The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role.
THEJOURNALOF BIOLOGICAL CHEMISTRY (c) 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 266, No. 19, Issue of July 5, pp. 12752-12758, 1991 Printed in U.S.A.
Plasminogen Activation by Receptor-boundUrokinase A KINETICSTUDYWITHBOTHCELL-ASSOCIATEDANDISOLATEDRECEPTOR* (Received for publication, February 22, 1991)
Vincent Ellis$, Niels Behrendt, andKeld Dan0 From the Finsen Laboratory, Rigshospitalet, Strandbouleuarden49, DK-2100 Copenhagen 0, Denmark
The specificcellular receptorforurokinase-type type plasminogen activator (uPA)’while intravascular fibrinplasminogen activator (uPA) is found on a variety of olysis is dependent upon tissue-type plasminogen activator. cell typesand hasbeen postulated to play a central role uPA is secreted by a diversityof cells of normal andneoplastic in the mediation of pericellular proteolytic activity. a number of origin, and its expression has been correlated to We have studied the kineticsof plasminogen (Plg) ac- physiological and pathological processes involving cell migrativationcatalyzedbyuPAspecificallyboundtoits tion and tissue remodeling, including tumour invasion and receptor on the human monocytoid cell-line U937 and metastasis (1-3). demonstrate this process to have properties differing A molecule proposed to be intimately involved with the role of uPA as mediator of pericellular proteolysis is the specific widely from those observed for uPA in solution. The solution-phase reaction was characterized by a K,,, of cellular receptor for uPA (4-7). This receptor molecule is 2 5 p~ and for thecell-associated reaction this fell40- found on a variety of cell types and has recently been extenfold to 0.67p ~ below , the physiological Plg concentra- sively characterized (8, 9). The high affinity binding of uPA tion of 2 p ~ A. concomitant 6-fold reduction in kc,, to uPARoccurs through interactions with the amino-terminal resulted in an increase in the overall catalytic effidomain of the enzyme which bears homology to epidermal been ciency, kCat/K,,,, of 5.7-fold. This high affinity Plg ac- growth factor (10) and uPA thus bound has qualitatively tivation wasabolished in the presence of a Plg-binding demonstrated to retain enzymatic activity (4, 5, 11, 12). In antagonist. In contrast to intact cells, purified uPA some adherent cell types, bound uPA hasbeen demonstrated t o be located at discrete sites of cell-cell and cell-substratum receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937 cells) was observed to partially contact (13-15). The cellular binding of plasminogen is an inhibit uPA-catalyzedPlg activation, although activityapparently widespread phenomenon which is characterized against low molecular weightsubstrates was retained. by a high capacity anda low affinity (16-18). Some cell types Therefore, the cellular binding of Plg appears to be of which bind plasminogen have also been demonstrated to bind critical importance for the efficient activationof Plg uPA (16). We have previously demonstrated that bindingof pro-uPA by receptor-bounduPA. Plasmin generated in the cellto uPAR on human monocytic U937 cells results in a rapid surface Plg activation system described here was also reciprocal observed to be protected from its principalphysiolog- acceleration of plasmin generation, through the activation of both zymogens (11).The accelerationis primarical inhibitor a-2-antiplasmin. Together, these data demonstrate that thecell surface constitutes the pref- ily due to an increase in thefeedback activation of pro-uPA cellular binding of erential site for Plg activation when uPA isbound to by the generated plasmin and requires the its specific cellular receptor, which therefore has the plasminogen. We present herea detailed kinetic studyof the effect of uPAR on the enzymatic activity of uPA and demnecessary characteristics to play an efficient role in onstrate that uPA-catalyzed plasminogen activation on the the generationof pericellular proteolytic activity. cell surface occurs with properties differing widely from those in solution and that, by the concomitant bindingof uPA and plasminogen, cell surfaces become preferential sites for plasminogen activation. Plasminogen activators are responsible for the conversion of the abundant extracellular zymogen plasminogen into the active proteinase plasmin. Plasmin is an enzyme of broad specificity and, as well as being responsible for the intravascular degradation of fibrin during blood clot dissolution, is capable of either directly or indirectly degrading all components of them extracellular matrix. This extravascular degradation is thought to be mediated primarily by urokinase-
EXPERIMENTALPROCEDURES
PurifiedProteins-Native Glu-plasminogenwaspurifiedfrom fresh human plasma as previously described (19). This was further separated into itstwo isoforms byelution from lysine-Sepharose with a linear gradient of 6-amino-hexanoic acid, and both isoforms gave single discrete bands on SDS-PAGE under nonreducing conditions. Lys-plasminogen was prepared by limited digestion of Glu-plasminThe abbreviations used are: uPA,urokinase-type plasminogen
* This work was supported by grants from the Danish Biotechnol- activator; uPAR, urokinase-typeplasminogen activator receptor; Gluogy Program and the Danish Cancer Society. 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. $ To whom correspondence should be addressed Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen 8, Denmark. Tel.: (45)35-45-58-76; Fax: (45) 31-38-54-50.
plasminogen, native plasminogen with Glul NH, terminus; Lys-plasminogen, plasmin-degraded plasminogen with primarily Lysv NHZterminal; DFP,diisopropyl fluorophosphate; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; BSA, fatty acid-free bovine serumalbumin; AMC, 7-amido-4-methylcoumarin;tranexamic acid, trans-4-(aminomethyl)-cyclohexanecarboxylicacid SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
12752
Kinetics of Plasminogen Activation ogen withplasmin(20).Plasmin was prepared by activation of plasminogen with uPA in 50% glycerol (19). The Glu-plasminogen mutant (Ser7"'+Ala), expressed in baby hamster kidney cells (21),' was a gift from ZymoGenetics, Seattle. uPA was obtained either by plasmin activation of pro-uPA (22) or as Ukidan (Serono), and both preparations were greaterthan 95% high molecularweight uPA determined by SDS-PAGE. The concentration of active uPA in these preparations was determined by active-site titration with p-nitrophenyl-p-guanidinobenzoate.DFP-inactivated uPA was prepared as described (6). uPAR was purified from lysates of PMA-stimulated U937 cells as previously described (8).The anticatalytic monoclonal antibody to uPAwas clone 2 from (23), and the monoclonal antibody inhibitory to plasmin activity (24) was the kind gift of Dr.Ross Stephens,University of Helsinki. a-2-Antiplasmin was purchased from BehringDiagnostics and its molar concentration determined by titration with plasminas described (25). Aprotinin was obtained from Boehringer Mannheim. Proteins were 1251-labeledwith IODO-GEN as previously described (6). Cell Culture-Cells of the hystiocytic lymphoma cell-line U937 (which have monocyte-like characteristics) were grown in suspension in RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum. PMA stimulation of U937 cells was performed at a cell density of0.5 X lo6 cells/ml with 150 nM PMA for 4days. The adherent cell population was harvested with a rubber scraper and resuspended in PBS. Measurement of Plasminogen Activation by Cell-bound uPA-Prior t o their use in kinetic experiments cells were washed three times in RPMI 1640 and then resuspended in RPMI 1640 containing 2 mg/ ml BSA. Cells were incubated with uPA (1.4 nM) a t a cell count of 2 X 107/ml for 20 min a t 37 "C, followed by three washes in PBS containing 2mg/mlBSA. Alternatively,prior to incubationwith uPA, cells were incubated for three min with0.05 M glycine-HC1, p H 3.0, 0.1 M NaC1, followed by neutralization to remove endogenously bound uPA (26). The cells were then incubated at a final concentration of 1 X lo6 cells/ml in 0.05 M Tris-HC1, p H 7.4, 0.1 M NaCl with varying concentrations of plasminogen and 0.2 mM of the plasminspecific fluorogenic substrate H-D-Val-Leu-Lys-AMC (Bachem). In some incubations tranexamic acid (Sigma) at a concentration of 1 mM was included as an antagonist of plasminogen cellular binding (27).Theseincubations were madein10-mmplasticfluorimeter cuvettes which were maintained a t 37 "C and gently stirred in a Perkin-Elmer Cetus spectrofluorimeter equipped with a micromagat 1-min neticstirrer.The fluorescence intensitywasmeasured intervals a t a n excitation wavelength of 380 nm and an emission wavelength of 480 nm, with both slits set to 5 nm. The plasminogen activator activity observed under these conditionswas demonstrated to be due solely to cell receptor-bound uPA by the criteria described previously (28), which include complete inhibitionby an anticatalytic monoclonal uPA antibody, competition for binding by DFP-inactivated uPA, and also by a polyclonal antibody to the purified uPAR (28). Quantification of Cell Receptor-bound uPA-The concentration of cell-bound uPAwas quantified by measurement of the bindingof '"1uPA in parallel incubations.1.4 nM '""IuPA was incubated with 2 X 10' cells/ml for 20 min at 37 "C, whichwere then washed as described above. Total cell-bound'2sII-uPAwas counted, as well as 1Z51-~PA released by subsequent acid-washing of the cells (this was never less than 90% of the total cell-bound radioactivity).'''I-DFP-inactivated uPA bound to a similar extent to active uPA, and the binding was greater than 85% inhibitable by preincubation with a 100-fold excess of cold DFP-inactivated uPA. The total uPA bound to the cells was examined by SDS-PAGE and autoradiography andwas identical to the addedligand, withno evidence of degradation or inhibitorcomplex formation. Total lY5I-uPA binding to the cellswas determined using acid-washedcells and was within the range 5,000-11,000 molecules/cell for unstimulated U937 cells and 30,000-65,000 molecules/cellfor PMA-stimulated U937 cells under the experimental conditions used. '"I-uPA binding to non-acid-washedcells was lower due to the presence of endogenously bound uPA. In various experiments this ranged from 15 to 35% of total uPA binding. Studies with Purified uPAR-uPAR waspurified fromPMAstimulated U937 cell lysates asdescribed previously (8). The concentration of the purified protein was estimated by amino acid analysis as described (8). All measurements of the effect of purified uPAR ' S . J. Busby, E. Mulvihill, D. Rao, A.A. Kumar, P. Lioubin, M. Heipel,C. Sprecher, L. Halfpap, D. Prunkard, J. Gambee, and D. Foster, manuscript submitted.
by Receptor-bound uPA
12753
were performed in 0.05 M Tris-HC1, pH 7.4, 0.1 M NaCl containing 0.1% Triton X-100. uPA (0.01 nM) was incubatedwith varying concentrations of uPAR (0.4-23 nM) in a total volume of 50 gl for 30 min at 37 "C. 15 gl of a solution containing Glu-plasminogen and Val-Leu-Lys-AMC was then added togive final concentrations of 0.3 p~ and 0.6 mM, respectively. Plasmin generated during 30 min of incubation was determined after additionof aprotinin (80 gg/ml) and subsequentmeasurement of fluorescence intensity. Residual uPA activity was calculated by reference to linear calibration curvesmade withvaryingdilutions of uPA.Subsequenttotheseexperiments chemical cross-linking of uPAR in the incubation mixtures to "'11labeled amino-terminal fragment of uPA was performed (8), to confirm that the plasmin activity generated during this assay procedure had not resulted in proteolytic degradation of the uPAR molecule. After SDS-PAGE and autoradiography, the cross-linked product was identical in apparent molecular weight and intensity to the starting uPAR preparation. Demonstration of Cell-associated Lys-Plasminogen Generation'""IGlu-plasminogen or "sII-Glu-plasminogen (Ser7"-Ala) (0.4 pg/ ml, approximately 0.7 MBq/ml) were incubated with U937 cells (2 X 106/ml, preincubated with uPA as described above) in 0.05 M TrisHC1, pH 7.4, 0.1 M NaC1, 2 mg/ml BSA. After incubation at 37 "C for varying times, the cells were pelleted by centrifugation, washed twice in PBS containing2 mg/ml BSA, and the bound plasminogen eluted by incubation in 0.05 M Tris-HC1, pH 7.4, 0.1 M NaCI, 2 mM tranexamic acid for 10 min. Eluates were prepared for electrophoresis by boiling in 2% SDS and then run on 7.5% SDS-PAGE using the Laemmli buffer system. Autoradiographywas performed on driedgels using Kodak XAR-5 x-rayfilm a t -80 "C. RESULTS
Plasminogen Activation by Receptor-bound uPA on U937 Cells"U937 cells were saturated with uPA and the activation of native Glu-plasminogenobserved by continuous hydrolysis of a plasmin-specific fluorogenic substrate. Thismethodology allows the detection of plasmin generation of as little as0.04 nM over a 15-min period. Measurements of plasmin generation overrelatively shorttimeperiods were necessary to ensure minimal dissociationof uPA from its cellular receptor (less than 5% dissociation in 15 min) and to avoid adverse effects of the generated plasmin upon thecells. Fig. 1 (panel A ) shows the primary data obtained ainsingle representative experiment performed with Glu-plasminogen concentrations between 0.09 and 1.73 WM. The rate of plasmin generation could be calculated directly by fitting these data atoquadratic equation, but to ensure that the generation of plasmin was linear with time the data were replotted as in Fig. 1 (panel B). When these data were plotted in a double-reciprocal manner (Fig. 2), the activation of plasminogen by receptor-bound uPA was found to follow apparently Michaelis-Menten type kinetics. The K,,, and kc,, for the reactionwere determined as 0.67 f 0.30 fitM and 0.12 f 0.06 s-', respectively. When these constants were compared to those for Glu-plasminogen activation by uPA in solution (TableI), it could be seen that the overall catalytic efficiency of the reaction, kC,JKm,was approximately 6-fold higher with receptor-bounduPA. Perhaps surprisingly thiswas due to ratherlarge and opposing effects on kc,, and K,,,,the latter beingdecreased 40-fold upon receptor bindingof uPA. To determine if the cellular binding of plasminogen was involved in the altered kinetics of plasminogen activation, the experiments were repeated in the presenceof the lysine analogue tranexamic acid,which hasbeen shown to abolish plasminogen binding to cells (27). Fig. 2 also shows the data obtainedinsuchanexperiment.Undertheseconditions, although the activation of plasminogen was clearly different fromthatintheabsence of tranexamic acid, it was not possible to accurately determine the individual kinetic constants due to the high K, of the reaction. However, such a
12754
Kinetics of Plasminogen Activation by Receptor-bound uPA
300 -
200 -
DO l
-
0
0
8
4
12
16
minutes
TABLEI Kinetic constants for Glu-plasminogen activation by receptor-bound uPa, compared to the solution-phase reaction Data are shown in the presence and absence of 1 mM tranexamic acid (Trx). The data shownfor U937 cells are the mean f S.D. from five independent experiments and from three experiments for PMAstimulatedandacid-washed cells. The mean k,,,/K,,, values were calculated from the slopes of the individual double-reciprocal plots. The constantsshown were derived with thecellular uPAR essentially saturated (see "Experimental Prcoedures"); however, in preliminary experiments lower degrees of receptor occupancy gave data quantitatively in agreement with those shown.
B
40 L
1
t
VLI
K,
kcat
NM
S-1
u937 -Trx +Trx
0.67 f 0.30 >15"
PMA-U937 -Trx +Trx
1.43 f 0.55 0.023 f 0.015 >15" >0.05"
Acid-washed U937 -Trx 0.11 f 0.06 >15" +Trx
minutes
0.12 f 0.06 >0.85"
0.22 +_ 0.15 >0.65"
kdKm NM" s"
0.165 f 0.048 0.056 f 0.011 0.016 f 0.007 0.004 & 0.003 2.23 f 0.88 0.042 f 0.015
FIG. 1. Plasmin generation by U937 cell receptor-bound uPA. U937 cells saturated with uPA were incubated with Val-Leu-
In solution 25 0.029 -Trx 0.73 Lys-AMC (0.2 mM) and Glu-plasminogen a t varying concentrations +Trx1.85 25 0.074 (0, 0.09; A, 0.15; 0, 0.22; X, 0.35; V, 0.52; 0.78; A, 1.16; B, 1.73 p ~ ) Continuous . substrate hydrolysis was measured byrecording Individual kinetic constants could not be determined accurately fluorescence intensities at 1-min intervals (data froma single repre- in the presenceof tranexamic acid as the double-reciprocal plots had sentativeexperiment shown in panel A ) . The rate of changein intercepts close to zero, therefore, lower limits for these constants are fluorescence between each time point, equivalent to plasmin concen-shown. (panel B ) . The slopes tration, was calculated and plotted against time of theselines were convertedtorates of plasmingeneration by lyzed by uPA in solution. These experiments demonstrate comparisonwithstandardcurvesconstructedusingactive-site-titrated plasmin. The lines shown in panel B intercept the ordinate at that the activation of Glu-plasminogen by receptor-bound values greater thanzero due to minorresidual plasmin activity in the uPA is markedly influenced by plasminogen binding to the plasminogen preparation, whichwas 7.7 X inthis case. cell surface and further suggest that this binding is directly
+,
- 2 0
2
4
6
l/[Glu-Plasminogen]
8
1 0 1 2
uM
FIG. 2. Kinetics of Glu-plasminogen activation by receptorbound uPA on U937 cells. The rates of plasmin generation calculated from theexperimentshownin Fig. 1 were plottedagainst plasminogen concentration in a double-reciprocal manner (W). The kinetic constants obtained from this experiment were K,,, 0.85 pM and Vmax0.85 X lo-' nM s-'. This value of V,,, a t a receptor-bound uPA concentration of 6.3 PM was equivalent to a katof 0.13 s-'. Also shown are data obtained froma single experiment performed in the presence of 1 mM tranexamic acid (0).The Vm.,/K, calculated from pL"l (nM s-I), equivalentto a k,.,/K,,, of the slopewas 0.39 X 0.064 p , ~ " s-'. The mean data from five separate experiments are shown In Table I.
reaction is adequately described in terms of its second-order catalytic efficiency ( kcat/Km). This parameter was found to be close to that for the same reaction catalyzed by uPA in solution (Table I), and furthermore both reactions have a similarly high K,,, (above 15 PM). The activation of solutionphase plasminogen by receptor-bound uPA therefore appears to have characteristics similar to those of the reaction cata-
responsible for the observed high affinity plasminogen activation. In an effort to elucidate further the role of the cellular binding of plasminogen, the kinetics of the activation of its two isolated glycosylation variants by receptor-bound uPA were studied, as these isoforms of Glu-plasminogen have been reported to bind to U937 cells with differing characteristics (29). However, the kinetic constants for their activation were found to be indistinguishable from each other or from the unfractionated plasminogen from which they were derived (data not shown). In some experiments in which the cells had been washed in acidic buffer to remove small amounts of endogenous uPA bound to the cells (26), prior to saturation with exogenously added uPA, it was observed that the activation of Glu-plasminogen was faster than in thepresence of non-acid-washed cells (Fig. 3). This was primarily due to a further reduction of the K,,,to 0.11 PM,which resulted in an overall 14-foldincrease in catalytic efficiency compared to non-acid-washed cells and 77-fold compared to the reaction in solution (Table I). In contrast the reaction in the presence of tranexamic acid was virtually unchanged from that of non-acid-washed cells (Table I). These observations suggest that acid washing of the cells leads to analteration in their plasminogen binding characteristics, and in support of this we have observed that this treatment causes an apparent increase in plasminogen binding,3 although the reason for this effect is unclear. Activation of Lys-Plasminogen by Receptor-bound uPAThe activation of the plasmin degraded form of plasminogen, Lys-plasminogen, by receptor-bound uPA was also studied V. Ellis, unpublished experiments.
Kinetics of Plasminogen Activation by Receptor-bound uPA
TABLEI1 Kinetic constants forLys-plasminogen activation by receptor-bound uPA, compared to the solution-phase reaction Data are shown in the presence and absence of 1 mM tranexamic acid (Trx). The datashown for U937 cells are the mean f S.D. from three independent experiments. The mean kcaI/K,,,value was calculated from theslopes of the individual double-reciprocal plots.
T
L
- 6 - 3 0 3 6 l/[Glu-plosminogen]
12755
9 uM
12
FIG. 3. Kinetics of Glu-plasminogen activation by receptorbound uPA on acid-washed U937 cells. The double-reciprocal ).( and plot of plasminogen activation rates obtained in the absence of 1 mM tranexamic acid is shown for cells which have presence (0) been acid washed to remove endogenously bound uPA and subsequently saturated with exogenously added uPA.The kinetic constants obtained in this single experiment were K,,, 0.18 p~ and V,,, 2.8 X lo-:%nM s-'. equivalentto a kcat of0.35 s-'. Inthepresence of tranexamic acid the V,,,../K,,, calculated from the slope was 0.22 X lo-:' (nM s-l), equivalent to a kCat/K,,,of 0.027 pM-' s-'. The mean data from three separate experiments areshown in Table I.
u937 -Trx +Trx
K"?
kcat
&M
S"
0.30 f 0.19 >lo"
In solution -Trx +Trx
0.2711.5 10.4
kcstIKm
pM"
s-'
*
0.45 f 0.24 >3.0"
1.50 0.39 0.27 f 0.06
3.1 3.2
0.31
" Individual kinetic constants could not be determined accurately in the presenceof tranexamic acid as the double-reciprocal plots had intercepts close to zero; therefore, lower limits for these constants are shown.
._ e,.
LT
6ot
50 I
0
5
[u-PA
10
15
20
I
25
receptor], nM
FIG. 5. Effect of purified uPAR on uPA catalyzed plasminogen activation. Purified uPAR was incubated with uPA prior to determination of residual plasminogen activating activity. The data absence FIG. 4. Kinetics of Lys-plasminogen activation by receptor- are expressed as percentage activity compared to uPA in the of uPAR. All data points shown are the meansof triplicate determibound uPA on U937 cells. The rates of plasmin generation obnations. Inset, the data are shown replotted in a double-reciprocal tained in the absence ).( and presence (0)of 1 mM tranexamic acid manner, with residual uPA activity expressed fractionally. The interare plotted against plasminogen concentration aindouble-reciprocal manner for a single experiment. The kinetic constants obtained from cepts of this plot give a maximum inhibitory effect a t infinite uPAR concentration of 41% and half-maximal inhibition a t a uPAR conthis experiment in the absence of tranexamic acid were K,,, 0.61 p~ and V,, 0.014 nM s-', equivalent to a kc,, of 0.75 s-'. In the presence centration of 1.6 nM, this parameter being equivalent to the Kd for the uPA-uPAR interaction. Thespecificity of these effects was demof tranexamic acid, the V,,,,,/K, calculated from the slope was 4.48 X lo-" p"' (nM s-'), equivalent toa k,.,/K,,, of 0.24 pM-' s-'. The mean onstrated by preincubation of uPAR with a 7.5-fold molar excess of DFP-inactivateduPA, which competedgreaterthan 70% of the data from three separate experiments are shown in Table11. inhibitory effect.
and the data obtained shown in Fig. 4. The cell-associated activation of Lys-plasminogen proceeded much more rapidly than that of Glu-plasminogen, a situation analogous to the reactions in solution. The K,,, was again greatlyreduced when both uPA and plasminogen were cell bound, and similarly there was a concomitant decrease in the kc,, when compared to the reaction in solution (Table 11), with the overall effect of a 5.5-fold increase inkcat/K,.In the presence of tranexamic acid, the activation of Lys-plasminogen by receptor-bound uPA was very similar to thatby uPA in solution (Table11). Plasminogen Activation on PMA-stimulated U937 CellsThe phorbol ester PMA, an inducerof U937 cell differentiation, has been shown to increase uPAR synthesis in these cells, giving anincreaseinuPAbindingtogetherwith a concomitant decrease in binding affinity (30). It was therefore of interest to determine whether these cells also displayed the high affinity plasminogen activation observed on the unstimulated cells. This was in fact the case, with the K,,, for Gluplasminogen activation only slightly changed from 0.67 to 1.43 FM (Table I). However the kcat/K,,,for the reaction was 10-fold lower than with unstimulated cells, as a further reductionin kc,, wasalsoobserved. That this high affinity
plasminogen activation was a consequence of plasminogen binding to the cells was again confirmed by a large increase in the K , in the presence of tranexamic acid, although the catalytic efficiency remained lower than that observed for unstimulated cells or in solution, primarily due atolow value for kcat (Table I). Plasminogen Activation inthe Presence of Purified uPARTodeterminewhetherthebinding of uPA to its specific cellular receptor has anyeffect on the activityof uPA in the absence of other cellular components, experimentswere performed using isolated uPAR, purified from PMA-stimulated U937 cells (8).Incubation of uPA with varying concentrations of purified uPAR (up to 23 nM) resulted in a concentrationdependent decrease in subsequent plasminogen activation by uPA (Fig. 5). This inhibition saturated within the range of uPAR concentrations used, with a maximum observed inhibition of 38%. The observations with isolated uPAR were essentiallyunchangedinthepresence of tranexamic acid (data not shown), in contrast to thelarge differences seen in the intact-cell system. These inhibitory effects appeared tobe steric in nature as in control experiments the active site of
Kinetics of Plasminogen Activation by Receptor-bound uPA
12756
A
The inhibition of Lys-plasminogen generation by aprotinin might suggest that plasmin generated by receptor-bound uPA was responsible for the conversion. However, it was found that Lys-plasminogen generation was not inhibited by preincubation of the cells with either an anticatalytic monoclonal antibody to uPA (50 pglml), theplasminogen activator inhibitor PAI-1 (100 nM), a-%antiplasmin (500 nM) or a monoclonal antibody inhibitoryto plasmin activity (50 pg/ml), and Glu was also resistant toelution by both acid-washing and lysine Lvs analogues. To demonstrate further that the generation of Lysplasminogen was independent of the generation of plasmin activity, similar experiments were performed using a Gluplasminogen active-site mutant Seri4"+Ala. This molecule, - + which is devoid of proteolytic activity upon activation by 60 plasminogen activators, was converted to the Lys-plasmino2 5 10 20 40 60 gen form upon binding to cells in a similar manner to the FIG. 6. Conversion of cell-associated Glu-plasminogen to natural molecule (Fig. 6B). Lys-plasminogen. "'I-Glu-plasminogen was incubated with U937 a-2-Antiplasmin Inhibition of Plasmin Generated by Cellcells (preincubated with uPA), in the presence (+) or absence (-) of aprotinin (100 pg/ml). Bound plasminogen was eluted from the cells bound uPA-The kinetics of plasminogen activation by receptor-bound uPA presented here stongly suggest that plasminat timed intervals (up to 60 min) and run on SDS-PAGE under nonreducing conditions, as described under "Experimental Proceogen is activated while actually bound to thecell surface, and two lanes of each therefore the plasmin generated is presumably also celldures." Also shown on the autoradiograph (the first panel) are 12sII-Glu-plasminogenand "'I-Lys-plasminogen, not incubated with cells and diluted 500-fold compared to the ligand present bound. While we have previously demonstrated that receptorbound uPA is liable to rapid inactivation by plasminogen in theabove incubations. Panel A shows data for normal plasminogen activator inhibitors (28), cell-bound plasmin has been postuand panel B for the plasminogen active-site mutant Ser'"'+Ala. lated to be protected to some degree from its primary physuPA remained fully accesible to low molecular weight uPA- iological inhibitor a-2-antiplasmin (16). Fig. 7A shows the specific peptide substrates and since plasmin activation of effect of addition of a-Z-antiplasmin to U937 cells with receppro-uPA was also inhibited: These observations demonstrate tor-bound uPA after theaddition of Glu-plasminogen. A slow a point fundamental to the understanding of this system, inhibition of plasmin activity was observed, although comwhich is that uPAR does not behave as a "cofactor" for uPA plete inhibition of plasmin activity was not achieved. This catalyzed plasminogen activation.The data could be replotted was in contrast to an enzyme half-life of approximately 1 s in a double-reciprocal manner toobtain a linear relationship determined at thisconcentration of inhibitor with plasmin in (Fig. 5, inset). The experimental conditions employed allow solution (data not shown). The inhibition of plasmin generthe intercept on the abscissa to be used as an approximation ated on the cells was, however, insensitive to variations in ato thedissociation constant for uPA binding to uPAR, which 2-antiplasminconcentration (see legend to Fig. 7). These was estimated to be 1.6 nM. This is slightly lower than the observations suggest that the slow inhibition observed may dissociation constant of 4-17 nM reported for the binding of be a consequence of the dissociation of plasmin from the cell the amino-terminal fragment of uPA to intact PMA-stimusurface and itssubsequent rapid inhibition. lated U937 cells (30). The interpretation of these observations was confirmed in Conversion of Cell-bound Glu-Plasminogen to Lys-Plasminexperiments using exogenous plasmin prebound to the cells ogen-We have demonstrated here that cell-bound Lys-plasminogen is activated with more favorable kinetics than the (Fig. 7B). Subsequent to the binding of plasmin to the cells, native Glu-plasminogen form by receptor-bound uPA. It is of plasmin activity was continuously measured both before and importancetherefore to know whetheractivation of Glu- after theaddition of a high concentration of a-2-antiplasmin. plasminogen on the cell surface results in the generation of In each experiment the addition of a-Z-antiplasmin led to a Lys-plasminogen by the action of the plasmin formed. We rapid decrease in plasmin activity. However, the magnitude have studied this using '2sI-Glu-plasminogen, which was in- of this decrease was proportional to thetime before addition cubated with U937 cells in the presence or absence of apro- of the inhibitor (data not shown) and thus reflects the inhitinin. The plasminogen was subsequently eluted from the bition of plasmin which has dissociated from the cells. The cells with tranexamic acid and the presence of Lys-plasmin- inhibition of the remaining (i.e. cell-associated) plasmin was ogen determined by SDS-PAGE under nonreducing condi- very slow. This inhibition was also due to dissociation of tions. Fig. 6A shows such an experiment; it can be seen that plasmin from the cells, as the data points during this slow Lys-plasminogen was generated upon incubation of the cells inhibition phase extrapolated close to 100% plasmin activity with Glu-plasminogen and that this conversion could be in- a t zero time i.e. 5 min before addition of inhibitor in the hibited by aprotinin. Lys-plasminogen was formed very rap- experiment shown in Fig. 7B. In contrast if a-2-antiplasmin idly, but the proportion of Lys- to Glu-plasminogen appeared was added together with 6-aminohexanoic acid, in order to to increase only slowly upon longer incubation. This was dissociate cell-bound plasmin, there was a rapid inhibitionof confirmed by counting radioactivity in the excised bands greater than 95% of the plasmin activity (Fig. 7B). This which demonstrated approximately 30% Lys-plasminogen at experiment also demonstrates that the reduction in plasmin 2 min of incubation, rising to approximately 55% at 60 min. No conversion of Glu-plasminogen was detected in the cell inhibition upon cellular binding is much more pronounced than thatcaused by lysine analogues (31), and indeed may be supernatants or in the absence of cells with similaruPA close to a complete protection from inhibition, the difference concentrations. in inhibition rates of free and cell-associated plasmin estimated from the datahere being a t least 4 orders of magnitude. N. Behrendt, unpublished experiments. -
T
-
T
-
r
"
T
-
7
-
7
" " "
Kinetics of Plasminogen Activation by Receptor-bound uPA A
12757
K,,, for plasminogen activation in the presence of cells, 0.67 p ~ is, within the range of Kd values determined for the equilibrium binding of plasminogen to this and other cell types, 0.3-2.8 p~ (18).The saturationof the cell surface with plasminogen therefore results in a saturation of the rate of plasminogen activation. Such an effect could arise either from the direct activation of cell-bound plasminogen or from an increased local concentration of plasminogen in the cellsurface micro-environment due to the high density of plasminogen bound to the cell. It is possible to discriminate 0 5 10 15 20 between these two mechanisms on the basis of the kinetic minutes data, as the lattermechanism would be expected to result in an alteration only in the K , for the reaction, whereas we also observe an effect on kcat. From this we can postulate that receptor-bound uPA is able to activate plasminogen while it is actually bound to the cell surface. The reduction in kcat, observed concomitantly with the reduction in K,, may therefore result from restrictions imposed upon the system by the relative immobilization of the reactants upon binding to the cell surface. The hypothesis that receptor binding does not alter the intrinsic enzymatic properties of uPA is consistent with the postulated domain structure of uPA (10) and also with our ‘0 5 x) 15 20 minutes previous observations that receptor binding has very little FIG. 7. Effect of a-2-antiplasmin on the plasmin activity effect on the inhibition of uPA by the specific plasminogen generated on U937 cells. Panel A , cells preincubated with uPA activator inhibitors PAI-1 and PAI-2 (28). To test this hywere incubated with 0.25 PM Glu-plasminogen and plasmin genera- pothesis further we studied the effect of purified uPAR on tion measured as described in the legend to Fig. l. After 8.5 min, a2-antiplasmin was added, and themeasurement of plasmin generation uPA catalyticactivity. Purified uPAR behaved as anapparent inhibitor of plasminogen activation but gave only a 40% continuedin both this incubation (0) and acontrol incubation without a-2-antiplasmin (B).The datashown were obtained using an inhibition at saturating concentrations of receptor. Although CY-2-antiplasmin concentration of 30 nM, but theinhibition of plasmin this effect was not analyzed kinetically, the lack of effect of activity was essentially the same using a-2-anti-plasmin concentra- tranexamic acid suggests that large changes similar to those tions of 10, 60, and 100 nM. Panel B shows an experiment in which with cell-associated uPAR are not occurring. The inhibitory cells were preincubated with plasmin (500 nM for 10 min), followed by two washes in PBS-BSA. Plasmin activity then was measured effect may berelated to thereduced efficiency of plasminogen continuously as above, and at 5 min a-2-antiplasmin (500 nM) was activation observed on PMA-stimulated cells. These cells added in the presence (A)or absence (B) of 6-aminohexanoic acid were the source of the purified uPAR, which is known to be (final concentration 10 mM). more heavily glycosylated than the molecule from unstimulated cells (8). This reduced efficiencywashowevermuch DISCUSSION larger with cell-associated uPAR, kcat/& being 10-14-fold less than on unstimulated cells. It is possible that other effects We have demonstrated that uPA bound to its specific cellular receptor molecule on U937 cells activates native Glu- contribute to thereduced efficiency of plasminogen activation plasminogen with kinetic characteristics differing widely from with PMA-stimulated cells, particularly effects causing a rethose observed for uPA in the solution phase. The primary duced accessibility of uPA to plasminogen, possibly asa effect was a reduction in the K , for plasminogen activation, consequence of the large changes in cellular morphology and which fell from 25 p~ in solution to 0.67 pM with cell receptor- uPAR distribution (32)seen upon PMA stimulation. We have bound uPA. This K , is significantly below the normal plasma also observed a differential in the rates of inhibition of recepconcentration of plasminogen (approximately 2 p M ) , which tor-bound uPA on these two types of cells by plasminogen suggests that this reaction may be of physiological signifi- activator inhibitors (28), and thebinding affinity for uPA has cance. Large reductions in K , were also observed with acid- also been shown to be decreased (30). However, further clarwashed cells, PMA-stimulated cells, and for the activation of ification of the properties of these two forms of the receptor must await purification of uPAR from unstimulated U937 Lys-plasminogen. The high affinity plasminogen activation by uPA bound via cells in sufficient quantity toenable detailed kinetic analysis. The datadiscussed here demonstrate that thecellular binduPAR to the cell surface was demonstrated to be dependent upon the cellular binding of plasminogen as it was abolished ing of plasminogen has the importantfunctional consequence by the plasminogen-binding antagonist tranexamic acid. In- that thecell surface becomes the preferential sitefor plasminclusion of tranexamic in the kineticdeterminations with ogen activation when uPA is receptor-bound. Howeverno unstimulated U937 cells completely reversed the effects specific cellular binding molecules for plasminogen have been caused by the presence of the cells, so that (within the demonstrated, although it has been postulated that plasminogen binds to both protein andlipid (ganglioside) moieties on constraints of determining the individual constants under these conditions) the kinetics were virtually indistinguishable the cell surface (33). The high binding capacity of many cell from those of the reaction in solution. Consequently, the types for plasminogen suggests that the interaction is relaaltered kinetics observed in thepresence of the cellular recep- tively nonspecific in terms of the binding molecules involved tor for uPA appear to be mediated through the plasminogen and the binding is known to be of relatively low affinity. binding capability of the cells, rather than to an alteration of These characteristicsmay beessential for cell-associated plasthe intrinsic enzymatic properties of uPA. In this respect it minogen activation as a large “reservoir” of plasminogen can is of particular interestto note that thekinetically determined be present on the cell surface and the low affinity binding,
12758
Kinetics of Plasminogen Activation Receptor-bound by
uPA
P., Nielsen, L. S. & Skriver, L. (1985) Adu. Cancer Res. 44, which suggests a rapid equilibrium of free and bound ligand, 139-266 may be necessary to maintaina continuing supply of plasmin3. Blasi, F., Vassalli, J.-D. & Dan@,K. (1987) J . Cell Biol. 104,801ogen t o receptor-bound uPA. Modulation of either of these 804 characteristics of plasminogen binding may therefore be ex4. Vassalli, J.-D., Baccino, D. & Belin, D. (1985) J . Cell Biol. 100, pected to alter the efficiency of plasminogen activation by 86-92 uPA bound to itscellular receptor. 5. Stoppelli, M. P., Corti, A,, Soffientini, A., Cassani, G., Blasi, F. & Associan, R. K. (1985) Proc. Natl. Acad. Sci. U. S.A. 8 2 , The conversion of native Glu-plasminogen to Lys-plasmin4939-4943 ogen upon binding toU937 cells has previously been reported Kellerman, G. M., Behrendt, N., Picone, R., Danb, L. S., (34). Our data confirm this observation and further demon- 6. Nielsen, K. & Blasi, F. (1988) J. Biol. Chem. 263, 2358-2363 strate that this limited proteolytic degradation is a rather 7. Estreicher, A., Wohlwend, A,, Belin, D., Schleuning, W.D. & rapid process, which is not affected by specific inhibitors of Vassalli, J.-D. (1989) J . Biol. Chem. 2 6 4 , 1180-1189 plasmin or plasmin generation.However, the inhibitionof the 8. Behrendt, N., R@nne,E., Ploug, M., Petri, T., L@ber,D., Nielsen, L. S., Schleuning, W.-D., Blasi, F., Appella, E. & Dan@,K. conversion by aprotinin verified that the event was proteolytic (1990) J . Bid. Chem. 2 6 5 , 6453-6460 in nature, rather than being due to preferential binding of 9. Roldan, A. L., Cubellis, M.V., Masucci, M. T., Behrendt, N., Lys-plasminogen possibly present as a contaminant in the Lund, L. R., Dan@,K., Appella, E. & Blasi, F. (1990) EMBO J. '"I-Glu-plasminogen preparation. The independence of this 9,467-474 proteolytic conversion from the generation of plasmin activity 10. Appella, E., Robinson, E. A., Ulrich, S. J., Stoppelli, M. P., Corti, A., Cassani, G. & Blasi, F. (1987) J. Biol. Chem. 2 6 2 , 4437was confirmed in experiments using a Glu-plasminogen ac4440 tive-site mutant. Therefore, the generationof Lys-plasminogen appears to be mediated by a cell-associated proteinase 11. Ellis, V., Scully, M. F. & Kakkar, V. V. (1989) J. Biol. Chem. 264,2185-2188 other than plasmin, as has been speculated for endothelial 12. Stephens, R. W., Pollanen, J., Tapivaara, H., Leung, K.-C., Sim, cells (35), althoughitcannotbe absolutelyexcluded that P.-S., Salonen, E -M., R@nne,E., Behrendt, N., Danb, K. & plasmin tightly bound to thecells and possibly derived from Vaheri, A. (1989) J. Cell Biol. 108, 1987-1995 the culture medium is involved in thisprocess. In spiteof the 13. Pollanen, J., Saksela, O., Salonen, E."., Andreasen, P., Nielsen, L., Dan@,K. & Vaheri, A. (1987) J. Cell Biol. 1 0 4 , 1085-1096 generation of cell-associated Lys-plasminogen, it is not clear whether thisis the primary substratefor receptor-bound uPA 14. Pollanen, J., Hedman, K., Nielsen, L. S., Dan@,K. & Vaheri, A. (1988) J. Cell Biol. 106,87-95 when Glu-plasminogen is added to thecells. The proportion- 15. Hebert, C. A. & Baker, J. B. (1988) J. Cell Biol. 106, 1241-1247 ality between the rates of Glu- and Lys-plasminogen activa- 16. Plow, E. F., Freaney, D. E., Plescia, J. & Miles, L. A. (1986) J. tion in solution is maintained on the cell surface (Tables I Cell Biol. 103,2411-2420 and 11) rather thanbeing decreased in the presenceof cells as 17. Hajjar, K. A,, Harpel, P. C., Jaffe, E. A. & Nachman, R. L. (1986) J. Biol. Chem. 2 6 1 , 11656-11662 may be expected if generated Lys-plasminogenwere the substrate. Furthermore, the generation of plasmin from added 18. Miles, L. A. & Plow, E. F. (1988) Fibrinolysis 2, 61-71 K. & Reich, E. (1979) Biochim. Biophys. Acta 566, 138Glu-plasminogen is linear with time. However, the plasmin 19. Dana, 151 present on thecell surface (determined by running gels such 20. Petersen, L. C., Lund, L. R., Nielsen, L. S., Danb, K. & Skriver, as inFig. 6 under reducing conditions) appears to be primarily L. (1988) J. Biol. Chem. 263, 11189-11195 in theLys-plasmin form, although theconversion in this case 21. Lijnen, H. R., Van Hoef, B., Nelles, L. & Collen, D. (1990) J. may have occurredsubsequent to plasminogen activation. The Biol. Chem. 265,5232-5236 exact nature of the plasminogen substrate for cell-receptor- 22. Ellis, V., Scully, M. F. & Kakkar, V. V. (1987) J . Biol. Chem. 262,14998-15003 bound uPA remains therefore to be determined. 23. Nielsen, L. S., Andreasen, P. A,, Grbndahl-Hansen, J., Huang, JWe have demonstrated previously that plasminogen/plasY., Kristensen, P. & Dan@,K. (1986) Thromb. Haemostasis55, minbindingto U937 cells is necessaryfor the feedback 206-212 amplification of plasmingeneration when receptor-bound 24. Sim, P-S., Fayle, D. R. H., Doe, W. F. & Stephens, R. W. (1986) Eur. J. Biochem. 158,537-542 uPA is initially inthesingle-chain proenzyme form (11). 25. Wiman, B. & Collen, D. (1978) Eur. J. Biochem. 84, 573-578 Togetherwiththeprotection of cell-boundplasminfrom M. P., Tacchetti, C., Cubellis, M. V., Corti, A., Hearing, inhibition by a-2-antiplasmin observed here, these data pro- 26. Stoppelli, V. J., Cassani, G., Appella, E. & Blasi, F. (1986) Cell 45, 657vide further evidence that the cell surface constitutes a pri684 mary sitefor plasmin generation and activity. The cell surface 27. Miles, L. A. & Plow, E. F. (1985) J. Biol. Chem. 260,4303-4311 therefore has the potential toefficiently generate proteolytic 28. Ellis, V., Wun, T.-C., Behrendt, N., Rbnne, E. & Dan@,K. (1990) J. Biol. Chem. 265,9904-9908 activitythroughthebinding of plasminogen, and also to regulate and direct this activity through the expression and 29. Gonzalez-Gronow, M., Edelberg, J. M. & Pizzo, S. V. (1989) Biochemistry 28, 2374--2377 discrete cellular localization of the specific receptor for uPA. 30. Picone, R., Kajtaniak, E. L., Nielsen, L. S., Behrendt, N., Mas-
Acknowledgments-We would like to thank Bente Johannessen and Marianne Val10 Nielsen for their excellent technical assistance, Dr. Ross Stephens for the gift of the anti-plasmin monoclonal antibody, and Zymogenetics Corporation and Dr. Lars-Christian Petersen (Novo-Nordisk) for the gift of Glu-plasminogen Ser74"-+Ala. REFERENCES 1. Reich, E. (1978) in Molecular Basis of Biological Degradatiue Processes (Berlin, R. D., Herrmann, H., Lepow, I. H. & Tanzer, J. M., eds) pp. 155-169, Academic Press, New York 2. Dan@,K., Andreasen, P. A., Grbndahl-Hansen, J., Kristensen,
31. 32. 33. 34. 35.
tronocola, M.R., Cubeilis, M.V., Stoppelli, M. P., Pedersen, S., Dan@,K. & Blasi, F. (1989) J . Cell Bid. 108, 693-702 Wiman, B., Boman, L. & Collen, D. (1978) Eur. J . Biochem. 87, 143-146 Hansen, S.H., Behrendt, N., Dan@,K. & Kristensen, P. (1990) Exp. Cell Res. 187,255-262 Miles, L. A., Dahlberg, C. M., Levin, E. G . & Plow, E. F. (1989) Biochemistry 28,9337-9343 Silverstein, R. L., Friedlander R. J., Nicholas, R. L. & Nachman, R. L. (1988) J . Clin. Inuest. 8 2 , 1948-1955 Hajjar, K. & Nachman, R. L. (1988) J. Clin. Inuest. 82, 17691778
