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Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types
THROMBOSIS RESEARCH 59; 339-350,199O 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
CHARACTERIZATION OF TISSUE PLASMINOGEN ACTIVATOR BINDING PROTEINS ISOLATED FROM ENDOTHELIAL CELLS AND OTHER CELL TYPES
D.P. BEEBE, L.L. WOOD, and M. MOOS Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, U.S.A.
(Received 21.8.1989; accepted in revised form 13.5.1990 by Editor N.U. Bang)
ABSTRACT Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between '?I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HWEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a tPA affinity column revealed binding of t-PA to 54,000 Several t-PA and 95,000 molecular weight proteins. binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase.
INTRODUCTION Components of the fibrinolytic pathway including plasminogen, urokinase (u-PA), and tissue-type plasminogen activator (t-PA) have been shown to bind to specific receptors on a variety of cell types (l-5). The resulting activation of plasmin on cell surfaces may contribute to preventing clot formation and/or removing clots in vessel walls in the case of endothelial cells or tissue restructuring and/or metastasis in the case of malignant cells. A t-PA receptor with low affinity (K, = 18-240 nM) has been characterized on human umbilical vein endothelial cells (HWEC) (3-5). The interaction was shown to involve a specific region of Key Words: t-PA, receptor, cell lines 339
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the finger domain (residues 7-20) (6). A t-PA binding protein (40 kDa) has also been identified in isolated cell membranes (7). A high affinity, low density receptor has also been described which is most likely the plasminogen activator inhibitor (PAI-1) (4-5, 8, 9). PAI- localizes in the endothelial cell matrix (ECM) and is not associated on the membrane surface (10). t-PA can penetrate to the ECM and bind to PAI- (10). In the present report, we describe the binding of t-PA to different cell types as well as the characterization of several t-PA binding proteins isolated from immunopurified cell lysates. MATERIALS AND METHODS Reagents: Purified recombinant t-PA was a generous gift of Genentech, Inc. (South San Francisco, CA). High molecular weight u-PA (Winkinase) was kindly provided by Dr. Gene Murano (CBER, Bethesda, MD). Purified recombinant scu-PA was generously provided by Dr. Jack Henkin (Abbott Laboratories, IL). Na1251was purchased from New England Nuclear (Boston, MA). Phosphate buffered saline (PBS), M199 and D-MEM were purchased from Quality Biological (Gaithersburg, Endothelial cell growth factor (ECGF) and Nu Serum was MD). purchased from Collaborative Research (Bedford, MA). Fetal bovine serum (FBS), trypsin, gentamycin, and glutamine were purchased from GIBCO (Grand Island, NY). Monoclonal antibodies to t-PA, u-PA, and PAI- were obtained from Monozyme ApS (Charlottenlund, Denmark). t-PA peptide 7-20 (>95% pure) was synthesized on an ABI 430a automated peptide synthesizer and was generously provided by J. Regan and Dr. K. Seamon (CBER, Bethesda, MD). Iodination of t-PA: t-PA (25 ug) was labelled by the "Iodo-gen" method with 1 mCi Na'? as previously described (3). The specific activity was calculated to be 2.73-3.2 X 10' cpm per ug. Cell Cultures: Primary cultures of HUVEC were a generous gift of Dr. Tom Lawley (NIH, Bethesda, MD) and were maintained as previously described (3). Human osteosarcoma cells (HOS) were kindly provided by Dr. Martin Ruta (CBER, Bethesda, MD). A-431 cells (human epidermoid carcinoma), L-132 cells (human embryonic lung), and Chang liver cells were obtained from American Type Tissue Collection (Rockville, MD) and cultured in D-MEM with 10% FBS and 100 mM glutamine. Binding of "'I-t-PA to Cultured Cells: Each cell type was grown to confluence in 24-well tissue culture plates (Co-star, Lincoln, MA). Binding was performed as previously described with 20 ul dilutions of t-PA per well and 20 ul 1'51-t-PA (3 nM) per well. After incubation at 4°C for 30 min the cells in each well were washed three times and lysed with buffer (10% SDS,
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0.1 M EDTA) and counted (Compugamma, McLean, VA). The data were analyzed according to the method of Scatchard using the LIGAND program (courtesy of Dr. Peter Munson, NIH, Bethesda, MD). Chemical Crosslinking
of t-PA to Cultured Cells:
Cells were plated into Nunclon tissue culture dishes (245 X 245 X 2 mm) (Nunc, Denmark) at a density of 1 X lo* cells per dish and incubated overnight. The cell monolayer was washed once with 0.05 M glycine buffer containing 0.1 M NaCl (pH 3.0) to remove endogenous protein from cell surfaces, once with 0.5 M HEPES buffer (0.1 M NaCl, pH 7.5), and three times with PBS. Excess '251-t-PAwas incubated with cells at 4°C for 30 min. The cell monolayer was rinsed three times with PBS and subsequently incubated with the homobifunctional cross linking agent 3,3dithiobis(sulfosuccinimidyl)propionate (DTSSP) (3-1OmM) for 15 min at room temperature to crosslink t-PA to the receptor. The monolayer was then rinsed three times with PBS and lysed with 3 ml Triton X buffer (0.1 M Tris-HCl, 0.1% Triton X-100, pH 8.1). Fractionation
by High Performance
Liquid Chromatography
(HPLC):
HPLC was performed on a Beckman Model 344 instrument using a TSK 2000 SW column (7.5 mm X 30 cm) equilibrated in Tris buffer (0.02 M Tris-HCl, 0.15 M NaCl, 5 mM EDTA, pH 6.95). A Flo-One\Beta Radioactive flow detector (Radiomatic Instruments, Tampa, FL) was connected to the HPLC for analyzing radioactive samples. Human albumin (2.5% HA) (Miles, Berkeley, CA) was used to calibrate the column for molecular weight determination. t-PA Affinity
Column:
t-PA (1 mg/ml) was coupled in 0.1 M NaHCO, buffer to CNBr activated The Sepharose (Pharmacia, Uppsala, Sweden) at pH 5.5 overnight. coupling efficiency was estimated to be 85%. The cells were lysed in Triton X buffer containing protease inhibitors leupeptin (10 ug/ml) and Trasylol (20 u/ml) for 15 min and spun to remove the nuclei in a microfuge (10,000 X G) for 15 min. Cell lysates were The passed through the column which was equilibrated in PBS. column was washed until the absorbance reached background. Bound protein was then eluted with 0.1 M glycine-HCl buffer pH 2.5 and 0.5 ml fractions were collected and concentrated by rotoevaporation (Speed-Vat Concentrator, Savant). SDS-PAGE,
Immunoblotting,
and Ligand Blotting:
Samples were electrophoresed on 10% SDS-PAGE slab gels in a Mighty Small gel slab unit (Hoefer, San Francisco, CA) using the method were proteins immunoblotting the For Laemml i of (11) * electrophoretically transferred to nitrocellulose paper for 2 hr The blot was then incubated with the specific at 400 mamp. antibodies for 2 hr at 25°C followed by peroxidase conjugated antigoat or anti-mouse antibody and developed with o-dianisidine as previously described (12). For ligand blotting the blot was incubated with '251-t-PAovernight, washed in PBS, 2.5% HA, 1% Tween80 4 times for one hour each. The blot was then autoradiographed.
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Microsequence Analysis: Samples were run on SDS-PAGE at neutral pH and electrophoretically transferred to PVDF (Immobilon) membranes. The stained band was excised and placed in an automated sequenator (Applied Biosystem Model 470A). Sequence and analysis of PTH derivatives were performed according to standard procedures (13). RESULTS t-PA Binding to Various Cell Lines: Four different cell lines were tested for the ability to bind tPA. The A-431 cells and HOS cells demonstrated quantitative binding of t-PA whereas binding to Chang and L-123 was poor in compa$?ison. Figure 1 depicts the various binding curves. Binding to A-431 and HOS was reversible as shown by the dissociation of bound ligand in excess buffer fnot shown) and the time course for binding was similar to that‘of HUVEC (3).
200
100 TPA
ADDED
300 (I-M)
Fig. 1. Binding curves of '?-t-PA to Cells. (+) A-431 cells, (h) HOS cells, (A) HUVEC, (+) L-123 cells, (0) Chang cells. Binding was routinely performed for 30 min at 4°C. Non-specific binding (in the presence of 6.7 uM unlabelled t-PA) averaged 10%. Plots of bound over free versus bound ligand (Scatchard plots) were linear. The binding constants for A-431 and HOS were calculated from the LIGAND program assuming one class of binding site and were &= 9.42 X lo-*M,5.2 X lo6 sites per cell and z
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Crosslinking:
?-t-PA was incubated with HUVEC, A-431, and HOS cells at a concentration to saturate the receptors (1 uM) at 4°C for 30 min. The cells were washed to remove unbound ligand. The ligandreceptor complex was then covalently crosslinked with the water soluble homobifunctional agent DTSSP. This reagent is membrane impermeable and, therefore, only membrane components should be crosslinked. After thorough washing to remove the coupling agent the cell membranes were solubliized with Tris-Triton buffer and the supernatents were analyzed by HPLC on a TSK-3000 gel filtration column. The t-PA-receptor complexes from HUVEC, HOS, and A-431 were each chromatographed. A representative plot of the chromatography of the A-431-receptor complex is shown in Figure 2. -
A.
Mr
130K
65K
I
I
t
10
15
20
25
30
Fraction Number
Fig. 2. Chromatography of l?-t-PA Receptor (A-431) Complex on TSK 2000. A. Unreduced sample B. Reduced sample Two radioactive peaks corresponding to molecular weight ranges of 130,000 and 65,000 respectively were present in the elution profile (Figure 2a). The first peak, which is approximately 40% of the recovered cpm, most likely corresponds to the covalent complex of "'I-t-PA and a membrane protein whereas the second peak is presumably uncomplexed t-PA. When the crosslinked receptor complex was treated with 1 mM DTT (37"C, 2 hr) to cleave the thio bond in the crosslinking reagent, most of the 125-I counts were recovered in the 65,000 molecular weight range indicating recovery of the originally complexed t-PA ligand (Figure 2b). The cross-linking studies were repeated with HUVEC in suspension (cells were scraped off the plates). Autoradiography of the resulting t-PA receptor complexes which were run on revealed bands SDS-PAGE two corresponding to molecular weights of 116,000 and 65,000 (not shown).
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with t-PA Peptide:
The results with coupling whole t-PA to the receptor indicated that the &of the receptor was 51-65,000 since the complex eluted in the molecular weight range of 116-130,000. To obtain additional molecular weight estimates, binding to HWEC was performed with "'1 labelled t-PA peptide 7-20 (molecular weight 1792). This peptide, which corresponds to a portion of the finger region of t-PA, had previously been shown to compete with whole t-PA binding to HWEC (6). Following crosslinking with DTSSP according to the previous procedure, the receptor complex was chromatographed on TSK 2000 SW. As shown in Figure 3, two radioactive peaks were recovered but in this instance the molecular weights corresponded to 48-65,000 and 1792 and most likely represent the receptor complex and uncomplexed peptide, respectively. 1500 A.
Mr
<26OK 1
130K 65K
I
i
1792
1
246
1
Fraction Number
Fig. 3. Chromatography of l?-t-PA Peptide-Receptor Complex on TSK2000 SW. A. Top Panel:Absorbance 280 nm (albumin) B. Middle Panel: cpm (Y-Peptide 7-20) C. Bottom Panel: cpm (l"SI-Peptide-Receptor complex). Table 1 lists the retention times of the radiolabelled peaks and the protein markers obtained from chromatographing t-PA-receptor complexes on TSK 2000 SW. Isolation and chromatography of complexes from each cell type was performed in at least three separate experiments each. Since the peptide is 1792 D, the molecular weight of the receptor was estimated to be 48-65,000.
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TABLE 1 Chromatography of Cell Receptor Complexes on TSK 2000 Retention Time (min) M, Sample
130,Oo'O
HWEC-tPA 13.7-14.4 HOS-tPA 14 A431-tPA 14.5 A431-tPA (red)* --HWEC-tPA-Peptide ----tPA-Peptide 2.5% HA dimer 14.6 --monomer Acetyl-Tryptophan ---
65,000
1,792
246
16.3-18.2 17.. l 17.9 17.5 16.9-18.2 ---
--------26.4-29.3 26.4-28.4
----_--------
--17.1 ---
--_----
----31.7
* reduced Isolation of t-PA Receptor: A-431, HOS, and HWECs (1 X 10" cells) were washed in PBS and disrupted with Tris-Triton lysis buffer. The supernatents were then passed over a t-PA-conjugated Sepharose affinity column. The column was washed with PBS, then eluted with glycine buffer, pH 2.5. A small protein peak eluted and when this peak was pooled, concentrated and run on SDS-PAGE, both major and minor bands of approximately 54,000 molecular weight were detected as well as several high mw bands by Coomassie Blue staining (Figure 4,1ane b).
97
k
66
k 54
42 k
35
k k
c d efgh a b Fig. 4. SDS-PAGE (Coomassie Blue stain) and Immunoblot of the t-PA Binding Protein(s).Samples were run under reducing conditions(BME).
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(a) High molecular weight standards (b) Binding protein(s) from A431 cells (c) Binding protein blotted with anti-PAI- (d) t-PA (e) Binding protein blotted with anti-u-PA (f) u-PA blotted with antiu-PA (g) Binding protein blotted with anti-t-PA blotted with antit-PA (h) t-PA blotted with anti-t-PA. Similar molecular weight bands were detected by autoradiography of SDS-PAGE gels when 9 labelled HWEC were lysed and fractionated over the t-PA column (not shown). The same band pattern was also demonstrated by Coomassie Blue staining of SDS-PAGE gels of affinity purified cell lysates when the t-PA column was substituted with an A-H Sepharose column to which the t-PA peptide (res. 7-20) was conjugated (not shown).When affinity purified cell lysates were immunoblotted with antibodies to t-PA, u-PA or PAI-1, reactivity appeared in the molecular weight range of 54,000 with both anti-UK and anti-PAI- while the higher molecular weight bands reacted with antibodies to t-PA, u-PA, and PAI- (Figure 4, lanes c,e,and g). The high mw bands might represent t-PA-or u-PA-PAI- complexes. The major 54,000 molecular weight band was shown to be tubulin beta chain by microsequence analysis (76.5% identity in 17 a.a. overlap) (13): M.R.Y.I.V.N.I.Q.A.G.N.F.G.N.F.G.N.Q.I.G.M.A.T. Ligand Blotting with "'I-t-PA: Receptor preparations isolated from either the t-PA or the t-PA peptide affinity column were run on SDS-PAGE along with purified preparations of t-PA and ret scu-PA. The proteins were electrophoretically transferred to nitrocellulose and probed with "'I-t-PA. Figure 5a shows an autoradiogram of a representative blot.
54K-
1
2
Figure 5. Ligand Blot of Affinity purified Cell Lysates and sou-
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PA. Samples were run on SDS-PAGE and transferred to nitrocellulose. The blot was incubated with 1'51-t-PA,washed and autoradiographed. (1) Binding protein from A-431 cells isolated from the t-PA peptide affinity column. (2) recombinant scu-PA. Reactivity of the labelled ligand was demonstrated to 95,000 and 54,000 molecular weight bands in the affinity purified cell lysate (lane 1) as well to purified scu-PA (lane 2). Similarly, significant binding of "'I-t-PA to purified t-PA was demonstrated (not shown). When the affinity purified cell lysates were tested in an amidolytic assay (14), significant plasminogen activator activity (dilutions out to 1:512,000) was demonstrated in the absence of fibrin promoter fragments (not shown), indicative of the presence of u-PA. Furthermore, chromatography of "'I-t-PA or '?-u-PA on the t-PA affinity column demonstrated considerable binding of labelled material to the column (nearly 50% in either case) as opposed to X0.1% of 12'I-HA.These data would indicate significant interaction between t-PA and u-PA as well as self-association of t-PA. DISCUSSION Endothelial cells have the ability to bind several components of the fibrinolytic system including plasminogen (15), u-PA (16), and t-PA (3-5). These proteins bind via regions other than the catalytic site so as to allow expression of enzyme activity on the cell surface. In addition these cells synthesize u-PA and t-PA as well as the plasminogen activator inhibitor (PAI-1) (17). The latter has been shown to be associated with the extracellular matrix in HWEC (7,17). PAI- inhibits both u-PA and t-PA. Thus, both activators and inhibitors as well as the substrate plasminogen can be localized on the endothelial cell surface or the extra cellular matrix. Both Sakata et al (18) and Schleef et al (10) have shown that t-PA binds to PAI- to form high molecular weight complexes which then are released into the supernatent of HWEC. Since the majority of PAI- is located in the endothelial cell matrix as a homogeneous carpet underneath the cells, the t-PA-PAI- interaction would presumably occur in the ECM rather than on the cell membrane. The fact that t-PA forms complexes with PAI- even in the presence of a cell monolayer (10) indicates that t-PA can penetrate to the ECM As suggested by Scheef et al, HWEC in culture may not form tight junctions normally asscociated with endothelial cells in viva. In HEP G2 cells binding of t-PA has been shown to occur solely through its interactions with PAI- which is localized in the substratum (19). t-PA-PAI- complexes are endocytosed by the cells and binding of complexes is not competed for by free t-PA. The question then arises as to whether or not t-PA binds to a membrane component that is distinct from PAI- on HWEC membranes. Hajjar and Hamel have recently demonstrated the binding of t-PA to a 40,000 molecular weight protein isolated from purified HWEC membranes (7). In the work described here, t-PA was shown to bind
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to a component in the molecular weight range of 48-65,000 (crosslinking studies), to proteins in the molecular weight range of 54,000 as well as 95,000 (ligand blotting) and specifically to tubulin (sequence analysis), PAI(immunoblotting), and SC-uPA (immunoblotting and ligand blotting). Since tubulin would normally not be located on the surface of cells, it is presumably not a true membrane binding component. Although is is not surprising that u-PA, t-PA, PAI-1, and tubulin were isolated from these cells (20,21), reversible interactions between each of these proteins and t-PA were not expected. The only possible surface oriented protein might be membrane bound SC-uPA. Thus, t-PA may be binding to u-PA which has bound to the u-PA receptor (21). PAI- may bind to either t-PA or u-PA subsequently. In fact this is known to occur with u-PA (22). t-PA or u-PA may also interact with PAI- in the cell substratum. Since active-site modified t-PA or u-PA still demonstrate binding to cells, the site of interaction among the various proteins is presumably located in heavy chain. Previous results have demonstrated a specific sequence in the finger region (residues 7-20) which inhibited binding of tPA to cells (6). Other modules such as kl and k2 have not been studied yet. The low affinity of t-PA binding to HUVEC, A-431, and HOS hampers efforts to purify what may be considered a specific membrane associated binding site for t-PA. It should be noted that binding of t-PA to a 40,000 molecular weight protein similar to that described by Hajjar was not observed in these studies. Work is currently underway to explore whether t-PA-PAIcomplexes bind to HWEC via a specific receptor similar to that demonstrated on HEPG2 cells. Furthermore, it remains to be determined whether the binding or the lack of quantitative binding to all the cell lines tested (A-431, HOS, L-123, and Chang) resulted from varying levels of PAI-1, t-PA or u-PA in the substratum or on the cell membranes. ACKNOWLEDGEMENTS We would like to thank Dr. Martin Ruta for helpful suggestions and Judy Regan (CBER, Bethesda, MD) for synthesizing t-PA peptide (720). REFERENCES 1. Miles, L.A., Plow, E,F. Plasminogen receptors:Ubiquitous sites for cellular regulation of fibrinolysis. Fibrinolysis, 2, 61-72, 1988. 2. Blasi, F. Surface receptors for urokinase plasminogen activator. Fibrinolvsis, 2, 73-90, 1988. 3. Beebe, D.P. Binding of tissue plasminogen activator to human umbilical vein endothelial cells. Thromb. Res. 46, 241-253, 1987. 4. Hajjar, K.A., Hamel, N.M., Harpel, P.C., Nachman, R.L. Binding of tissue plasminogen activator to cultured human endothelial cells. J. Clin. Invest. 80, 1712-1719, 1987.
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5. Barnathan, E.S., Kuo, A., Van der Keyl, H., McCrae, K.R., Larsen, G.R., Cines, D.B. Tissue-type plasminogen activator binding to human endothelial cells. J. Biol. Chem. 253, 7792-7799, 1988. 6. Beebe, D-P., Miles, L.A., Plow, E.F. Tissue plasminogen activator binding to endothelial cells: inhibition by a peptide from the finger region (abstract #2033). Circulation, 78, 11-509, 1988. 7. Hajjar, K.A., and Hamel, N.M. Identification and characterization of human endothelial cell membranebinding sites for tissue plasminogen activator and urokinase. J. Biol. Chem., 265, 2908-2916, 1990. 8. Russell, M.E., Quertermous, T., Declerck, P.J., Collen, D., Haber, E., Homey, C.J. Binding of tissue type plasminogen activaotr with human endothelial cell monolayers. J. Biol. Chem., 265, 25692575, 1990. Ramakrishnan, V., Sinicrop, D.V., Dere, R., Darbonne, W.C., Bechtol, K.B., Baker, J.B. Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activaotr with human umbilical vain endothelial cell monolayers. J. Biol. Chem., 265, 2755-2762, 1990. 9.
10. Schleef, R.R., Podor, T-J., Dunne, E Mimuro, J., and Luskutoff, D.J. 'The majority of Type 1 Pi&minogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissuetype plasminogen activator. J. Cell Biol., 110, 155-163, 1990. 11. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685, 1970. 12. Karcher, D., Lowenthal, A., Thormer, H., and Noppe, M. J. Serological identification of viral antigens after electrophoretic transfer. Immunol. Methods, 43, 175-179, 1981. 13. Moos, M., Jr., Nguyen, N.Y., and Liu, T-Y. Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem., 263, 6005-6008, 1988. 14. Wienberg, J.B., Hobbs, M.M., and Pizza, S.V. Microassay for plasminogen photometric quantitation of cell-associated the activator using a chromogenic tripeptide substrate. J. Immunol. Methods, 75, 289-294, 1984. 15. Hajjar, K.A., Harpel, P.C., Jaffe, E-A., Nachman, R.L. Binding of plasminogen to cultured human endothelial cells. J. Biol. Chem. 261, 11656-11660, 1986. 16. Miles, L.A., Levin, E.G., Plescia, J., Collen, D., Plow, E.F. Plasminogen receptors, urokinase receptors and their modulation on human endothelial cells. Blood, in press, 1989.
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17. Loskutoff, D.J. The fibrinolytic system of cultured endothelial cells: Deciphering the balance between plasminogen activation and inhibition. In: Progress in Fibrinolvsis. J.F. Davidson et al (Eds.1, 7, 15-22, 1985. 18. Sakata, Y., Okada, M., Noro, A., and Matsuda, M. Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells. J. Biol. Chem., 263, 1960-1969, 1988. 19. Owensby, D.A., Morton, P.A., and Schwartz, A.L. Interactions between tissue-type plasminogen activator ad extracellular matrixassociated plasminogen activator inhibitor type-l in the human hepatoma cell line HepG2. J. Bio. Chem., 264, 18180-18187, 1989.17. 20. Saksela, O., Vaheri, A., Schleuning, W.D., Mignatti, P., Barlati, S. Plasminogen activators, activation inhibitors and alpha-2 macroglobulin produced by cultured normal and malignant human cells. Int. J. Cancer, 33, 609-616, 1984. 21. Nielson, L.S., Kellerman, G.M., Behrendt, N., Picone, R., Dano, K Blasi, F. A, 55,000-60,000 M receptor for urokinase-type pi&.minogen activator. J. Biol. Chem. 263, 2358-2363, 1988. 22. Cubellis, M.V., Andreasen, P., Ragno, P., Mayer, M., Dano, K., and Blasi, F. Accessibility of receptor-bound urokinase to type-l plasminogen activator inhibitor. proc. Natl. Acad. Sci. 86, 48284832, 1989.
CHARACTERIZATION OF TISSUE PLASMINOGEN ACTIVATOR BINDING PROTEINS ISOLATED FROM ENDOTHELIAL CELLS AND OTHER CELL TYPES
D.P. BEEBE, L.L. WOOD, and M. MOOS Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, U.S.A.
(Received 21.8.1989; accepted in revised form 13.5.1990 by Editor N.U. Bang)
ABSTRACT Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between '?I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HWEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a tPA affinity column revealed binding of t-PA to 54,000 Several t-PA and 95,000 molecular weight proteins. binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase.
INTRODUCTION Components of the fibrinolytic pathway including plasminogen, urokinase (u-PA), and tissue-type plasminogen activator (t-PA) have been shown to bind to specific receptors on a variety of cell types (l-5). The resulting activation of plasmin on cell surfaces may contribute to preventing clot formation and/or removing clots in vessel walls in the case of endothelial cells or tissue restructuring and/or metastasis in the case of malignant cells. A t-PA receptor with low affinity (K, = 18-240 nM) has been characterized on human umbilical vein endothelial cells (HWEC) (3-5). The interaction was shown to involve a specific region of Key Words: t-PA, receptor, cell lines 339
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the finger domain (residues 7-20) (6). A t-PA binding protein (40 kDa) has also been identified in isolated cell membranes (7). A high affinity, low density receptor has also been described which is most likely the plasminogen activator inhibitor (PAI-1) (4-5, 8, 9). PAI- localizes in the endothelial cell matrix (ECM) and is not associated on the membrane surface (10). t-PA can penetrate to the ECM and bind to PAI- (10). In the present report, we describe the binding of t-PA to different cell types as well as the characterization of several t-PA binding proteins isolated from immunopurified cell lysates. MATERIALS AND METHODS Reagents: Purified recombinant t-PA was a generous gift of Genentech, Inc. (South San Francisco, CA). High molecular weight u-PA (Winkinase) was kindly provided by Dr. Gene Murano (CBER, Bethesda, MD). Purified recombinant scu-PA was generously provided by Dr. Jack Henkin (Abbott Laboratories, IL). Na1251was purchased from New England Nuclear (Boston, MA). Phosphate buffered saline (PBS), M199 and D-MEM were purchased from Quality Biological (Gaithersburg, Endothelial cell growth factor (ECGF) and Nu Serum was MD). purchased from Collaborative Research (Bedford, MA). Fetal bovine serum (FBS), trypsin, gentamycin, and glutamine were purchased from GIBCO (Grand Island, NY). Monoclonal antibodies to t-PA, u-PA, and PAI- were obtained from Monozyme ApS (Charlottenlund, Denmark). t-PA peptide 7-20 (>95% pure) was synthesized on an ABI 430a automated peptide synthesizer and was generously provided by J. Regan and Dr. K. Seamon (CBER, Bethesda, MD). Iodination of t-PA: t-PA (25 ug) was labelled by the "Iodo-gen" method with 1 mCi Na'? as previously described (3). The specific activity was calculated to be 2.73-3.2 X 10' cpm per ug. Cell Cultures: Primary cultures of HUVEC were a generous gift of Dr. Tom Lawley (NIH, Bethesda, MD) and were maintained as previously described (3). Human osteosarcoma cells (HOS) were kindly provided by Dr. Martin Ruta (CBER, Bethesda, MD). A-431 cells (human epidermoid carcinoma), L-132 cells (human embryonic lung), and Chang liver cells were obtained from American Type Tissue Collection (Rockville, MD) and cultured in D-MEM with 10% FBS and 100 mM glutamine. Binding of "'I-t-PA to Cultured Cells: Each cell type was grown to confluence in 24-well tissue culture plates (Co-star, Lincoln, MA). Binding was performed as previously described with 20 ul dilutions of t-PA per well and 20 ul 1'51-t-PA (3 nM) per well. After incubation at 4°C for 30 min the cells in each well were washed three times and lysed with buffer (10% SDS,
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0.1 M EDTA) and counted (Compugamma, McLean, VA). The data were analyzed according to the method of Scatchard using the LIGAND program (courtesy of Dr. Peter Munson, NIH, Bethesda, MD). Chemical Crosslinking
of t-PA to Cultured Cells:
Cells were plated into Nunclon tissue culture dishes (245 X 245 X 2 mm) (Nunc, Denmark) at a density of 1 X lo* cells per dish and incubated overnight. The cell monolayer was washed once with 0.05 M glycine buffer containing 0.1 M NaCl (pH 3.0) to remove endogenous protein from cell surfaces, once with 0.5 M HEPES buffer (0.1 M NaCl, pH 7.5), and three times with PBS. Excess '251-t-PAwas incubated with cells at 4°C for 30 min. The cell monolayer was rinsed three times with PBS and subsequently incubated with the homobifunctional cross linking agent 3,3dithiobis(sulfosuccinimidyl)propionate (DTSSP) (3-1OmM) for 15 min at room temperature to crosslink t-PA to the receptor. The monolayer was then rinsed three times with PBS and lysed with 3 ml Triton X buffer (0.1 M Tris-HCl, 0.1% Triton X-100, pH 8.1). Fractionation
by High Performance
Liquid Chromatography
(HPLC):
HPLC was performed on a Beckman Model 344 instrument using a TSK 2000 SW column (7.5 mm X 30 cm) equilibrated in Tris buffer (0.02 M Tris-HCl, 0.15 M NaCl, 5 mM EDTA, pH 6.95). A Flo-One\Beta Radioactive flow detector (Radiomatic Instruments, Tampa, FL) was connected to the HPLC for analyzing radioactive samples. Human albumin (2.5% HA) (Miles, Berkeley, CA) was used to calibrate the column for molecular weight determination. t-PA Affinity
Column:
t-PA (1 mg/ml) was coupled in 0.1 M NaHCO, buffer to CNBr activated The Sepharose (Pharmacia, Uppsala, Sweden) at pH 5.5 overnight. coupling efficiency was estimated to be 85%. The cells were lysed in Triton X buffer containing protease inhibitors leupeptin (10 ug/ml) and Trasylol (20 u/ml) for 15 min and spun to remove the nuclei in a microfuge (10,000 X G) for 15 min. Cell lysates were The passed through the column which was equilibrated in PBS. column was washed until the absorbance reached background. Bound protein was then eluted with 0.1 M glycine-HCl buffer pH 2.5 and 0.5 ml fractions were collected and concentrated by rotoevaporation (Speed-Vat Concentrator, Savant). SDS-PAGE,
Immunoblotting,
and Ligand Blotting:
Samples were electrophoresed on 10% SDS-PAGE slab gels in a Mighty Small gel slab unit (Hoefer, San Francisco, CA) using the method were proteins immunoblotting the For Laemml i of (11) * electrophoretically transferred to nitrocellulose paper for 2 hr The blot was then incubated with the specific at 400 mamp. antibodies for 2 hr at 25°C followed by peroxidase conjugated antigoat or anti-mouse antibody and developed with o-dianisidine as previously described (12). For ligand blotting the blot was incubated with '251-t-PAovernight, washed in PBS, 2.5% HA, 1% Tween80 4 times for one hour each. The blot was then autoradiographed.
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Microsequence Analysis: Samples were run on SDS-PAGE at neutral pH and electrophoretically transferred to PVDF (Immobilon) membranes. The stained band was excised and placed in an automated sequenator (Applied Biosystem Model 470A). Sequence and analysis of PTH derivatives were performed according to standard procedures (13). RESULTS t-PA Binding to Various Cell Lines: Four different cell lines were tested for the ability to bind tPA. The A-431 cells and HOS cells demonstrated quantitative binding of t-PA whereas binding to Chang and L-123 was poor in compa$?ison. Figure 1 depicts the various binding curves. Binding to A-431 and HOS was reversible as shown by the dissociation of bound ligand in excess buffer fnot shown) and the time course for binding was similar to that‘of HUVEC (3).
200
100 TPA
ADDED
300 (I-M)
Fig. 1. Binding curves of '?-t-PA to Cells. (+) A-431 cells, (h) HOS cells, (A) HUVEC, (+) L-123 cells, (0) Chang cells. Binding was routinely performed for 30 min at 4°C. Non-specific binding (in the presence of 6.7 uM unlabelled t-PA) averaged 10%. Plots of bound over free versus bound ligand (Scatchard plots) were linear. The binding constants for A-431 and HOS were calculated from the LIGAND program assuming one class of binding site and were &= 9.42 X lo-*M,5.2 X lo6 sites per cell and z
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Crosslinking:
?-t-PA was incubated with HUVEC, A-431, and HOS cells at a concentration to saturate the receptors (1 uM) at 4°C for 30 min. The cells were washed to remove unbound ligand. The ligandreceptor complex was then covalently crosslinked with the water soluble homobifunctional agent DTSSP. This reagent is membrane impermeable and, therefore, only membrane components should be crosslinked. After thorough washing to remove the coupling agent the cell membranes were solubliized with Tris-Triton buffer and the supernatents were analyzed by HPLC on a TSK-3000 gel filtration column. The t-PA-receptor complexes from HUVEC, HOS, and A-431 were each chromatographed. A representative plot of the chromatography of the A-431-receptor complex is shown in Figure 2. -
A.
Mr
130K
65K
I
I
t
10
15
20
25
30
Fraction Number
Fig. 2. Chromatography of l?-t-PA Receptor (A-431) Complex on TSK 2000. A. Unreduced sample B. Reduced sample Two radioactive peaks corresponding to molecular weight ranges of 130,000 and 65,000 respectively were present in the elution profile (Figure 2a). The first peak, which is approximately 40% of the recovered cpm, most likely corresponds to the covalent complex of "'I-t-PA and a membrane protein whereas the second peak is presumably uncomplexed t-PA. When the crosslinked receptor complex was treated with 1 mM DTT (37"C, 2 hr) to cleave the thio bond in the crosslinking reagent, most of the 125-I counts were recovered in the 65,000 molecular weight range indicating recovery of the originally complexed t-PA ligand (Figure 2b). The cross-linking studies were repeated with HUVEC in suspension (cells were scraped off the plates). Autoradiography of the resulting t-PA receptor complexes which were run on revealed bands SDS-PAGE two corresponding to molecular weights of 116,000 and 65,000 (not shown).
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with t-PA Peptide:
The results with coupling whole t-PA to the receptor indicated that the &of the receptor was 51-65,000 since the complex eluted in the molecular weight range of 116-130,000. To obtain additional molecular weight estimates, binding to HWEC was performed with "'1 labelled t-PA peptide 7-20 (molecular weight 1792). This peptide, which corresponds to a portion of the finger region of t-PA, had previously been shown to compete with whole t-PA binding to HWEC (6). Following crosslinking with DTSSP according to the previous procedure, the receptor complex was chromatographed on TSK 2000 SW. As shown in Figure 3, two radioactive peaks were recovered but in this instance the molecular weights corresponded to 48-65,000 and 1792 and most likely represent the receptor complex and uncomplexed peptide, respectively. 1500 A.
Mr
<26OK 1
130K 65K
I
i
1792
1
246
1
Fraction Number
Fig. 3. Chromatography of l?-t-PA Peptide-Receptor Complex on TSK2000 SW. A. Top Panel:Absorbance 280 nm (albumin) B. Middle Panel: cpm (Y-Peptide 7-20) C. Bottom Panel: cpm (l"SI-Peptide-Receptor complex). Table 1 lists the retention times of the radiolabelled peaks and the protein markers obtained from chromatographing t-PA-receptor complexes on TSK 2000 SW. Isolation and chromatography of complexes from each cell type was performed in at least three separate experiments each. Since the peptide is 1792 D, the molecular weight of the receptor was estimated to be 48-65,000.
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TABLE 1 Chromatography of Cell Receptor Complexes on TSK 2000 Retention Time (min) M, Sample
130,Oo'O
HWEC-tPA 13.7-14.4 HOS-tPA 14 A431-tPA 14.5 A431-tPA (red)* --HWEC-tPA-Peptide ----tPA-Peptide 2.5% HA dimer 14.6 --monomer Acetyl-Tryptophan ---
65,000
1,792
246
16.3-18.2 17.. l 17.9 17.5 16.9-18.2 ---
--------26.4-29.3 26.4-28.4
----_--------
--17.1 ---
--_----
----31.7
* reduced Isolation of t-PA Receptor: A-431, HOS, and HWECs (1 X 10" cells) were washed in PBS and disrupted with Tris-Triton lysis buffer. The supernatents were then passed over a t-PA-conjugated Sepharose affinity column. The column was washed with PBS, then eluted with glycine buffer, pH 2.5. A small protein peak eluted and when this peak was pooled, concentrated and run on SDS-PAGE, both major and minor bands of approximately 54,000 molecular weight were detected as well as several high mw bands by Coomassie Blue staining (Figure 4,1ane b).
97
k
66
k 54
42 k
35
k k
c d efgh a b Fig. 4. SDS-PAGE (Coomassie Blue stain) and Immunoblot of the t-PA Binding Protein(s).Samples were run under reducing conditions(BME).
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(a) High molecular weight standards (b) Binding protein(s) from A431 cells (c) Binding protein blotted with anti-PAI- (d) t-PA (e) Binding protein blotted with anti-u-PA (f) u-PA blotted with antiu-PA (g) Binding protein blotted with anti-t-PA blotted with antit-PA (h) t-PA blotted with anti-t-PA. Similar molecular weight bands were detected by autoradiography of SDS-PAGE gels when 9 labelled HWEC were lysed and fractionated over the t-PA column (not shown). The same band pattern was also demonstrated by Coomassie Blue staining of SDS-PAGE gels of affinity purified cell lysates when the t-PA column was substituted with an A-H Sepharose column to which the t-PA peptide (res. 7-20) was conjugated (not shown).When affinity purified cell lysates were immunoblotted with antibodies to t-PA, u-PA or PAI-1, reactivity appeared in the molecular weight range of 54,000 with both anti-UK and anti-PAI- while the higher molecular weight bands reacted with antibodies to t-PA, u-PA, and PAI- (Figure 4, lanes c,e,and g). The high mw bands might represent t-PA-or u-PA-PAI- complexes. The major 54,000 molecular weight band was shown to be tubulin beta chain by microsequence analysis (76.5% identity in 17 a.a. overlap) (13): M.R.Y.I.V.N.I.Q.A.G.N.F.G.N.F.G.N.Q.I.G.M.A.T. Ligand Blotting with "'I-t-PA: Receptor preparations isolated from either the t-PA or the t-PA peptide affinity column were run on SDS-PAGE along with purified preparations of t-PA and ret scu-PA. The proteins were electrophoretically transferred to nitrocellulose and probed with "'I-t-PA. Figure 5a shows an autoradiogram of a representative blot.
54K-
1
2
Figure 5. Ligand Blot of Affinity purified Cell Lysates and sou-
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PA. Samples were run on SDS-PAGE and transferred to nitrocellulose. The blot was incubated with 1'51-t-PA,washed and autoradiographed. (1) Binding protein from A-431 cells isolated from the t-PA peptide affinity column. (2) recombinant scu-PA. Reactivity of the labelled ligand was demonstrated to 95,000 and 54,000 molecular weight bands in the affinity purified cell lysate (lane 1) as well to purified scu-PA (lane 2). Similarly, significant binding of "'I-t-PA to purified t-PA was demonstrated (not shown). When the affinity purified cell lysates were tested in an amidolytic assay (14), significant plasminogen activator activity (dilutions out to 1:512,000) was demonstrated in the absence of fibrin promoter fragments (not shown), indicative of the presence of u-PA. Furthermore, chromatography of "'I-t-PA or '?-u-PA on the t-PA affinity column demonstrated considerable binding of labelled material to the column (nearly 50% in either case) as opposed to X0.1% of 12'I-HA.These data would indicate significant interaction between t-PA and u-PA as well as self-association of t-PA. DISCUSSION Endothelial cells have the ability to bind several components of the fibrinolytic system including plasminogen (15), u-PA (16), and t-PA (3-5). These proteins bind via regions other than the catalytic site so as to allow expression of enzyme activity on the cell surface. In addition these cells synthesize u-PA and t-PA as well as the plasminogen activator inhibitor (PAI-1) (17). The latter has been shown to be associated with the extracellular matrix in HWEC (7,17). PAI- inhibits both u-PA and t-PA. Thus, both activators and inhibitors as well as the substrate plasminogen can be localized on the endothelial cell surface or the extra cellular matrix. Both Sakata et al (18) and Schleef et al (10) have shown that t-PA binds to PAI- to form high molecular weight complexes which then are released into the supernatent of HWEC. Since the majority of PAI- is located in the endothelial cell matrix as a homogeneous carpet underneath the cells, the t-PA-PAI- interaction would presumably occur in the ECM rather than on the cell membrane. The fact that t-PA forms complexes with PAI- even in the presence of a cell monolayer (10) indicates that t-PA can penetrate to the ECM As suggested by Scheef et al, HWEC in culture may not form tight junctions normally asscociated with endothelial cells in viva. In HEP G2 cells binding of t-PA has been shown to occur solely through its interactions with PAI- which is localized in the substratum (19). t-PA-PAI- complexes are endocytosed by the cells and binding of complexes is not competed for by free t-PA. The question then arises as to whether or not t-PA binds to a membrane component that is distinct from PAI- on HWEC membranes. Hajjar and Hamel have recently demonstrated the binding of t-PA to a 40,000 molecular weight protein isolated from purified HWEC membranes (7). In the work described here, t-PA was shown to bind
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to a component in the molecular weight range of 48-65,000 (crosslinking studies), to proteins in the molecular weight range of 54,000 as well as 95,000 (ligand blotting) and specifically to tubulin (sequence analysis), PAI(immunoblotting), and SC-uPA (immunoblotting and ligand blotting). Since tubulin would normally not be located on the surface of cells, it is presumably not a true membrane binding component. Although is is not surprising that u-PA, t-PA, PAI-1, and tubulin were isolated from these cells (20,21), reversible interactions between each of these proteins and t-PA were not expected. The only possible surface oriented protein might be membrane bound SC-uPA. Thus, t-PA may be binding to u-PA which has bound to the u-PA receptor (21). PAI- may bind to either t-PA or u-PA subsequently. In fact this is known to occur with u-PA (22). t-PA or u-PA may also interact with PAI- in the cell substratum. Since active-site modified t-PA or u-PA still demonstrate binding to cells, the site of interaction among the various proteins is presumably located in heavy chain. Previous results have demonstrated a specific sequence in the finger region (residues 7-20) which inhibited binding of tPA to cells (6). Other modules such as kl and k2 have not been studied yet. The low affinity of t-PA binding to HUVEC, A-431, and HOS hampers efforts to purify what may be considered a specific membrane associated binding site for t-PA. It should be noted that binding of t-PA to a 40,000 molecular weight protein similar to that described by Hajjar was not observed in these studies. Work is currently underway to explore whether t-PA-PAIcomplexes bind to HWEC via a specific receptor similar to that demonstrated on HEPG2 cells. Furthermore, it remains to be determined whether the binding or the lack of quantitative binding to all the cell lines tested (A-431, HOS, L-123, and Chang) resulted from varying levels of PAI-1, t-PA or u-PA in the substratum or on the cell membranes. ACKNOWLEDGEMENTS We would like to thank Dr. Martin Ruta for helpful suggestions and Judy Regan (CBER, Bethesda, MD) for synthesizing t-PA peptide (720). REFERENCES 1. Miles, L.A., Plow, E,F. Plasminogen receptors:Ubiquitous sites for cellular regulation of fibrinolysis. Fibrinolysis, 2, 61-72, 1988. 2. Blasi, F. Surface receptors for urokinase plasminogen activator. Fibrinolvsis, 2, 73-90, 1988. 3. Beebe, D.P. Binding of tissue plasminogen activator to human umbilical vein endothelial cells. Thromb. Res. 46, 241-253, 1987. 4. Hajjar, K.A., Hamel, N.M., Harpel, P.C., Nachman, R.L. Binding of tissue plasminogen activator to cultured human endothelial cells. J. Clin. Invest. 80, 1712-1719, 1987.
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5. Barnathan, E.S., Kuo, A., Van der Keyl, H., McCrae, K.R., Larsen, G.R., Cines, D.B. Tissue-type plasminogen activator binding to human endothelial cells. J. Biol. Chem. 253, 7792-7799, 1988. 6. Beebe, D-P., Miles, L.A., Plow, E.F. Tissue plasminogen activator binding to endothelial cells: inhibition by a peptide from the finger region (abstract #2033). Circulation, 78, 11-509, 1988. 7. Hajjar, K.A., and Hamel, N.M. Identification and characterization of human endothelial cell membranebinding sites for tissue plasminogen activator and urokinase. J. Biol. Chem., 265, 2908-2916, 1990. 8. Russell, M.E., Quertermous, T., Declerck, P.J., Collen, D., Haber, E., Homey, C.J. Binding of tissue type plasminogen activaotr with human endothelial cell monolayers. J. Biol. Chem., 265, 25692575, 1990. Ramakrishnan, V., Sinicrop, D.V., Dere, R., Darbonne, W.C., Bechtol, K.B., Baker, J.B. Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activaotr with human umbilical vain endothelial cell monolayers. J. Biol. Chem., 265, 2755-2762, 1990. 9.
10. Schleef, R.R., Podor, T-J., Dunne, E Mimuro, J., and Luskutoff, D.J. 'The majority of Type 1 Pi&minogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissuetype plasminogen activator. J. Cell Biol., 110, 155-163, 1990. 11. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685, 1970. 12. Karcher, D., Lowenthal, A., Thormer, H., and Noppe, M. J. Serological identification of viral antigens after electrophoretic transfer. Immunol. Methods, 43, 175-179, 1981. 13. Moos, M., Jr., Nguyen, N.Y., and Liu, T-Y. Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem., 263, 6005-6008, 1988. 14. Wienberg, J.B., Hobbs, M.M., and Pizza, S.V. Microassay for plasminogen photometric quantitation of cell-associated the activator using a chromogenic tripeptide substrate. J. Immunol. Methods, 75, 289-294, 1984. 15. Hajjar, K.A., Harpel, P.C., Jaffe, E-A., Nachman, R.L. Binding of plasminogen to cultured human endothelial cells. J. Biol. Chem. 261, 11656-11660, 1986. 16. Miles, L.A., Levin, E.G., Plescia, J., Collen, D., Plow, E.F. Plasminogen receptors, urokinase receptors and their modulation on human endothelial cells. Blood, in press, 1989.
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17. Loskutoff, D.J. The fibrinolytic system of cultured endothelial cells: Deciphering the balance between plasminogen activation and inhibition. In: Progress in Fibrinolvsis. J.F. Davidson et al (Eds.1, 7, 15-22, 1985. 18. Sakata, Y., Okada, M., Noro, A., and Matsuda, M. Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells. J. Biol. Chem., 263, 1960-1969, 1988. 19. Owensby, D.A., Morton, P.A., and Schwartz, A.L. Interactions between tissue-type plasminogen activator ad extracellular matrixassociated plasminogen activator inhibitor type-l in the human hepatoma cell line HepG2. J. Bio. Chem., 264, 18180-18187, 1989.17. 20. Saksela, O., Vaheri, A., Schleuning, W.D., Mignatti, P., Barlati, S. Plasminogen activators, activation inhibitors and alpha-2 macroglobulin produced by cultured normal and malignant human cells. Int. J. Cancer, 33, 609-616, 1984. 21. Nielson, L.S., Kellerman, G.M., Behrendt, N., Picone, R., Dano, K Blasi, F. A, 55,000-60,000 M receptor for urokinase-type pi&.minogen activator. J. Biol. Chem. 263, 2358-2363, 1988. 22. Cubellis, M.V., Andreasen, P., Ragno, P., Mayer, M., Dano, K., and Blasi, F. Accessibility of receptor-bound urokinase to type-l plasminogen activator inhibitor. proc. Natl. Acad. Sci. 86, 48284832, 1989.