plex or of urokinase shortened the lag hase, whereas catalytic amounts (5% molar ratio) of the plasmin in- hibitor a2-antiplasmin delayed active site generation.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 268, No. 11, Issue of April 15,pp. 8284-8289, 1993 Printed in U.S. A.
0 1993 by The American Society for Biochemistry and Molecular Biology, Inc
On the Mechanismof the Activation of Human Plasminogenby Recombinant Staphylokinase" (Received for publication, October 12, 1992)
Desire CollenSQ,Bernhard SchlottV, Yves EngelborghsII, Berthe Van HoefS, Manfred Hartmannq, H. Roger LijnenS, and Detlev BehnkeV From the $Centerfor Thrombosis and Vascular Research and the IlLuboratory of Physical Biochemistry, University of Leuuen, B-3000Leuuen, Belgium and the llznstitute for Molecular Biotechnology, 0-6900 Jena, Germany
The mechanism of activation of human plasminogen Staphylokinase (STA),' a 136-amino acid protein produced by recombinant staphylokinase (STAR) was studied by Staphylococcus aureus is long since known to have profiusing the activesite titrant p-nitrophenyl-p'-guanidi- brinolytic properties (1,2). Its mechanism of action (3,4), its nobenzoate (NPGB). NPGB prevented active site exin vitro fibrinolytic properties (5, 6), and its in vivo thromposureinequimolarmixtures of plasminogen and bolytic properties in experimental animal models (7, 8) have STAR but reacted stoichiometrically with mixtures been evaluated to some extent. We have recently initiated a preincubated in the absence of titrant. Active site gen- series of systematic studies on the thrombolytic propertiesof eration occurred progressively, with a marked initial recombinant STA (STAR) in animal models (9, 10) and in lag phase followed by an exponential growth phase, and wasassociated with the conversion of single-chain man.' It was demonstrated that STA,like streptokinase, is not an plasminogen to two-chain plasmin. Incubation of mixa stoichiometric complex with plastures of plasminogen and STAR with catalytic amountsenzyme but that it forms minogen that then activates otherplasminogen molecules (3, (~0.2% molar ratio)of preformed plasmin.STAR complex or of urokinase shortened the lag hase, whereas 4). With the use of the active site titrant p-nitropheny1-p'catalytic amounts (5% molar ratio) of the plasmin in- guanidinobenzoate (NPGB) it has been shown that streptokinase rapidly forms a 1:lstoichiometric complex with human site generation. hibitor a2-antiplasmin delayed active The following kinetic model for the activation of plasminogen which then undergoes a slower intramolecular transition ( t 1 I 2 of4.5 min at 5 "C) withoutpeptidebond plasminogen (P) by STAR (S) fits the experimental cleavage, which results in exposure of a titratable active site data, in the plasminogen molecule (11).Surprisingly, we observed that similar titrations with NPGB of equimolar mixtures of (Model A l ) STAR and plasminogen did not reveal active site exposure when the titrantwas added at the timewhen the components were mixed, whereas titrationof preincubated equimolarmixand is described by tures yielded 1:l stoichiometric amountsof active site. Therefore, the mechanism of human plasminogen activation by STAR, as revealed by titration with NPGB, was studied in more detail. Our results indicate that active site exposure in or the plasminogen. STARcomplex requires conversion to plasmin, a rate-limiting stepwhich is very sensitive toacceleration by catalyticamounts of plasminogen activatorsandto In this model, plasminogen and STAR produce an suppression by plasmin inhibitors. inactive complex (P.S), in which active plasmin. STAR EXPERIMENTALPROCEDURES (p. S) is generated in a rate limiting step, which is accelerated by plasminogen activators and delayed by Reagents-STAR was purified from thecell extract of Escherichia coli transduced with an expression vector containing thesak42D gene plasmin inhibitors. At room temperature in a 0.1 M Verona1 buffer, pH cloned from bacteriophage q542D (12, 13) by chromatography on S8.3,containing 0.1 M arginine, the data are adequately Sepharose and phenyl-Sepharose. The purified material was homogeneous on SDS-gel electrophoresis and had an apparent M , of 18,000. fitted by the integrated equation withkl = 4.0 % 10" Native human plasminogen (NH2-terminal glutamic acid) and aZs-l and k2 = 1.3 X p"' s-'. The kl value could be antiplasmin were purifiedfrom plasma and characterized as described explained by contamination of the plasminogen prep- elsewhere (14, 15). Plasmin was obtained by activation of Glu-plasaration with3 ppm plasmin, converted byS to p.S. minogen with urokinase, as described elsewhere (16), and Lys-plasIt is concluded that STAR activates plasminogen via minogen wasprepared by treatment of Glu-plasminogen with plasmin a mechanism which differs in several essential aspects (17). Recombinant plasminogen with the active site residue Ser741 mutagenized to Ala (rPlg-Ala7")was obtained byexpression in from thatof streptokinase. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 To whomcorrespondenceshouldbeaddressed:Centerfor Thrombosis and Vascular Research, University of Leuven, Campus Gasthuisberg, 0 & N, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-72; Fax: 32-16-34-59-90.
The abbreviations used are: STA, staphylokinase; STAR, recombinantSTAobtained byexpression of thestaphylokinase gene (sak42D) cloned from bacteriophage $42D, in E. coli cells; PAGE, polyacrylamide gel electrophoresis; NPGB, p-nitrophenyl-p'-guanidinobenzoate; rPlg-AlaY4', recombinant plasminogen with the active site Ser741 mutagenizedto Ala; rPli-Ala'", inactive two-chain plasmin moiety obtained by treatment of rPlg-Ala741with urokinase. Collen, D., and Van de Werf, F. (1993) Circulation, in press.
8284
Staphylokinase-PlasminogenTitration with NPGB Chinese hamster ovary cells of the plasmid PLG 251a/219b (18) (kindly provided by Zymogenetics) and was characterized as described elsewhere (19). It was converted to an inactive two-chain plasminlike moiety by treatment with urokinase (16). Streptokinase devoid of albumin was purchased from Boehringer Mannheim, Germany, and NPGB from E. Merck, Darmstadt, Germany. NPGB was dissolved in dimethy1formamide:acetonitrile(1:4) to a concentration of 10 mM. Aprotinin-Sepharose was prepared by coupling aprotinin (Trasylol@, Bayer, Leverkusen, Germany) to CNBr-activated Sepharose 4B. The low molecular weight calibration kit for SDS-gel electrophoresis was purchased from Pharmacia, Uppsala, Sweden. Methods-SDS-PAGE was performed with the Phast System'" (Pharmacia) using either 20% high density gels for estimation of the M,of STAR or 10-15% gradient gels for the analysis of the chain composition of plasminogen. Reduction of samples was performed by heating a t 100 "C for 3 min in the presence of 1% SDS and 1% dithioerythritol. The fibrinolytic activity of the STARpreparation,determined with a clot lysis assay (20), was 210,000 arbitrary units (home units) (by comparison with an in-house standard of purified natural staphylokinase (STAN)(batchSTAN5) which was assigned an activity of 100,000 home units) per mg of protein (as determined by amino acid composition) (21). Proteinconcentration was measured by the method of Bradford (22), using the STAN standard as a primary standard and bovine serum albumin as a secondary reference. NH2terminal amino acid sequence analysis,performedon an Applied Biosystems 477A ProteinSequencer with identification of amino acids by HPLC (courtesy of Dr. J. Van Damme and P. Proost, Rega Institute, University of Leuven, Leuven, Belgium), revealed a single main sequence Ser-Ser-Ser-. Plasmin contamination of the plasminogen preparations was measured by incubation of plasminogen solution (final concentration 10 p ~ with ) 1 mM chromogenic substrate S-2251 and determination of the absorbance at 405 nm. In this assay, 1 nM plasmin yields a AA of 12 mA/min. The time dependence of active site generation was determined by active site titration with NPGB in 1-ml cuvettes in a Pye Unicam SP1800 spectrophotometer at 410 nm. A 0.1 M Veronal buffer, pH 8.3, containing 0.1 M 6-aminohexanoic acid or 0.1 M arginine was used, following the instructions of Chase and Shaw (23) and McClintock and Bell (11). Stocksolutions of plasminogen (150 p M ) , STAR (350 p ~ ) and , accelerating orinhibiting substances were diluted in buffer in the cuvette to a final volume of 1 ml. After different incubation times, 10 pl of the 10 mM NPGB stock solution , the absorbance was was added (final concentration, 100 p ~ ) and measured. The concentration of active site was determined from the "burst" ofp-nitrophenol, using amolar extinction coefficient of 16,700 (20). The experimental data, expressed as active site in p~ uersu.9 time in seconds were fitted using the program Sigmaplot (Jandel Scientific GmbH, Erkrath, Germany). Initial simulations were performed using the program ISIM (Ladybridge, Manchester, United Kingdom).
r
8285
I
smin
FIG. 1. Active site titration with NPGB at room temperature in 0.1 M Veronal buffer, pH 8.3, containing 0.1 M arginine. Mixtures of 9 pM plasminogen and 10 p~ STAR or of 9 pM plasminogen and 20 p~ streptokinase were used. The graphs represent A:;,",, of a mixture of plasminogen and staphylokinase with immediate addition of NPGB (tracing 1), a mixture of plasminogen and STARwith NPGB added after incubation for 10 min (tracing 2 ) , and a mixture of plasminogen, streptokinase, and NPGB (tracing 3 ) . The inset shows SDS-PAGE under nonreducing ( A ) or reducing ( B ) conditions of samples taken from the cuvette at the end of the experiment (5 min after addition of NPGB). Lane S represents a 97,0001, protein calibration mixture consisting of phosphorylase b (M, 67,000), ovalbumin (M, 45,000), carbonic anhydrase (M, albumin (M, 30,000), trypsin inhibitor (M, 20,100), and a-lactalbumin (M, 14,400).
followed by an exponential acceleration. Similar curves were obtained in 0.1 M Veronal buffer, pH 8.3, containing either 0.1 M arginine (Fig. 2) or 0.1 M 6-aminohexanoic acid (not RESULTS shown), which were required tomaintain clearsolutions. Fig. 1shows that in mixturesof plasminogen (final concen- Pretreatment of plasminogen with aprotinin-Sepharose, tration, 9 p ~ and ) NPGB (final concentration, 100 p ~ to) which removed trace contaminantsof plasmin (approximately which a molar excess of streptokinase was added (final con- 0.007% molar ratio as determined with the chromogenic subof a titratable strate D-valyl-leucyl-lysyl-p-nitroanilide), centration, 20 p M ) , aprogressiveexposure prolonged the lag active site occurred, resulting in exposure of one active site phase of the curvesignificantly, but thefinal 1:l stoichiometper plasminogen molecule, whereas addition of STAR (final ric exposure of active site was unaffected. SDS-gel electroconcentration, 10 p ~ did) not trigger release of p-nitrophenol. phoresis revealed that exposure of an active site was dependIn contrast, a stoichiometric 1:1 recovery of active site was ent on, or a t least associated with, conversion of single-chain observed in preincubated mixtures (Fig. 1).SDS-gel electro- plasminogen to two-chain plasmin and was also associated phoresis revealed intact plasminogen, streptokinase,and with a conversion of M , 18,000 STAR toa M , 16,500 derivative STAR moieties at the end of experiments in which NPGB (Fig. 2, inset). was added at the start, whereas in the preincubated mixture The shapeof the active site concentrationuersus time curve of plasminogen and STAR, plasminogen was quantitatively and theassociated structural changesin the plasmin molecule converted toplasmin and staphylokinase to a lower M , deriv- suggested the following mechanism of activation of plasminative (Fig 1, inset). ogen (P) by STAR (S), Fig. 2 shows that inequimolar mixtures of plasminogen and STAR incubatedfor various time intervals a t room temperaP + s P.S 2 p . s (Model 1 ture before addition of NPGB, a progressive exposure of a n active site occurred, characterized by a significant lag phase k,
*
t,
Staphylokinase-PlasminogenTitration withNPGB
8286 A 10
I
I
I
I
I
A S 0 30 60 90 120 -.
8
.
6 c \
I
a
v
B al
.e
4
S
u)
0 30 60 90 120
e,
> .c
0
Q : 2
0
0
50
100
150
Time (s)
B 10
a
6 CI
I
a
kl.
v
W
.Y
200
According to thismodel, plasminogen reacts with STAR to form an inactive 1:l stoichiometric complex, which is activated by conversion of plasminogen to plasmin (p). This conversion is accelerated by generated p . S complex. Fitting of the experimental data(Fig. 2 4 ) to the integrated equation, yielded kl = 2.7 f 3.4 X s-' and k p = 1.1 & 0.2 x lo-' p"' s-' with an amplitude (maximal concentration of active site) A = 8.8 & 0.2 p~ and a residual sum of squares of 0.6. After pretreatment with aprotinin-Sepharose,correspondthe s-' and k2 = 0.8 f 0.1 X ing values were kl = 1.6 & 2.1 X lo-' p ~ "s" with A = 8.1 f 0.5 p ~ with , a residual sum of squares of 1.5. Surprisingly, although Fig. 2A suggested marked differences before and after treatment with aprotininSepharose, thevalues of kl and k2 were not markedly different. Therefore fitting of the data was performed with the integrated rate equation inwhich either kl or k2 were fixed at the s-' and kp = 0.95 X mean of the fitted values ( k , = 2.2 X lo-' p"' s-'). With kl fixed, the fitted values were kz = 1.1 f 0.03 X lo-' ~ M - I s-' and A = 8.8 k 0.2 p~ before treatment with aprotinin-Sepharose and k2 = 0.72 f 0.05 X lo-' ph4-I s-' and A = 8.2 f 0.4 FM after aprotinin-Sepharose. Conversely, when k2 was fixed a t 0.95 X lo-' p"'1 s-', fitted s-' and A = 8.9 k 1.8 pM values were kl = 5.8 f 1.3 X before and kl = 0.4 k 0.19 X p"' s" and A = 7.8 f 3.1 p~ afteraprotinin-Sepharose.Thus,the differencein the rates of active site exposure before and after treatment of plasminogenwith aprotinin-Sepharosecan be explained either by a 30% reduction of k2 with a fixed kl or by a 15-fold reduction of kl with a fixed kz. Because it is unlikely that removal of traces of plasmin from the plasminogen preparation would alter the rate constant of the conversion of P. S to p - S by the p.S complex, we believe that the experimental data in Fig. 2A are best explained by the 15-fold reduction in
4
kl could represent, at least in part, activationof P. S by p . S generated by reaction of S with a trace contamination of
plasmin in the plasminogen preparation. Because aprotinin treatment removed the 0.007% molar ratio of plasmin from 0 the plasminogen preparation (corresponding to a concentra< 2 tion of 0.6 nM at theplasminogen concentration of 9 pM which was used for titration), kl before treatment with aprotinin would include a term kz[p.S], = 0.95 X lo-' X 0.6 X lo-' s" = 0.6 X s-'. This would onlyexplain about10% of the 0 observed value of k l . However, if the ratio kl = kz x 0.6 x 0 50 100 150 is fixed (required to fully explain kl by the presence of 200 contaminating plasmin), fittedvalues were kz = 1.3 k 0.03 X Time (s) lo-' p"' s-l and A = 8.8 f 0.15 p ~ with , a residual sum of FIG.2. Time course of active site generation in mixtures of squares of 0.62 for the experimental data pointsbefore aproplasminogen and STAR preincubated in the absence of NPGB. kl = 1.3 X Plasminogen (9 p~ final concentration) either before (0)or after ( 0 ) tinin-Sepharose. These valuescorrespondwith = 7.8 X lod6s-'. With k2 fixed a t 1.3 x lo-' treatment with aprotinin-Sepharose was incubated with STAR (10 lo-' x 0.6 X pL"' s-', values of kl = 4.0 f 2.8 X low7s-' and A = 7.5 & 0.4 p~ final concentration) at room temperature for different time periods (0-240 s) before addition of NPGB (100 p~ final concentration). p ~ with , a residual sum of squares of 1.9, were obtained for The inset shows SDS-PAGE under reducing conditions of samples the data points after aprotinin-Sepharose. The residual plastaken from the cuvettes at the end of the experiment (5.min after min contamination which could explain the value of kl after addition of NPGB), using 10-15% gradient gels ( A ) or 20% homogeneous gels ( B ) .Lane S, protein calibration mixture. The experi- aprotinin-Sepharose would be 30 PM (or 3 ppm in the plasminogen preparation). Fig. 2B, which represents the experimental data were fitted with the following integrated rate equation (see Equation 2 under "Results"). In A, no restrictions on k1 and kt mental data points fitted as described above, suggests that were imposed. In B, kl = kl X 0.6 X was imposed to fully explain the integrated rate equation with these values for kl and k2 k1 by contaminating plasmin in theplasminogen preparation. adequately fits the data pointsbefore and after treatment of plasminogen with aprotinin-Sepharose. which is described by the following rate equation, The model can be tested in several control experiments. First, if plasminogen andSTAR form a relatively stable (Eq. 1) complex, the time course of active site exposure should be which in its integrated form yields the following. directly proportional to the concentration of the P. S complex. In equimolar mixtures of plasminogen and STAR (2.25-20 p~ plasminogen), the durationof the lag of active site expo(0
e,
> .e
Staphylokinase-PlasminogenTitration with NPGB sureis indeedinversely proportional to the concentration (Fig. 3A). At each concentration of reagents, active site exposure amounted to 0.8-0.9 active sites/plasminogen molecule. However, when STAR was incubated for different time periods with a %"-fold molar excess of plasminogen before addition of NPGB, thetimecourse of exposureandthe concentration of active site was proportional to the plasminA
I
I
1
1
200
300
400
0
16 14
12 n
I
10
a
v
8
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.Y
m 0)
6
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4
8287
ogen and not to the staphylokinase concentration (Fig. 3B). This apparant inconsistency with the model can, however, be explained by the rapid conversion of excess plasminogen to plasmin by generated plasmin. STAR complex. Indeed, incubation of plasminogen (final concentration 18 p M ) with an equimolar mixture of preincubated ( 5 min) plasmin. STAR ) in exposure of 7.8 pM active site complex (2.5 p ~ resulted within 20 s and 14 p~ active site within 60 s (results not shown). The model would also predict that the lag phase would depend on the initial concentration of contaminating plasmin and consequently be proportional to the plasminogen concentration. The lag phases are relatively invariate in the experimentswith 9 p~ plasminogen butsomewhat more 18 p~ plasminogen(Fig. variable in the experiments with 3B). It is unclear whether this variability is due to experimental error or to a possibly higher reactivity of p . S with P . S than with P. Second, if the generation of p . S is responsible for the acceleration of active site exposure (the productof the reactionisalsoitsenzyme),addition of catalyticamounts of preformed p . S complex should accelerate the activation by reduction of the lag phase. As shown in Fig. 44, active site exposure was indeed enhanced in a concentration-dependent
2
0
0
100
500
Time (s)
B
20
I
I
I
I
,
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-
0.05
0.10
0.15
0.20
Molar ratio ( percent)
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.-al
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Time (s) 250 300 FIG. 3. Time course of active site generation as a function 200 1500 100 50 of the concentration of reagents in equimolar mixtures of Time ( s ) plasminogen and STAR ( A ) or in mixtures of STAR with excess plasminogen ( B ) .In A, equimolar mixtures of plasminogen FIG. 4. Effect of catalytic amounts of plasminogen activator (2.25, 4.5, 9, and 18 PM final concentration) and STAR (2.5, 5, 10, on the rate of active site exposure in equimolar mixtures of and 20 PM final concentration), respectively, were incubated a t room plasminogen and STAR. A, the active siteconcentration was
temperature for different time periods before addition of NPGB. The symbols used are W, 0, 0, and 0, respectively. In B , plasminogen (9 or 18 p~ final concentration) was incubated with varying concentration of STAR (2.5,5, 10, or 20 pM final concentrations).The symbols used are A, A, and V with 9 p~ plasminogen and 2.5, 5, or 10 PM STAR and W, 0, 0, and 0 with 18 P M plasminogen and 2.5, 5, 10, and 20 PM STAR.
measured after 45 s by addition of NPGB tomixtures of plasminogen (9 FM final concentration) and STAR (10 PM final concentration) containing catalytic amounts (0-0.2% molar ratio) of preformed plasmin.STAR complex (0)or urokinase (W). B, the time course of active site exposure was measured after addition of NPGB tomixtures of plasminogen (9 FM) and STAR (10 PM) incubated with buffer ( 0 ) . 0.05% urokinase (W), 0.5% plasmin.STAR (+I, or 0.5% plasmin (A).
8288
Staphylokinase-Plasminogen with Titration
manner by addition of catalytic amounts of urokinase or of preformed p .S complex to equimolar mixtures of plasminogen and STAR. The approximately 10-fold higher efficacyof urokinase over preformed p . S complex was confirmed by measuring the time course of active site exposure with 0.05% urokinase and 0.5% p . S or plasmin (Fig. 4B). Third, if p . S generation is the rate-limiting step of the reaction, catalyticamounts of inhibitors of this complex should delay activation. As shown in Fig. 5 , addition of a2antiplasmin (0.5 pM final concentration) to equimolar mixtures of plasminogen (9 p~ final concentration) and STAR (10 pM final concentration) indeed results in prolongation of the lag phase of active site exposure, without significantly affecting the total concentration of active sites generated. Fourth, if the conversion of plasminogen to active plasmin is required for activation of plasminogen, addition of preformed complexes of staphylokinase with an active site mutant of plasminogen should not affect the activation kinetics. Addition of catalytic amounts of rPlg-Ala741,rPli-Ala741,or their preformed equimolar complexes with STAR (0.09 p~ final concentration) to mixtures of plasminogen (9 p~ final concentration) and STAR(10 p~ final concentration) indeed did not affect the rate of active site exposure, as determined by addition of NPGB after preincubation for 60 s (data not shown). Finally, the assumption that conversion of native Gluplasminogen to Lys-plasminogen (cf. SDS-PAGE shown in Fig. 2) is an epiphenomenon of the activation process can be tested by control experiments with these reagents. As shown in Fig. 6, the time course of active site generation in equimolar mixtures of plasminogen and STAR was very similar, irrespective of whether Glu-plasminogen or Lys-plasminogen was used. Indeed, although the lag phase observed with Lysplasminogen was shorter than that of Glu-plasminogen, it remained quite significant.
10
NPGB
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0
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100
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8
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4
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150
200
Time (s)
FIG.6. Effect of the molecular form of plasminogen on the time course of active site generation in equimolar mixtures with STAR. Glu-plasminogen (0)or Lys-plasminogen (0)(9 P M final concentration) were incubated with STAR (10PM final concentration) for different time periods before measurement of the active site concentration by titration with NPGB. Both plasminogen preparations were pretreated with aprotinin-Sepharose. DISCUSSION
The present study was initiated by the surprising observation that the active site titrant NPGB inhibits active site exposure in plasminogen by STAR but not by streptokinase. The results indicate that there are indeed significant differences between the mechanisms of activation of plasminogen by these two substances. McClintock and Bell (11) have 10 I I I I I demonstrated that streptokinase and plasminogen combine 0 in a 1:1 molar ratio to yield a complex which undergoes a time- and temperature-dependent change, resulting in expo8 sure of an active site which can be titrated with NPGB. This phenomenon occurs in the absence of proteolytic cleavage and does not require plasmin for its initiation. In contrast, with STAR, generation of plasmin appears to 6 be a necessary step for the exposure of a reactive site in its I complex with plasminogen. Initially, this occurs in a rateY. v limiting step,either by traces of plasmin presentin the W plasminogen preparation or, possibly, by a slow spontaneous .-ul 4 conversion of plasminogen to plasmin in the plasminogen. W STAR complex.However, as plasmin is formed and com> .+ plexed with STAR, this complex constitutes a potent plas9 2 minogen activator, which explains that the activation accelerates exponentially until exhaustion of the substrate and full exposure of the active site in 1:l stoichiometric amounts. Several control experimentsdesigned to test this hypothesis 0 appear to support the model. Thus, addition of catalytic 0 50 100 150 200 250 amounts(up to 0.2% molar ratio) of preformed plasmin. STAR complex or of urokinase dramatically reduce the initial Time (s) lag phase, whereas the addition of 1%molar ratio of preformed FIG.5. Effect of catalytic amounts of a2-antiplasmin on the complexes of STAR with rPlg-Ala741or rPli-Ala741has no time course of active site exposure in equimolar mixtures of effect. Furthermore, addition of a2-antiplasmin, which inhibplasminogen and STAR. Plasminogen (9 PM final concentration) its bothplasmin and theplasmin. STARcomplex, reduces the and STAR (10 PM finalconcentration) were incubated at room rate of activation. The inhibitor is, however, clearly less temperature in the absence (0)or the presence (0)of cu2-antiplasmin (0.5 PM final concentration)for different time periods before addition potent in delaying the activation than the activators are in accelerating it. This apparent inconsistency can, however, be of NPGB.
r\
Y
Staphylokinase-Plasminogen with Titration explained in two ways.First, the inactive plasminogen. STAR complex, which is a substrate for the plasmin. STARenzyme complex, competes with the inhibitors and, at thelarge molar excesses of substrate over inhibitor in our experiments, reduces the efficacy of the latter. Second, other studies from our laboratory3 indicate that STAR dissociates from the plasmin. STAR complex after neutralizationof the enzyme by a'antiplasmin and that released STAR is recycled to other plasminogen molecules. The model also assumes that theconversion of native high M, STAR ( M , 18,000) tothe M, 16,500 derivative is an epiphenomenon and not an essential step of the activation process. This hypothesis is supported by the similar specific activities and plasminogen activating capacity of both forms (21,24) andcould in principle be confirmed by direct titration with each of these moieties, if they were available in sufficient amounts. Finally, although conversion of plasminogen to plasmin is an essential step for active site exposure, conversion of native Glu-plasminogen to partially degraded Lys-plasminogen also appears to be an epiphenomenon of the activation process. The rate equation of the model adequately fits the experimental data with values of kl = 7.8 X s-' and k2 = 1.3 X lo-' p"' s-' at 22 "C and pH 8.3. These values indicate that the apparent rate constantof the initial rate limiting step of the reaction is 2,000 times lower than that of the plasmin. STAR-catalyzed step. This initial rate-limitingactivation can be fully explained by the trace contamination of our plasminogen preparation with 0.007% molar ratio of plasmin. The initial lag phase is prolonged when plasminogen is pretreated with aprotinin-Sepharose, reducing kl to 4 x s-l (20-fold reduction), which can be fully explained by a residual concentration of p . s of 30 pM (3 ppm plasmin in the plasminogen preparation). In reaction mixtures with excess plasminogen (P) over staphylokinase ( S ) the generated p .S complex will also convert the excess P to p, whereby the reaction model expands to the following, P
1.
+s
P.S
-% p . s
8289
equation using values of kl and kt of 4 X s-' and 1.3 x lo-' p ~ - 's-', respectively. The present study may be of significance for the explanation of the fibrin specificity of STAR in a plasma milieu (25, 26). Indeed, several mechanisms may contribute to this phenomenon. First, we have demonstrated previously that the plasmin.STAR complex is rapidly neutralized by a'-antiplasmin (apparent second order rate constant 2 X lo6 M-' s-') but that this reaction is 130 times slower when the lysine binding sites of plasmin in the complex are either removed or saturated by binding to 6-aminohexanoic acid or to fibrin (25). This would result in a protection of fibrin-bound complex against rapid inhibition and restriction of the plasminogen activation phenomenon to theclot surface. Second, in experiments to be reported elsewhere: we have obtained evidence that STAR dissociates in active form fromthe plasmin. STAR complex following its inhibition by as-antiplasmin and that released STAR molecules are recycled to other plasminogen molecules. Third, as demonstrated in the present study, the plasminogen. STAR complex is inactive and unable to convert to active plasmin. STAR complex at appreciable rates in the presence of excess NPGB. This last observation may provide some explanation for the remarkable stability of plasminogen in plasma in the presence of relatively high concentrations of STAR (25, 26). What remains to be investigated is the mechanism by which a fibrin clot disturbs this stability producing fibrin-specific clot lysis both in in vitro systems (25, 26) as well as in in vivo situations, including experimental animal thrombosis models (9,lO) and patients with acute myocardial infarction.' REFERENCES 1. Lack, C. H. (1948) Nature 161,559-560 2. Lewis, J. H., and Ferguson, J. H. (1951) A m . J. Physiol. 166,594-603 3. Kowalska-Loth, B., and Zakrzewski, K. (1975) Acta Biochim. Pol. 2 2 , 327am
4. 5. 6. 7.
Erygon, R. (1977) Chern. Abstr. 86 202 (Abstr. 673862) Sweet, B., McNicol G. P., and Douilas, A. S. (1965) Clin. Sci. 29,375-382 Lewis, J. H., and Shirakawa, M. (1964) A m . J. Physiol. 207,1049-1052 Lewis, J. H., Kerber, C. W., and Wilson, J. H. (1964) A m . J. Physiol207, 1 nAA-1 nAA
8. Kanai, K. (1986) Biol. Abstr. 8 1 , 748 (abstract 65436) 9. Collen, D., De Cock, F., Vanlinthout, I., Declerck, P. J., and Lijnen, H. R. (1992) Fibrinolysis 6,232-242 10. Collen, D., De Cock, F., and Stassen J. M. (1992) Circulation, in ress 11. McClintock, D. K.,and Bell, P. H. (i971) Biochem. Biophys. Res. &mmun.
- ."
A", 2 GQA-7ll3 -1
(Model 2)
k,
P
12. Behnke, D., and Gerlach, D. (1987) Mol. & Gen. Genet. 210,528-534 Mikro13. Gerlach, D., Kraft. R.. and Behnke. D. (1988) Zentralbl. Bakteriol .. . . . .. . biol. Hyg. Ser. A: 289,314-322 14. Deutsch, D. G., and Mertz, E. T. (1970) Science 1 7 0 , 1095-1096 15. Wiman, B. (1980) Biochem. J. 1 9 1 , 229-232 16. Nelles, L., Lijnen, H. R., Collen, D., and Holmes, W. E. (1987) J. Biol. Chem. 2 6 2 , 10855-10862 17. Lijnen, H. R., Van Hoef, B., De Cock, F., and Collen, D. (1990) Thromb. Huemostmu 64. .-,fil-fix .- .18. Bushy, S. J., Mulvihill, E., Rao, D., Kumar, A. A,, Liouhin, P., Heipel, M., Sprecher, c . , Halfpap, L., Prunkard, D., Gambee, J., and Foster, D.C. (1991) J. Biol. Chem. 2 6 6 , 15286-15292 19. Lijnen, H. R.,Van Hoef, B., Nelles,L., and Collen, D. (1990) J. Biol. Chem. '
which, provided complex formation between P and S is rapid and quantitative, is described by the following rate equation. d[p
NPGB
p'sl = ( k + ~ k&.S])[P.S] dt
+
+ k,[p.S][P]
(Eq. 3)
This rate equation has no simple analytical solution but can be integrated numerically, taking the following restrictions into account: [p. SI= [PI when [PI < [SI, and [p. SI= [SI, when [PI > [SI,. The experimental data of Fig. 3B could adequately be simulated by this numerically integrated rate Silence, K., Collen, D., and Lijnen, H.R. (1993) J. Bioi. C&m.
268, in press.
'%x%5323-532G "-, """
'
'
" "
20. Gaffney, P. J., and Curtis, A. D. (1985) Thromb. Huemostmls 53,134-136 21. Collen, D., Silence,K., Demarsin, E., De Mol, M., and Lijnen, H. R. (1992) Fibrinolysis 6,203-213 22. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 23. Chase, T., Jr., and Shaw, E. (1969) Btochemrstry 8,2212-2224 24. Lijnen, H.R., Van Hoef, B., Vandenbossche, L.,and Collen, D. (1992) Fibrinolvsis 6 . 214-225 25. Lijnen, H-R., VanHoef,-B., De Cock, F., Okada, K., Ueshirna, S., Matsuo, O., and Collen, D. (1991) J. Biol. Chem. 266,11826-11832 26. Matsuo, 0.Okada, K., Fukao, H., Tomioka, Y., Ueshima, S., Watanuki, M., and gakai, M. (1990) Blood 76,925-929
