THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry

and Molecular Biology, Inc.

Vol. 265, No. 9, Issue of March 25, pp. 5232-5236,199O Printed in U.S. A.

Plasminogen Plasminogen

Activation with Single-chain Activator (scu-PA)

PLASMINOGEN

Urokinase-type
( Ser7@-+Ala) AND

STUDIES WITH ACTIVE SITE MUTAGENIZED PLASMIN-RESISTANT scu-PA (Ly~~*+Glu)*

(Received for publication, H. Roger

From

July 10, 1989)

LijnenS,
for

Berthe
Thrombosis

Van Hoef,
and Vascular

Luc Nelles,
Research,

and D&sir&

Collen
B-3000 Leuven, Belgium

the Center

University

of Leuven,

The mechanism of the activation of plasminogen by single-chain urokinase-type plasminogen activator (single-chain u-PA, scu-PA) was studied using rscuPA-G1ul, a recombinant plasmin-resistant mutant of human scu-PA obtained by site-specific mutagenesis of LysS8 to Glu, and rPlg-Ala, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active-site Ser740 to Ala. Conversion of 251-labeled single-chain plasminogen to two-chain plasmin was quantitated on reduced sodium dodecyl sulfate-gel electrophoresis combined with autoradiography and radioisotope counting of gel bands. The catalytic efficiencies of both rscu-PA-Glu68 and rscuPA for the activation of rPlg-Ala740 and of natural plasminogen were comparable and were 250-500-fold lower than that of recombinant two-chain u-PA (rtcuPA) for rscu-PA-Glu and lOO-200-fold lower for rscu-PA. Pretreatment of rscu-PA-Glu* or rscu-PA with excess az-antiplasmin, which efficiently neutralizes all contaminating rtcu-PA, did not significantly reduce the catalytic efficiency of these single-chain moieties, indicating that they have a low but significant intrinsic plasminogen activating potential. The low intrinsic catalytic efficiency of rscu-PA for the conversion of plasminogen to plasmin may be sufficient to generate trace amounts of plasmin, which may regulate plasminogen activation by converting poorly active rscu-PA to very active rtcu-PA.

min or kallikrein. scu-PA has a very low reactivity toward low M, synthetic substrates or active site inhibitors that are very reactive toward tcu-PA (for references, see Ref. 1). Plasminogen, the substrate of u-PA, is a single-chain glycoprotein with an M, of 92,000. It is converted to the twochain serine protease plasmin by specific cleavage of the Arg560-Va1561 peptide bond; the heavy (NH*-terminal) chain of M, 60,000 is connected with the light (COOH-terminal) chain of M, 25,000 through two disulfide bonds. The active center of the enzyme, located in the light chain, is composed of His602, ASPIRE, and Ser7*. Native plasminogen has NH*terminal glutamic acid (Glu-plasminogen), but is easily converted to modified forms with NH&erminal lysine, valine, or methionine (commonly designated Lys-plasminogen), by plasmic cleavage of the Arg67-Met68, Ly~~~-Lys~~, or LYS~~Va17 peptide bonds. Activation of Glu-plasminogen by tcuPA obeys Michaelis-Menten kinetics, with reported kinetic parameters between 1.4 and 200 pM for the Michaelis constant (K,) and between 0.26 and 1.5 s- for the catalytic rate constant ( k2). Lys-plasminogen is activated 3-lo-fold more efficiently than Glu-plasminogen by all known plasminogen activators (for references, see Ref. 2). Estimations of the catalytic efficiency of scu-PA for the activation of plasminogen have varied widely. Initially we reported that scu-PA has a catalytic efficiency similar to that of tcu-PA (3). Several groups have reported a lower but significant catalytic efficiency ranging between 0.4 and 6% of that of tcu-PA (4, 5) while other authors have reported that scu-PA is a genuine proenzyme without enzymatic activity

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Urokinase-type plasminogen activator (u-PA) can be obtained as a single-chain molecule (single-chain u-PA, scu-PA) or as a two-chain proteolytic derivative (tcu-PA, urokinase) following cleavage of the Lys58-Ile59 peptide bond with plas* This study was supported in part by the Geconcerteerde Onderzoeksacties (project 85-90/78). The costs of publication of this article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: Center for Thrombosis and Vascular Research, K. U. Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. The abbreviations used are: u-PA, urokinase-type plasminogen activator; scu-PA, sinple-chain u-PA; rscu-PA, recombinant scu-PA obtainedby expression of its cDNA in Chinese hamster ovary cells; rscu-PA-Glu58. rscu-PA with Lvs~ reDlaced bv Glu bv site-directed mutagenesis; t&-PA, two-chain-u-PA; ;tcu-PA; recombinant tcu-PA obtained from rscu-PA by cleavage of the Lys58-Ile9 peptide bond with plasmin; nPlg, native plasminogen purified from human plasma; rPlg-kla7, recombinant piasminog& obtained by site-specific mutagenesis of SerT4 to Ala; SDS-PAGE, sodium dodecvl sulfate-polyaciylamide gel electroph&esis; ELISA, enzyme-linked immun&rbent assay.

(6-8).
We have proposed that in mixtures of plasminogen and scu-PA the conversion of plasminogen to plasmin and of scuPA to tcu-PA can quantitatively be described by a sequence of three reactions each of which obeys Michaelis-Menten kinetics (3, 9). In the first reaction, scu-PA directly activates plasminogen to plasmin, then plasmin converts scu-PA to tcu-PA, and finally residual plasminogen is activated by tcuPA. This mechanism attributes some intrinsic plasminogen activating potential to scu-PA, which initially was found to be very high (3) but which was subsequently shown to be of the order of 1% of that of tcu-PA (9, 10). Several methodological difficulties, however, hamper the quantitative investigation of the activation of plasminogen by scu-PA. These include variability in kinetic properties of scuPA obtained from different sources (9, ll), generation of more readily activatable Lys-plasminogen forms (12), and efficient conversion of scu-PA to tcu-PA by generated plasmin during the activation process (4-11). To investigate the significance of the conversion of scu-PA to tcu-PA for the activation of plasminogen, we have previously constructed plasmin-resist-

5232

Plasminogen Activation
ant mutants of scu-PA by site-specific mutagenesis of Lysi5s to Glu or to Gly, thereby destroying the plasmic cleavage site for conversion to tcu-PA (13). These mutants still activate plasminogen, albeit with a catalytic efficiency which is at least an order of magnitude lower than that observed with wildtype scu-PA, which suggests that conversion of scu-PA to tcuPA is not a prerequisite for plasminogen activation. In the present study we have further improved the methodology for studying the mechanism of plasminogen activation by scu-PA by using a recombinant plasminogen mutant obtained by site-specific mutagenesis of the active site Ser to Ala (rPlg-Alar4) whereby the catalytic site of plasmin is destroyed (14). The catalytic efficiency of rscu-PA, rscu-PAGlui5, and rtcu-PA for the conversion of rPlg-Alar* and of natural plasminogen (nPlg) to their two-chain derivatives was determined by reduced SDS-gel electrophoresis.
MATERIALS AND METHODS

with scu-PA

5233

The chromogenic substrates pyroglutamyl-glycyl-arginine-p-nitroanilide (S-2444) for tcu-PA and D-valyl-leucyl-lysine-p-nitroanilide (S-2251) for plasmin were purchased from KabiVitrum (Brussels, Belgium). The synthetic inhibitors D-Val-Phe-Lys-CH&l for plasmin (20), Glu-Gly-Arg-CH&l for tcu-PA, and D-Ik?-Pro-Arg-C&Cl for thrombin (21), were custom-synthesized at UCB (Brussels, Belgium). Aprotinin (Trasylol) was from Bayer (Leverkusen, FRG). Benzamidine-Sepharose and 1ysineSepharose were obtained by coupling ofpaminobenzamidine or lysine to CNBr-activated Sepharose 4B. Analytical Techniques-Plasminogen concentration was determined from the absorbance at 280 nm using Acls, lem) = 16.1 (22). uPA concentrations were determined with a specific ELISA (23). SDSPAGE was performed on lo-15% gradient gels after reduction with dithioerythritol using the Phast System (Pharmacia, Uppsala, Sweden). Lys-plasmin(ogen) was measured with a specific ELISA, using monoclonal antibody MA-LPml, which does not react with Gluplasminogen (24). tcu-PA was quantitated with an ELISA specific for tcu-PA, using MA-12E6A8 which reacts 15,000-fold better with tcu-PA than with scu-PA (31).
Activation

of Plasminogen

with

TWX-PA,

rscu-PA-Gh?,

and rtcu-

Recombinant scu-PA (rscu-PA), prepared by expression of cDNA encoding human scu-PA in Escherichia coli was a gift from Griinenthal AG (Aachen, Federal Republic of Germany). Its specific activity after chromatography on benzamidine-Sepharose (9), determined by comparison with the International Reference Preparation for Urokinase (66/46, obtained from the National Institute for Biological Standards and Control, London, United Kingdom) was 180,000 + 15,000 IU/mg (mean + S.D.; n = 3) on fibrin plates (15) and <250 IU/mg as measured with the chromogenic substrate S-2444. rtcu-PA was obtained by treatment of rscu-PA (final concentration = 18 fiM) with plasmin (2% molar ratio) for 20 min at 37 C in 0.05 M TrisHCl buffer, pH 7.4, containing 0.038 M NaCl and 0.01% Tween 80, followed by chromatography on benzamidine-Sepharose as described previously (9). Complete conversion of u-PA from one-chain to twochain molecules was confirmed by SDS-PAGE on lo-15% gradient gels after reduction with dithioerythritol (not shown). The specific fibrinolytic activity of &u-PA was 180,000 f 15,000 IU/mg and the specific amidolytic activity was 100,000 + 17,000 IU/mg (mean f S.D.; n = 3). rscu-PA-Glu58, obtained by site-specific mutagenesis of Lys to Glu, was prepared and characterized as described elsewhere (13). To remove trace amounts of rscu-PA or of active rtcu-PA, the preparation of rscu-PA-Glu58 (f ma 1 concentration = 14 PM) was treated with plasmin (1% on a molar basis) for 30 min at 37 C. Plasmin was then neutralized by addition of aprotinin (final concentration = 2 FM) and D-Val-Phe-Lys-CH&l (final concentration = lo-M) and rtcu-PA by addition of Glu-Gly-Arg-CH&l (final concentration = lo- M). Excess inhibitor was then removed by extensive washing on a Centricon 30 microconcentrator (Amicon). The specific fibrinolytic activity of rscu-PA-Glu was about 500 IU/mg and the specific amidolytic activity was about 160 IU/mg. Recombinant plasminogen with Se? mutagenized to Ala (rPlgAla7) was obtained by expression in Chinese hamster ovary cells of the plasmid plg 251a/219b (14) using the vector Zem229 (16). These materials were kindly provided by ZymoGenetics. Cells were cultured as described (13), and rPlg-Ala40 was purified from the conditioned medium by affinity chromatography on lysine-Sepharose and elution with 6-aminohexanoic acid (17). Recoveries of rPlg-AlaT40 were 0.64 + 0.21 mg/liter in three preparations from approximately lo-liter batches of cultured medium. Before use, 6-aminohexanoic acid was removed by extensive dialysis against 0.05 M Tris-HCl buffer, pH 7.4, containing 0.038 M NaCl and 0.01% Tween 80. Purified rPla-Ala was homogeneous on SDS-PAGE and migrated with an M, of about 90,000. It was quantitatively converted to a two-chain plasmin molecule (M, 60,000 and 25,000) by treatment with tcu-PA, as monitored on SDS-PAGE after reduction with dithioerythritol (not shown). Conversion of rPlg-Ala7 to a two-chain molecule was, however, not associated with the generation of enzymatic activity. This was shown by the absence of amidolytic activity against S-2251 (final concentration = 1 mM) using the two-chain mutant at a final concentration of 250 nM (detection limit = 0.2% under the conditions used). Native human plasminogen (nPlg) was purified from human plasma and characterized as described (9, 17). Plasminogen preparations were labeled with 1 using the IODO-GEN method (18), to a specific activity of 6-9 X lo6 cpm/pg. Human a2-antiplasmin was purified from plasma and its activity determined by titration with plasmin (19).

PA-nPlg or rPlg-Ala 740(final concentration = 1.5 PM, Containing approximately 5 X lo6 cpm of Y-labeled plasminogen/ml) were incubated at 37 C in 0.05 M Tris-HCl buffer, pH 7.4, containing 0.038 M NaCI, 0.01% Tween 80, and 20 pM aprotinin, with rscu-PA (final concentration = 75, 150, or 300 nM), rscu-PA-Glu58 (final concentration = 75, 150, or 300 nM) or rtcu-PA (final concentration = 0.37, 0.75, or 1.50 nM). At different time intervals (O-60 min) samples were removed from the incubation mixtures and reduced immediately by heating at 100 C for 3 min in the presence of 1% SDS and 1% dithioerythritol. SDS-PAGE was performed on lo-15% gradient gels followed by overnight autoradiography using intensifying screens. Radioactive bands were cut from the gel and their 1 content measured. The plasmin concentration in each sample was determined from the ratio of the total radioactivity and the sum of the radioactivity recovered in the gel bands corresponding to the plasmin heavy and light chains. When plasminogen was fully converted to plasmin, the sum of the radioactivity in the plasmin heavy and light chains was 91 + 11 or 82 f 5% of that in the single band for nPlg or rPlg-Ala40 (not shown). Generation of Lys-plasmin(ogen) and of tcu-PA during the experiment was monitored by ELISA using samples collected into Glu-Gly-Arg-CH&l (final concentration = 10e4 M). In additional experiments, rscu-PA, rscu-PA-Glu (final concentration = 300 nM each), or rtcu-PA (final concentration = 1.5 nM) were preincubated with a2-antiplasmin (final concentration = 3 pM) for 2 h at 37 C before addition of rPlg-Ala740 (final concentration = 1.5 pM) and aprotinin (final concentration = 20 FM).
RESULTS

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In mixtures of rPlg-Ala740 (1.5 PM, containing approximately 5 x lo6 cpm iz51-labeled rPlg-Ala740/ml) and rscu-PA, rscu-PA-Gluas, or &u-PA, the labeled single-chain material was progressively converted to two-chain material as revealed by reduced SDS-gel electrophoresis and autoradiography (Fig. 1). The amount of two-chain material quantitated by measuring the iz51 content of the corresponding bands of the SDSgel increased linearly with time for all three u-PA moieties studied (Fig. 2). A very similar response was obtained using nPlg instead of rPlg-Ala740 (not shown). The catalytic efficiencies (ka/K,) of the three u-PA moieties for the activation of nPlg or rPlg-Ala740 are calculated from the initial rate of plasmin generation, the concentration of plasminogen and u-PA used, using the formula u/[u-PA] x [Plg] = Izz/(K,,, + [Plg]). For &u-PA and rscu-PA-Gl@s, [Plg] can be neglected as compared to K, (3, 9, 10, 13), whereas for rscu-PA, [Plg] of 1.5 PM is of the same order of magnitude as the K,,, of approximately 1 pM (9,lO). Therefore, the values of k,/K,,, for rscu-PA (Table I) are corrected. The catalytic efficiencies of rscu-PA, rscu-PA-Glui5, and rtcu-PA for the activation of both nPlg and rPlg-Alar4 were comparable. However, the catalytic efficiencies of rscu-PA were lOO200-fold lower and those of rscu-PA-Glui5s were 250-500-fold lower than those of &u-PA.

5234 FIG. 1. Autoradiography of reduced SDS-PAGEfollowing activation of rPlpAla740 PM, conkking (1.5 5 X lo6 cpk 251-labeled rPlg-AlaS4/ml)with 300 nM rscu-PA (A), with 300 nM rscu-PAGlu (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 (lane 6), and 60 min (lane 7). Arrows, apparent M, of plasminogen (M, =: 90,000) and of the plasmin heavy chain (M, = 60,000) and light chain (M, z 25,000).

Plasminogen A
1234567

Activation

with scu-PA B
1234567 1234567

1
l n

200 n

;r/

ix

FIG. 2. Activation of rPlg-Ala40(1.5 containing a trace amount of lz51rPlg-Ala40) with rscu-PA (A), rscu-PAGluLSR (B), or rtcu-PA (C). Concentrations used are 75 nM (O), 150 nM (*), or 300 nM (m) for rscu-PA or rscu-PAGl@ and 0.37 nM (*), 0.75 nM (A), and 1.50 nM (V) for rtcu-PA. The concentration of two chain material, determined from SDS gels, is plotted versus time. The data were fitted by linear regression analysis with forced intercept; correlation coefficients were between 0.94 and 0.99.
FM,

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TIME

( min)

Catalytic u-PA moiety

TABLE I efficiency (k*/KJ of mu-PA, rscu-PA-Glu58, for the activation of nPb or rPb-AlaT Concentration used W&i nPlg
,,,M-.s-

or r&u-PA

rPlg-Ala 0.00037 0.00032 0.00032 0.00034 ~0.00003 0.00013 0.00013 0.00010 0.00012 ~0.00002 0.062 0.075 0.070 0.069 kO.006

rscu-PA

75 nM 150 nM 300 nM Mean

kS.D.

0.00045 0.00045 0.00050 0.00047 ~0.00003 0.00015 0.00015 0.00018 0.00016 +0.00002 0.045 0.045 0.033 0.041 f0.007

either with nPlg or with rPlg-Ala740. Preincubation of rscu-PA or rscu-PA-Glu58 with az-antiplasmin, under conditions where up to 0.5% contaminating rtcu-PA is completely neutralized, did not abolish the activation of rPlg-Ala740 (Fig. 3). The catalytic efficiencies for the activation of rPlg-Ala740 by rscu-PA or by rscu-PA-Glu58 in the presence of az-antiplasmin were, respectively, 13 or 27% (mean of two independent experiments) lower than in the absence of a2-antiplasmin, whereas &u-PA was totally inactivated by preincubation with az-antiplasmin.
DISCUSSION

rscu-PAGlu=

75 nM 150 nM 300 nM Mean &S.D.

rtcu-PA

0.37 nM 0.75 nM Mean +S.D.

1.50 nM

Monitoring of Lys-plasmin(ogen) levels during incubation confirmed the absence of Lys-forms in the experiments with rPlg-Ala740 (<0.2% at 60 min with the highest concentration of rscu-PA, rscu-PA-Glu15*, or rtcu-PA used), whereas in the experiments with nPlg the concentration of Lys-forms reached a maximum of 7, 0.8, and 2.5% at 60 min using the highest concentration of rscu-PA, rscu-PA-Glu58, and rtcuPA, respectively. Monitoring of rtcu-PA levels in the experiments with rscu-PA and rscu-PA-Glu58 confirmed that no active two-chain u-PA was generated during the experiments (~0.5% at 60 min at the highest concentration of u-PA),

Elucidation of the activation mechanism of plasminogen by scu-PA is complicated by the fact that generated plasmin may convert scu-PA to tcu-PA, which has a much higher catalytic efficiency for plasminogen, or that it may degrade Glu-plasminogen to Lys-plasminogen with a much greater sensitivity to activation. Both phenomena indeed would result in marked acceleration of plasminogen activation during the experiment. We have previously measured plasminogen activation by scuPA with a coupled substrate assay, in which excess plasmin substrate S-2251 was used to measure plasmin and at the same time to prevent or reduce conversion of scu-PA to tcuPA (3,9). In addition, we have usedplasmin-resistant mutants of scu-PA (rscu-PA-Glu58) which are not converted to active tcu-PA moieties by plasmin (13). These experiments have suggested that scu-PA has a very low, but probably significant plasminogen-activating potential. In the experimental systems used, it was, however, impossible to exclude trace contamination of the reagents with plasmin and/or tcu-PA and to totally prevent conversion of scu-PA to tcu-PA by traces of generated plasmin. In the present study, we have measured the catalytic efficiency of scu-PA for plasminogen in a system which is intrin-

Plasminogen

Activation

with scu-PA

5235

A
1234567 01234567

B
1234561

FIG. 3. Autoradiography of reduced SDS-PAGE following activation of rPlg-Ala40 (1.5 pM, containing a trace amount. 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 preincubation of the u-PA moieties with cu2-antiplasmin (3 pM) for 2 h at 37 C. Samples were taken at times 0 (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 (lane 7). Arrows. aonarent M. of olasminogen CM, = 90.000) and of the plasmin heavy chain (M, = 60,000) and light chain .. (M,- 25,000). sically

devoid of tcu-PA or plasmin activity. This is achieved by using a plasmin-resistant mutant of rscu-PA (rscu-PAGlu) and an active site mutant of recombinant plasminogen (rPlg-Ala740) which is totally inactive after conversion to a two-chain molecule by plasminogen activators (14). Generaof plasmin was quantitated by autoradiography after

tion

of plasminogen.

This positive

feedback

mechanism

has

tion

SDS-PAGE under reducing conditions. In addition, the experiments were performed in the presence of excess aprotinin and the generation of rtcu-PA and Lys-plasmin(ogen) was monitored with specific ELISAs. Our results indicate that the catalytic efficiencies (KJK,) of both rscu-PA and rscu-PA-Glu for rPlg-Ala7 and nPlg are comparable but 100-200-fold lower than those of rtcu-PA for rscu-PA and 250-500-fold lower for rscu-PA-Glu58. This indicates that the catalytic efficiency of the present E. coliderived rscu-PA for the activation of Glu-plasminogen is between 0.5 and 1.0% of that of rtcu-PA. These values are comparable to the value of ~1% obtained previously in a coupled substrate assay with natural scu-PA obtained from CALU-3 cells (9) or with rscu-PA obtained from Chinese hamster ovary cells (13). In a previous study, the catalytic efficiency of rscu-PA from E. coli, provided by Genentech was, 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. A catalytic efficiency of scu-PA for plasminogen of about 0.4% of that of tcu-PA was reported previously by Pannell and Gurewich (4), whereas Ellis et al. (5) found a catalytic efficiency of 4-6% of that of tcu-PA. In contrast, other authors have reported that scu-PA has no plasminogen-activating potential (6-8). The 3-fold difference in catalytic efficiency for plasminogen activation of rscu-PA and rscu-PA-Glu58 found in the present study contrasts with the lo-fold higher value for rscu-PA as compared to rscu-PA-Glu5s reported previously (13). This may be due to the fact that the previously used coupled substrate assay (3, 9) does not totally prevent conversion of scu-PA to tcu-PA, resulting in overestimation of the intrinsic activity of scu-PA. The methodology used in the present study allows exclusion of the generation of both tcu-PA and plasmin activity during the experiment and thereby establishes that rscu-PA has a low but significant intrinsic plasminogen activating potential. This may be important for the regulation of the mechanism of plasminogen activation by scu-PA via generation of a small amount of plasmin which in turn converts poorly active scuPA to very active tcu-PA, resulting in an accelerated activa-

also been implied in fibrin clot dissolution by scu-PA in human plasma in vitro (10). Furthermore, it was suggested that conversion of scu-PA to tcu-PA by plasmin occurs mainly at the fibrin surface without extensive systemic conversion in the surrounding plasma (31). It has been demonstrated previously that certain zymogens, such as chymotrypsinogen and trypsinogen, are not entirely enzymatically inactive but have a very low intrinsic activity which, upon activation, increases by several orders of magnitude (25-27). The single-chain form of tissue-type plasminogen activator (t-PA) has significant enzymatic activity toward low molecular weight synthetic substrates (lo-15% of that of the two-chain form) (28) whereas its plasminogen activating potential is comparable to that of the two-chain form (29). The structural basis of this high activity as compared to other single-chain forms of serine proteases has not yet been explained. The intrinsic enzymatic activity of scu-PA appears to be intermediary to these extremes. The mechanism of plasminogen activation in purified systems and of fibrin dissolution in human plasma in vitro presented here may, however, not be identical to the physiological mechanism of action of scu-PA. It was indeed found that plasmin-resistant mutants of scu-PA (i.e. rscu-PAGlu) have only a 3-5-fold lower in uiuo thrombolytic potency as compared to wild-type scu-PA (30), suggesting that for in uiuo thrombolysis, conversion of scu-PA to tcu-PA may play a less important role.
Acknowledgments-We are very grateful to Dr. S. Busby and G. Parker (ZymoGenetics, Seattle, WA) for supplying the plasmid plg 251a/219b and the vector Zem 229.

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249-301

3. Collen, D., Zamarron, C., Lijnen, H. R., and Hoylaerts, M. (1986) J. Biol. Chem. 261, 1259-1266 4. Pannell, R., and Gurewich, V. (1987) Blood 69,22-26 5. Ellis, V., Scully, M. F., and Kakkar, V. V. (1987) J. Biol. Chem. 262,14998-15003 6. Kasai, S., Arimura, H., Nishida, M., and Suyama, T. (1985) J.
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7. Petersen, L. C., Lund, L. R., Nielsen, L. S., Dano, K., and Skriver, L. (1988) J. Biol. Chem. 263,11189-11195 8. Urano, T., de Serrano, V. S., Gaffney, P. J., and Castellino, F. J.
(1988) Arch. Biochem. Biophys. 264, 222-230

5236

Plasminogen Activation

with scu-PA

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