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1.
Biochemistry (Mosc) ; 89(Suppl 1): S14-S33, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38621742

ABSTRACT

Reactive oxygen species (ROS) are constantly generated in a living organism. An imbalance between the amount of generated reactive species in the body and their destruction leads to the development of oxidative stress. Proteins are extremely vulnerable targets for ROS molecules, which can cause oxidative modifications of amino acid residues, thus altering structure and function of intra- and extracellular proteins. The current review considers the effect of oxidation on the structural rearrangements and functional activity of hemostasis proteins: coagulation system proteins such as fibrinogen, prothrombin/thrombin, factor VII/VIIa; anticoagulant proteins - thrombomodulin and protein C; proteins of the fibrinolytic system such as plasminogen, tissue plasminogen activator and plasminogen activator inhibitor-1. Structure and function of the proteins, oxidative modifications, and their detrimental consequences resulting from the induced oxidation or oxidative stress in vivo are described. Possible effects of oxidative modifications of proteins in vitro and in vivo leading to disruption of the coagulation and fibrinolysis processes are summarized and systematized, and the possibility of a compensatory mechanism in maintaining hemostasis under oxidative stress is analyzed.


Subject(s)
Hemostasis , Tissue Plasminogen Activator , Tissue Plasminogen Activator/metabolism , Reactive Oxygen Species , Blood Coagulation , Blood Coagulation Factors/metabolism , Oxidative Stress
2.
Int J Biol Macromol ; 260(Pt 2): 129514, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38237825

ABSTRACT

Electrospun biomimetic materials based on polyester of natural origin poly-3-hudroxybutyrate (PHB) modified with hemin (Hmi) and fibrinogen (Fbg) represent a great interest and are potentially applicable in various fields. Here, we describe formulation of the new fibrous PHB-Fbg and PHB-Hmi-Fbg materials with complex structure for biomedical application. The average diameter of the fibers was 3.5 µm and 1.8 µm respectively. Hmi presence increased porosity from 80 % to 94 %, significantly reduced the number of defects, ensured the formation of a larger number of open pores, and improved mechanical properties. Hmi presence significantly improved the molding properties of the material. Hmi facilitated effective Fbg adsorption on the of the PHB wound-healing material, ensuring uniform localization of the protein on the surface of the fibers. Next, we evaluated cytocompatibility, cell behavior, and open wound healing in mice. The results demonstrated that PHB-Fbg and PHB-Hmi-Fbg electrospun materials had pronounced properties and may be promising for early-stage wound healing - the PHB-Hmi-Fbg sample accelerated wound closure by 35 % on the 3rd day, and PHB-Hmi showed 45 % more effective wound closure on the 15th day.


Subject(s)
Biomimetic Materials , Hemostatics , Mice , Animals , Fibrinogen , Wound Healing , Biomimetic Materials/pharmacology , Polyesters/chemistry
3.
Biophys Rev ; 15(3): 367-383, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37396452

ABSTRACT

Significant evidence suggests that reversible oxidation of methionine residues provides a mechanism capable of scavenging reactive species, thus creating a cycle with catalytic efficiency to counteract or mitigate deleterious effects of ROS on other functionally important amino acid residues. Because of the absence of MSRs in the blood plasma, oxidation of methionines in extracellular proteins is effectively irreversible and, therefore, the ability of methionines to serve as interceptors of oxidant molecules without impairment of the structure and function of plasma proteins is still debatable. This review presents data on the oxidative modification of both intracellular and extracellular proteins that differ drastically in their spatial structures and functions indicating that the proteins contain antioxidant methionines/the oxidation of which does not affect (or has a minor effect) on their functional properties. The functional consequences of methionine oxidation in proteins have been mainly identified from studies in vitro and, to a very limited extent, in vivo. Hence, much of the functioning of plasma proteins constantly subjected to oxidative stress remains unclear and requires further research to understand the evolutionary role of methionine oxidation in proteins for the maintenance of homeostasis and risk factors affecting the development of ROS-related pathologies. Data presented in this review contribute to increased evidence of antioxidant role of surface-exposed methionines and can be useful for understanding a possible mechanism that supports or impairs structure-function relationships of proteins subjected to oxidative stress.

4.
Biochim Biophys Acta Gen Subj ; 1865(10): 129970, 2021 10.
Article in English | MEDLINE | ID: mdl-34339807

ABSTRACT

BACKGROUND: Human fibrinogen, which plays a key role in plasma haemostasis, is a highly vulnerable target for oxidants. Fibrinogen undergoes posttranslational modifications that can potentially disrupt protein structure and function. METHODS: For the first time, by differential scanning calorimetry, dynamic and elastic light scattering and confocal laser scanning microscopy, the consequences of HOCl/-OCl-induced oxidation of fibrinogen on its thermal denaturation, molecular size distribution and fibrin clot network have been explored. RESULTS: Within a wide range of HOCl/-OCl concentrations (50-300 µM), the molecular size distribution remained unimodal; however, the average size of the hydrated molecules decreased. HOCl/-OCl-induced oxidation of fibrinogen resulted in the diminished thermal stability of regions D and E. As evidenced by elastic light scattering and confocal laser scanning microscopy, HOCl/-OCl caused the formation of abnormal fibrin with a decreased diameter of individual fibres. CONCLUSIONS: The current results along with data from previous studies enable one to conclude that the effect of HOCl/-OCl-mediated oxidation on the thermal stability of region D is influenced directly by oxidative damage to the D region structure. Since the E region is not subjected to oxidative modification, its structural damage is likely to be mediated by the oxidation of other protein structures, in particular α-helical coiled-coils. GENERAL SIGNIFICANCE: The experimental findings acquired in the current study could help to elucidate the consequences of oxidative stress in vivo on damage to the structure of fibrinogen/fibrin under the action of different ROS species.


Subject(s)
Fibrin/antagonists & inhibitors , Fibrinogen/antagonists & inhibitors , Hypochlorous Acid/pharmacology , Temperature , Adult , Fibrin/chemistry , Fibrin/metabolism , Fibrinogen/chemistry , Fibrinogen/metabolism , Humans , Oxidation-Reduction , Oxidative Stress/drug effects , Protein Denaturation/drug effects
5.
Thromb Haemost ; 119(6): 906-915, 2019 Jun.
Article in English | MEDLINE | ID: mdl-30934104

ABSTRACT

Factor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. It is known to bind to procoagulant platelets. In contrast, the zymogen fXIII interaction with platelets is not well characterized. We investigated the interaction of zymogen fXIII with activated platelet subpopulations. Confocal microscopy and flow cytometry using fluorescently labelled factors and antibodies. Phosphatidylserine (PS)-positive activated platelets bound 700 to 800 molecules/cell of fXIII at 100 nM, while both PS-negative activated platelets and resting platelets bound 200 to 400 molecules/cell. The binding was reversible, calcium-independent and linear within the fXIII concentration range of up to 1,000 nM. fXIII predominantly bound to the caps of procoagulant platelets and co-localized with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS-negative platelets; this effect was abolished by the integrin αIIbß3 antagonist monafram. The fXIII binding was 1.5- to 3-fold decreased for platelets from four patients with grey platelet syndrome, and was variable for platelets from six patients with Glanzmann's thrombasthenia. Strong platelet stimulation, fibrinogen and αIIbß3 play essential roles in fXIII binding, without any of them fXIII does not bind to platelets. The preferential binding in the cap-like structures might be important for increasing local fXIII concentration in platelet thrombi.


Subject(s)
Afibrinogenemia/metabolism , Blood Platelets/physiology , Factor VIII/metabolism , Fibrinogen/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Thrombasthenia/metabolism , Afibrinogenemia/genetics , Blood Coagulation , Enzyme Precursors , Fibrinolysis , Humans , Platelet Activation , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Protein Binding
6.
Free Radic Res ; 52(1): 14-38, 2018 Jan.
Article in English | MEDLINE | ID: mdl-29284315

ABSTRACT

Proteins represent extremely susceptible targets for oxidants. Oxidative modifications of proteins may bring about violation of their structure and functionality. It implies that the structures of proteins are not infallible in terms of their antioxidant defence. The protection mechanisms in preventing oxidative damages for proteins within cells are mainly related to a large variety of antioxidant enzymatic systems. In contrast, plasma proteins are scarcely protected by these systems, so the mechanism that provides their functioning in the conditions of generating reactive oxygen species (ROS) seems to be much more complicated. Oxidation of many proteins was long considered as a random process. However, the highly site-specific oxidation processes was convincingly demonstrated for some proteins, indicating that protein structure could be adapted to oxidation. According to our hypothesis, some of the structural elements present in proteins are capable of scavenging ROS to protect other protein structures against ROS toxicity. Various antioxidant elements (distinct subdomains, domains, regions, and polypeptide chains) may act as ROS interceptors, thus mitigating the ROS action on functionally crucial amino acid residues of proteins. In the review, the oxidative modifications of certain plasma proteins, such as α2-macroglobulin, serum human albumin, fibrinogen, and fibrin-stabilising factor, which differ drastically in their spatial structures and functions, are analysed. The arguments that demonstrate the possibility of existing hypothetical antioxidant structures are presented. For the first time, the emphasis is being placed on the programmed mechanism of protein oxidation.


Subject(s)
Antioxidants/metabolism , Fibrinogen/metabolism , Proteins/metabolism , Humans , Oxidation-Reduction
7.
Free Radic Biol Med ; 95: 55-64, 2016 06.
Article in English | MEDLINE | ID: mdl-26969792

ABSTRACT

Fibrinogen is extremely susceptible to attack by reactive oxygen species (ROS). Having been suffered an oxidative modification, the fibrinogen molecules, now with altered spatial structure and function of fibrin network, affect hemostasis differently. However, the potential effects of the oxidative stress on the early stages of the fibrin self-assembly process remain unexplored. To clarify the damaging influence of ROS on the knob 'A': hole 'a' and the D:D interactions, the both are operating on the early stages of the fibrin polymerization, we have used a novel approach based on exploration of FXIIIa-mediated self-assembly of the cross-linked fibrin oligomers dissolved in the moderately concentrated urea solutions. The oligomers were composed of monomeric desA fibrin molecules created by cleaving the fibrinopeptides A off the fibrinogen molecules with a thrombin-like enzyme, reptilase. According to the UV-absorbance and fluorescence measurements data, the employed low ozone/fibrinogen ratios have induced only a slight fibrinogen oxidative modification that was accompanied by modest chemical transformations of the aromatic amino acid residues of the protein. Else, a slight consumption of the accessible tyrosine residues has been observed due to intermolecular dityrosine cross-links formation. The set of experimental data gathered with the aid of electrophoresis, elastic light scattering and analytical centrifugation has clearly witnessed that the oxidation can serve as an effective promoter for the observed enhanced self-assembly of the covalently cross-linked oligomers. At urea concentration of 1.20M, the pristine and oxidized fibrin oligomers were found to comprise a heterogeneous set of the double-stranded protofibrils that are cross-linked only by γ-γ dimers and the fibers consisting on average of four strands that are additionally linked by α polymers. The amounts of the oxidized protofibrils and the fibers accumulated in the system were higher than those of the non-oxidized counterparts. Moreover, the γ and α polypeptide chains of the oxidized molecules were more readily crosslinked by the FXIIIa. Upon increasing the urea solution concentration to 4.20M, the cross-linked double-stranded desA fibrin protofibrils have dissociated into the single-stranded fibrin oligomers, whereas the fibers dissociated into both the double-stranded desA fibrin oligomers, the structural integrity of the latter being maintained by means of the intermolecular α polymers, and the single-stranded fibrin oligomers cross-linked only by γ-γ dimers. The data we have obtained in this study indicate that the FXIIIa-mediated process of assembling the cross-linked protofibrils and the fibers constructed from the oxidized monomeric fibrin molecules was facilitated due to the strengthening of D:D interactions. The findings infer that the enhanced longitudinal D:D interactions become more essential in the assembly of soluble protofibrils when the interactions knobs 'A': holes 'a' are injured by oxidation. The new experimental findings presented here could be of help for elucidating the essential adaptive molecular mechanisms capable of mitigating the detrimental action of ROS in the oxidatively damaged fibrin self-assemblage processes.


Subject(s)
Fibrin/chemistry , Fibrinogen/chemistry , Hemostasis , Reactive Oxygen Species/metabolism , Factor XIII/chemistry , Factor XIII/metabolism , Fibrin/metabolism , Fibrin Fibrinogen Degradation Products/chemistry , Fibrin Fibrinogen Degradation Products/metabolism , Fibrinogen/metabolism , Humans , Oxidation-Reduction , Polymers/chemistry , Protein Binding , Protein Multimerization , Thrombin/chemistry , Thrombin/metabolism , Urea/pharmacology
8.
Biochem Biophys Res Commun ; 461(2): 408-12, 2015 May 29.
Article in English | MEDLINE | ID: mdl-25896761

ABSTRACT

FXIIIa-mediated isopeptide γ-γ bonds are produced between γ polypeptide chains of adjacent monomeric fibrin. Despite the use of the different methodological approaches there are apparently conflicting ideas regarding the orientation of γ-γ bonds. To identify the orientation of these bonds a novel approach has been applied. It was based on self-assembly of soluble cross-linked fibrin protofibrils ongoing in the urea solution of moderate concentrations followed by dissociation of protofibrils in the conditions of increasing urea concentration. The oligomers were composed of monomeric desA fibrin molecules created by cleavage of the fibrinopeptides A from fibrinogen molecules with thrombin-like enzyme, reptilase. The results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated an emergence of the double-stranded rod-like fibrin protofibrils. For the first time, the protofibrils are proved to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures. Since no accumulation of α polymers has been found the covalent structure of soluble single-stranded fibrin oligomers is entirely brought about by γ-γ bonds. The results of this study provide an extra evidence to support the model of the longitudinal γ-γ bonds that form between the γ chains end-to-end within the same strand of a protofibril.


Subject(s)
Factor XIIIa/metabolism , Fibrin Fibrinogen Degradation Products/chemistry , Fibrin/chemistry , Fibrin/metabolism , Fibrin Fibrinogen Degradation Products/metabolism , Humans , Hydrodynamics , Light , Protein Multimerization , Scattering, Radiation , Solubility , Ultracentrifugation , Urea/chemistry
9.
Free Radic Biol Med ; 77: 106-20, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25224034

ABSTRACT

Native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing cross-linked fibrin clots under the influence of thrombin and fibrin-stabilizing factor (FXIIIa). The aim of this study was to investigate mechanisms of impairment of both the molecular structure and the spatial organization of fibrinogen under ozone-induced oxidation. FTIR analysis showed that ozone treatment of the whole fibrinogen molecule results in the growth of hydroxyl, carbonyl, and carboxyl group content. A similar analysis of fibrinogen D and E fragments isolated from the oxidized protein also revealed transformation of distinct important functional groups. In particular, a remarkable decay of N-H groups within the peptide backbone was observed along with a lowering of the content of C-H groups belonging to either the aromatic moieties or the aliphatic chain CH2 and CH3 units. The model experiments performed showed that the rather unexpected decay of the aliphatic CH units might be caused by the action of hydroxyl radicals, these being produced in the water solution from ozone. The observed dissimilarities in the shapes of amide I bands of the fibrinogen D and E fragments before and after ozone treatment are interpreted in terms of feasible local conformational changes affecting the secondary structure of the protein. Taken as a whole, the FTIR data suggests that the terminal D fragments of fibrinogen are markedly more susceptible to the ozone-induced oxidation than the central E fragment. The data on elastic and dynamic light scattering provide evidence that, in the presence of FXIIIa, both the unoxidized and the oxidized fibrinogen molecules bind to one another in an "end-to-end" fashion to form the flexible covalently cross-linked fibrinogen homopolymers. The γ and α polypeptide chains of the oxidized fibrinogen proved to be involved in the enzymatic cross-linking more readily than those of unaffected fibrinogen. The experimental data on fibrinogen oxidation acquired in the present study, combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some reactive oxygen species actions detrimental to the protein function.


Subject(s)
Fibrinogen/chemistry , Ozone/chemistry , Humans , Kinetics , Oxidation-Reduction , Phthalic Acids/chemistry , Protein Multimerization , Protein Structure, Tertiary , Reactive Oxygen Species/chemistry , Spectroscopy, Fourier Transform Infrared
10.
Biochim Biophys Acta ; 1834(12): 2470-9, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23948453

ABSTRACT

The plasma fibrin-stabilizing factor (pFXIII) function is to maintain a hemostasis by the fibrin clot stabilization. The conversion of pFXIII to the active form of the enzyme (FXIIIа) is a multistage process. Ozone-induced oxidation of pFXIII has been investigated at different stages of its enzyme activation. The biochemical results point to a decrease of an enzymatic activity of FXIIIа depending largely on the stage of the pFXIII conversion into FXIIIа at which oxidation was carried out. UV-, FTIR- and Raman spectroscopy demonstrated that chemical transformation of cyclic, NH, SH and S-S groups mainly determines the oxidation of amino acid residues of pFXIII polypeptide chains. Conversion of pFXIII to FXIIIa proved to increase protein sensitivity to oxidation in the order: pFXIII

Subject(s)
Factor XIIIa/chemistry , Free Radical Scavengers/chemistry , Oxidants, Photochemical/chemistry , Ozone/chemistry , Factor XIIIa/metabolism , Free Radical Scavengers/metabolism , Free Radicals/chemistry , Free Radicals/metabolism , Humans , Oxidation-Reduction
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