©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Enhancement of the Enzymatic Activity of Single-chain Urokinase Plasminogen Activator by Soluble Urokinase Receptor (*)

(Received for publication, April 25, 1995)

Abd Al-Roof Higazi (1)(§) Robert L. Cohen (2) Jack Henkin (3) Douglas Kniss (4) Bradford S. Schwartz (5) Douglas B. Cines (1)

From the  (1)Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104, (2)Genentech, South San Francisco, California 94080, (3)Abbott Laboratories, Abbott Park, Illinois 60064-3500, the (4)Department of Obstetrics and Gynecology, Ohio State University, Columbus, Ohio 43210, and the (5)Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Single-chain urokinase (scuPA), the unique form of urokinase secreted by cells, is converted to an active two-chain molecule through the cleavage of a single peptide bond by plasmin and other specific proteinases. Although scuPA may express limited enzymatic activity, its contribution to plasminogen activation on cell surfaces remains uncertain. Further, although it is well known that scuPA binds to a specific extracellular urokinase-type plasminogen activator receptor, the effect of this interaction on the enzymatic activity of scuPA has not been described. In the present paper we report that the binding of scuPA to cellular and to recombinant soluble urokinase-type plasminogen activator receptors (suPAR) increases its catalytic activity as measured by the cleavage of a urokinase-specific chromogenic substrate. suPAR increased the V of scuPA 5-fold with little change in its K. suPAR also stimulated the plasminogen activator activity of scuPA by decreasing its K for Glu-plasminogen from 1.15 µM to 0.022 µM and by increasing the k of this reaction from 0.0015 to 0.022 s. Preincubation of scuPA with suPAR also enhances its susceptibility to inhibition by plasminogen activator inhibitor type 1, consistent with exposure of its catalytic site. The activity of scuPA bound to suPAR is not accompanied by cleavage of scuPA, which continues to migrate as a single band in SDS-polyacrylamide gel electrophoresis under reducing conditions. Moreover, suPAR increases the plasminogen activator activity of a plasmin-insensitive variant, scuPA (scuPA-Glu), as well. Enhancement of scuPA activity by suPAR is both prevented and reversed by its amino-terminal fragment (amino acids 1-135), which competes for receptor binding, suggesting that continued binding to the receptor is required for expression of scuPA's enzymatic activity. Thus, our data suggest that scuPA may undergo a reversible transformation between a latent and an active state. The urokinase receptor may induce or stabilize scuPA in its active conformation, thereby contributing to the initiation of plasminogen activation on cell surfaces.


INTRODUCTION

Plasmin has been implicated in a variety of physiologic and pathophysiologic processes by virtue of its capacity to proteolyze diverse substrates. Plasmin, which is formed from plasminogen by cleavage of the Arg-Val bond by two-chain urokinase (tcuPA)()and other plasminogen activators(1) , in turn cleaves the Lys-Ile bond in scuPA to form tcuPA(2) . Although the capacity of plasmin and tcuPA to mutually reinforce the production of each other is well established, the initial steps in the conversion of plasminogen to plasmin are less clear.

The cell surface may regulate these initial steps by providing specific binding sites for urokinase and plasminogen. Several groups have reported that the binding of scuPA to uPAR in the presence of plasminogen markedly accelerates plasmin formation(3, 4, 5) . Plasmin formation is also accelerated in systems where the scuPA has been linked directly to the cell surface by a glycosyl phosphatidylinositol anchor (6) or when complexed with plasminogen by specific monoclonal anti-uPA antibodies(7) . Based on these data, it has been postulated that the enhancement of plasmin generation may be a consequence of the increased local concentration of single-chain urokinase (scuPA) and plasminogen on cell surfaces relative to plasma(6) .

However, it is also possible that cell surface receptors may modulate the induction or stability of critical intermediates in plasmin formation, which have been described to date only in fluid phase systems. For example, there is evidence to suggest that the limited plasminogen activator activity of scuPA (8, 9) may be stimulated when lysine or fibrin binds to its substrate, Glu-plasminogen(10) . In addition, a transitional state of scuPA has been described to occur after it has been cleaved by plasmin or kallikrein in which the molecule has greater catalytic efficacy against Glu-plasminogen than does tcuPA(11) .

Despite these observations, soluble scuPA is generally considered to contribute little to plasminogen activator activity relative to tcuPA in most settings. However, there may be specific circumstances in which the intrinsic enzymatic activity of scuPA is enhanced. In this study, we asked whether the binding of scuPA to its receptor (uPAR) is one such situation. Our studies indicate that binding of scuPA to soluble uPAR is sufficient to expose the catalytic site of scuPA and cause an approximately 50-fold reduction in its K and an approximately 850-fold increase in its catalytic efficiency.


EXPERIMENTAL PROCEDURES

Materials

scuPA and its amino-terminal fragment (amino acids 1-132) and recombinant soluble uPA receptor were prepared and isolated as described(4, 12, 13) . PAI-1 was a gift of Dr. Christopher Reilly (Merck Sharpe and Dohme) and was reactivated with guanidine(4) . The chromogenic substrate of urokinase, Spectrozyme UK (carbobenzoxy-L--glutamyl-(-t-butoxyl)-glycyl-arginine-p-nitroanilide diacetate salt), Glu-plasminogen, Lys-plasminogen, the chromogenic substrate of plasmin, Spectrozyme PL (H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide diacetate salt), plasmin, and high molecular weight two-chain urokinase were provided by American Diagnostica Inc. (Greenwich, CT). -Antiplasmin was obtained from Sigma.

Variant scuPA

The cDNA for scuPA-Glu (kindly supplied by Dr. H. R. Lijnen, Leuven, Belgium) (14) was ligated into the HindIII site of pcDNA 3 (Invitrogen) and amplified in DH5 cells, and its nucleotide sequence was verified (Sequenase, U. S. Biochemical Corp.). Transient transfection in COS-7 cells was performed using DEAE-dextran. Cell supernatants were collected in Dulbecco's modified Eagle's medium containing 2 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, and 10 mM benzamidine. scuPA-Glu was isolated by affinity chromatography using a monoclonal antibody directed at its kringle immobilized on Sepharose (Pharmacia Biotech Inc.). scuPA-Glu migrated as a single 55-kDa protein on a SDS-polyacrylamide gel under reducing conditions before and after incubation with plasmin-Sepharose, whereas native scuPA migrated as a 33- and a 20-kDa protein after the same exposure to plasmin. No scuPA was detected in supernatants from COS cells transfected with vector alone. Purified scuPA-Glu was then incubated sequentially with plasmin-Sepharose, diisopropyl fluorophosphate, and Glu-Gly-Arg-chloromethyl ketone prior to use as described (5) to eliminate any native scuPA that might have been present but have gone undetected.

Measurement of scuPA Amidolytic Activity

The amidolytic activity of scuPA was monitored by the release of p-nitroaniline from Spectrozyme UK, measured by the rate of change in light absorbance at 405 nm. The reaction mixture contained 60 nM scuPA and 58 µM Spectrozyme UK in phosphate-buffered saline, pH 7.4. In some experiments equimolar concentrations of suPAR (60 nM) were added simultaneously with scuPA and the chromogenic substrate, whereas in others scuPA and suPAR were preincubated for various times before the chromogenic substrate was added. The reaction was monitored continuously for the indicated times, and the reaction rate (V) and the K were calculated from the linear portions of the activity curves as described(15) .

The amidolytic activity induced by the binding of scuPA to its receptor was also determined on cell surfaces. First trimester human trophoblastic cells were obtained and characterized as described previously(16) . Confluent cultures grown in 48-well tissue culture plates (Falcon) were washed twice with phosphate-buffered saline, incubated for 5 min with glycine buffer, pH 3, to elute endogenous uPA, and washed two additional times. The cells were then incubated with phosphate-buffered saline containing 300 µM Spectrozyme UK and 10 mM aprotinin in the presence and the absence of 10 nM scuPA for 3 h at 37 °C, and the A was determined. In other experiments, the incubation was performed in an identical manner in the presence of scuPA and ATF (100 nM).

Measurement of Plasminogen Activator Activity

The plasminogen activator activity of scuPA and scuPA-Glu were measured in the presence of plasminogen using the plasmin substrate Spectrozyme PL. 2 nM scuPA or 1 nM scuPA-Glu were preincubated with or without equimolar concentrations of suPAR for 50 min. 1 µM Glu- or Lys-plasminogen and 500 µM chromogenic substrate were added in phosphate-buffered saline, pH 7.4, for various periods of time, and the reaction was monitored at 405 nm. A high concentration of the chromogenic substrate (1724-fold higher than the K) was used to minimize activation of scuPA by plasmin and thereby isolate the stimulatory effect of suPAR. In one set of experiments, scuPA alone or scuPA and suPAR were preincubated for 50 min before plasminogen and the chromogenic substrate were added. In another set of experiments, 10-fold molar excess ATF was added to preformed suPARscuPA complexes immediately prior to or 4 min after adding plasminogen and the chromogenic substrate.

Kinetic analysis of plasminogen activation was performed as described (17) . Briefly, 10 nM scuPA in the presence and the absence of an equimolar concentration of suPAR was incubated with increasing concentrations of Glu-plasminogen and 500 µM amidolytic substrate of plasmin in phosphate-buffered saline at room temperature, and the A was measured continuously over time. The A generated in the absence of added scuPA at each time point determined in parallel was subtracted. The concentration of plasmin generated during each interval was calculated from the rate of change in A using a standard curve made with known concentrations of plasmin. The kinetic parameters of plasmin generation were then determined using a double-reciprocal plot of the rates of plasmin generation versus plasminogen concentration(17) .

Effect of PAI-1, ATF, and -Antiplasmin

In some experiments, 400-600 nM ATF or 200 nM -antiplasmin were added to scuPA alone or to preformed suPARscuPA complexes during the preincubation step. To test the reversibility of the reaction, scuPA and suPAR were preincubated for 50 min as above and 10-fold molar excess ATF was then added for 0-60 min before the amidolytic activity was measured. In other experiments, 120 nM PAI-1 was incubated with scuPA or with preformed suPARscuPA complexes for 5 min before the chromogenic substrate was added.

Gel Electrophoresis

Samples of 200 nM scuPA alone, 40 nM suPAR alone, or 200 nM scuPA preincubated with equimolar concentrations of suPAR for either 60 min or 24 h at 37 °C were mixed with equal volumes of 10% SDS and 10% 2-mercaptoethanol. The mixture was heated at 100 °C for 5 min. 20-µl aliquots were then applied to a 10% SDS/12% polyacrylamide gel(18) , and the protein bands were identified by staining with Coomassie blue.


RESULTS

Soluble uPAR augments the enzymatic activity of scuPA, as measured by the cleavage of the chromogenic substrate Spectrozyme UK (Fig. 1). The expression of enzymatic activity by scuPA in the presence of its receptor followed a time-dependent pattern in which three phases could be discerned (Fig. 1, inset). When suPAR, scuPA, and the chromogenic substrate were added contemporaneously, no amidolytic activity was detectable for the first 19 min, after which the enzymatic activity was stimulated approximately 220% compared with scuPA alone. Beginning at approximately 50 min, an additional 2-fold increase in the velocity of the reaction became evident.


Figure 1: suPAR stimulates the amidolytic activity of scuPA. 60 nM scuPA was incubated with () or without (⊡) equimolar concentrations of suPAR and the urokinase chromogenic substrate Spectrozyme UK for 100 min in phosphate-buffered saline, pH 7.4, and the chromogenic activity (A) was monitored. suPAR alone had no activity (). The mean ± S.D. of three separate experiments is shown. Inset, the triphasic pattern of the amidolytic activity of the suPARscuPA complexes is illustrated.



We hypothesized that this lag phase reflects the time required for scuPA within the suPARscuPA complex to undergo structural isomerization to a more active form. To examine this possibility, scuPA was preincubated with suPAR for 50 min prior to adding chromogenic substrate. Under these conditions, the amidolytic activity was evident immediately (Fig. 2), and the velocity of the reaction was similar to the third phase of activity seen when the preincubation step was omitted (Fig. 2, inset). The third phase of activity was linear and stable over time, because incubating scuPA with suPAR in the presence and the absence of substrate for longer periods did not enhance its activity further. The enzyme kinetic properties were then studied in greater detail under the conditions of preincubation. The V of scuPA preincubated with suPAR for the chromogenic substrate increased more than 5-fold, whereas the K increased only 30% compared with the activity of scuPA alone (Fig. 3, A and B).


Figure 2: Preincubation of scuPA with suPAR stimulates its amidolytic activity. 60 nM scuPA was preincubated with () or without (⊡) equimolar concentrations of suPAR for 50 min before the addition of the urokinase chromogenic substrate as described in the legend to Fig. 1. The mean ± S.D. of three separate experiments is shown. Inset, the third phase of the amidolytic activity of suPARscuPA complexes shown in Fig. 1(top curve) is compared with the activity measured after preincubation of scuPA with suPAR (Fig. 2, bottom curve).




Figure 3: Kinetics of scuPA activation by suPAR. A, 60 nM scuPA was preincubated with () or without (⊡) equimolar concentrations of suPAR for 50 min before increasing concentrations of the urokinase chromogenic substrate were added. B, analysis of the data presented in A using a double-reciprocal plot. The results from one of two such experiments are shown.



We then examined whether suPAR also stimulated the plasminogen activator activity of scuPA using its physiologic substrates Glu- and Lys-plasminogen. Kinetic analysis of plasminogen activator activity shows that the interaction of scuPA with suPAR decreases its K toward Glu-plasminogen from 1.15 to 0.022 µM and increases the k from 0.0015 to 0.022 s. This corresponds to an increase in enzyme efficiency (k/K) from 0.0013 to 1.1 µM s (Fig. 4). At approximate physiologic concentrations of scuPA (2 nM) and Glu-plasminogen (1 µM), suPAR increased the PA activity 79.3-fold at 16.5 min compared with scuPA alone (Fig. 5A). Lys-plasminogen was cleaved more efficiently by scuPA than was Glu-plasminogen (Fig. 5B), as expected(10) . However, the plasminogen activator activity of scuPA for Lys-plasminogen was enhanced 8.9-fold following preincubation with suPAR under the same experimental conditions described for Glu-plasminogen (Fig. 5B).


Figure 4: Effect of suPAR on the kinetics of Glu-plasminogen activation by scuPA. A, 10 nM scuPA was incubated with varying concentrations of Glu-plasminogen (0.1-0.65 µM). The initial rates of plasmin generation, determined as described under ``Experimental Procedures,'' were plotted against plasminogen concentration as a double-reciprocal plot. B, 10 nM scuPA was incubated with equimolar concentrations of suPAR and varying concentrations of Glu-plasminogen (0.004-0.030 µM), and the data were analyzed as above. In both panels, the results of an experiment representative of two so performed are shown.




Figure 5: suPAR stimulates the plasminogen activator activity of scuPA. A, cleavage of Glu-plasminogen by suPARscuPA complexes. 2 nM scuPA was preincubated for 50 min in the absence () or the presence (⊡) of equimolar concentrations of suPAR. 1 µM Glu-plasminogen and 500 µM plasmin chromogenic substrate Spectrozyme PL were added for the indicated times, and the A was monitored. The mean ± S.D. of five separate experiments is shown. B, the experiment was performed as described in A except that 1 µM Lys-plasminogen was used as the substrate instead of Glu-plasminogen. The mean ± S.D. of three separate experiments is shown.



The next series of experiments was performed to determine whether this stimulation of scuPA activity by suPAR was indeed dependent on persistent binding to its receptor, required only initial contact between scuPA and suPAR, or was the result of its conversion to two-chain urokinase by enzymes such as plasmin. First, we examined the effect of ATF, which competes for receptor binding but has no enzymatic activity. The results shown in Fig. 6indicate that the presence of ATF during the preincubation step totally prevented the stimulatory effect of suPAR on the amidolytic activity of scuPA. Second, in contrast, -antiplasmin had no inhibitory effect, excluding contamination of either preparation by plasmin. Third, the expression of amidolytic activity was not due to the conversion of scuPA to tcuPA by another enzyme (Fig. 7A). Rather, scuPA remained as a single-chain molecule throughout the experiment, and no two-chain uPA was detected even when incubations were continued for as long as 24 h (Fig. 7B). Fourth, the enhancement of the amidolytic activity of scuPA by suPAR was totally reversible, because it was abolished by ATF even when added 50 min after suPARscuPA complexes had been allowed to form (Fig. 8). ATF also abolished the stimulatory effect of suPAR on scuPA activity using Glu-plasminogen as the substrate (Fig. 9). Fifth, to address the possibility that ATF might have inhibited scuPA activation to tcuPA by an unknown mechanism, we added ATF after the stimulatory effect of suPAR had begun. When ATF was added to suPARscuPA complexes 20 min after the addition of the urokinase chromogenic substrate, the amidolytic activity was suppressed to the level seen with scuPA alone (Fig. 8). Further, when ATF was added 4 min after the addition of suPARscuPA to Glu-plasminogen at a point when plasmin formation had already begun, the cleavage of the chromogenic substrate of plasmin became linear over time. The latter result is consistent with ATF preventing further stimulation of plasmin formation by suPARscuPA. In contrast, under the same experimental conditions, neither ATF, suPAR, nor a combination of ATF and suPAR affected the amidolytic or PA activity of two-chain urokinase or the enzymatic activity of plasmin over 1 h (not shown), indicating that the inhibition of scuPA activity described above was due to competition for binding to suPAR. Finally, to further exclude any possible contribution of plasmin-mediated conversion of scuPA to tcuPA, we analyzed the capacity of suPAR to augment the PA activity of a variant scuPA (scuPA-Glu) that cannot be cleaved by plasmin(5, 14) . suPAR stimulated the PA activity of the mutant scuPA approximately 115-fold in the presence of 1 mM Glu-plasminogen (not shown).


Figure 6: ATF inhibits the stimulation of scuPA amidolytic activity by suPAR. 60 nM scuPA was incubated for 50 min alone (open bar), with equimolar concentrations of suPAR (bar with dots), with suPAR and 6.6-fold molar excess ATF (bar with squares), or with suPAR and 200 nM -antiplasmin (hatched bar). Each mixture was then incubated with the urokinase chromogenic substrate for an additional 40 min, and the A was determined. The mean ± S.D. of three separate experiments is shown.




Figure 7: Migration of scuPA in the absence and the presence of suPAR on SDS-polyacrylamide gel electrophoresis. 200 nM scuPA was incubated with 200 nM suPAR for 1 h (A) and for 24 h (B). Lanes 1, molecular mass markers (fourth band from top corresponds to protein, M = 46 kDa); lanes 2, suPAR alone; lane 3, in A, suPAR and scuPA incubated for 60 min; lane 3 in B, suPAR and scuPA incubated for 24 h; lanes 4, scuPA alone.




Figure 8: The stimulation of scuPA amidolytic activity is reversible by ATF. Preformed suPARscuPA complexes were incubated with the urokinase chromogenic substrate in the absence () or in the presence of 10-fold molar excess ATF added either coincident with () or 20 min after () the addition of substrate. The amidolytic activity seen when ATF and the chromogenic were added to suPARscuPA complexes at the same time cannot be distinguished from the activity of scuPA alone (not shown). The mean ± S.D. of three separate experiments is shown.




Figure 9: The stimulation of scuPA plasminogen activator activity is reversible by ATF. Preformed suPARscuPA complexes were incubated with Glu-plasminogen as described in Fig. 5in the absence of ATF (⊡) or in the presence of 10-fold molar excess ATF added either coincident with () or 4 min after () the addition of Glu-plasminogen. The plasminogen activator activity seen when ATF and the Glu-plasminogen were added to suPARscuPA complexes at the same time cannot be distinguished from the activity of scuPA alone (not shown). The mean ± S.D. of three separate experiments is shown.



We then asked whether binding of scuPA to cellular uPA receptors stimulated plasminogen activator activity as well. In these experiments 10 µM aprotinin was added to prevent endogenous serine protease activity that might be directed at either scuPA itself or its substrate. In the absence of added scuPA, no amidolytic activity was detected. Addition of scuPA to the cells stimulated its PA activity approximately 6-fold (not shown). This stimulation was almost completely (>90%) inhibited by the addition of ATF, showing that binding of scuPA to cellular uPAR was required.

It has been reported that PAI-1 shows an approximate 40% reduction in its capacity to inhibit tcuPA bound to its cellular receptor compared with the free enzyme(19) . Therefore, we examined the capacity of PAI-1 to inhibit the intrinsic activity expressed by scuPA and scuPA bound to suPAR. As shown in Fig. 10, the amidolytic activity of suPARscuPA complexes was totally inhibited by a 3-fold molar excess of PAI-1, whereas scuPA was virtually insensitive to inhibition under the same conditions.


Figure 10: Inhibition of scuPA and scuPAsuPAR by PAI-1. The amidolytic activity of scuPA and scuPAsuPAR were determined as in Fig. 5in the presence or the absence of a 5-min incubation with 120 nM PAI-1. Black bar, scuPA alone; hatched bar, scuPA preincubated with PAI-1; shaded bar, scuPA preincubated with suPAR for 50 min; white bar, scuPA preincubated with suPAR for 50 min and an additional 5 min with PAI-1 (not evident). The mean ± S.D. of three separate experiments is shown.




DISCUSSION

The results of this study indicate that the interaction of scuPA with its receptor stimulates its amidolytic activity using a direct assay of urokinase activity and an indirect assay that depends upon plasminogen activation. The requirement for the physical interaction between scuPA and its receptor is evidenced by the lag time before enzymatic activity was observed, by the fact that the lag time was abolished when scuPA was preincubated with suPAR, and by the fact that stimulation of the enzymatic activity was completely abolished by ATF. Preincubation of scuPA with soluble, recombinant uPAR increased the V of the reaction on its chromogenic substrate approximately 500% with minimal change in the K, compatible with an increase in the total amount of enzymatic activity. Further, suPAR increased the catalytic efficiency of scuPA approximately 850-fold when the cleavage of Glu-plasminogen was studied.

The generation of enzymatic activity was not due to its conversion to two-chain urokinase and was thus intrinsic to the scuPA molecule itself. This conclusion is supported by several observations. First, scuPA continued to migrate as a single-chain molecule in SDS-polyacrylamide gel electrophoresis. Second, ATF both prevented and reversed the stimulatory effect of suPAR even after stimulation had begun. Third, suPAR stimulated the plasminogen activator activity of a variant scuPA molecule that is unable to be cleaved by plasmin. The kinetic parameters of the resultant suPARscuPA complexes described here differ from those reported for high molecular weight uPA, low molecular weight uPA(20) , and scuPA itself, suggesting the formation of a previously undescribed activity state of scuPA.

Taken together, the data are most compatible with a model in which scuPA bound to its receptor undergoes a conformational change that exposes its catalytic site to substrate. This hypothesis is supported further by the increased susceptibility of suPARscuPA complexes to inhibition by PAI-1 compared with scuPA alone. Thus, scuPA may be better thought of as a regulatory enzyme rather than solely as a proenzyme. It is likely that the increase in enzymatic activity is induced by binding to suPAR as a result of a conformational change in scuPA. In support of this concept, we have observed that preincubation of scuPA with suPAR also promotes its binding to vitronectin, that this binding of suPARscuPA complexes is no longer inhibitable by scuPA alone, and that the binding of scuPA to -macroglobulin receptor/lipoprotein receptor-related protein is inhibited by suPAR as well (manuscript submitted). Our data do not exclude the possibility that soluble scuPA undergoes a reversible transition between an active and an inactive state and that the receptor stabilizes the active conformation as proposed (5) or, theoretically, binds preferentially to the enzymatically active form of the molecule. Thus, the presence of suPAR may change the equilibrium of the reaction favoring the generation of scuPA molecules that are in the active conformation. However, the relationship of the conformation of scuPA bound to its receptor to the intermediates that develop during its conversion to tcuPA (11) remains to be determined. In either case, it is clear that the transformation of scuPA to an active enzyme is a reversible process dependent on continued binding to suPAR, because the addition of ATF, even after scuPA's enzymatic activity had already been stimulated, abrogated any subsequent receptor-dependent augmentation. The physiologic relevance of this reaction is supported both by the fact that the cleavage of Glu-plasminogen by single-chain urokinase was stimulated by suPAR as well by the fact that binding of scuPA to cellular uPA receptors stimulated its intrinsic catalytic activity.

The observation that the interaction of scuPA with its receptor enhances its enzymatic activity is compatible with previous observations concerning the role of uPAR in accelerating the rate of plasmin formation on cell surfaces. The effect of uPAR has generally been attributed to its role in increasing the local concentrations of scuPA (or tcuPA) and plasminogen, although it has been reported that the rate of plasminogen activation by a glycolipid-anchored tcuPA molecule is retarded relative to its fluid phase counterpart(6) . This observation raises the possibility that the receptor activates scuPA, as suggested previously(5) . The results of this study make it likely that the binding of scuPA to its receptor increases plasmin generation above that which can be attributed to a concentration effect alone. Thus, the initiation of plasminogen activator activity attendant on the binding of scuPA to its receptor may comprise an important initial step in plasmin formation and the subsequent generation of tcuPA on cell surfaces.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL40387, HL50970, and HL49517 and a grant from the Southeast Pennsylvania Chapter of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Pathology and Laboratory Medicine, 7 Founders Pavilion, Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104.

The abbreviations used are: tcuPA, two-chain urokinase-type plasminogen activator; uPA, urokinase-type plasminogen activator; scuPA, single-chain urokinase plasminogen activator; ATF, amino-terminal fragment of urokinase; uPAR, urokinase-type plasminogen activator receptor; suPAR, recombinant soluble urokinase-type plasminogen activator receptor; PAI-1, plasminogen activator inhibitor type 1; PA, plasminogen activator.


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