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THE JOUR NAL OF BIOLOGICAL C HEM ISTRY0 19 90 by The American Society for Biochemistry and Molecular Biology, Inc. Vol . 265, No. 9, Issue of March 25, pp. 5232-5236,199 OPrinted i n U .S . A.

Plasminogen Activation with Single-chain Urokinase-typePlasminogen Activator (scu-PA)STUDIES WITH ACTIVE SITE MUTAG ENIZED PLASMINOGEN ( Ser7@-+Ala) ANDPLAS MIN-RE SISTAN T scu-PA (Ly~~*+Glu)*

(Received for publicat ion, July 10, 1989)H. Roger Li jnenS, Berthe Van Hoef, Luc Nelles, and D&sir& C ollenFrom the Center for Thrombosis and Vascular Research, University of Leuven, B-3000 L euven, Belg ium

The mechan ism of the act ivat ion of plasminogen bysingle-chain urokinase-type plasminogen act ivator(single-chain u-PA, scu-PA) w as studied using rscu-PA-G1ul, a recombinant plasmin-resistant mutant ofhuman scu-PA obtained by site-specif ic mutagenesis ofLysS8 to Glu, and rPlg-Ala, a recombinant humanplasminogen in which the catalyt ic site is destroyed bymutagenesis of the act ive-site Ser740 to Ala. Conversionof 251-labeled single-chain plasminog en to two-cha inplasmin was quant itated on reduced sodium dodecylsulfate-gel electrophoresis combined with autoradiog-raphy and radioisotope count ing of gel bands. Thecatalyt ic eff ic iencies of both rscu-PA-Glu68 and rscu-PA for the act ivat ion of rPlg-Ala740 and of naturalplasminogen were comparable and were 250-500-foldlower than that of recombinant two-chain u-PA (rtcu-PA) for rscu-PA-G lu and lOO-200-fold lower forrscu-PA. Pretreatment of rscu-PA-Glu* or rscu-PAwith excess az-ant iplasmin, which eff ic ient ly neutral-izes al l contaminat ing rtcu-PA, did not signif icant lyreduce the catalyt ic eff ic iency of these single-chainmoiet ies, indicating that they have a low but signif i-cant intr insic plasminogen act ivat ing potent ial. Thelow intr insic catalyt ic eff ic iency of rscu-PA for theconversion of plasminogen to plasmin may be suff ic ientto generate trace amounts of plasmin, which may reg-ulate plasminogen act ivat ion by convert ing poorly ac-t ive rscu-PA to very act ive rtcu-PA.

Urokinase-type plasminogen act ivator (u-PA) can be ob-tained as a single-chain molecule (single-chain u-PA, scu-PA)or as a two-chain proteolyt ic derivat ive (tcu-PA, urokinase)fol lowing cleavage of the Lys58-I le59 pept ide bond with plas-

* This study was supported in part by the Geconcerteerde Onder-zoeksact ies (project 85-90/78). The co sts of publication of this articlewere defrayed in part by the payment of page charges. This art ic lemust therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Center forThrombosis and Vascular Research, K. U. Leuven, Campus Gasthuis-berg, Herestraat 49, B-3000 Leuve n, B elgium .

The abbreviations used are: u-PA, urokinase-type plasmino genactivator; scu-PA, sinple-chain u-PA; rscu-PA, recombinant scu-PAobtainedby expression of its cDNA in Chinese hamster ovary cells;rscu-PA-Glu58. rscu-PA with Lvs~ reDlaced bv Glu bv site-directedmutagenesis; t&-PA, two-chain-u-PA; ;tcu-PA; recombinant tcu-PAobtained from rscu-PA by cleavage of the Lys58-Ile9 peptide bondwith plasmin; nPlg, native plasminogen purif ied from human plasma;rPlg-kla7, recombinant piasmin og& obtained by site-specific mu-tagenesis of SerT4 to Ala; SDS-P AGE, sodium dodecvl sulfate-poly-aciylamide gel electroph&esis; ELISA , enzyme-linked immun&r-bent assay.

min or kall ikrein. scu-PA has a very low react iv ity towardlo w M, synthet ic substrates or act ive site inhibitors that arevery react ive toward tcu-PA (for references, see Ref. 1).

Plasminogen, the substrate of u-PA, is a single-chain gly-coprotein with an M, of 92,000. I t is converted to the two-chain serine protease plasmin by specif ic cleavage of theArg560-Va1561 pept ide bond; the heavy (NH*-terminal) chainof M, 60,000 is connected with the l ight (COOH-terminal)chain of M, 25,000 through two disulfide bonds. The act ivecenter of the enzym e, located in the l ight chain, is composedof His602, ASPIRE , and Ser7*. Nat ive plasminogen has NH*-terminal glutamic acid (Glu-plasminogen), but is easily con-verted to modif ied forms with NH&erminal lysine, valine, ormethionine (commo nly designated Lys-plasminogen), byplasmic cleavage of the Arg67-Met68, Ly~~~-Lys~~, or LYS~~-Va17 pept ide bonds. Act ivat ion of Glu-plasminogen by tcu-PA obeys Michaelis-Menten kinet ics, with reported kinet icparameters between 1.4 and 200 pM for the Michaelis constant(K,) and between 0.26 and 1.5 s- for the catalyt ic rateconstant ( k2). Lys-plasminogen is act ivated 3-lo-fold moreeff ic ient ly than Glu-plasminogen by al l known plasminogenact ivators (for references, see Ref. 2).Est imations of the catalyt ic eff ic iency of scu-PA for theact ivat ion of plasminogen have varied widely. Init ial ly wereported that scu-PA has a catalyt ic eff ic iency similar to thatof tcu-PA (3). Several groups have reported a lower bu tsignif icant catalyt ic eff ic iency ranging between 0.4 and 6% ofthat of tcu-PA (4, 5) while other authors have reported thatscu-PA is a genuine proen zyme without enzym atic act iv ity(6-8).We have proposed that in mixtures of plasminogen andscu-PA the conversion of plasminogen to plasmin and of scu-PA to tcu-PA can quant itat ively be described by a sequenceof three react ions each of which obeys Michaelis-Mentenkinet ics (3, 9). In the f irst react ion, scu-PA direct ly act ivatesplasminogen to plasmin, then plasmin converts scu-PA totcu-PA, and f inally residual plasminogen is act ivated by tcu-PA. This mechan ism attr ibutes some intr insic plasminogenact ivat ing potent ial to scu-PA, which init ial ly was found tobe very high (3) but which was subsequently shown to be ofthe order of 1% of that of tcu-PA (9, 10).

Several methodological dif f iculties, however, hamper thequant itat ive invest igation of the act ivat ion of plasminogen byscu-PA. These include variabili ty in kinet ic propert ies of scu-PA obtained from dif ferent sources (9, l l) , generat ion of morereadily act ivatable Lys-plasminogen forms (12), and eff ic ientconversion of scu-PA to tcu-PA by generated plasmin duringthe act ivat ion process (4-11). To invest igate the signif icanceof the conversion of scu-PA to tcu-PA for the act ivat ion ofplasminogen, we have previously constructed plasmin-resist-

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Plasminogen Activation with scu-PA 5233ant mutants of scu-PA by site-specif ic mutagenesis of Lysi5sto Glu or to Gly, thereby destroying the plasmic cleavage sitefor conversion to tcu-PA (13). These mutan ts st i l l act ivateplasminogen, albeit with a catalyt ic eff ic iency which is at leastan order of magnitude lower than that observed with wild-type scu-PA, which suggests that conversion of scu-PA to tcu-PA is not a prerequisite for plasminogen act ivat ion.

The chromogenic substrates pyroglutamyl-glycyl-arginine-p-ni-troanilide (S-2444) for tcu-PA and D-valyl-leucyl-lysine-p-n itroani-l ide (S-2251) for plasmin were purchased from KabiVitrum (Brussels,Belgium). The synthet ic inhibitors D-Val-Phe-Lys-CH&l for plasmin

In the present study we have further improved the meth-odology for studying the mechanism of plasminogen act iva-t ion by scu-PA by using a recombinant plasminogen mutantobtained by site-specif ic mutagenesis of the act ive site Ser toAla (rPlg-Alar4) where by the cata lytic site of plasmin isdestroyed (14). The catalyt ic eff ic iency of rscu-PA, rscu-PA-Glui5, and rtcu-PA for the convers ion of rPlg-Alar* and ofnatural plasminogen (nPlg) to their two-chain derivat ives wasdetermined by reduced SDS-gel electrophoresis.

MATERIALS AND METHODSRecombinant scu-PA (rscu-PA), prepared by expression of cDNAencoding human scu-PA in Escherichia coli was a gif t f rom Gri inen-thal AG (Aachen, Federal Republic of Germany). I ts specif ic act iv ityafter chromatography on benzamidine-Sepharose (9), determined bycomparison with the International Reference Preparation for Uroki-nase (66/46, obtained from the National Inst i tute for BiologicalStandards and Control, London, United Kingdom) was 180,000 +15,000 IU/mg (mean + S.D.; n = 3) on fibrin plates (15) and

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5234 Plasminogen Activation with scu-PAFIG. 1. Autoradiography of reducedSDS-PAG E ollowing activation of rPlp-Ala740 1.5 PM, conkking 5 X lo6 cpk251-labeledPlg-AlaS4/ml)with 300 nMrscu-PA (A), with 300 nM rscu-PA-Glu (B), or with 1.5 nM rtcu-PA (C).Sampleswere taken at time 0 (lane I),10 min (lane 2), 20 min (lane 3), 30 min(lane 4), 40 min (lane 5), 50 min (lane6), and 60 min (lane 7).Arrows, apparentM, of plasminogen (M, =: 90,000) and ofthe plasmin heavy chain (M, = 60,000)and light chain (M, z 25,000).

A B1234567 1234567

Al

1200

n

B

n

;r/ ix

TIME ( min)

TABLE I either with nPlg or with rPlg-Ala740.Catalytic efficiency (k*/KJ of mu-PA , rscu-PA-Glu58, or r&u-PA

for the activation of nPb or rPb-AlaT Preincubation of rscu-PA or rscu-PA-Glu 58 with az-anti-plasmin, under conditions where up to 0.5% contaminatingrtcu-PA is completely neutralized, did not abolish the acti-vation of rPlg-Ala740 (Fig. 3). The ca talytic efficiencies for theactivation of rPlg-Ala740 by rscu-PA or by rscu-PA-G lu58 inthe presence of az-antiplasmin were, respectively , 13 or 27%(mean of two independent experiments) lower than in theabsence of a2-antiplasmin, whereas &u-PA was totally in-activated by preincubation with az-antiplasmin.

u-PA Concentrationmoiety used

W& inPlg rPlg-Ala

, , ,M- .s-rscu-PA 75 nM 0.00045 0.00037150 nM 0.00045 0.00032300 nM 0.00050 0.00032Mean 0.00047 0.00034

kS.D. ~0.00003 ~0.00003rscu-PA-

Glu=75 nM 0.00015

150 nM 0.00015300 nM 0.00018Mean 0.00016&S.D. +0.00002

0.000130.000130.000100.00012

~0.00002

rtcu-PA 0.37 nM 0.045 0.0620.75 nM 0.045 0.0751.50 nM 0.033 0.070Mean 0.041 0.069+S.D. f0.007 kO.006

Monitoring of Lys-plasmin(ogen) levels during incubationconfirmed the absence of Lys-forms in the experiments withrPlg-Ala740 (

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5235A B C

1234567 01234567 1234561

Plasminogen Activation with scu-PA

FIG. 3. Autoradiography of reduced SDS-PAGE fol lowing act ivat ion of rPlg-Ala40 (1.5 pM , containing a traceamoun t. of I-rPln-Ala740) with 300 nM rscu-PA (A) with 300 nM rscu-PA-Glu58 (B) or with 1.5 nM rtcu-PA (C)after preincubat ion of the u-PA m oiet ies with cu2-ant iplasmin (3 pM ) for 2 h at 37 C. Samples were take n at t imes0 (lane I), 10 min (lane 2), 20 min (lane 3). 30 min (lane 4), 40 min (lane .5), 50 min (lane 6), and 60 min ( l ane 7) .Arrows. aonarent M. of olasminogen CM , = 90.000) and of the plasmin heavy chain (M, = 60,000) and l ight chain.(M,- 25,000). - -

sical ly devoid of tcu-PA or plasmin activity. This is achievedby using a plasmin-resistant mutant of rscu-PA (rscu-PA-Glu ) and an active site mutant of recombinant plasminogen(rPlg-Ala740) which is totally inactive after conversion to atwo-chain molecule by plasminogen activators (14). Genera-t ion of plasmin was quant itated by autoradiography afterSDS -PAG E under reducing conditions. In addition, the ex-periments were performed in the presence of excess aprotininand the generation of rtcu-PA and Lys-plasmin(ogen) wasmonitored with specific ELISAs.Our results indicate that the catalytic efficiencies (KJK,)of both rscu-PA and rscu-PA-Glu for rPlg-Ala7 and nPlgare comparable but 100-200-fold lower than those of rtcu-PAfor rscu-PA and 250-500-fold lower for rscu-PA-Glu58. Thisindicates that the catalytic efficiency of the present E. coli-derived rscu-PA for the activation of Glu-plasminogen isbetween 0.5 and 1.0% of that of rtcu-P A. These values arecomparable to the value of ~1% obtained previously in acoupled substrate assay with natural scu-PA obtained fromCALU-3 cells (9) or with rscu-PA obtained from Chinesehamster ovary cells (13). In a previous study, the catalyticefficiency of rscu-PA from E. coli, provided by Genentechwas, however, found to be comparable to that of rtcu-PA (3);the molecular basis of this discrepancy with rscu-PA from E.coli obtained from an alternative source remains unclear. Acatalytic efficiency of scu-PA for plasminogen of about 0.4%of that of tcu-PA was reported previously by Pannell andGurewich (4), whereas Elli s et al. (5) found a catalytic effi-ciency of 4-6% of that of tcu-PA. In contrast, other authorshave reported that scu-PA has no plasminogen-activatingpotential (6-8).The 3-fold difference in catalytic efficiency for plasminogenactivation of rscu-PA and rscu-PA-Glu58 found in the presentstudy contrasts with the lo-fold higher value for rscu-PA ascompared to rscu-PA-Glu5s reported previously (13). Thismay be due to the fact that the previously used coupledsubstrate assay (3, 9) does not totally prevent conversion ofscu-PA to tcu-PA, resulting in overestimation of the intrinsicactivity of scu-PA.The methodology used in the present study allows exclus ionof the generation of both tcu-P A and plasmin activ ity duringthe experiment and thereby establishes that rscu-PA has alow but significant intrinsic plasminogen activating potential.This may be important for the regulation of the mechanismof plasminogen activation by scu-PA via generation of a smallamount of plasmin which in turn converts poorly active scu-PA to very active tcu-PA, resulting in an accelerated activa-

t ion of plasminogen. This posit ive feedback mecha nism hasalso been implied in fibrin clot dissolution by scu-PA inhuman plasma in vitro (10). Furthermore, it was suggestedthat conversion of scu-PA to tcu-PA by plasmin occurs mainlyat the fibrin surface without extensive systemic conversion inthe surrounding plasma (31).It has been demonstrated previously that certain zymogens,such as chymotrypsinogen and trypsinogen, are not entirelyenzymatically inactive but have a very low intrinsic activitywhich, upon activation, increases by several orders of magni-tude (25-27). The single-chain form of tissue-type plasmino-gen activator (t-PA) has significant enzymatic activity towardlow molecular weight synthetic substrates (lo-15% of that ofthe two-chain form) (28) whereas its plasminogen activatingpotential is comparable to that of the two-chain form (29).The structural basis of this high activity as compared to othersingle-chain forms of serine proteases has not yet been ex-plained. The intrinsic enzymatic activity of scu-PA appearsto be intermediary to these extremes.The mechanism of plasminogen activation in purified sys -tems and of fibrin dissolut ion in human plasma in vitropresented here may, however, not be identica l to the physio-logical mechanism of action of scu-PA. It was indeed foundthat plasmin-resistant mutants of scu-PA (i.e. rscu-PA-Glu) have only a 3-5-fold lower in uiuo thrombolytic po-tency as compared to wild-type scu-PA (30), suggesting thatfor in uiuo thrombolysis, conversion of scu-PA to tcu-PA mayplay a less important role.

Acknowledgments-We are very grateful to Dr. S. Busby and G.Parker (ZymoG enetics, Seatt le, WA) for supplying the plasmid plg251a/219b and the vector Zem 229.

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