Molecular and Cellular Biochemistry 267: 141–146, 2004.c© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Peroxynitrite and fibrinolytic system: The effectof peroxynitrite on plasmin activity

Pawel Nowak, Joanna Ko �lodziejczyk and Barbara WachowiczDepartment of General Biochemistry, University of Lodz, Poland

Received 23 January 2004; accepted 3 June 2004

Abstract

We have shown that peroxynitrite (ONOO−) inhibits streptokinase-induced conversion of plasminogen to plasmin in aconcentration-dependent manner and reduces both amidolytic (IC50∼280µM at 10 µM 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: 141–146, 2004)

Key words: plasminogen/plasmin inactivation, fibrinolytic system, tyrosine nitration, peroxynitrite

Abbreviation: PLG – plasminogen, PL – plasmin, FG – fibrinogen, FDP – fibrinogen degradation products

Introduction

Peroxynitrite (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 [7–9]. 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 [10–12]. 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 nitrogen

Address 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)

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].

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 activators–bacterial 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 α-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.

142

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.

Materials and methods

Materials

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.

Protein preparation

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 µM PLG with 10 µl 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; FG—15.5, respectively).

Synthesis of ONOO− and treatment of PL/PLGwith ONOO−

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 contained80–100 mM peroxynitrite as determined spectrophotometri-cally at 302 nm in 0.1 M NaOH (ε302nm = 1679 M−1.cm−1).

PL/PLG preparations (at the concentration of 10 µM) 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).

Plasmin amidolytic activity assays

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 µlreaction 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].

Plasmin proteolytic activity assays

Fibrinogen (10 µM) 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 µl of incubation mixtures were dissolved in 2× loadingbuffer and separated on 6.5% SDS-PAGE under non-reducingconditions [25].

Spectrophotometric detection of nitrotyrosine

The amount of nitrotyrosine present in the plasminogen aftertreatment with peroxynitrite was determined spectrophoto-metrically (at pH 11.5, ε430nm = 4400M−1.cm−1) [13].

Measurement of dityrosine

Formation of dityrosine was monitored by fluorescence mea-surements (λexcitation at 325 nm and λemission at 415 nm) [26].

Gel electrophoresis and Western blot analysis

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

143

coupled with peroxidase and visualized with a chemilumi-nescence kit (Amersham) [28].

Statistical analysis

Data are expressed as mean ±S.D. Comparisons betweendata were performed by the Student’s test (two-tailed) forunpaired samples.

Results and discussion

Exposure of plasmin to increasing concentrations of perox-ynitrite resulted in a concentration-dependent inhibition ofplasmin amidolytic activity, with IC50 about 280 µM at theenzyme concentration of 10 µM (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 µM 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.

Fig. 1. Inactivation of plasmin by peroxynitrite. Plasmin (10 µM) was incu-bated with ONOO− (at the final concentration of 0.015–1 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.

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.

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(0.015–1 mM; data not shown).

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).

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

144

Fig. 3. Effect of ONOO− on conversion of plasminogen to plasmin andnitration of tyrosine residues in PLG. Plasminogen (10 µM) was exposed toONOO− (0.015–1 mM) in the presence of 100-mM potassium phosphatebuffer, pH 7.4, for 15 min at 25 ◦C. To 20 µl of PLG in microplate well,220 µl of 50-mM phosphate buffer, pH 7.4; 20 µl of streptokinase (10000U/ml) and 40 µl 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 (ε430nm = 4400M−1cm−1.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 µM) 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.

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].

Plasmin is relatively resistant to inactivation by mostoxidants at micromolar concentrations. However, incubationof plasmin (90 nM) with 5 µM Cu2+ in the presence ofthe reducing agent ascorbic acid resulted in a loss of its

Fig. 4. The effects of antioxidants on: (A) peroxynitrite-induced inhibitionof plasminogen conversion to plasmin, (B) peroxynitrite-induced tyrosinenitration in plasminogen. Plasminogen (10 µM) was incubated with 0.5 mMONOO− in the presence of 100-mM phosphate buffer, pH 7.4, for 15 min at25 ◦C. Antioxidants (glutathione–GSH, deferoxamine - DFO, at the concen-tration 0.005; 0.05, and 0, 5 mM) were added 5 min prior to ONOO−. Afteraddition streptokinase and chromogenic substrate to reaction mixtures, theactivities of generated plasmin were measured as described in Fig. 3. Theresults (panel A) are expressed as% of plasmin activity generated in controlsamples. Panel B shows the determination of tyrosine nitration with a mon-oclonal antibody against nitrotyrosine after treatment of plasminogen withONOO−in the presence of scavengers: lane 1 – control (without ONOO−);lane 2 – 0.005 mM DFO + 0.5 mM ONOO−; lane 3 – 0.05 mM DFO + 0.5mM ONOO−; lane 4 – 0.5 mM DFO + 0.5 mM ONOO−; lane 5 – 0.005mM GSH + 0.5 mM ONOO−; lane 6 – 0.05 mM GSH + 0.5 mM ONOO−;lane 7 – 0.5 mM GSH + 0.5 mM ONOO−; lane 8 – 0.5 mM ONOO−. Theresults are representative of three independent experiments and in panel Aare expressed as mean ±S.D.

proteolytic and amidolytic activity [19]. It seems that Hisresidue in the active of PL may be the target of oxidativeinactivation [19].

Recently, Gugliucci [31] suggested that impairment ofhemostasis by the inhibition of fibrinolytic system in dia-betic patients may be caused by action of peroxynitrite onplasminogen.

Peroxynitrite causes tyrosine nitration in different proteinsin vivo and in vitro [32, 33]. Although peroxynitrite hasbeen shown to cause other modifications of proteins suchas oxidation of amino acids to form carbonyl derivatives

145

and dityrosine cross-links [13], in our experiments only littleor no dityrosine formation was observed. It indicates thatthe inhibitory effects of peroxynitrite on the conversion ofplasminogen to plasmin and on plasmin activities are mostlydue to tyrosine nitration but not the formation of dityrosinecross-links.

The molecular basis for enzyme inactivation by tyrosinenitration is still not well understood. Yakamura et al. [34]found that inactivation of human Mn-SOD by peroxynitritewas caused by exclusive nitration of Tyr34. Thus, nitration ofa single key residue in or near the active site of an enzyme issufficient to disrupt its biological activity.

It is possible that ONOO− induced nitration and charg-ing of some tyrosine residues in plasminogen might causeconformational changes in the kringle domains that in turnhamper proteinase activity. Moreover, nitration of Tyr614 inthe active site of PL may take place causing the inhibitionof this activity. However, additional studies are required toprove this point definitively.

In conclusion, the presented results indicate that un-der particular pathological conditions when peroxynitrite isgenerated, the fibrinolytic system may be impaired due to in-hibited conversion of plasminogen to plasmin and decreasedactivity of plasmin.

Acknowledgements

This work was supported by a grant from University of Lodz(505/447).

References

1. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA: Ap-parent hydroxyl radical production by peroxynitrite: Implications forendothelial injury from nitric oxide and superoxide. Proc Natl Acad SciUSA 87: 1620–1624, 1990

2. Huie RE, Padmaja S: The reaction of no with superoxide. Free RadicRes Commun 18: 195–199, 1993

3. Koppenol WH, Moreno JJ, Pryor WA, Ischiropoulos H, Beckman JS:Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide.Chem Res Toxicol 5: 834–842, 1992

4. Zhu L, Gunn C, Beckman JS: Bactericidal activity of peroxynitrite. ArchBiochem Biophys 298: 452–457, 1992

5. Beckman JS: Ischaemic injury mediator. Nature 345: 27–28, 19906. Ischiropoulos H, Al Mehdi AB, Fisher AB: Reactive species in ischemic

rat lung injury: Contribution of peroxynitrite. Am J Physiol 269: L158–L164, 1995

7. Beckman JS, Crow JP: Pathological implications of nitric oxide, super-oxide and peroxynitrite formation. Biochem Soc Trans 21: 330–334,1993

8. Cazevieille C, Muller A, Meynier F, Bonne C: Superoxide and nitric ox-ide cooperation in hypoxia/reoxygenation-induced neuron injury. FreeRadic Biol Med 14: 389–395, 1993

9. Villa LM, Salas E, Darley-Usmar VM, Radomski MW, Moncada S:Peroxynitrite induces both vasodilatation and impaired vascular relax-ation in the isolated perfused rat heart. Proc Natl Acad Sci USA 91:12383–12387, 1994

10. Ischiropoulos H: Biological tyrosine nitration: A pathophysiologicalfunction of nitric oxide and reactive oxygen species. Arch BiochemBiophys 356: 1–11, 1998

11. Berlett BS, Stadtman ER: Protein oxidation in aging, disease, and ox-idative stress. J Biol Chem 272: 20313–20316, 1997

12. Davies MJ, Fu S, Wang H, Dean RT: Stable markers of oxidant damageto proteins and their application in the study of human disease. FreeRadic Biol Med 27: 1151–1163, 1999

13. Ischiropoulos H, Al Mehdi AB: Peroxynitrite-mediated oxidative pro-tein modifications. FEBS Lett 364: 279–282, 1995

14. Patel RP, McAndrew J, Sellak H, White CR, Jo H, Freeman BA, Darley-Usmar VM: Biological aspects of reactive nitrogen species. BiochimBiophys Acta 1411: 385–400, 1999

15. Pignatelli B, Li CQ, Boffetta P, Chen Q, Ahrens W, Nyberg F, MukeriaA, Bruske-Hohlfeld I, Fortes C, Constantinescu V, Ischiropoulos H,Ohshima H: Nitrated and oxidized plasma proteins in smokers and lungcancer patients. Cancer Res 61: 778–784, 2001

16. Lijnen HR, Collen D: Mechanisms of physiological fibrinolysis. Bail-lieres Clin Haematol 8: 277–290, 1995

17. Fukuyama N, Ichimori K, Su Z, Ishida H, Nakazawa H: Peroxyni-trite formation from activated human leukocytes. Biochem Biophys ResCommun 224: 414–419, 1996

18. Nowak P, Wachowicz B: Peroxynitrite-mediated modification of fib-rinogen affects platelet aggregation and adhesion. Platelets 13: 293–299,2002

19. Lind SE, McDonagh JR, Smith CJ: Oxidative inactivation of plasminand other serine proteases by copper and ascorbate. Blood 82: 1522–1531, 1993

20. Deutsch DG, Mertz ET: Plasminogen: purification from hu-man plasma by affinity chromatography. Science 170: 1095–1096,1970

21. Cuatrecasas P, Wilchek M, Anfinsen CB: Selective enzyme purificationby affinity chromatography. Proc Natl Acad Sci USA 61: 636–643,1968

22. Doolittle RF, Schubert D, Schwartz SA: Amino acid sequencestudies on artiodactyl fibrinopeptides. I. Dromedary camel, muledeer, and cape buffalo. Arch Biochem Biophys 118: 456–467,1967

23. Pryor WA, Cueto R, Jin X, Koppenol WH, Ngu-Schwemlein M,Squadrito GL, Uppu PL, Uppu RM: A practical method for prepar-ing peroxynitrite solutions of low ionic strength and free of hydrogenperoxide. Free Radic Biol Med 18: 75–83, 1995

24. Wohl RC, Summaria L, Robbins KC: Kinetics of activation of humanplasminogen by different activator species at pH 7.4 and 37 degrees C.J Biol Chem 255: 2005–2013, 1980

25. Krajewski T, Nowak P, Cierniewski C: Terminal plasmin degradationproducts D and E of duck fibrinogen and their effect on polymerizationof fibrin. Acta Biochim Pol 32: 144–154, 1985

26. Davies KJ, Delsignore ME, Lin SW: Protein damage and degradationby oxygen radicals. II. Modification of amino acids. J Biol Chem 262:9902–9907, 1987

27. Laemmli UK: Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227: 680–685, 1970

28. Nowak P, Wachowicz B: Peroxynitrite-mediated modification of fib-rinogen affects platelet aggregation and adhesion. Platelets 13: 293–299,2002

29. Beckman JS, Chen J, Ischiropoulos H, Crow JP: Oxidative chemistryof peroxynitrite. Methods Enzymol 233: 229–240, 1994

146

30. Collen D: The plasminogen (fibrinolytic) system. Thromb Haemost 82:259–270, 1999

31. Gugliucci A: Human plasminogen is highly susceptible to peroxynitriteinactivation. Clin Chem Lab Med 41: 1064–1068, 2003

32. Greenacre SA, Ischiropoulos H: Tyrosine nitration: localisation, quan-tification, consequences for protein function and signal transduction.Free Radic Res 34: 541–581, 2001

33. Reiter CD, Teng RJ, Beckman JS: Superoxide reacts with nitric oxideto nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem275: 32460–32466, 2000

34. Yamakura F, Taka H, Fujimura T, Murayama K: Inactivation of hu-man manganese-superoxide dismutase by peroxynitrite is caused byexclusive nitration of tyrosine 34 to 3-nitrotyrosine. J Biol Chem 273:14085–14089, 1998