Ozone-induced oxidative modification of fibrinogen molecules

Ozone-induced oxidation of fibrinogen has been investigated. The conversion of oxidized fibrinogen to fibrin catalyzed either by thrombin or by a reptilase-like enzyme, ancistron, in both cases is accompanied by production of gels characterized by a higher weight/length ratio of fibrils in comparison with the native fibrin gels. IR spectra of the D and E fragments isolated from unoxidized and oxidized fibrinogen suggest a noticeable transformation of functional groups by oxidation. A decrease in content of N-H groups in the peptide backbone and in the number of C-H bonds in aromatic structures, as well as a decrease in the intensity of the C-H valence vibrations in aliphatic fragments CH₂ and CH₃ were found. The appearance in the differential spectra of the D fragments of rather intense peaks in the interval of 1200–800 cm^?1 clearly indicates the interaction of ozone with amino acid residues of methionine, tryptophan, histidine, and phenylalanine. Comparison of the differential spectra for the D and E fragments suggests that fibrinogen fragment D is more sensitive to the oxidant action than fragment E. Using EPR spectroscopy, differences are found in the spectra of spin labels bound with degradation products of oxidized and unoxidized fibrinogen, the D and E fragments, caused by structural and dynamical modifications of the protein molecules in the areas of localization of the spin labels. The relationship between the molecular mechanism of oxidation of fibrinogen and its three-dimensional structure is discussed.     

The strengthening role of <Emphasis Type="Italic">D</Emphasis>:<Emphasis Type="Italic">D</Emphasis> interactions in fibrin self-assembly under oxidation

The effect on ozone-induced oxidation on the self-assembly of fibrin in the presence of fibrin-stabilizing factor FXIIIa of soluble cross-linked fibrin oligomers was studied in a medium containing moderate urea concentrations. It is established that fibrin oligomers were formed by the protofibrils cross-linked through γ-γ dimers and the fibrils additionally cross-linked by through α-polymers. The oxidation promoted both the accumulation of greater amounts of γ-γ dimers and the formation of protofibrils, fibrils, and their dissociation products emerging with increasing urea concentrations, which have a high molecular weight. It is concluded that the oxidation enhances the axial interactions between D-regions of fibrin molecules.     

Mechanism of site-specific DNA damage induced by ozone

Ozone has been shown to induce lung tumors in mice. The reactivity of ozone with DNA in an aqueous solution was investigated by a DNA sequencing technique using 32P-labeled DNA fragments. Ozone induced cleavages in the deoxyribose-phosphate backbone of double-stranded DNA, which were reduced by hydroxyl radical scavengers, suggesting the participation of hydroxyl radicals in the cleavages. The ozone-induced DNA cleavages were enhanced with piperidine treatment, which induces cleavages at sites of base modification, but the inhibitory effect of hydroxyl radical scavengers on the piperidine-induced cleavages was limited. Main piperidine-labile sites were guanine and thymine residues. Cleavages at some guanine and thymine residues after piperidine treatment became more predominant with denatured single-stranded DNA. Exposure of calf thymus DNA to ozone resulted in a dose-dependent increase of the 8-oxo-7,8-dihydro-2'-deoxyguanosine formation, which was partially inhibited by hydroxyl radical scavengers. ESR studies using 5,5-dimethylpyrroline-N-oxide (DMPO) showed that aqueous ozone produced the hydroxyl radical adduct of DMPO. In addition, the fluorescein-dependent chemiluminescence was detected during the decomposition of ozone in a buffer solution and the enhancing effect of D2O was observed, suggesting the formation of singlet oxygen. However, no or little enhancing effect of D2O on the ozone-induced DNA damage was observed. These results suggest that DNA backbone cleavages were caused by ozone via the production of hydroxyl radicals, while DNA base modifications were mainly caused by ozone itself and the participation of hydroxyl radicals and/or singlet oxygen in base modifications is small, if any. A possible link of ozone-induced DNA damage to inflammation-associated carcinogenesis as well as air pollution-related carcinogenesis is discussed.     

The oxidative modification of cellular fibrin-stabilizing factor

For the first time, the induced oxidative modification of cellular fibrin-stabilizing factor (cFXIII) has been studied. According to the electrophoresis analysis, the conversion of oxidized cFXIII into FXIIIa resulted in producing the enzyme that significantly lost the initial enzymatic activity. At the same time, FXIIIa subjected to induced oxidation was completely devoid of enzymatic activity. The results of FTIR spectroscopy showed that the oxidation of cFXIII or FXIIIa was accompanied by profound changes both in chemical and spatial structures of the protein. The results of this study are in good agreement with our earlier assumption regarding the antioxidant role of the regulatory subunits B of plasma fibrin-stabilizing factor.     

Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation

The mechanism of self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation has been studied by the methods of elastic and dynamic light-scattering and viscosimetry. Fibrin obtained from oxidized fibrinogen exhibits higher average fiber mass/length ratio compared with native fibrin. Fibrinogen ozonation sharply reduced the latent period preceding aggregation of protein molecules; however, the mechanism of self-assembly of ozonated and non-ozonated fibrinogen cluster was identical. In both cases flexible polymers are formed and reaching a certain critical length they form densely packed structures and aggregate. Using infrared spectroscopy, it has been shown that free radical oxidation of amino acid residues of fibrinogen polypeptide chains catalyzed by ozone results in formation of carbonyl, hydroxyl, and ether groups. It is concluded that fibrinogen peripheral D-domains are the most sensitive to ozonation, which causes local conformational changes in them. On one hand, these changes inhibit the reaction of longitudinal polymerization of monomeric fibrin molecules; on the other hand, they expose reaction centers responsible for self-assembly of fibrinogen clusters.     

Plant defence systems induced by ozone

Recent advances in the understanding of the molecular basis of plant response to ozone attack are reviewed. Plants grown in elevated atmospheric ozone are known to undergo several biochemical changes before any actual damage can be detected. These reactions include increases in the activities of enzymes associated with general plant defence mechanisms. Ozone exposure often causes a surge in the production of the plant hormone ethylene, as well as changes in polyamine metabolism and increases in the activities of several phenylpropanoid and flavonoid pathway enzymes. The activities of superoxide dismutase and peroxidases that protect cells from the oxidative damage caused by hydroxyl radicals, H₂O₂ and superoxides also increase. However, ozone-induced changes in plant cells at the gene level are almost unknown. The limited data available suggest close similarities between ozone-induced and pathogen-induced defence responses in plants. Several general defence genes that have been cloned in other studies will soon be applied to studies of gene expression in ozone-exposed plants. The use of molecular biological tools in ozone research should enable the development of highly specific and sensitive molecular markers for biomonitoring ozone-induced injuries in plants.     

Uric acid protects membranes and linolenic acid from ozone-induced oxidation

Aqueous preparations of linolenic acid, bovine serum albumin, and bovine erythrocyte membrane fragments were bubbled with ozone in the presence or absence of uric acid. Ozonation of the membrane fragments or the bovine serum albumin did not result in protein degradation. After 15 min of ozonation, the absorbance of the thiobarbituric acid-reactive material increased by 0.34 in the linolenic acid preparation and by 0.08 in the suspension of membrane fragments. In the presence of uric acid, these changes in absorbance were reduced to 0.14 for the fatty acid and to 0.01 for the membrane fragments. This result indicates that uric acid protects lipids from ozone-induced oxidation.     

The Role of Hydroxyl Radicals in the Degradation of DNA By Ozone

The degradation of the nucleotides dAMP, dGMP, dCMP and dTMP and of calf thymus DNA by ozone was studied. In all cases both base and sugar moiety were degraded. Furthermore, strand breaks were induced in calf thymus DNA. Hydroxyl radicals were probably involved in the oxidation of the base in dAMP and of the deoxyribose ring, but not in the degradation of the other bases. This indicates that ozone-induced DNA damage proceeds both directly via ozone molecules and indirectly via hydroxyl radicals.     

The Role of Hydroxyl Radicals in the Degradation of DNA By Ozone

The degradation of the nucleotides dAMP, dGMP, dCMP and dTMP and of calf thymus DNA by ozone was studied. In all cases both base and sugar moiety were degraded. Furthermore, strand breaks were induced in calf thymus DNA. Hydroxyl radicals were probably involved in the oxidation of the base in dAMP and of the deoxyribose ring, but not in the degradation of the other bases. This indicates that ozone-induced DNA damage proceeds both directly via ozone molecules and indirectly via hydroxyl radicals.     

Inhibition of ozone-induced SP-A oxidation by plant polyphenols

Surfactant protein-A (SP-A) is the best studied and most abundant of the protein components of lung surfactant and plays an important role in host defense of the lung. It has been shown that ozone-induced oxidation of SP-A protein changes its functional and biochemical properties. In the present study, eight plant polyphenols (three flavonoids, three hydroxycinnamic acids, and two hydroxybenzoic acids) known as strong antioxidants, were tested for their ability to inhibit ozone-induced SP-A oxidation as a mechanism for chemoprevention against lung damage. SP-A isolated from alveolar proteinosis patients was exposed to ozone (1 ppm) for 4 h. The flavonoids protected SP-A from oxidation in a dose dependent manner. ( - )-Epicatechin was the most potent flavonoid and exhibited inhibition of ozone-induced formation of carbonyls by 35% at a concentration as low as 5 μM. Hydroxybenzoic acids inhibited SP-A oxidation in a dose-dependent manner although they were less potent than flavonoids. On the other hand, hydroxycinnamic acids exhibited a different inhibitory pattern. Inhibition was observed only at medium concentrations. The results indicate that inhibition of SP-A oxidation by plant polyphenols may be a mechanism accounting for the protective activity of natural antioxidants against the effects of ozone exposure on lungs.     

Interaction of fibrinogen and its derivatives with fibrin

The binding between complementary polymerization sites of fibrin monomers plays an essential role in the formation of the fibrin clot. One set of polymerization sites involved in the interaction of fibrin monomers is believed to pre-exist in fibrinogen, while the complementary set of binding sites is exposed after the cleavage of fibrinopeptides from fibrinogen. The polymerization sites present in fibrinogen and its derivatives mediate their binding to fibrin. Although the binding of fibrinogen and its derivatives to fibrin have been qualitatively studied, there has been no systematic, quantitative investigation of their interaction with forming or preformed clots. In the present study, the binding of fibrinogen and fragments DD, D1, and E1 was measured using a sonicated suspension of plasminogen- and thrombin-free human cross-linked fibrin as a model of a preformed clot. Dissociation constants of 0.056, 0.19, and 2.44 microM, and the number of binding sites corresponding to 0.10, 0.21, and 0.13/fibrin monomer unit of fibrin polymer were found for fibrinogen, fragment DD, and fragment D1, respectively. Fragment E1 did not bind to sonicated noncross-linked or cross-linked fibrin suspensions. However, it was bound to forming fibrin clots as well as to fibrin-Celite, suggesting that the binding sites on fibrin involved in the interaction with fragment E1 may have been altered upon sonication. Affinity chromatography of various fibrinogen derivatives on a fibrin-Celite column showed that only part of the bound fragment DD was displaced by arginine, whereas fragments D1 and E1 were completely eluted under the same conditions. The results indicate that interaction of fibrinogen with the preformed fibrin clots is characterized by affinity in the nanomolar range and that binding between fibrin monomers, in the process of clot formation, could be characterized by even a higher affinity.     

Biomarkers of oxidative stress study V: Ozone exposure of rats and its effect on lipids,proteins, and DNA in plasma and urine

Ozone exposure effect on free radical-catalyzed oxidation products of lipids, proteins, and DNA in the plasma and urine of rats was studied as a continuation of the international Biomarker of Oxidative Stress Study (BOSS) sponsored by NIEHS/NIH. The goal was to identify a biomarker for ozone-induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods for measuring the various types of oxidative products. The time- and dose-dependent effects of ozone exposure on rat plasma lipid hydroperoxides, malondialdehyde, F₂-isoprostanes, protein carbonyls, methionine oxidation, and tyrosine- and phenylalanine oxidation products, as well as urinary malondialdehyde and F₂-isoprostanes were investigated with various techniques. The criterion used to recognize a marker in the model of ozone exposure was that a significant effect could be identified and measured in a biological fluid seen at both doses at more than one time point. No statistically significant differences between the experimental and the control groups at either ozone dose and time point studied could be identified in this study. Tissue samples were not included. Despite all the work accomplished in the BOSS study of ozone, no available product of oxidation in biological fluid has yet met the required criteria of being a biomarker. The current negative findings as a consequence of ozone exposure are of great importance, because they document that in complex systems, as the present in vivo experiment, the assays used may not provide meaningful data of ozone oxidation, especially in human studies.     

The role of ligand-ligand interactions in competition by fibrinogen and fibrin degradation products for fibrinogen binding to human platelets

Binding to human platelets of radioiodinated human fibrinogen and fragments X, Y, D, D₁ dimer and E was studied to determine the domain of the fibrinogen molecule responsible for binding to the platelet receptor. Although the fragments did not bind, some wer able to complete for the binding of fibrinogen to platelets. It was postulated that the fragments bound to fibrinogen and subsequently interfered with its binding to the receptor. Two approaches were developed to test this hypothesis. In the first technique, molecular exclusion on Sephacryl S-200 superfine was utilized to examine the interaction of radiolabeled fragments with fibrinogen. In the second seties of studies, fibrinogen-Sepharose was prepared and the binding of degradation products directly determined. A spin dialysis apparatus was employed in each case to achieve rapid separation of bound and free radioligand. These studies demonstrated that fragments D and E bind to fibrinogen. Therefore, the mechanism by which degradation products interfere with fibrinogen binding to the platelet receptor is ligand-ligand interaction rather than binding of the fragments to the receptor. Since none of the radiolabeled degradation products bound to platelets, it appears that receptor recognition requires the intact molecule.     

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.     

20-HETE Mediates Ozone-Induced,Neutrophil-Independent Airway Hyper-Responsiveness in Mice

Background

Ozone, a pollutant known to induce airway hyper-responsiveness (AHR), increases morbidity and mortality in patients with obstructive airway diseases and asthma. We postulate oxidized lipids mediate in vivo ozone-induced AHR in murine airways.

Methodology/Principal Findings

Male BALB/c mice were exposed to ozone (3 or 6 ppm) or filtered air (controls) for 2 h. Precision cut lung slices (PCLS; 250 µm thickness) containing an intrapulmonary airway (∼0.01 mm² lumen area) were prepared immediately after exposure or 16 h later. After 24 h, airways were contracted to carbachol (CCh). Log EC₅₀ and E_max values were then calculated by measuring the airway lumen area with respect to baseline. In parallel studies, dexamethasone (2.5 mg/kg), or 1-aminobenzotriazol (ABT) (50 mg/kg) were given intraperitoneal injection to naïve mice 18 h prior to ozone exposure. Indomethacin (10 mg/kg) was administered 2 h prior. Cell counts, cytokine levels and liquid chromatography-mass spectrometry (LC-MS) for lipid analysis were assessed in bronchoalveolar lavage (BAL) fluid from ozone exposed and control mice. Ozone acutely induced AHR to CCh. Dexamethasone or indomethacin had little effect on the ozone-induced AHR; while, ABT, a cytochrome P450 inhibitor, markedly attenuated airway sensitivity. BAL fluid from ozone exposed animals, which did not contain an increase in neutrophils or interleukin (IL)-6 levels, increased airway sensitivity following in vitro incubation with a naïve PCLS. In parallel, significant increases in oxidized lipids were also identified using LC-MS with increases of 20-HETE that were decreased following ABT treatment.

Conclusions/Significance

These data show that ozone acutely induces AHR to CCh independent of inflammation and is insensitive to steroid treatment or cyclooxygenase (COX) inhibition. BAL fluid from ozone exposed mice mimicked the effects of in vivo ozone exposure that were associated with marked increases in oxidized lipids. 20-HETE plays a pivotal role in mediating acute ozone-induced AHR.     

Degradation of oxidized proteins by the 20S proteasome

Oxidatively modified proteins are continuously produced in cells by reactive oxygen and nitrogen species generated as a consequence of aerobic metabolism. During periods of oxidative stress, protein oxidation is significantly increased and may become a threat to cell survival. In eucaryotic cells the proteasome has been shown (by purification of enzymatic activity, by immunoprecipitation, and by antisense oligonucleotide studies) to selectively recognize and degrade mildly oxidized proteins in the cytosol, nucleus, and endoplasmic reticulum, thus minimizing their cytotoxicity. From in vitro studies it is evident that the 20S proteasome complex actively recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP and a reconstituted functional ubiquitinylating system, is not very effective. Furthermore, relatively mild oxidative stress rapidly (but reversibly) inactivates both the ubiquitin activating/conjugating system and 26S proteasome activity in intact cells, but does not affect 20S proteasome activity. Since mild oxidative stress actually increases proteasome-dependent proteolysis (of oxidized protein substrates) the 20S 'core' proteasome complex would appear to be responsible. Finally, new experiments indicate that conditional mutational inactivation of the E1 ubiquitin-activating enzyme does not affect the degradation of oxidized proteins, further strengthening the hypothesis that oxidatively modified proteins are degraded in an ATP-independent, and ubiquitin-independent, manner by the 20S proteasome. More severe oxidative stress causes extensive protein oxidation, directly generating protein fragments, and cross-linked and aggregated proteins, that become progressively resistant to proteolytic digestion. In fact these aggregated, cross-linked, oxidized proteins actually bind to the 20S proteasome and act as irreversible inhibitors. It is proposed that aging, and various degenerative diseases, involve increased oxidative stress (largely from damaged and electron 'leaky' mitochondria), and elevated levels of protein oxidation, cross-linking, and aggregation. Since these products of severe oxidative stress inhibit the 20S proteasome, they cause a vicious cycle of progressively worsening accumulation of cytotoxic protein oxidation products.     

Immunochemical mapping of the conformation of human fibrinogen. The gamma 95-264 segment in inaccessible to antibody in native fibrinogen but progressively exposed by plasmic cleavage

The accessibility of the gamma 95-264 sequence to specific antibody probes in the native fibrinogen molecule and its plasmic cleavage fragments have been investigated. The gamma 95-264 segment was generated by cyanogen bromide cleavage of the gamma chain and isolated by gel filtration and ion exchange chromatography. Rabbit antisera to this peptide and to gamma chain recognized at least five antigenic loci uniformly distributed throughout this segment. In primary binding assays, antibodies to gamma 95-264 bound gamma 95-264, free gamma chain, and fibrinogen fragment D, but not native fibrinogen. Also, gamma 95-264 was bound by antibodies to gamma chain and fibrinogen fragment D, but not by antibodies generated to native fibrinogen. Thus, the gamma 95-264 sequence was not accessible to antibody in the native structure. In competitive equilibrium radioimmunoassays, neither native fibrinogen nor highly soluble fibrinogen fraction I-9 inhibited the binding of gamma 95-264 by its antiserum or anti-gamma chain. With plasmic cleavage, however, the gamma 95-264 sequence became accessible to antibody and the series of fragments D greater than Y greater than D:E = X describes the relative reactivity of the gamma chain sequence in fibrinogen degradation products. Differential expression of gamma 95-264 antigenic loci was also observed with D fragments differing in molecular weight. Plasmic cleavage of cross-linked and noncross-linked fibrin generated D fragments which did not express gamma 95-264 as well as fibrinogen D derivatives, indicating that the D domains of fibrinogen and fibrin are immunochemically distinguishable. These findings indicate that the central segment of the gamma chain is inaccessible to antibody in native fibrinogen, but that proper surface orientation is achieved upon plasmic degradation.     

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay

Activated factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS)-based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetic assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting only the final deacylation portion of the transglutaminase reaction. With the MALDI–TOF MS assay, glutamine-containing peptides derived from α₂-antiplasmin, Staphylococcus aureus fibronectin binding protein A, and thrombin-activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P₋₁ substrate position is sensitive to charge character, and the P₋₂ and P₋₃ substrate positions are sensitive to the broad FXIIIa substrate specificity pockets. The more distant P₋₈ to P₋₁₁ region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.     

The putative role of fibrin fragments in the biosynthesis of fibrinogen by hepatoma cells

The current study reinvestigated whether pure plasminolytic fragments D and E from fibrin directly increase fibrinogen synthesized by the hepatocyte. Fibrinogen protein levels and fibrinogen mRNA levels in an interleukin-6 (IL-6) responsive rat hepatoma cell line (FAZA) were measured quantitatively by [35S]-methionine pulse-chase experiments and Northern hybridizations, respectively. The results demonstrate that neither fragments D nor E, alone or in combination, in the presence or absence of dexamethasone, had any influence on the production of fibrinogen.