binding and activation of plasminogen on the surface of osteosarcoma cells

JOURNAL OF CELLULAR PHYSIOLOGY 159:l-10 (19941

Binding and Activation of Plasminogen on the -

Surface of Osteosarcoma Cells PHIL G. CAMPBELL,* KAREN WINES, THOMAS B. YANOSICK, AND JOSEF F. NOVAK

Orthopaedic Research Laboratory, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania 15272

Plasmin (Pm) is a broad action serine protease implicated in numerous physiological functions. In bone, Pm may play a role in growth, resorption, metastasis, and the activation of growth factors. The various components of the Pm system are known to bind and function on the cell surface of various cell types, but no pertinent data are available describing membrane-bound Pm or its zymogen, plasminogen (Pg), in either normal or neoplastic bone cells. We report here that Pg binds to the surface of the human osteosarcoma cell line MG-63 and i s activated to Pm by endogenous urokinase plasminogen activator (uPA). These conclusions are based on experiments utilizing radiolabeled compounds and a cell surface proteolytic assay measuring amidolytic activity of Pm. I2%Pg binding to cells was time dependent, saturable, reversible, and specific. Binding was characterized by a relatively low affinity (Kd -0.9 FM) and a high capacity (-7.5 x lo6 siteskell). The binding of 12s1-Pg was associated with lysine binding sites of the plasrninogen molecule. Activation of 1251-Pg to 1251-Pm occurred on the cell surface and was dependent upon cell bound uPA, a5 determined by inhibitory antibodies. Binding of Pg to MG-63 monolayers represented -80% bound specifically to the cell surface and the remainder to the surrounding extracellular matrix. Either co-incubation with uPA or pre-incubation with Pm resulted in increased 1251-Pg binding to osteosarcoma cells. Cell surface Pm proteolytic activity was confirmed by an amidolytic chromogenic assay. Both Pm and Pg bound to cells with Pg being activated by endogenous uPA. Plasmin activated on the cell surface was partially protected from inhibition by a2-antiPm (requiring Pm lysine binding site interaction) but inhibited by aprotinin, (interacting directly with the Pm catalytic site). Resistance of cell bound Pm to a2-antiPm inhibition suggests that cell surface proteolysis can occur in the presence of a soluble Pm inhibitor known to exist in the extracellular space. Based on these results, we speculate that the various bone physiological processes implicating Pm may occur at or near the bone cell surface. o 1994 Wiley-Liss, Inc.

The plasmin (Pm) system, although classically associated with fribinolysis, is also involved in many other cell/extracellular matrix interactions, including mor- phogenesis, ovulation, mammary gland involution, dif- ferentiation, and metastasis (Dano et al., 1985; Sak- sela, 1985; Saksela and Rifkin, 1988). In bone, Pm is implicated in resorption (Hamilton et al., 1985; Allen et al., 1986; Pfeilschifer et al., 1990, Grills et al., 1990; Allan et al., 1991; Fukumoto et al., 1992; Hoekman et al., 1991; Leloup et al., 1991). Secretion of plasminogen activators (PAS) by osteosarcoma cells suggests a possible role in metastasis (Hamilton et al., 1985; Allen et al., 1986; Hoekman et al., 1991; Fukumoto et al., 1992; Allan et al., 1991). Plasmin activated by endogenous PAS is also known to activate growth factors in bone, including transforming growth factor @, insulin-like growth factor-I (IGF-I), and IGF-I1 (Novak et al., 1991; Allan et al., 1991; Campbell et al., 1992). Furthermore, activation of IGF-I is responsible for the growth stimu- latory response of Pm in osteosarcoma cells (Campbell and Novak ,1991). 0 1994 WLLEY-LISS, INC.

Plasminogen (Pg) is the zymogen for Pm which occurs in plasma and in the interstitial space, and is activated by PAS. In vivo, bone is exposed to an abundant amount of Pg present in the bone marrow (Barnhart and Riddle, 1963). In addition, bone is known to secrete the two principle PAS, urokinase PA (uPA) and tissue- type PA (tPA) (Leloup et al., 1991). Bone, therefore, contains all primary components necessary for Pm generation.

There has been an increasing interest concerning the localization of Pm proteolytic activity at specific sites of action (i.e., the extracellular matrix (ECM) and cell surface). Emerging evidence indicates that Pm forms at the cell surface or in association with ECM components (Osada et al., 1991; Ellis et al., 1991; Gonzalez-Gronow et al., 1991; Plow et al., 1986; Stephens et al., 1989;

Received October 9,1992; accepted October 27,1993. *To whom reprint requestsicorrespondence should be addressed.

2 CAMPBELL ET AL.

Miles and Plow, 1985). Plasmin formed at these sites is dependent upon lysine binding sites in the heavy chain region of the Pm molecule and is resistant to inhibition by such inhibitors as a2-antiPm (Plow et al., 1986; Stephens et al., 1989; Ellis et al., 1991; Hall et al., 1991) and a2-macroglobulin (Hall et al., 1991). Distinct cellular receptors exist for PmlPg, uPA, and tPA at the cell surface of numerous cell types (Miles and Plow, 1988; Saksela and Rifkin, 1988). Urokinase receptors have been identified on osteosarcoma cells (Rabbani et al., 19901, but whether bone cells localize Pm activity upon their surface remains unknown.

We report here that Pg binds to the human osteosarcoma cell line, Mg-63 and is activated to proteolytically active Pm by endogenous uPA, and that Pm on the surface of bone cells is resistant to inhibition by a2- antiPm. Cell surface Pg activation by PAS may represent the site of physiologically relevant Pm action in bone.

Limited aspects of this work were presented at the 2nd International Workshop on Insulin-Like Growth Factor Binding Proteins, Opio, France (Campbell et al., 1993).

MATERIALS AND METHODS Materials

Glu-plasminogen was purified from fresh frozen human plasma using lysine-Sepharose (Deutsch and Mertz, 1970) in the presence of 1 mM benzamidine, 50 p,g/mol trypsin inhibitor, and 20 KIU/ml aprotinin. Glu-plasminogen was >95% pure and >98% activatable by tPA and had no apparent Lys-Pg contamination as determined by SDS-PAGE under reducing conditions. No measurable Pm contamination was observed using S-2551 chromogenic assay and SDS-PAGE under reducing conditions. Plasmin, single chain uPA (scuPA), aa-antiPm, and inhibitory antibodies to PAS were purchased from American Diagnostica (Green- wich, CT), Two-chain activated uPA and tPA were purchased from Calbiochem (La Jolla, CA). Lysine ana- logues E-aminocaproic acid (EACA) and tranexamic acid were purchased from Sigma (St. Louis, MO). Pg was iodinated using the Chloramine-T method to 1.5-4 p,Ci/pg protein (Miles and Plow, 1985). Aprotinin was purchased from Miles Inc. (Kankakee, IL).

Cell culture Human osteosarcoma MG-63 cells were obtained

from ATCC (Rockville, MD). MG-63 cells were main- tained in alpha minimum essential medium/Ham's F-12 Nutrient Mixture ( 0 1 2 , 1/11 containing 10% heat inactivated fetal bovine serum (FBS), penicillin/ streptomycin (50 U/m1/50 pg/ml), and 2 mM glutamine

ECM IGF-I

2 n tPA *A a2-antiPm EACA

Ab breuiations

extracellular matrix insulin-like growth factor-I plasminogen plasmin tissue-type plasminogen activator urokinase plasminogen activator a-anti-plasmin eaminocaproic acid.

in humidified 5% CO, atmospheres at 37°C. All experiments utilized passages 92-102. Cells were released with trypsidEDTA, and cell number was determined by either hemocytometer or Coulter Counter (Coulter Electronics, Hialeah, FL). Cell viability was determined by trypan blue exclusion.

lZ5I-Pg binding studies Cells were grown in 24-well plates (Flow Laborato-

ries, McLean, VA) in serum containing a/F12. At confluence, serum containing media was aspirated, and monolayers were washed twice with PBS and preincu- bated for a t least 2 h, 37"C, with serum-free 0 1 2 media containing 100 pg/ml BSA (RIA grade, Sigma, St. Louis, MO) and 10 pg/ml transferrin (BT-medium). BT- media was aspirated and monolayers were rinsed with PBS. Forty nanomoles 1251-Pg was incubated with additions as described in the figure legends in a total incubation volume of 0.25 ml binding buffer (0.1 M HEPES, 0.12 M NaCl, 5 mM KC1,1.2 mM MgS04, 9 mM glucose, and 1% BSA, pH 7.4) at 37°C for indicated times. Incu- bations were terminated by rinsing all monolayers three times with ice-cold PBS. Cells were solubilized in 1 N NaOH and the digest counted for radioactivity. In certain experiments, bound radioactivity was extracted by 1% deoxycholate to determine binding to the cell layer, while binding to the ECM was determined by extraction of the deoxycholate insoluble fraction with 4% SDS (McKeown-Longo and Mosher, 1983).

Activation of "'I-Pg to 1251-Pm Cells were grown to confluence in 12-well plates

(Flow Laboratories, McLean, VA) in serum containing media. lZ5I-Pg was equilibrated with MG-63 cells as described above with additions as described in figure legends. Incubations were terminated by washing cells three times with PBS and extracing bound 1251-Pg or 1251-Pm with 200 mM EACA for 10 min, 23"C, with gentle agitation. Aliquots of EACA washes were diluted in reducing Laemmli sample buffer and run over 11% SDS-PAGE (Laemmli, 1970), dried, and autoradio- graphed.

$3-2251 chromogenic assay for cell bound P m Cells were grown to confluence in serum containing

media in 12-well plates. At confluence, serum containing media were aspirated and cells were washed once with PBS. Cells were incubated in BT-medium for 24 h, 37°C. BT-medium was aspirated and cells were washed once with PBS. Cells were incubated with unlabeled PM or Pg in binding buffer as described in figure legends. Incubation conditions were 1 h, 37°C for Pm and 3 h, 37°C for Pg. Treatment additions are described in figure legends. Incubations were terminated by aspiration of treatments and rinsing cells three times with PBS. The chromogenic substrate D-Val-L-Leu-L-Lys-p- nitroanilide hydrochloride (S-2251; KabiVitrum, Inc., Franklin, OH) was added at a final concentration of 0.18 mM in 525 pl binding buffer. Unless otherwise stated, after 1 h, 37"C, a 200 p1 aliquot from each well was read a t in a Molecular Devices microplate reader (Menlo Park, CA). The concentration of Pm bound directly to or generated on the cell surface from

PLASMIN ACTIVITY ON OSTEOSARCOMA CELLS 3

- I

N - 0.0 0 15 30 45 60 75 90 105120

Time, Min

Fig. 1. Binding of 1251-Pg to MG-63 cells, time dependency, and competition by unlabeled Pg. MG-63 cells were prepared for radiolabel binding experiments as described in Materials and Methods. The time dependency of 1z61-Pg binding (A) was determined by incubating monolayers with 40 nM '"I-Pg in binding buffer for indicated times at 37°C; incubation buffer was aspirated, and cells were rinsed three times with ice-cold PBS. Cells were solubilized in 1 N NaOH and counted for radioactivity. Total binding (0) represents the addition of IZ5I-Pg alone, and nonspecific binding (V) was determined by the in-

Pg was determined against a Pm standard curve under identical but cell-free conditions.

Statistical analysis Statistical analysis was performed by analysis of

variance followed by Tukey's HSD test using multivari- ate general linear hypothesis (Wilkinson, 1990). Where appropriate, Student's t-test was used to compare two means. Probability values less than 0.05 were considered significant.

RESULTS 1251-Pg binding experiments

1251-Pg binding to MG-63 cells was time dependent, equilibrating by 1 h at 37°C (Fig. 1A). Binding was saturable with an ED,, of 0.6 KM for unlabeled Pg (mean of six experiments) competing against 12,1-Pg (Fig. 1B). Plasminogen binding was specific as competition for "'I-Pg did not occur with the addition of immu- noglobulin, transferrin, fibrinogen, or ovalbumin up to concentrationsof 10 pM (data not shown). Competition by eACA for the cellular lysine residues resulted in maximal inhibition at 0.4 mM maintaining a constant BlBo of 0.07 or 7% above nonspecific binding (40 pM unlabeled Pg). Binding isotherms were constructed using a constant concentration of 1251-Pg (40 nM) equilibrated against an increasing concentration of unlabeled Pg. Scatchard plots were generated using LIGAND program as originally developed by P.J. Mun- son and D. Robard and modified for microcomputers by McPherson (1985). MG-63 cells exhibited Pg binding which was of low affinity and high capacity, with a Kd of 0.92 * 0.30 pM and 7.4 * 3.7 x lo6 sites/cell

0.6 - 0 e m

0.4 -

0.2 -

0.01 0.1 1 10 100

Plasminogen, pM

clusion of 40 pM unlabelled Pg during incubations. Individual symbols represent the mean 2 SEM of duplicate determinations. Compe- tition for 1z51-Pg binding by unlabeled Pg (B) was determined by equilibrating cells with 40 nM 1251-Pg in the presence of increasing concentrations of unlabeled Pg for 1 h, 37°C. Incubations were terminated as described above. Symbols depict the binding of '"I-Pg corrected for nonspecific binding (40 pM unlabeled Pg) and respresent the mean * SEM of six separate experiments.

(mean +- SEM of five separate experiments). A representative Scatchard plot is presented in Figure 2. These data are in agreement with those from numerous other cell types which suggest that Pg binding to cells forms a substantial pool of Pg a t the cell surface (Miles and Plow, 1988).

12'I-Pg binding to MG-63 cells was reversible (Fig. 3). After equilibration of 1251-Pg with MG63 cells for 1 h, 37"C, unbound 1251-Pg was removed by rinsing with PBS. Cells were incubated with either binding buffer or 20 p M unlabeled Pg at 1 h, 37°C for indicated times. By 1 h, 91% of bound 1251-Pg was dissociated from cells. Dissociation of 1251-Pg also occurred in the presence of binding buffer alone with a BlBo a t -45% of initial binding. The loss of 12'I-Pg under buffer conditions probably reflects the relatively low affinity of the Pg receptor. Similar dissociations have been previously reported for other cell types (Miles and Plow, 1985; Plow et al. 1986).

Plasminogen is cleaved from a single chain protein molecule to the active two chain Pm molecule, upon activation by PAS. It is therefore possible to differenti- ate between cell bound Pg and Pm using reducin SDS- PAGE (Miles and Plow, 1985; Plow et al., 1986). F251-Pg bound to the MG-63 cell monolayers was activated to lZ5I-Pm (Fig. 4). 12,1-Pg used in these studies did not contain Pm (i.e., no radioactivity migrated at a M, of 58 kDa which represents the heavy chain (HC) portion of Pm). When bound to MG-63 cells, 1251-Pg was activated to Pm as evidenced by the appearance of the 58 kDa band of radioactivity. Using densimetric scans of auto- radiographs to quantitate Pm formation, Pm HC formation in the presence of uPA antibodies was

4

l o I

CAMPBELL ET AL.

0 2 4 6 8 1 0

Plasmmogen. UM

0'03 0.02 t \" k rn

0 2 4 6 8 10

Plasminogen Bound, pmole/lO cells 6

Fig. 2. Scatchard plot for the binding of 1251-Pg to MG-63 cells. Bind- ing isotherms for Pg were determined by incubating increasing concentrations of labeled Pg with a constant concentration of '"I-Pg. Incubations were for 1 h, 37°C. Incubations were terminated, Pg bound was determined (see Materials and Methods), and Scatchard plots were developed. The specific binding isotherm is presented in the insert.

28.2 * 16.5% of control (mean ? SEM of three experiments; P < 0.05, Tukey's HSD test), suggesting that the activation of lZ5I-Pg to lZ5I-Pm was dependent upon uPA. Antibodies to tPA were without affect with HC formation at 76.3 5 25% of control (mean * SEM of three experiments; P = 0.784, Tukey's HSD test). Co- incubation of lZ5I-Pg with EACA resulted in minimal 1251-Pg binding and no HC formation. Co-incubation of lZ5I-Pg with aprotinin resulted in a significant reduc- tion in HC formation (20.6 * 13.6% of control, mean * SEM of three experiments; P < 0.05, Tukey's HSD test). A higher M, for the HC was observed in the aprotinin treatment lane. Aprotinin does not block the activation of Pg to Pm but binds to the active catalytic site of Pm inhibiting is activity. Plasmin converts Glu-Pg to Lys-Pg, and the resulting Lys-Pm exhibits a HC M, of -10 kDa less than Glu-Pm (Robbins, 1978). In the present study, aprotinin apparent1 inhibits the

appearance of an apparent HC radioactive band at -68 kDa reflects the activation of 1251-G1~-Pg to 1251-Glu- Pm, presumably by cell surface active uPA. This is supported by our observation that a substantial portion of uPA in the conditioned media surrounding MG-63 cells is active (unpublished results from our laboratory). It is most likely that uPA was activated by cell surface Pm produced during exposure of cells to serum media containing activatable Pg. These data are also consistent with our previous observation that MG-63 cells secrete primarily uPA (Campbell et al., 1992) and that osteosarcoma cells exhibit uPA receptors (Rabbani

formation of 1251-Lys-Pg and resulting l2 B I-Lys-Pm. The

0-4 t I

0.2

0.0 0 15 30 45 60 75 90 105 120

Time, min

Fig. 3. Reversibility of lZ5I-Pg binding to MG-63 cells. MG-63 cells were equilibrated with Iz5I-Pg as described in Materials and Methods. After 1 h, unbound lZ5I-Pg was removed by rinsing with PBS. Fresh binding buffer containing 20 pM unlabeled Pg (W or not (0 ) was added and samples were incubated at 37°C. At indicated timepoints, incubations were terminated and 1251-Pg remaining bound to cells was determined. Individual points represent mean i SEM of duplicate determinations.

et al., 1990), including MG-63 cells (unpublished results from our laboratory).

The binding of lZ5I-Pg was influenced by both its activator or its activated product. Co-incubation of '"I-Pg with 30 nM active two chain uPA resulted in an increase in lZ5I-Pg binding to MG-63 cells of 371 2 84% of control (mean * SEM of seven experiments). The inclusion of aprotinin with uPA did not consistently inhibit the uPA effect (75 * 14% of uPA control; mean * SEM of three experiments). Furthermore, single chain precursor (scuPA) at 15 nM resulted in increased binding as well (268 ? 20% of control; mean ? SEM of two experiments). Increased binding of Pg does not necessarily represent increased formation of lZ5I-Pg, as catalytically inactivated uPA also increases Pg binding in other cell types (Plow et al., 1986). Our current observations support a similar con- clusion as stimulation of Pg binding occurred with scuPA and the inclusion of aprotinin with uPA. How- ever, limited proteolysis of cells with Pm is known to increase subsequent binding of Pg (Camacho et al., 1989; Gonzalez-Gronow et al., 1991). The inclusion of uPA with lZ5I-Pg results in cell bound 1251-Pm in MG-63 cells. We therefore considered the effects of Pm on "'I-Pg binding to MG-63 cells. Pre-incubation of MG-63 cells with Pm for 1 h, 37°C increased lZ5I-Pg binding (Table 11, suggesting that formation of Pm on the cell surface upregulates cell surface capacity to bind additional Pg. The increase in '"I-Pg binding by Pm pre-treatment represented proteolysis and an apparent increase in available lysine binding sites, as the inclusion of aprotinin inhibited the Pm effect by 91%. To determine if the increase in lZ5I-Pg binding by either


TABLE 1. Pre-treatment of MG-63 cells with plasmin increases the bindinn of '251-Pe

Fig. 4. Activation of lZ5I-Pg at the cell surface of MG-63 cells. MG-63 cells were incubated with "'I-Pg for 3 h a t 37°C in the presence of EACA (200 mM), uPA inactivating antibody (10 pg/ml), tPA inactivating antibody (10 pg/ml), aprotinin (20 pg/ml), or no additions. Treat- ments were terminated by aspiration of the media and the cells washed three times with PBS. Bound I2"I-Pg and/or '261-Pm was extracted from cell surface receptors by incubating cells with 200 mM EACA for 10 min, 23°C. Aliquots of the tACA wash were run over 11% SDS-PAGE, reduced conditions. Glu-Pg represents -92,000 daltons, HC represents heavy chain portion of Pm at -58,000 daltons, and LC represents light chain portion of Pm at -28,000 daltons.

Pm or uPA represented a change in binding affinity or receptor number, Scatchard analyses of lZ5I-Pg binding under control, uPA co-incubation, and I'm pre-treatment were performed (Fig. 5). The resulting Kds were 1.64 pM, 1.86 pM, and 1.52 pM for control, Pm, and uPA, respectively. Receptor numbers were 16.1 x lo", 44.7 x lo6, and 34.9 x lo6 siteskell for control, Pm, and uPA, respectively. These data indicate that Pm pre-treatment of MG-63 cells resulted in an increase in receptor number by 2.8-fold and that uPA co-treatment increased receptor number by 2.2-fold. The increase in receptor number with Pm pre-treatment is consistent with that observed with monocytoid cells (Gonzalez- Gronow et al., 1991). In contrast, Plow et al. (1986) observed an increase in receptor affinity with uPA co- treatment of fibroblasts using disisopropylphosphoryl- uPA (DIP-uPA) at 1 pM. In the present study, active

~

% of Control Plasmin (ng/ml) Experiment 1 Experiment 2

1,000 234.8 k 14.5* 235.6 2 11.7* 100 213.0 k 6.1* 208.4 6.3* 10 144.3 * 6.6* 168.5 f 15.8* 1 95.1 k 5.7 114.3 ? 2.7

~~ ~~

'MG63 cells were prepared for radiolabeled binding experiments as described in Mate- rials and Methods. Monolayers were pre-incubated with Pm at the indicated concentrations for 1 h, 37°C. Monolayers were then wsshed with PBS and incubated with 1 mM tranexamic acid, 10 min, 23°C with gentle agitation to remove cell bound Pm. Cells were then incubated with 40 nM Iz5I-Pg for 1 h, 37°C. Cells were rinsed three times with ice-cold PBS and lysed in 1 N NaOH. Nonspecific binding was determined by the addition of 40 pM unlabeled Pg. The binding of l2%Pgt0 MG63 is expressed in percent of non-Pm pre-incubated control carrected for nonspecific binding. Within each experiment. values represent the mean iSEM of quadruplicate determinations. *Within the individual experiment, values with an asterisk differ from the nonPm pre-incubated control (Tukey's HSD test, P < 0.05).

o.20 I 0.15 &

Plasmmagen. uM

0.10

0.05

0.00 0 10 20 30 40 50 60 70 80

Plasminogen Bound, pmole/l 0 cells 6

Fig. 5. The effects of Pm and uPA on the binding of Iz5I-Pg to MG-63 cells. Cells were either pre-incubated with 100 ng/ml Pm (W as described in Table 1, co-incubated with 30 nM uPA (W as detailed in Results, or non-treatment control (0) as detailed in Materials and Methods. IZ5I-Pg bound to MG-63 cells was determined and Scatchard analyses performed. Specific binding isotherms are presented in the insert.

uPA at 30 nM was used, with activation of Pg to Pm allowed to occur. Under these conditions, uPA interaction with lZ51-Pg results in lZ5I-Pm, and presumably both uPA and Pm interact resulting in an increase in receptor number in osteosarcoma cells.

Binding of lZ51-Pg was differentiated between that 1251-Pg bound to the cell surface and that bound to the ECM (Table 2). 1251-Pg was equilibrated with MG-63 cells after pre-treatment with Pm, with co-treatment with uPA, or with the no treatment control. Cell bound 1251-Pg was extracted with deoxycholate, and the remaining deoxycholate insoluble fraction was extracted

CAMPBELL ET AL. 6

TABLE 2. Deoxycholate extraction of lZ5I-Pg bound to MG-63 monolayers'

Plasminogen associated with cells (%)

Emeriment 1 Emeriment 2

Control 78.6 i 7.2 Plasmin 67.6 ? 1.0 uPA 75.5 k 0.7

77.0 i 7.1 60.8 ? 3.5 71.9 _c 1.2

'Confluent monolayers were pre-incubated with Pm (100 ng/well) as described in Table 1 and coincubated with uPA (30 Uiwell) as described in Table 1. Radiolabeling with '%Pg was performed a8 described in Materials and Methods. "'I-Pg associated with the cell layer was determined by extraction of monolayers with 18 deoxycbolate, 30 min, 23°C. '%Pg associated with the deoxycholate insoluble fraction, representing bindin to the extracellular matrix, was determined by extraction with 46 SDS, 30 min, 23%. "I-F&bound to respective fractions was corrected for nonspecific binding, and the percent of I-Pg associated with cells (deoycholate soluble fraction) is presented above. Values represent the mean 2 SEM of triplicate determinations. Differences between treatments within an experiment were not different as determined by ANOVA (experiment 1: P = 0.242: experiment 2: P = 0.114).

with 4% SDS and represents the ECM bound 1251-Pg. Neither Pm pre-treatment nor uPA co-treatment al- tered Pg binding to the cell fraction. MG-63 cells secrete substantial matrix proteins including thrombospondin (Clezardin et al., 19911, which together with other matrix proteins may possibly explain Pg binding to MG-63 ECM. Although both cell and ECM probably contribute to osteosarcoma cell Pm activity, cell associated PgPm remains the primary contributor at 77.8%.

Pg and Pm binding MG-63 cells determined by amidolytic activity

The 1251-Pg binding experiments demonstrate the binding of Pg and its activation to Pm on the cell surface of MG-63 cells but did not illustrate the proteolytic activity of cell bound Pm. We made use of the chromogenic substrate, S2251, to further characterize the binding and activation of Pg. Similar assays have been developed to measure cell surface activation of Pg (Stephens et al., 1989; Osada et al., 1991; Gonzalez- Gronow et al., 19911, but Gonzalez-Gronow et al. (1991) is the only other study to our knowledge to measure Pm directly on the cell. Neither Pg, Pm, nor S2251 affected cell attachment, morphology, or viability during the experimental period, as determined by trypan blue exclusion and phase contrast microscopy (data not shown). A negative control (binding bdfer alone) was used to estimate nonspecific hydrolysis of substrate which was performed in each experiment and sub- tracted from all treatment groups. There was no nonspecific hydrolysis associated with cells as the negative controls under cell and cell-free conditions both indicate G0.05 O.D. units. Both Pm and Pg bound to cells in a concentration-dependent manner, as represented by cell surface amidolytic activity (Fig. 6). The binding of Pm was measured directly in contrast to Pg, which is ineffective against S2251. Activation of Pg to Pm first must occur, and the amount of cell associated Pm is dependent upon cell surface PA and exogenous Pg. This explains the difference in cell associated Pm between cells incubated with Pm and Pg. However, the increase in "'I-Pg binding observed following Pm pretreatment suggests direct modulation of the cell surface properties by Pm.

Table 3 demonstrates that the binding and activation of Pg on MG-63 cells were dependent upon endogenous

uPA, while tPA appears uninvolved. This data con- firms the activation of lZ5I-Pg as present in Figure 4. Co-incubation of Pg with PAI-I blocked activation of Pg, further confirming a role for uPA. Pm inhibitor requiring interaction with the lysine binding site of Pm ta2-antiPm) was ineffective in inhibiting cell surface Pm activity, while the inhibitor interacting directly with the catalytic site of Pm (aprotinin) inhibited cell surface Pm activity. The concentration of a 2 - a n t i h used here maximally inhibits Pm in solution at Pm concentrations up to 6 pmoles, a mass tenfold greater than the mass of Pm generated in the cell surface by endogenous uPA for 0.43 pM Pg incubated. The occurrence of Pm on the cell surface, in the presence of 012- antiPm, is consistent with the view that Pg binds to the cell surface prior to activation (Stephens et al., 1989).

Cell associated Pm activity was reversible as demonstrated in Figure 7. Pm bound to MG-63 cells was dissociated by EACA in a concentration-dependent manner (Fig. 7A). When Pm was equilibrated with MG-63 cells and then washed with EACA, essentially all Pm activity was eluted from cells (Fig. 7B), producing results consistent with 1261-Pg experiments. Cell associated Pm activity could also be associated from cells with another lysine analog, tranexamic acid (data not shown).

Cell surface Pm activity was resistant to the soluble protease inhibitor, a2-antiPm (Table 4). Pm associated with the cell surface exhibited activity in the presence of a2-antiPm -35% of the non-2-antiPm control, representing a 14-fold increase over similar Pm concentrations under cell-free conditions. The loss of cell surface Pm activity in the presence of a2-antiPm is probably a result of the dissociation of Pm from the cell surface, which is then followed b Pmla2-antiPm complex formation. Incubation of l2 I-Pm bound to cells with a2- antiPm does not result in cell surface Pda2-antiPm complex formation (data not shown). Resistance to proteolytic inhibition was dependent upon the interaction of Pm to its cell surface binding site, as Pm dissociated from cells was readily inhibited by 1x2-antiPm. Resis- tance of cell associated Pm to soluble protease inhibitors is observed in numerous cell types and appears to be a characteristic of the Pg receptor(s) (Plow et al., 1986; Stephens et al., 1989; Ellis et al., 1991; Hall et al., 1991).

DISCUSSION These data demonstrate the existence of an active Pm

system localized upon the surface of osteosarcoma cells and may represent functional pericellular proteolytic activity for osteosarcoma cells. This is supported by several observations. First, Pg binds to MG-63 cells in a specific, saturable manner. Second, Pg is activated to Pm on the cell surface by endogenous uPA occurring on the cell surface. Third, cell associated Pm is resistant to a primary protease inhibitor, a2-antiPm. Fourth, cell surface Pm activity is not internalized, but reversible by competition for lysine binding sites on the Pm molecule. Lastly, Pm under the conditions used here does not adversely affect the viability of osteosarcoma cells.

The binding of Pg to MG-63 cells requires unoccupied lysine binding sites which are associated with the kringle 1 region of the Pg molecule (Lerch et al., 1980).

B

PLASMIN ACTIVITY ON OSTEOSARCOMA CELLS

3.0

2.5

2.0

1.5

1.0

7

-

-

-

-

-

0.4 - A

w 0.3 - - 0

5 U c 0' 0.2 - m

E

EL 0.1 -

C

v) m

.-

0.0 0.0 0.5 0 kL--- 25 50 75 100 125 0 50 100 150 200 250

Plasminogen, nM

Fig. 6. Amidolytic activity of Pm on the surface of MG-63 cells. Ir- creasing concentrations of unlabeled Pg (A) or Pm (B) were incubated with MG-63 cells in binding buffer, 37°C. Pg treatments were incubated for 3 h, 37"C, and Pm treatments were incubated for 1 h, 27°C. Cell bound Pm amidolytic activity was determined by S2251 chro-

TABLE 3. Cell surface activation of plasminogen: Effects of various inhibitors of the plasmin system components'

Q Pg Control Treatment Experiment 1 Experiment 2

Pg + uPA antibodies 13.7 2 9.8* 10.5 2 4.2* Pg + tPA antibodies 102.4 ? 18.9 107.5 % 2.3 Pg + PAL1 29.0 ? 5.4* N.D. Pg + aprotinin 3.7 ? 2.2* 2.5 5 1.4* Pg + a2-antih 85.5 2 22.0 92.5 5 8.2

'MG-63 cells were grown to confluence in 12-well plates, and 40 pgiml Pg was incubated with additions a8 indicated for 3 h, 37°C. Treatments were removed, plates rinsed, and bound F'm determined by chromogenic assay (Materials and Methods). Antibodies to PAS and aprotinin were added at 10 pgiml. PAI-1 was added at lpg/ml, and a2-antiPm was added at 2 pg/ml. Treatment groups were compared to percent of Pg control (40 pg/ml) corrected for the none Pg control. Values represent the mean * SEM for six determinations in experiment 1 and four determinations in experiment 2. N.D. not determined. *Values with asterisk differ from their respective Pg control within an experiment (Tukey's HSD test, P < 0.05).

Kringle 1 is identical for both Pg and Pm molecules. Both Pm and Pg bind to the same receptor(s) (Miles and Plow, 1985; Plow et al., 1986; Camacho et al., 1989). There may be differences in affinity, as Pg and Pm bind with equal affinity to the platelets, U937 monocytic cells (Miles and Plow, 1985; Plow et al., 19861, and as the present study suggests for osteosarcoma cells. How- ever, Pm has a greater affinity than Pg to the colonic cancer cell line SWll l (Camacho et al., 1989). The magnitude of binding sites on osteosarcoma cells indicates that the Pg/Pm receptor(s) must comprise a significant portion of the plasma membrane components. As such, u-enolase is localized on the cell surface, presents available lysine groups, and binds Pg (Miles et al., 1991). These authors also reported a Pg receptor in MG-63 cells which shares similar characteristics as u-enolase as determined by ligand blot assay to lZ5I-Pg

Plasrnin, nM

mogenic assay (see Materials and Methods). Individual symbols represent the mean & SEM of duplicate determinations. This figure is a representative experiment for at least three similar experiments each for Pg and Pm.

(Miles et al., 1991). Additionally, thrombospondin binds to Pg and Pm (Silverstein et al., 1984) and is known to be synthesized by osteoblasts and osteosarcoma cells, including MG-63 cells (Clezardin et al., 1989). "hrombospondin binds to the surface of MG-63 cells and occurs in the ECM (Clezardin et al., 1991) and therefore may represent a receptor candidate on the cell surface and/or in the ECM. However, PgPm receptors may not be restricted to protein molecules, as ganglio- sides associated with the plasma membrane also bind Pg (Miles et al., 1989). The binding of Pg/Pm to non- protein molecules (Miles et al., 1989) and the broad concentration range for EACA inhibition of Pg binding (Miles and Plow, 1985; Plow et al. 1986) suggest that the binding of Pg or Pm is more complicated than merely a direct interaction of the high affinity lysine binding site of Pg/Pm to exposed cell surface lysine sites. The upregulation of Pg binding by Pm and uPA in MG-63 cells suggests PA/Pg interaction in Pg binding. Enhanced Pg activation occurs when Pg is bound to the cell membrane (Miles and Plow, 1985). This may in part be associated with a 40-fold lower K, value for Pg activation by receptor bound uPA as compared to K, for Pg activation in solution (Ellis et al., 1991). These observations imply not a "static" Pg to receptor interaction, but a dynamic interaction involving Pg, I'm, pro- and active forms of PAS, and immediate association of the PgPm receptor(s1 and the PA receptors.

In Mg-63 cells although -80% of Pg is associated with the cell surface, -20% is associated with the ECM. Whether Pg is bound to cells or ECM is dependent upon lysine binding sites on either extracellular location. Preliminary immunohistochemical evidence from our laboratory indicates that Pg associated with the ECM of MG-63 cells is directly under the ventral surface of the cells. We hypothesize that both cell surface Pg and

8 CAMPBELL ET AL

120 r

loo[ e, t \ Lc

0

40

A

Fig. 7. The binding of Pm to MG-63 cells involves interaction with cellular lysine binding sites. The reversibility of Pm binding to cells (A) was determined by incubating 122 nM Pm in binding buffer with cells for 1 h, 37°C. Incubation media was aspirated and cells were washed three times with PBS. Cells were washed with increasing concentrations of EACA for 10 min, 37°C with gentle agitation. EACA treatments were aspirated and cells were washed three times with PBS. Pm remaining bound to cells was determined by chromogenic assay (see Materials and Methods). Individual symbols represent the

1.2

1.0

2 0.8 - d 2 0.6 - rn

-

- aJ 0 -

C

C .- 0.4

0.2 -

- m h

B

0.0 0 25 50 75 100 125

Plasmin, nM

mean -+ SEM of duplicated determinations. Dependency of Pm activity in the assay upon cell bound Pm (B) was demonstrated by binding 122 nM Pm to cells for 1 h, 37°C. Incubation media were aspirated and cells were washed three times with PBS. Cells were either washed with 200 mM EACA, 10 min, 23°C to extract bound Pm or not. Amido- lytic activity for bound Pm to EACA washed cells (V), unwashed cells (o), and Pm in the EACA wash (V) was determined by chromogenic assay. Individual symbols represent the mean * SEM of duplicate determinations.

TABLE 4. Cell bound plasmin is protected from soluble a2-antiPm

Cell bound' Cell free' Treatment Dmole % Control Dmole % Control

Control 2.16 f 0.16 100 2.40 2 0.04 100 a2-antiPm 0.74 t 0.03 34.1 * 1.4 0.06 * 0.03 2.5 k 1.3

'MG-63 celle were grown to confluence in 12-well plates and equilibrated with Pm (10 pg/ml) for 1 h, 37°C. Cells were rinsed three times with PBS to remove unbound Pm. Cell bound Pm activity was determined by chromogenic assay (see Materials and Methods) in the presence or absence of 2 p g / d a2-antiPm. Cell bound Pm activity is represented in pmoles of Pm bound (quantitated against a Pm standard curve under identical hut cell-free conditions), or pmoles Pm hound was adjusted to percent of control not incubated with a2-antiPm. Values represent the mean -+ SEM of three experiments. a2-antiPm treatment differs from its respective control (Student's t-test, P < 0.05). 'Pm at an equivalent concentration to the Pm hound to cells was placed in the chromogenic assay under identical hut cell-free conditions as for the cell hound experiments. Pm activity is represented in pmoles of Pm (quantitated against a Pm standard curve under identical conditions), or pmoles Pm was adjusted to percent ofcontrol not incubated with a2-antiPm. Values represent the mean f SEM of two experiments. a2-antiPm treatment differs from its respective control (Student's t-test, P i 0.05).

ECM bound Pg are available to affect the immediate extracellular environment of osteosarcoma cells.

Occupany of Pg/Pm receptors on bone cells in vivo cannot be speculated upon, as the concentration of Pg in the bone cell pericellular environment is unclear. How- ever, the bone marrow is a ready source of Pg (Barnhart and Riddle, 1963), and Pg/Pm occurs interstitially on the surface of tumors (Burtin et al., 1987; Costantini et al., 19911, confirming the availability of Pg outside of the bloodstream. Interestingly, the maximal concentration of Pg used in the amidolytic assay was -0.4 pM, well below the -2 pM in plasma (Miyashita et al., 1988).

The formation of Pm on the osteosarcoma cell surface by endogenous cell bound uPA occurs in the presence of an inhibitory concentration of soluble a2-antiPm. This, coupled with the relative resistance to protease inhibitors of receptor bound Pm compared to Pm in solution, enhances the attractiveness of cell surface activation of Pg in vitro and in vivo. The secretion of a2-macroglobu-

lin by osteosarcoma cells (Grofova et al., 19881, availability of 1x2-antiPm interstitially to tumor cells (Bur- tin et al. 1987; Costantini et al. 1991), and presumed abundance of other protease inhibitors in milieu surrounding bone cells suggest that relevant Pm activity is probably associated with the cell surface or the ECM. uPA mutagenic studies further underscore the func- tionality of cell surface Pm activity in neoplastic cells. Cell surface Pm can be displaced by exogenously added active site inhibited uPA mutants. This results in inhibiting basal membrane degradation, which is be- lieved to limit the metastatic potential of tumor cells (Cohen et al. 1991).

The osteoblast or osteosarcoma surface Pm activity may control cell specific proteolysis regulating resorption (Hamilton et al., 1985; Allen et al., 1986; Pfeilschi- fer et al., 1990; Grills et al., 1990; Allan et al., 1991; Fukumoto et al., 1992; Hoekman et al., 1991; Leloup et al., 1991), growth factor activation (Novak et al., 1991; Allan et al., 1991; Campbell et al., 1992) and growth


(Campbell and Novak, 1991). Recently, we demonstrated that cell surface Pm can regulate proteolysis of IGFBPs, destroy their ability to bind IGFs, and therefore may represent the principal site of Pm induced activation of IGFs (Campbell et al., 1993). Amplifica- tion of cell surface Pm activity is suggested to occur on breast cancer cells (Costantini et al., 1991). Such an enhanced surface proteolytic activity in osteosarcoma would bestow cell specific metastic ability and enhanced extraction of available nutrients (i.e., growth factors).

ACKNOWLEDGMENTS This work was supported in part by NIH grant

CA54363 and ASRI grant 91-009-1. A preliminary report of this work was presented at the First Interna- tional Meeting on Interdisciplinary Research in Os- teosarcoma, Pittsburgh, Pennsylvania, 1991. We thank Teresa Hentosz for her technical assistance in cell culture and Laurie Iannuzzi for her help in the prepara- tion of this manuscript.

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