Peroxynitrite and fibrinolytic system: The effect of peroxynitrite on plasmin activity

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<ul><li><p>Molecular and Cellular Biochemistry 267: 141146, 2004.c 2004 Kluwer Academic Publishers. Printed in the Netherlands.</p><p>Peroxynitrite and fibrinolytic system: The effectof peroxynitrite on plasmin activity</p><p>Pawel Nowak, Joanna Ko lodziejczyk and Barbara WachowiczDepartment of General Biochemistry, University of Lodz, Poland</p><p>Received 23 January 2004; accepted 3 June 2004</p><p>Abstract</p><p>We have shown that peroxynitrite (ONOO) inhibits streptokinase-induced conversion of plasminogen to plasmin in aconcentration-dependent manner and reduces both amidolytic (IC50280M at 10 M concentration of enzyme) and pro-teolytic activity of plasmin. Spectrophotometric and immunoblot analysis of peroxynitrite-treated plasminogen demonstratesa concentration-dependent increase in its nitrotyrosine residues that correlates with a decreased generation of active plasmin.Peroxynitrite (1 mM) causes the nitration of 2.9 tyrosines per plasminogen molecule. Glutathione, like deferoxamine, partiallyprotects plasminogen from peroxynitrite-induced inactivation and reduces the extent of tyrosine nitration. These data suggestthat nitration of plasminogen tyrosine residues by peroxynitrite might play an important role in the inhibition of plasmin catalyticactivity. (Mol Cell Biochem 267: 141146, 2004)</p><p>Key words: plasminogen/plasmin inactivation, fibrinolytic system, tyrosine nitration, peroxynitrite</p><p>Abbreviation: PLG plasminogen, PL plasmin, FG fibrinogen, FDP fibrinogen degradation products</p><p>IntroductionPeroxynitrite (ONOO) is a potent and a relatively long-livedcytotoxic oxidant formed in vivo in the rapid reaction betweennitric oxide and superoxide anion [1, 2]. It is believed to con-tribute to the bactericidal action of the phagocytes [3, 4], andthe major compound responsible for ischaemia-reperfusioninjury [5, 6] and tissue damage by inflammation [79]. Apathogenic role of peroxynitrite has been investigated in var-ious human diseases [10]. It is well known that proteins are amajor target for oxidative and nitrative damage in vivo [1012]. Exposure of proteins to peroxynitrite results in modifica-tion of amino acid residues, altering the protein structure andfunction [10, 11]. It has been demonstrated that reaction ofprotein with ONOO resulted in the oxidation of tryptophan,cysteine and methionine, tyrosine nitration, dityrosine forma-tion, carbonyl formation and protein fragmentation [13]. Freeand protein-bound 3-nitrotyrosine, a stable product of tyro-sine nitration, may be measured as an indicator of proteindamage induced by peroxynitrite and other reactive nitrogenAddress for offprints: P. Nowak, Department of General Biochemistry, University of Lodz, 90-237 Lodz, Banacha 12/16, Poland (E-mail: pnowak@biol.uni.lodz.pl)</p><p>species [10, 14]. A recent study identified fibrinogen, ceru-loplasmin, transferrin, and plasminogen as nitrated proteinspresent in human plasma from smokers and lung cancer pa-tients [15].</p><p>The blood fibrinolytic system comprises an inactive proen-zyme plasminogen (PLG) that can be converted to theactive enzyme plasmin (PL) degrading insoluble fibrininto soluble fibrin degradation products. Two immunologi-cally distinct physiological plasminogen activators (PA) havebeen identified: the tissue-type PA (t-PA) and the urokinase-type PA (u-PA). For therapeutic use two potent activatorsbacterial proteins: streptokinase (SK) and staphylokinase(SAK) have been utilized. Fibrinolytic activity can be reg-ulated on two levels: by specific plasminogen activator in-hibitiors (PAI-1 and PAI-2), or at the level of plasmin,mainly by -antiplasmin [16]. Under normal physiologi-cal conditions between coagulation process and fibrinoly-sis an equilibrium state exists, but under certain conditions(for example after oxidative stress) this equilibrium can bealtered.</p></li><li><p>142</p><p>It is well recognized that thrombi develop at the site of in-flammation where concurrent production of nitric oxide andsuperoxide anion can result in the formation of peroxynitrite[7, 17]. We have recently found that the reaction of perox-ynitrite with fibrinogen resulted in structural and functionalmodifications of fibrinogen [18]. Although plasmin is knownto be relatively resistant to inactivation by most oxidants [19],it is possible that the life span of thrombi that form at sitesof inflammation could be affected by peroxynitrite, whichis generated by activated inflammatory cells in the vicinityof the clot. Therefore the aim of our studies was to assessthe effects of peroxynitrite in vitro on proteins of fibrinolyticsystem. We estimated the conversion of plasminogen to plas-min after treatment of plasminogen with peroxynitrite and thechanges of amidolytic and proteolytic activities of plasmininduced by peroxynitrite. We studied whether the observedfunctional changes of plasminogen are associated with nitra-tion of tyrosine residues in plasminogen.</p><p>Materials and methodsMaterials</p><p>Chemical reagents were obtained from Sigma-Aldrich (strep-tokinase, Lys-Sepharose, glutathione, deferoxamine). Chro-mozym PL (Tosyl-Gly-Pro-Lys-4-nitranilide acetate) was ob-tained from Roche. Mouse anti-nitrotyrosine monoclonal an-tibodies were from Alexis.</p><p>Protein preparation</p><p>Plasminogen was isolated by affinity chromatography onlysine-Sepharose [20], Streptokinase (SK) was coupled toCNBr-activated Sepharose 4B [21], and used to generate plas-min by incubation of 1 ml of 10 M PLG with 10 l of SK-Sepharose in 100-mM potassium phosphate buffer, pH 7.4,for 2 h at 25 C followed by centrifugation to remove the resin.Fibrinogen (FG) was prepared from citrated human plasmaby the cold ethanol precipitation technique [22]. Protein con-centrations were evaluated spectrophotometrically (A1% 280nm: PLG-16.1; FG15.5, respectively).</p><p>Synthesis of ONOO and treatment of PL/PLGwith ONOO</p><p>Peroxynitrite was synthesized according to the method ofPryor et al. [23]. Freeze fractionation (70 C) of the per-oxynitrite solution formed a yellow top layer, which was re-tained for further studies. The top layer typically contained80100 mM peroxynitrite as determined spectrophotometri-cally at 302 nm in 0.1 M NaOH (302nm = 1679 M1.cm1).</p><p>PL/PLG preparations (at the concentration of 10 M) wereexposed to ONOO in the presence of 100-mM potassiumphosphate buffer, pH 7.4, for 15 min at 25 C. The reactionwas initiated by placing a small drop of peroxynitrite in theside of tube containing the plasmin/plasminogen solution im-mediately followed by vigorous vortexing. The highest molarratio of protein to peroxynitrite was 1:100. Some experimentswere also performed with decomposed ONOO, which wasprepared by allowing the ONOO to decompose at neutralpH (7.4) in 100-mM potassium phosphate buffer (15 min,25 C).</p><p>Plasmin amidolytic activity assays</p><p>Assays were performed at 25 C in 96 well-polystyreneflat-bottom plates. Liberation of p-nitroaniline from chro-mogenic substrate (Chromozym PL) by plasmin in 300 lreaction mixtures of 50-mM phosphate buffer, pH 7.4, wasmeasured in a microplate reader (Bio-Rad Microplate Reader,model 550). For coupled reactions, the velocity of the reac-tion was determined by plotting the initial rate of change ofthe optical density at 415 nm against time squared [24].</p><p>Plasmin proteolytic activity assays</p><p>Fibrinogen (10 M) in 100-mM phosphate buffer, pH 7.4was incubated with native and peroxynitrite-treated plasminfor 0, 15, 30, 60, and 120 min at 37 C. The molar ratioof enzyme to substrate was 1:100. At the indicated times,50 l of incubation mixtures were dissolved in 2 loadingbuffer and separated on 6.5% SDS-PAGE under non-reducingconditions [25].</p><p>Spectrophotometric detection of nitrotyrosine</p><p>The amount of nitrotyrosine present in the plasminogen aftertreatment with peroxynitrite was determined spectrophoto-metrically (at pH 11.5, 430nm = 4400M1.cm1) [13].</p><p>Measurement of dityrosine</p><p>Formation of dityrosine was monitored by fluorescence mea-surements (excitation at 325 nm and emission at 415 nm) [26].</p><p>Gel electrophoresis and Western blot analysis</p><p>Samples of control and peroxynitrite-treated plasmin/plas-minogen were separated on 8% SDS-PAGE [27], and thentransferred to Immobilon at 370 mA for 90 min. Nitrotyro-sine was detected by incubation of the blots with mouse anti-nitrotyrosine antibodies, followed by anti-mouse antibodies</p></li><li><p>143</p><p>coupled with peroxidase and visualized with a chemilumi-nescence kit (Amersham) [28].</p><p>Statistical analysis</p><p>Data are expressed as mean S.D. Comparisons betweendata were performed by the Students test (two-tailed) forunpaired samples.</p><p>Results and discussion</p><p>Exposure of plasmin to increasing concentrations of perox-ynitrite resulted in a concentration-dependent inhibition ofplasmin amidolytic activity, with IC50 about 280 M at theenzyme concentration of 10 M (Fig. 1). Decomposed per-oxynitrite (15 min, 25 C, pH 7.4) had no effect on plasminamidolytic activity (Fig. 1). Proteolysis of fibrinogen by plas-min after treatment with peroxynitrite was also inhibited, asassessed by SDS-PAGE. As shown in Fig. 2, incubation ofplasmin with 1 mM peroxynitrite resulted in a complete lossof its proteolytic ability effect on fibrinogen. The concentra-tions of peroxynitrite used in our experiments were relativelyhigh. However, it has been estimated that the bolus additionof 0.25 mM ONOO is roughly equivalent to a steady-statelevel of 1 M maintained for 7 min [29]. These concentra-tions could be readily formed at sites of inflammation, whereproduction of rates of NO and superoxide radicals consider-ably increases.</p><p>Fig. 1. Inactivation of plasmin by peroxynitrite. Plasmin (10 M) was incu-bated with ONOO (at the final concentration of 0.0151 mM) in 100-mMpotassium phosphate buffer, pH 7.4 for 15 min at 25 C. Enzyme sampleswere diluted 12-fold, and then immediately plasmin amidolytic activity wasassayed as described in Materials and methods. The addition of decom-posed ONOO was also tested. The results represent the means S.D. ofthree independent experiments carried out in duplicate.</p><p>Fig. 2. Inhibition of proteolytic activity of plasmin by peroxynitrite. Fib-rinogen was incubated with plasmin (before control and after treatment ofthe enzyme with 0.062, 0, 25, and 1 mM peroxynitrite) for 0, 15, 30, 60,and 120 min at 37 C. At the indicated times, samples of the incubationmixtures were dissolved in 2 loading buffer and separated on 6.5% SDS-PAGE under non-reducing conditions. FG intact fibrinogen, FDP-fibrinogendegradation products. The data are representative of two independentexperiments.</p><p>To determine whether the zymogene was also sensitiveto peroxynitrite, plasminogen was incubated with differentconcentrations of ONOO. Conversion of plasminogen toplasmin was accomplished by the addition of streptokinase.As shown in Fig. 3 (panel A), exposure of plasminogen toperoxynitrite reduced the generation of active plasmin afterthe addition of streptokinase. Consistent with the ability ofperoxynitrite to nitrate protein tyrosine residues, exposureof plasminogen to peroxynitrite resulted in a concentration-dependent increase of nitrotyrosine in the treated plasmino-gen, as determined by monitoring of absorbance at 430 nm(Fig. 3, panel A) and by Western blot analysis (Fig. 3, panelB). We have found a good correlation between the decreasedability of proenzyme to generate active plasmin and nitrationof tyrosines measured spectrophotometrically or immuno-chemically with a nitrotyrosine-specific monoclonal antibod-ies. Increasing concentrations of ONOO led to reduced gen-eration of active plasmin as well as to increased nitration ofplasminogen tyrosine residues. We have shown that peroxyni-trite (1 mM) nitrated 2.9 tyrosines per proenzyme molecule(Fig. 3, panel A). Analysis of peroxynitrite-treated plasmino-gen for presence of dityrosine showed only little or no dityro-sine formation at any of the peroxynitrite concentration used(0.0151 mM; data not shown).</p><p>The inhibitory effects of peroxynitrite on the conversionof plasminogen to plasmin were partially reduced by an-tioxidants (glutathione, deferoxamine) (Fig. 4, panel A),which significantly decreased tyrosine nitration of plasmino-gen (Fig. 4, panel B).</p><p>Molecule of human plasminogen consists of 791 aminoacid residues and is organized into seven domains: amino-terminal preactivation peptide, five kringle domains and oneprotease domain with catalytic triad: His603, Asp646, Ser741.Kringle domains are responsible for binding plasminogen</p></li><li><p>144</p><p>Fig. 3. Effect of ONOO on conversion of plasminogen to plasmin andnitration of tyrosine residues in PLG. Plasminogen (10 M) was exposed toONOO (0.0151 mM) in the presence of 100-mM potassium phosphatebuffer, pH 7.4, for 15 min at 25 C. To 20 l of PLG in microplate well,220 l of 50-mM phosphate buffer, pH 7.4; 20 l of streptokinase (10000U/ml) and 40 l of chromogenic substrate (3 mM) were added. The rate ofhydrolysis of chromozym PL was measured at 415 nm, and the velocity ofreaction (plasmin activity) was determined by plotting the optical density ofthe solution against time squared. The plasmin activity generated in controlsamples (without peroxynitrite) was expressed as 100% (panel A, left axis).In samples of the remaining plasminogen the tyrosine nitration was mea-sured spectrophotometrically at 430 nm after adjusted the pH to 11.5, and theextent of tyrosine nitration was calculated using (430nm = 4400M1cm1.The results are expressed as number of tyrosines nitrated per plasminogenmolecule (panel A, right axis). The B panel shows the determination oftyrosine nitration with a monoclonal antibody against nitrotyrosine aftertreatment of PLG (10 M) with ONOO in concentration of 0 mM (lane 1),0.031 mM (lane 2), 0.062 mM (lane 3), 0.125 mM (lane 4), 0.250 mM (lane5), 0.5 mM (lane 6), and 1 mM (lane 7). The results are representative ofthree independent experiments and in panel A are expressed as mean S.D.</p><p>with fibrin or cell surface. Plasminogen can be activated intoplasmin by cleavage Arg561/Val562 peptide bond. A two-chainmolecule is formed in which the heavy chain containing thefive kringle domains remains linked to the light chain con-taining catalytic domain through two disulfide bonds. Thisactivation can occur on fibrin or cell by either of two specificactivators, the tissue PLG activator (t-PA) and the urokinasePLG activator (u-Pa). Plasminogen molecule contains 30 ty-rosine residues, 11 of which are adjacent to disulfide bondsin the kringle domains and one (Tyr614) is in the plasmincatalytic domain [30].</p><p>Plasmin is relatively resistant to inactivation by mostoxidants at micromolar concentrations. However, incubationof plasmin (90 nM) with 5 M Cu2+ in the presence ofthe reducing agent ascorbic acid resulted in a loss of its</p><p>Fig. 4. The effects of antioxidants on: (A) peroxynitrite-induced inhibitionof plasminogen conversion to plasmin, (B) peroxynitrite-induced tyrosinenitration in plasminogen. Plasminogen (10 M) was incubated with 0.5 mMONOO in the presence of 100-mM phosphate buffer, pH 7.4, for 15 min at25 C. Antioxidants (glutathioneGSH, deferoxamine -...</p></li></ul>

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