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Thrombosis Research (2007) 121, 163–174

REGULAR ARTICLE

Different vulnerability of fibrinogen subunits tooxidative/nitrative modifications induced byperoxynitrite: Functional consequences

Pawel Nowak ⁎, Halina M. Zbikowska, Michal Ponczek,Joanna Kolodziejczyk, Barbara Wachowicz

Department of General Biochemistry, University of Lodz, Banacha 12/16 Street, 90-237 Lodz, Poland

Received 15 November 2006; received in revised form 24 January 2007; accepted 20 March 2007Available online 27 April 2007

Abbreviations: Fg, fibrinogen; PN,dinitrophenylhydrazine; PDVF, polyvisodium dodecyl sulfate; TCA, trichloroa⁎ Correspondingauthor.Tel.: +4842635E-mail address: pnowak@biol.uni.lo

0049-3848/$ - see front matter © 200doi:10.1016/j.thromres.2007.03.017

Abstract Based on previous studies suggesting that fibrinogen (Fg) might be apotential target for peroxynitrite (PN) action in plasma, we investigated the effectsof PN on structure and hemostatic function of Fg in vitro. Using fluorescence andspectrophotometric methods, we estimated that about 0.5, 2 and 8 tyrosine residuesper molecule were nitrated following the reaction of Fg at concentration 5.88 μMwith 10, 100 and 1000 μM PN, respectively. At the samemolar ratios of Fg to PN, about0.01, 0.19 and 0.34 of tyrosine residues per molecule were oxidized to dityrosine andthe amount of carbonyl groups in Fg increased 1.3-, 2,3- and 3.6-fold when comparedto control Fg. SDS-PAGE analysis of PN-modified Fg suggests that inter- andintramolecular dityrosine cross-links occur between Aα chains of Fg. Vulnerabilityof Fg subunits to oxidative/nitrative modifications induced by PN was different.Within the Fg molecule, mainly αC domains as well as D domains (contrary to Edomain) undergo the majority of the modifications. Low extent of nitration andoxidation of Fg molecule (induced by 10 μM PN) did not affect its clotting activity andsusceptibility to degradation by plasmin. Modification of Fg induced by higher PNconcentrations decreased these properties.© 2007 Elsevier Ltd. All rights reserved.

KEYWORDSFibrinogen;Peroxynitrite;Nitrotyrosine;Dityrosine;Protein carbonyl

peroxynitrite; DNPH, 2,4-nylidene difluoride; SDS,cetic acid.4482; fax:+48426354484.dz.pl (P. Nowak).

7 Elsevier Ltd. All rights reserv

Introduction

Increasing evidences indicate that oxidative andnitrative modification of plasma proteins involvedin blood coagulation may lead to the alteration ofhemostatic process [1–7] Among plasma proteinsfibrinogen seems to be highly susceptible to oxidantattack [8–10].

ed.

164 P. Nowak et al.

Peroxynitrite (ONOO−) is a powerful oxidant formedin vivo by the diffusion-controlled reaction betweennitric oxide (

UNO) and O2

U− radicals [11]. It is believedto contribute to the bactericidal action of thephagocytes [12], and themajor compound responsiblefor ischaemia–reperfusion injury [13,14] and tissuedamageby inflammation [15–18]. Exposureofproteinsto peroxynitrite (PN) results in modification ofdifferent amino acid residues, mainly in oxidation ofcysteine, methionine or tryptophan, nitration oftyrosine and formation of dityrosine and carbonylgroups [19]. In biological systems, particularly in bloodplasma, a major constituent which reacts with PN, iscarbon dioxide. The reaction with carbon dioxide isfast (k=5.7·104 M−1 s−1) and yields a short-livedintermediate, nitrosoperoxycarbonate (ONOOCO2

−),which homolyzes to carbonate radical (CO3

U−) andUNO2 in ∼35% yields. This reaction protects proteinmolecules from various peroxynitrite-induced oxida-tive damages but at the same time, it increasesnitration of tyrosine residues in proteins [20]. Nitrationof tyrosine residues in proteins may arise as the resultof PN activity in vivo. The level of nitrated proteins inplasma, though normally low, increases severely inpatients with diseases associated with inflammationand hyperoxia [21,22]. Moreover, nitrotyrosine resi-dues have been detected in atherosclerotic plaques,suggesting that PN plays a role in atherosclerosis [23].

An earlier investigation of the differential suscep-tibility of plasma proteins to oxidative modificationindicated that Fg is highly susceptible to oxidantattack [8]. Oxidized Fg has been shown to inhibitthrombus formation [1,8,24], and platelet aggrega-tion [25]. Oxidized and nitrated Fg is present inplasma of lung cancer patients and smokers [9]. Fgmodified by nitration was also found in the plasma ofpatients with acute respiratory distress syndrome(ARDS) and coronary artery disease [6,26]. Moreover,in diabetic patients with atherosclerotic complica-tions, intravascular fibrous deposits were shown tocontain, in addition to oxidized LDL, a fibrin(ogen)-like material (FLM). It has been suggested that FLMformation is associated with the formation ofdityrosine cross-links between Aα chains of Fg [27].

There has been conflicting data concerning theeffects of peroxynitrite-induced Fg modifications onits clotting activity. Peroxynitrite has been found toeither enhance [26] or diminish [28] the clottingactivity of Fg. Addition of NaHCO3 decreased theinhibitory effect of PN on Fg clotting, suggesting thatthe reactive species formed by the reaction of CO2

with PN were less efficient oxidants than PN itself[28]. Recently, Vadseth et al. [6] reported thattyrosine nitration of Fg significantly accelerates clotformation and factor XIII cross-linking, whereasexposure of Fg to non-nitrating oxidants decelerates

clot formation. In sum, these previous investigationssupport the possibility that ONOO− could eitherenhance or diminish hemostatic function of Fg.

The purpose of this study was to establish thestructural changes of the Fg molecule caused bydifferent concentrations of PN in the presence ofcarbon dioxide. The extent of peroxynitrite-inducednitrative/oxidative modifications in Fg moleculewere determined by Western blot analysis andfluorescence and spectrophotometric measure-ments. Furthermore, the effects of these changeson Fg clotting activity and degradation by plasminwere estimated. We demonstrated that within theFg molecule, the C-terminus of Aα chain and Ddomain (contrary to E domain) predominantlyunderwent nitrative/oxidative modifications. Lowextent of nitration and oxidation of Fg molecule didnot affect its clotting activity and susceptibility todegradation by plasmin while higher extent de-creased these properties.

Materials and methods

Materials

Fg was isolated from pooled citrated human plasma bythe cold ethanol precipitation technique followed byammonium sulphate fractionation at 26% saturation at4 °C, according to Doolittle [29]. Its concentrationwasdetermined spectrophotometrically at 280nmusing anextinction coefficient 1.55 for 1 mg/ml solution. Thecoagulability of Fg preparations used in the thrombintest was N95%. Preparation of Fg obtained by thismethod always contains a slight amount of Fg420 anddegradated form of Fg340. Sheep anti-nitrotyrosinepolyclonal antibodies were from Oxis (Portland, USA).Human plasmin (10 U) was obtained from Calbiochem.2,4-dinitrophenylhydrazine (DNPH) was purchasedfrom POCh S.A. (Poland). All other reagents wereobtained from Sigma-Aldrich.

Peroxynitrite preparation

Peroxynitrite was synthesized according to Pryor andSquadrito method [30]. The solution of PN was storedfor 2–3 months (−70 °C) with negligible changes in itsconcentration. The freeze-fractionated PN solutionforms a yellow top layer,whichwas retained to furtherstudies: it typically contained 80–100 mM PN asdetermined spectrophotometrically at 302 nm in0.1 M NaOH (ε=1679 M−1 cm−1). Immediately beforeuse, the stock solution was diluted in 0.1 M NaOH and,during experiments, was maintained in an ice bath.Some experiments were also performed with decom-posed ONOO−, which was prepared by allowing the PN

165Different vulnerability of fibrinogen subunits to oxidative/nitrative modifications

solution to decompose at neutral pH (7.4) in 100 mMpotassium phosphate buffer (60 min).

Exposure of fibrinogen to peroxynitrite

Samples of human Fg (5.88 μM) in 100 mM potassiumphosphate buffer, 25 mM bicarbonate, pH 7.4 wereexposed to PN at a final concentration between 10–1000 μM. The reaction was initiated by placing asmall drop of PN in the side of tube containing thesample solution immediately followed by vigorousvortexing. The controls were samples treated withthe same volume of 0.1 M NaOH. The protectingeffect of uric acid against the peroxynitrite-medi-ated Fg modification was studied, when testedantioxidant (100 μM) to the Fg sample 10 s beforePN (100 μM) was added. In control experiment uricacid 10 s after PN was added. The concentrations ofperoxynitrite used in our experiments were rela-tively high. However, because the half-life of ONOO−

is approx. 1 s in aqueous buffers, the understandingof peroxynitrite toxicity requires a consideration ofnot only the concentration but also the time ofexposure with the species involved. Exposure to abolus of 250 μM peroxynitrite is equivalent toexposure to a steady-state concentration of 1 μMONOO− for only 7 min. These concentrations couldbe readily formed at sites of inflammation, whereproduction rates of NO and superoxide radicals areconsiderably increased [31].

Gel electrophoresis and Western blotimmunoassays with anti-nitrotyrosine andanti-DNP antibodies

Samples were prepared for electrophoresis inLaemmli sample buffer [32] in the absence or pres-ence of β-mercaptoetanol and were separated onSDS-PAGE using a Mini-Protean Electrophoresis Cell(Bio-Rad, Hercules, CA). Protein was stained withCoomassie Blue R250. Electrophoretic transfer ontopolyvinylidene difluoride (PDVF) membranes wasperformed with a Mini Transfer-Blot Cell (Bio-Rad).The carbonyl groups in the protein side chains werederivatized to 2,4 nitrophenylohydrazone by reactionwith 2,4-dinitrophenylhydrazine. The membraneswere blocking 2 h with 5% non-fat dry milk solutionin 50 mM Tris/HCl, 150 mM NaCl, 0.05% Tween 20, pH7.4 (TBS-T), and incubated for 2 h with goatpolyclonal anti-nitrotyrosine (diluted 1:1000) orrabbit anti-DNP antibodies (diluted 1:2000) in TBS-Twith 5% non-fat dry milk. After washing six times,5 min each, with TBS-T, the membranes wereincubated for 1 h with horseradish peroxidase-labeled anti-goat/rabbit IgG antibodies (diluted

1:5000) in TBS-Twith 5% non-fat dry milk. The blotswere then washed six times, 5 min each, with TBS-T.Bands containing nitrotyrosine/carbonyl groups werevisualized by luminol-enhanced chemiluminescence(ECL) system and exposure to X-ray film.

Spectrophotometric quantification offibrinogen nitration and oxidation

The concentration of nitrotyrosine in Fg solution(5.7 μM) after treatment with PN was estimatedspectrophotometrically at 302 nm, at pH 11.5(molar absorption coefficient for nitrotyrosine,ε=4400 M−1 cm−1) [19]. The yield of nitrationwas presented as mol of nitrotyrosine per mol offibrinogen.

Fg samples for carbonyl groups detection (0.5 ml,protein concentration 2 mg/ml) were precipitatedon ice with cold trichloroacetic acid (TCA, finalconcentration 20%), centrifuged for 5 min and then1 ml of 10 mM DNPH in 2 M HCl was added to obtain1 mg/ml solution of protein. To correspondingsamples containing reagent blanks 1 ml of 2 M HClwas inserted. The samples were placed in the darkfor 1 h at room temperature, mixed every 10 min.Then they were precipitated with TCA to finalconcentration of 10% and centrifuged for 5 min.Supernatants were removed and a sediment ofproteins was washed with 10% TCA and then washedthree times with 1 ml of ethanol/ethyl acetatemixture (v/v, 1:1) to remove unbound DNPH.Samples were resuspended in 6 M guanidine hydro-chloride (in 2 M HCl) for 15 min with vortex mixing.Carbonyl content was determined at 366 nm(ε=22,000 M−1 cm−1) [33].

Measurement of dityrosine

Formation of dityrosine was monitored by fluo-rescence measurements (λexcitation at 325 nm andλemission at 415 nm). The final concentration ofdityrosine was calculated from a fluorescencecalibration curve constructed using authenticdityrosine [34].

Plasmin degradation of fibrinogen

Control and PN-treated Fg were extensively dialyzedagainst 100 mM Tris/HCl, pH 7.4, diluted to give afinal concentration of 1 mg/ml, and 5 mM CaCl2 wasadded. Proteolysis was initiated by the addition ofplasmin to a final concentration of 0.02 U/ml. Atspecified times, aliquots were removed and thereaction was stopped by mixing with Laemmli samplebuffer containing 8 M urea.

166 P. Nowak et al.

Trombin-catalyzed fibrin polymerization

Polymerization of fibrin was monitored at 595 nm in a96-wellmicrotiter plate reader (Bio-Radmodel 550) atambient temperature. To each reaction well wasadded 240 μl of control or PN-treated Fg (1 mg/ml)in 100mM Tris/HCl, 5mM CaCl2, pH 7.4. To initiate thepolymerization reaction, 60 μl of thrombin (at a finalconcentration of 0.25 U/ml) was added to all reactionwells with a multichannel pipette such that allreactions began simultaneously. Immediately afterthe addition of enzyme, the samples were automixedby the instrument for 4 s. Turbidity was monitoredevery 25 s for 20 min. Turbidity curves werecharacterized by two parameters— a lag phase duringwhich soluble fibrin oligomers did not change theabsorbance at 595 nm, and a growth phase where theslope depended primarily on the velocity of lateralprotofibrils association. A tangent line was drawnthrough the steepest slope of the turbidity curve. Theintersection of this tangent line with the absorbance

Figure 1 Effects of peroxynitrite on the electrophoretic pattetreated fibrinogen were separated on 7.5% SDS-PAGE gel under novisualized by staining with Coomassie Blue R250. The positions ofAα, Bβ, and γ) and additional high molecular weight (HMW) baapplied to each lane. Molecular masses are shown on the right. Tquantified by densitometry (C). Values represent means±SD focontrol Aα chain and Aα chain of fibrinogen treated with peroxy

baseline was denoted as lag time whereas Vmax wascalculated as degrees α=arctangent (ΔA595 nm/s).

Statistics

The significance of differences between the meanvalues of various perameters was analyzed by one-way ANOVA followed by Dunnet's and Tykey's post hoctests. A level pb0.05 was accepted as statisticallysignificant.

Results

Different susceptibility of fibrinogen subunitsto nitrative/oxidative modifications

The exposure of purified Fg at physiological concen-tration (5.88 μM) to PN (10–1000 μM) caused distinctchanges in its electrophoretic pattern (Fig. 1). SDS-PAGE analysis under non-reducing conditions

rn of human fibrinogen. Samples of native and peroxynitrite-n-reducing (A) or reducing conditions (B). Protein bands werethe non-reduced fibrinogen (Fg), fibrinogen chains (Aα110 kDa,nds are indicated. Approximately 15 μg of fibrinogen werehe band intensity of Aα, Bβ, and γ chains of fibrinogen werer three independent determinations. ⁎pb0.05 between thenitrite after analysis of variance using Tukey's post hoc test.

Figure 2 Detection of nitrotyrosine after treatment of fibrinogen with peroxynitrite. Samples of native and peroxynitrite-treated fibrinogen were separated on 7.5% SDS-PAGE gel under non-reducing (A) or reducing conditions (B), electrotransferredto PVDF membranes and probed with polyclonal anti-nitrotyrosine antibody as described in Materials and methods. Thepositions of the relevant non-reduced fibrinogen (Fg), fibrinogen chains (Aα110 kDa, Aα, Bβ, and γ) and additional high molecularweight (HMW) bands are indicated. Molecular masses are shown on the right.

Figure 3 Detection of oxidized fibrinogen subunits byWestern blot immunoassay. Samples of native and peroxyni-trite-treated fibrinogenwere derivatizedwith DNPH in SDS andtrifluoroacetic acid as described in Materials and methods.Samples were subjected to SDS-PAGE (7.5% gel) in the presenceof 2-mercaptoethanol and electrotransferred to PDVF mem-branes. Protein-associated carbonyls were detected by immu-noassay using anti-DNPH antibodies. The positions of therelevant fibrinogen chains are indicated. Molecular massesare shown on the right.

167Different vulnerability of fibrinogen subunits to oxidative/nitrative modifications

demonstrated that some amount of aggregatedprotein on the top of the gel in PN-treated Fg waspresent (Fig. 1A). Under reducing conditions (Fig. 1B)in PN-treated Fg not only bands corresponding toAα110, Aα, Bβ and γ chains but also some new high-molecular-weight (HMW) bands (mainly between 120–140 kDa) were observed. Moreover, densitometricscanning of reduced gel showed a decrease in the Aαsubunit of Fg together with an increase in HMWaggre-gates at increasing PN concentration. The band inten-sities of Bβ and γ chains were not changed (Fig. 1C).This may suggest that inter- and intramolecular cross-links between Aα chains of Fg were formed.

Western blot analysis of PN-treated Fg indicatedthat the amount of nitrotyrosine residues in Fgmolecule increased with PN concentration in a dose-dependent manner (Fig. 2). Moreover, the Fg subunitsshowed the different susceptibility to tyrosine nitra-tion. The Aα chain as well as the Aα chain-derivedHMW aggregates were first and the most intenselynitrated, while the chains Bβ and γ much less.

We have found that the Aα chain was not onlypreferably nitrated but it also showed high suscepti-bility to oxidative modifications induced by PN asanalyzed by Western blot immunoassay with anti-DNPantibodies (Fig. 3). The presence of carbonyl groups inadditional bands derived from Aα chain was alsoobserved (Fig. 3). The small amount of carbonyl groupspresent in native Fg is in agreement with the datadescribed by others [8].

Uric acid, the PN scavenger, added to Fg 10 s beforePN inhibited both, the formation of the HMWaggregates (Fig. 4A) and Fg nitration (Fig. 4B).Contrary, the inhibitory effect of uric acid wassignificantly weaker when the scavenger was added

10 s after PN. Decomposed PN did not induce anyoxidative/nitrativemodification in Fgmolecule (lane3in Fig. 4).

Formation of nitrotyrosine, dityrosine, andcarbonyl groups in oxidized fibrinogen

PN-induced oxidative/nitrative modifications in Fgmolecule were confirmed by fluorescence andspectrophotometrically quantitative determinations.

Figure 4 The effect of uric acid on peroxynitrite-inducedchanges in the electrophoretic pattern of fibrinogen and onfibrinogen tyrosine nitration. Fibrinogen (5.88 μM) wasexposed to 100 μM peroxynitrite in 100 mM potassium buffer,25 mM bicarbonate, pH 7.4, without or with 100 μM uric acid.(A) Protein bands visualization by staining with Coomassie BlueR250. (B) Western blot analysis with anti-nitrotyrosine anti-bodies. Lane 1 - control fibrinogen; lane 2— fibrinogen+100μMperoxynitrite; lane 3 — fibrinogen+100 μM decomposedperoxynitrite; lane 4 — fibrinogen+100 μM uric acid+100 μMperoxynitrite; lane 5 — fibrinogen+100 μM peroxynitrite+100 μM uric acid. Molecular masses are shown on the right.

Figure 5 The effect of peroxynitrite on the nitration andoxidation of human fibrinogen. Fibrinogen (5.88 μM) wasexposed to peroxynitrite in 100 mM potassium phosphate buffer,25mMbicarbonate, pH7.4. (A) The yield of nitrationdeterminedby measuring the absorbance at 430 nm at pH 11.5.(B) Formationofdityrosinemonitoredbychanges in fluorescence(λexcitation at 325 nm and λemission at 415 nm). (C) Formation ofDNPH-reactive carbonyl determined spectrophotometrically at366 nm. Values represent means±SD for five independentexperiments. The effects of the three concentrations ofONOO− were statistically significant according to the one-wayANOVA (A) — pb0.002; (B) — pb0.02; (C) — pb0.05.

168 P. Nowak et al.

We observed a dose-dependent increase in formationof nitrotyrosine, dityrosine and carbonyl groups(Fig. 5A–C). In native Fg molecule a backgroundlevel of carbonyl groups was found (0.56+0.09 molcarbonyl/mol Fg) while nitrotyrosine and dityrosinewere not present (Fig. 5A–C). Human Fg moleculecontains exactly 100 tyrosine residues. We estimatedthat about 0.5, 2 and 8 tyrosine residues permoleculewere nitrated following the reaction of Fg atconcentration 5.88 μM with 10, 100 and 1000 μMPN, respectively (Fig. 5A). At the samemolar ratios ofFg toPNabout 0.01, 0.19and 0.34 of tyrosine residuesper molecule were oxidized to dityrosine (Fig. 5B).The amount of carbonyl groups in Fg treated with 10,100 and 1000 μM PN were found about 1.3-, 2,3- and3.6-fold higher than in control Fg, respectively.

Plasmic digestion of peroxynitrite-modifiedfibrinogen

Comparison of plasmin digestion of native and PN-treated Fg by SDS-PAGE revealed the production of thesame conventional Fg degradation products [35]:

fragments PreX (α1–583, β1–461, γ1–411), X (α1–221,β43–461, γ1–411), Y (α1–78 and α105−196, β44–132

and β133–461, γ1–62 and γ1–411), D (α111–198, β133–461,γ95–411) and E (α20–78, β53–122, γ1–53) (Fig. 6A).

169Different vulnerability of fibrinogen subunits to oxidative/nitrative modifications

However, the first steps of plasmin degradation of thePN-modified Fg occurred slower than in control (Fig.6A). Based on the densitometry scanning analysis ofthe gels presented in Fig. 6A it has been noticed thatin PN-modified Fg the rates of fragment X disappear-ance and the subsequent appearance of fragment Dwere slower than in control Fg (data not shown). Atthe lowest concentration of PN only Fg and formedpreX fragment with partially degradated αC domainwere nitrated, while fragment X which lost αCdomain and other plasmin degradation products(fragments Y, D and E) did not contain nitratedtyrosines (Fig. 6B). At higher concentrations of PNfragments X, Y and D (contrary to fragment E) werealso nitrated (Fig. 6B).

Clotting activity of peroxynitrite-modifiedfibrinogen

Polymerization of Fg was monitored as the change inturbidity at 595 nm as described in Materials andmethods. To eliminate the effect of different pH inthe samples, control and PN-treated fibrinogenswere extensively dialyzed against 100 mM Tris/HClbuffer, pH 7.4, and diluted to give a final concen-tration of 1 mg/ml. Representative curves ofthrombin-catalyzed polymerization of the controland PN-modified Fg are shown in Fig. 7. We foundthat with the increasing PN concentration, theability of Fg to undergo clotting was diminished.The curve for Fg treated with decomposed PN(100 μM) was very similar to the curve for control Fg(not shown). From the curves in Fig. 7, wedetermined the lag time (time required for theprotofibrils formation), and the maximum slope(Vmax), that represents the rate of lateral proto-fibrils association. The results, presented in Table 1,demonstrated that only the higher concentrationsof PN (above 50 μM) significantly increased the lagperiod and decreased the maximum rate of lateralaggregation when compared to control Fg. Minorchanges caused by the lower PN concentrationswere not statistically significant (pN0.05).

Discussion

There is substantial evidence that oxidative stressassociated with inflammation or postischemicreperfusion [1,2,6,10,17,26,36] may lead to oxida-tively/nitratively modified fibrinogen and change ofits functional properties. However, there has beenconflicting data on the specific structural modifica-tion and biological consequences of PN-mediatednitration/oxidation of this protein. PN has beenfound to both enhance [26] or diminish [28] the

hemostatic activity of Fg. This promoted us toreexamine the action of PN on the structural andfunctional properties of Fg in vitro.

Our results confirmed a high susceptibility of Fg tonitrative/oxidative modifications induced by PN[26,28,37]. In our experiments for incubation withPN we used Fg at physiological concentration(5.88 μM). We have found that even the lowest PNconcentration (10 μM) induced distinct structuralchanges in Fg molecule resulted in nitration andoxidation of tyrosine residues (∼0.5 mol of nitrotyr-osine and ∼0.01 mol of dityrosine per mol of Fg,respectively) and in formation of carbonyl group(∼0.2 mol carbonyl/mol Fg). Vulnerability of Fgsubunits to oxidative/nitrative modifications inducedby PN was found to be different. We have shown thatwithin the Fg molecule, the C-terminus of Aα-chains(αC domains) as well as D domains (contrary to Edomain) undergo the majority of the modifications(Figs. 1–3, 6). This finding has been supported by thefollowing observations: (1) at lower PN concentration(10 μM) the Aα chain was the first and foremostnitrated/oxidized Fg subunit, (2) whenαC domain wasremoved from the Fg treated with 10 μM PN theobtained fragment X did not contain nitrotyrosineresidues, (3) fragment D and LMW degradationproducts (derived from αC domain – that are visiblein 10% PAGE – not shown) were the main nitratedterminal degradation products of PN-treated Fg,(4) covalent cross-linking between Aα subunits of PN-treatedFgwasobserved.The reasonof thedifferentialvulnerability of Fg subunits to oxidative/nitrativemodifications derives probably from specific confor-mation of the molecule. The structural studies,together with biochemical and immunochemical datahave demonstrated that the Bβ- and γ-chains arehighly intertwined in tertiary structure with very littlefree polypeptide exposed in solution. Contrary, the αCdomains form polar structures that are exposed on thesurface of Fgmolecule andmay bemore susceptible toPN attack. The described above observations remainconsistent with the results of Shacter et al. [8]obtained for Fg exposed to the Fe+3/ascorbateoxidative system. Those authors showed that withinthe Fg molecule, the Aα-chain incurs the majority ofthe modification. Similar profiles of Fg subunitsoxidation were obtained for γ-irradiated [38] andphotooxidated Fg in the presence of methylene blue(MB) [39].

A high increase of nitrotyrosine level aftertreatment of Fg with PN indicates that this proteinis a good substrate for tyrosine nitration and may beeasily cross-linked through covalent dityrosinebonds. Dimeric human Fg molecule contains exactly100 tyrosine residues (9 in Aα chain, 21 in Bβ chainand 20 in γ chain, respectively), but only 20 to 30 are

170 P. Nowak et al.

exposed to the solvent phase [40]. We havecalculated that following Fg reaction with PN (10,100 and 1000 μM), about 0.5, 2 and 8% of tyrosineresidues were nitrated and 0.01, 0.19 and 0.34% oftyrosine residues were oxidized to dityrosine,respectively (Fig. 5A–B). The local environment ofrelatively surface-exposed tyrosine residues appearsto determine the sites of nitration. Previously,Ischiropoulos [41] suggested that the presence ofneighbouring acidic residues plays a role in increas-ing the susceptibility of these tyrosines to nitration.In primary sequence of Fg Aα chain; Tyr76, Tyr178,Tyr409 and Tyr570 are flanked by glutamate and/orasparate residues. Tyr 76 and Tyr178 are placed indomain E and D, respectively. The two later tyrosinesare located in the αC domain and may be the firsttarget for PN attack. Subsequently, tyrosines in βand γ chains of the D domain would undergonitration. Our findings are consistent with theresults described by Pignatelli et al. [9]. Thoseauthors have identified in plasma of lung cancer

Figure 6 Plasmin digestion of native and peroxynitrite-treatdescribed under Materials and methods. At the indicated times,under non-reducing conditions and stained with Coomassie BlueWestern blot with anti-nitrotyrosine antibodies. Blots were visuaPositions of fibrinogen (Fg) and fragments preX, X, Y, D, and E ar

patients and smokers two nitrated and oxidizedbands at Mr 65 and 55 kDa, corresponding to the Aαand Bβ chains of Fg. Contrary, Gole et al. [26] havefound the Bβchain of Fg as the major modified bynitration subunit in acute respiratory distress syn-drome (ARDS). The observed discrepancies mayresult from a high susceptibility of αC domain of Fgto proteolytic degradation.

Fg exposure to PN also led to the formation ofSDS- and heat-stable Aα-chain aggregates resultedfrom the formation of dityrosine cross-linking(Figs. 1–3, 5B). Although nitration of tyrosineresidues by PN has been reported for severalproteins, formation of dityrosine has been detectedonly in a few proteins such as serum albumin,manganese–superoxide dismutase, epidermalgrowth factor receptor, α-synulein, tau protein,and lens proteins [19,42–46]. The formation of atyrosyl radical and its reaction with a tyrosyl radicalon another αC-domain of Fg may be the basis fordityrosine covalent cross-linking. Similarly, the

ed fibrinogen. Fibrinogens were incubated with plasmin assamples of a plasmin digest were separated on 7% SDS-PAGER250 (A). Nitrotyrosine-containing protein were detected bylized by chemiluminescence and exposure of X-ray film (B).e indicated.

Figure 7 The effect of peroxynitrite on fibrinogen clottinginduced by thrombin. Fibrinogen (5.88 μM) was exposed toperoxynitrite as described in Materials and methods. Afterdialysis against 100 mM Tris/HCl buffer, pH 7.4, fibrin clotformation in the presence of 5 mM CaCl2 was monitored byturbidity changes at 595 nm over time after the addition ofthrombin (0.25 U/ml). Representative polymerization curvesfrom three independent experiments are shown.

Table 1 Parameters of thrombin-catalyzedpolymerizationof native and peroxynitrite-modified fibrinogen

Samples Lag time [s] Vmax (s−1)×10−4

Fg 12.4±1.1 18.16±0.16Fg+10 μM ONOO− 13.2±1.3 17.94±0.53Fg+20 μM ONOO− 15.6±1.8 17.24±0.24Fg+50 μM ONOO− 18.3±1.4 ⁎ 14.21±0.55 ⁎

Fg+100 μM ONOO− 21.4±1.1 ⁎ 10.98±0.47 ⁎

Fg+1000 μM ONOO− 32.2±4.4 ⁎ 6.33±0.54 ⁎

Fg+decom 100 μM ONOO− 12.8±1.1 18.02±0.24

Data are an average of three independent experiments±SD.⁎, pb0.05 between the control and modified fibrinogen afteranalysis of variance using Tukey's post hoc test.

171Different vulnerability of fibrinogen subunits to oxidative/nitrative modifications

reported formation of HMW aggregates after expo-sure of Fg to oxidants (metal/ascorbate system,photooxidation induced by MB/light treatment) maybe due to formation of dityrosine between αC-domains [8,25,39]. All these results demonstratethat the propensity of αC-domains to form inter-and intramolecular dityrosine cross-links is a prop-erty of Fg molecule and may explain, at least inpart, the formation of Fg aggregates in pathologicallesions. The ability of Fg to form Fg depositions inthe presence of oxidizing/nitrating agents wasobserved in patients with atherosclerotic complica-tions [27] and in ARDS patients [26].

To investigate the potential effect of PN-medi-ated modifications of Fg on its functional proper-ties, thrombin-induced fibrin polymerization and arate of Fg degradation by plasmin were measured.

The data presented here indicated that polymer-ization of PN-modified Fg was impaired compared tocontrol Fg, with significantly increased lag time,lower rate of lateral protofibrils aggregation, andnoticeable decrease in final turbidity (Fig. 7,Table 1). However, these changes (in the clottingproperties of fibrinogen) were observed only at thehigher concentrations of PN (above 20 μM) althoughafter treatment of the protein with lower concen-tration of PN significant nitration and oxidation of Fgmolecule have occurred (Figs. 2, 3, 5). These findings

are partly consistent with the previous reportsshowing that PN-mediated modifications of Fgstrongly inhibit thrombin-catalyzed clot formationbut do not affect the release of fibrinopeptides [28].Contrary to our study, authors of this paper used Fg atmore then ten-fold lower concentration (0.5 μM) forincubation with PN. Thus, inhibitory effect of PN onthe clotting properties of Fg was more stronger (IC50at 22 μM PN concentration) than in our study (IC50 at120 μM PN concentration). On the other hand, ourfindings are opposite to the previous studies of Goleat al. [26] who have shown that exposure of Fg to1mMPN resulted in approx. 2-fold acceleration of theclot formation. This discrepancy is likely to be due tothe different experimental conditions. The authorsused 2.5-fold lower molecular ratio of PN to Fg and10-fold lower dose of thrombin to induce Fgpolymerization than we used in our experiments.Because of that any comparison of the measuredparameters was not possible. Moreover, in our studywe used the different wavelength (λ=595 nm,instead of 360 nm) to avoid the potential interferencewith the maximum absorption for nitrotyrosine(λ=302 nm).

Recent study of Vadseth et al. [6] has shown thatvery low nitration of Fg (∼45–65 μmol nitrotyr-osine/mol tyrosine) caused by nitrating oxidantssignificantly increased fibrin generation and dis-tinctly changed clot architecture as compared withnative fibrinogen. The authors proposed that even afew nitrated Fg molecules were capable to enhancethe prothrombic-activity of Fg.

It has been suggested that Fg, an acute phaseprotein which level in plasma distinctly increased inresponse to inflammatory agents, might act as animportant physiological antioxidant protectingother molecules from free radical mediated oxida-tion [47,48]. Moreover, quite recently Selmeci et al.have shown the correlation between levels of bloodplasma Fg and AOPP (advanced oxidation proteinproducts — a novel oxidative stress biomarker) in

172 P. Nowak et al.

patients with various peripheral vascular and car-diovascular diseases [10]. The authors have alsoimplicated that higher Fg concentrations wereassociated with more oxidatively transformedgroups on the Fg molecule [10]. If Fg acts as asacrificial antioxidant, this should lead in conse-quence to disturbances of its clotting activity.Indeed, in in vitro studies oxidized Fg looses itscoagulation abilities. It is well known that formationof ∼2 mol of carbonyl groups per mol of Fg orselectively oxidation of His, Trp or Met residuesmarkedly decreased the polymerization activity ofFg [1,8,24,49]. Exposure of Fg to PN caused tyrosinenitration in conjunction with oxidation of otheramino acid residues. Thus, PN-induced changes in Fgcoagulation properties seem to be a result of theoverall net effects of oxidation and nitration processoccurring in Fgmolecule, mainly in the Aα C domain.

The fibrin(ogen) αC-domains are involved inlateral aggregation of protofibrils [50,51], in con-trolling activation of factor XIII [52], and in celladhesion via their Aα572-574 RGD sequence [53].They also contain binding sites for t-PA and plasmin-ogen and cross-linking sites for α2-antiplasmin andPAI-2 [54–56], which play a role in regulation offibrinolysis. It is well known that the moleculardefects in the αC-domains of several congenitallyabnormal fibrinogens result in disfibrinogenemiaassociated with defective thrombolysis [57,58].

Impaired lateral association of protofibrils ob-served in our study indicates that the consequenceof Fg modification by PN might be a generation ofthe clot of abnormal structure. The decrease inlateral aggregation commonly results in a decreasein the maximal rate of assembly, an increase in thelag period, the number of fibers and the fiberlength, as well as a decrease in the maximumaverage fiber size [59]. Clots composed of the thinfibers and small pores are more thrombogenic andare associated with coronary artery disease [60,61].The biochemical reasons for the formation of theseabnormal fibrin gels is not known but was notrelated to fibrinogen concentration and was postu-lated to result from unidentified posttranslationalmodifications of fibrinogen [62].

We also observed that modification of Fg by PNconcentrations above 10 μM decreased the rate of theFg αC-domain degradation by plasmin. This maysuggest that the alterations may be due to formationof dityrosine between αC-domains which make the Fgmore resistant to plasmin. Similarly, introduction ofdityrosine cross-links into albumin made it moreresistant to ATP-independent proteolysis [63]. Veryrecently, Monaco et al. [64] showed that Fg reactedwith PN displayed a similar proteolytic processing byMMP-2, producing a similar fragments as that observed

for native Fg. While analysing the cleavage mechan-isms it was found that PN-induced oxidation of Fgresulted in a structural alteration of themolecule thatrendered itmore easily recognized by the protease butmore resistant to the cleavage [64]. For effective clotdigestion plasmin has to degrade fibrin in the coiled-coil region between the D- and E-regions. This processmaybe impairedwhendegradationof theαCdomain isinhibited. Thus, the modified Fg may form patholog-ical fibrin more resistant to plasmin.

Lipinski [65] suggested that oxidatively modifiedFg with cross-linked dityrosine coating on tumorcells might protect these cells against immunekilling. Moreover, inefficient removal and degrada-tion of oxidatively modified Fg forming intravascu-lar fibrin-like deposits in diabetic patients may leadto endothelial damage and initiation of atherogen-esis [27].

In conclusion, our results strongly suggest that infibrinogen molecule AαC domain is the most vulner-able to PN-induced modifications. The consequenceof these modifications may be the alteration in fibrinstructure associated with a prothrombic effect.

Acknowledgement

The work was supported by a grant from University ofLodz (506/810).

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