investigated. The oxidation sites were shown to be Met, Trp, and His residues. Using the DLS method, it was found that the oxidative modification of fibrinogen results in the change of microrheological characteristics of fibrin network. The fibrinogen oxidation diminishes its tolerance to plasmin hydrolysis and deteriorates the factor XIIIa ability to stabilize the fibrin gel.
Subject(s)
Fibrin/chemistry , Fibrinogen/chemistry , Hydrogen Peroxide/pharmacology , Oxidants/pharmacology , Factor XIIIa/metabolism , Fibrinogen/drug effects , Fibrinogen/metabolism , Fibrinolysin/metabolism , Humans , Oxidation-Reduction , Structure-Activity RelationshipABSTRACT
Plasminogen, the precursor of plasmin, is a serine protease that plays a fundamental role in the intravascular thrombolysis. For the first time, by using high-resolution mass spectrometry, data on the oxidative modifications of the plasminogen molecule under induced oxidation were obtained. The FTIR data show that, under oxidation on the protein, its secondary structure also undergoes the rearrangements. The high tolerance of plasminogen to oxidation can be due to both the closed conformation and the ability of some Met residues to serve as ROS trap.
Subject(s)
Hypochlorous Acid/chemistry , Models, Chemical , Plasminogen/chemistry , Humans , Oxidation-Reduction , Spectroscopy, Fourier Transform InfraredABSTRACT
The oxidative modification of human hemoglobin (Hb) treated with hydrogen peroxide was investigated. Using the mass spectrometry method, the oxidized amino acid residues of the hemoglobin molecule were detected: αTrp14, αTyr24, αArg31, αMet32, αTyr42, αHis45, αHis72, αMet76, αPro77, αLys90, αCys104, αTyr140, ßHis2, ßTrp15, ßTrp37, ßMet55, ßCys93, ßCys112, ßTyr130, ßLys144, and ßHis146. The antioxidant potential of the Hb molecule in the intracellular space and in the blood plasma is discussed.
Subject(s)
Hemoglobins/metabolism , Hydrogen Peroxide/pharmacology , Humans , Oxidation-Reduction/drug effects , Oxidative Stress/drug effectsABSTRACT
Oxidation of fibrinogen with hypochlorite inhibited the fibrin network self-assembly even at the lowest concentration of the oxidant. The analysis of the results of protein electrophoresis at this hypochlorite concentration showed the absence of fragmentation of the protein and covalent cross-linking of its chains. The study of the areas responsible for the conversion of fibrinogen into fibrin by mass spectrometry showed that they are not subject to oxidative damage. However, we identified oxidized amino acid residues, which could affect the protofibril aggregation.