- Email: [email protected]
Detection of vitronectin by ligand blotting with type 1 plasminogen activator inhibitor
026%9499/9010004-0197/$10.00
Fibrinolysis (1990) 4,197-202 01990 Longman Group UK Ltd
Detection of Vitronectin
by Ligand Blotting with Type 1 Plasminogen
Activator Inhibitor
D. Seiffert, J. Mimuro, D. J. Loskutoff SUMMARY. Ligand blotting procedures were developed for the detection of type 1 plasminogen activator inhibitor (PAI-1) binding protein(s) (BPS) transferred to nitrocellulose sheets after sodium dodecyl sulfate polyacrylamide gel electropboresis (SDS-PAGE). Purified vitronectin (Vn), a well cbaracterised PAI- BP, was employed to optimise this assay system. After blocking with casein, the sheets were washed and then incubated with either 1251-labelled PAI- (direct assay), or with unlabelled PAI- followed by a polyclonal antiserum to PAI- (indirect assay). In the latter case, the bound antibody was detected by using an 1251-labelled second antibody. Binding was dose-dependent with respect to both Vn and PAI-1, and only active PAI- bound to Vn (i.e. latent PAI-1 and PAI-1 in complex with tissue-type plasminogen activator (t-PA), did not bind to Vn in this system). Moreover, t-PA, arginine and acidic conditions dissociated PAI- from Vn adsorbed to nitrocellulose. Analysis of bovine plasma by these techniques revealed the presence of a single PAI- BP, and this protein was recognised by antisera to Vn. These results indicate that Vn previously fractionated by SDS-PAGE and transferred to nitrocellulose, continues to bind to PAI- in a manner that resembles its behaviour in plasma and on extracellular matrix. These ligand blotting procedures may thus represent useful new approaches for the detection of other SDS-stable PAI- binding proteins in biological samples. KEYWORDS. Type 1 plasminogen Vitronectin Ligand blotting.
activator.
Type
1 plasminogen
activator
inhibitor
binding
protein.
recent demonstration that PAI- is a component of the extracellular matrix (ECM) of a variety of cells,7-11 and that the bound form is fully active,sr9,11 raises the possibility that PAl-1 binding proteins (PAl-1 BPS) also may regulate fibrinolytic activity by stabilising and localising PAI-1.l PAl-1 binds to plasma vitronectin (Vn), and interaction that may again stabilise PAl-1 against its spontaneous loss of activity,l’-I6 and Vn competes with ECM for PAZ-1 binding. These results suggest that PAI- in ECM is bound to Vn.14 Quantitative assays that reveal the presence of PAl-1 BP(s) in complex biological samples will be important for the further characterisation of these proteins. We have developed ligand blotting assays for detecting PAl-1 BP(s). These assays utilise 1251labelled PAl-1 (direct assay) or unlabelled PAl-1 in conjunction with 1251-labelled antibodies (indirect assay) to detect PAl-1 BP(s) transferred to nitrocellulose after SDS-PAGE. The characteristics of this assay and its utility for the analysis of PAI- BP(s) in plasma is described.
The initiation of vascular fibrinolysis is a complex process, regulated not only by tissue-type plasminogen activator (t-PA) and urokinase-type PA (u-PA), but also through the interaction of these PAS with cell surface receptors, with components of the cell matrix, and with plasma cofactors.’ However, the primary regulatory element of this system may well be type 1 PA inhibitor (PAI-l), a physiological inhibitor of both t-PA and u-PA.lm3 Active PAl-1 is a relatively unstable molecule .in solution where it rapidly decays into an inactive, latent form.“6 The D. Seiffert, D. J. Loskutoff, Committee on Vascular Biology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA, J. Mimuro, Division of Hemostasis and Thrombosis, Institute of Hematology, Jichi Medical School, Tochigi-Ken, 329-04, Japan. This work was supported in part by Grant HL319.50-06 to D. J. Loskutoff from the National Institutes of health, and was performed during the tenure of a fellowship to D. Seiffert from Deutsche Forschungsgemeinschaft, Bonn, West Germany and a fellowship to J. Mimuro from the American Heart Association, California Affiliate, with funds contributed by the Central Mission Trails Chapter. 197
198 Detection of Vitronectin by Ligand Blotting with Type 1 Plasminogen Activator Inhibitor
Vitronectin (ngl
1
500
0 50 lb0
Vitronectin
(ng)
Fig. 1 Ligand blotting: Dose-response of Vn. Increasing amounts of either purified Vn (non-reduced [O] and reduced [0]) or BSA (U) were fractionated by SDS-PAGE, transferred to nitrocellulose sheets, and analysed by the indirect ligand blotting assay using lOOng/ml guanidine-activated PAI- (see ‘Methods’). Autoradiograms were prepared and the portions of the nitrocellulose containing PAI- (as revealed by the presence of bound 1251-labelled antibody) were excised and the cpm in them determined by using a gamma counter. The inset shows the actual autoradiograms obtained using non-reduced (N.R.) and reduced (R.) Vn.
MATERIALS AND METHODS Proteins Bovine PAI- was purified from the conditioned medium of bovine aortic endothelial cells (BAEs) as described previously.6 Purified PAI- was labelled with 125I using the Bolton-Hunter procedure,l’ and its specific ‘radioactivity was 160000 cpm/kg.18 Radiolabelled and unlabelled PAI- were activated with guanidine-hydrochloride ‘19and titrated against the international u-PA standard (66/46).6 Antiserum against PAIwas raised in rabbits.20 t-PA was isolated from the conditioned medium of human melanoma cells ,2l and t-PA/PAI-1 complexes were isolated using lysine sepharose.22 Vn was purified from pooled bovine plasma as described.23 The final product consisted of a single peptide of M, 80000 when analysed by SDS-PAGE under reducing conditions.
Ligand Blotting Assay SDS-PAGE was performed on slab gels according to Laemmli24 with reagents obtained from Bio-Rad laboratory (Richmond, CA, USA). The upper stacking gel contained 4% acrylamide and the lower separating gel contained 9% acrylamide. After SDSPAGE, the proteins in the gel were electrophoretitally transferred to nitrocellulose sheets (Schleicher
and Schuell, Keene, NH, USA).9,25 The sheets were incubated for 1 h at 25°C in a 5% casein solution (pH 7.4) to block nonspecific protein binding sites, and then soaked (25”C, 20min) in phosphate buffered saline (PBS) containing 0.25% Triton X-100 to neutralise any remaining SDS. The nitrocellulose sheets were incubated (4”C, 16h) either with guanidine-activated 1251-labelled PAI- (direct assay) or with guanidine activated unlabelied PAI- (indirect assay) in PBS containing 1% Tween 80, and then were washed with PBS containing 1% casein and 0.1% Triton X-100 (PBS/casein/Triton). Nitrocellulose sheets to be used in the direct ligand blotting assay then were washed with 2M NaCl in PBS, dried and exposed to X-ray film (Kodak XAR5, Rochester, NY, USA) at -70°C with an intensifying screen. The sheets to be employed for the indirect ligand blotting assay were incubated (25”C, 1 h) with rabbit anti-PAI- antiserum (in PBS/casein/Triton), washed, and then incubated (25”C, 1 h) with 1251labelled donkey anti-rabbit IgG (200000 cpm/ml in PBS/casein/Triton; Amersham, Arlington Heights, IL, USA). Finally, the sheets were washed with 2M NaCl, dried and exposed to X-ray film as described above. Control sheets for the indirect assay were treated identically except that the PAI- incubation step was omitted. In both assays, the amount of PAI- bound to Vn was determined by excising the portion of the nitrocellulose sheet corresponding to the specific signal and subjecting it to analysis by y-counting.
Fibrinolysis
199
2 nl
1.0
0.5
5.0
PAL1 (nglml)
0.5
5
1
10
50
100
PAI1 (nglml) Fig. 2 Ligand blotting: Dose-response of PAI-1. Non-reduced Vn (500ng/lane) was fractionated by SDS-PAGE, transferred to nitrocellulose sheets, and then incubated in the presence of increasing concentrations of guanidine-activated PAI-I. The amount of PAI- was quantified as in Figure 1. The inset shows the actual autoradiogram from this experiment.
RESULTS PAI- Binding Properties of Vn Immobilised Nitrocellulose after SDS-PAGE
on
PAI- binds to Vn in plasma.‘2-14 Experiments were performed to determine whether this interaction still occurs after SDS-PAGE. Increasing amounts of Vn were fractionated by SDS-PAGE, transferred to nitrocellulose sheets, and then incubated with a constant amount of quanidine-activated PAI(lOOng/ml). Under these conditions, the binding of PAI- to Vn increased linearly in a dose-dependent manner (Fig. 1). Although chemically reduced Vn could also bind to PAI- under these conditions, its binding capability was reduced approximately 6-fold. The PAI- binding capacity of Vn (up to 1 pg) was totally abolished upon boiling in sample buffer (not shown), and no binding of PAI- to BSA (up to 1 Fg) was observed (Fig. 1). Experiments were performed to determine whether the extent of binding also was dependent on the PAI- concentration. A constant amount of non-reduced Vn (500ng) on nitrocellulose sheets was incubated with increasing concentrations of activated PAI-1, and the extent of binding was determined (Fig. 2). Binding again increased in a dose-dependent manner, reaching a plateau at a PAI- concentration of lO-50ng/ml (Fig. 2). The results shown in Figures 1 and 2 indicate that the interaction between PAI- and Vn is dose-dependent with respect to both Vn and PAI- concentrations, and suggest that the indirect assay may be
useful for detecting and quantitating SDS-stable PAI- BPS in various samples. Experiments were performed to compare the sensitivity and specificity of the indirect assay with that of the direct assay since the latter assay is much simpler. In these experiments purified Vn and whole plasma were fractionated by SDS-PAGE, the proteins were transferred to nitrocellulose and then analysed by both the direct and the indirect ligand blotting assays (Fig. 3). A single band was detected when Vn was analysed by both procedures (compare panel A, lane 1 and panel B, lane 1). Moreover, both techniques also revealed a single PAI- BP in plasma (panel A, lane 2 and panel B, lane 2) which comigrated with purified Vn. This PAI- BP was not observed in Vn-depleted plasma prepared by passing bovine plasma over an immunoaffinity column of anti-Vn IgG coupled to Sepharose 4Bi4 (not shown). The positive signal observed in the indirect assay was PAI- dependent since no radioactive band was observed in control experiments in which the PAI- incubation step was omitted (Fig. 3, panel B, lane 3). The high molecular weight band seen in the indirect assay (panel B, lane 2) was also apparent when the PAI- preincubation step was omitted (panel B, lane 3) and when the antibodies to PAI- were omitted (not shown). This band thus appears to represent binding of the second antibody to IgG present in plasma. The autoradiograms obtained from the direct assay (Fig. 3, panel A, lanes 1 and 2) required 160h exposure time while those from the indirect assay were obtained after only 6h.
200
Detection of Vitronectin by Ligand Blotting with Type 1 Plasminogen Activator Inhibitor
97K68K43K-
29K12
12
3
4
5
Fig. 3 Comparison of the direct and indirect ligand blotting assay for PAI- BPS. Purified Vn (500na: lane 1, nanels A and B) and plasma (0.5 pl; lane 2, panels A aid B) were subjected to ’ SDS-PAGE and analysed by direct (panel A) and indirect (panel B) ligand blotting as described in ‘Methods’. Lane 3, panel B shows a control for the indirect ligand blot. In this case, after the plasma proteins were transferred to nitrocellulose, the membrane was processed as above but without the PAI- preincubation step. The autoradiograms for the direct ligand blot (160 h exposure) and for the indirect ligand blot (6 h exposure) are shown. Panel B, lane 4 and 5 show a nitrocellulose sheet containing 500ng non-reduced Vn incubated with either latent PAI- (lOOng/ml, lane 4) or with t-PA/PAI-1 complexes (equivalent to lOOng/ml PAI-1, lane 5). The positions of the molecular weight protein standards are indicated.
Thus, the indirect assay is considerably tive than the direct assay.
more sensi-
Additional Properties of Nitrocellulose-associated Vn A series of experiments were performed to determine whether Vn subjected to SDS-PAGE and immobilised on nitrocellulose retains properties of native Vn. After electrophoresis and transfer, the nitrocellulosebound Vn was incubated in the presence of guanidine-reactivated PAI-1, latent PAI-1, or t-PA/PAI-1 complexes, and the amount of bound PAI- was determined. Only active PAI- bound to Vn (Fig. 3, panels A and B, lane l), consistent with the observation that latent PAI- (Fig. 3, panel B, lane 4) and t-PA/PAI-1 complexes (Fig. 3, panel B, lane 5) bind poorly to Vn in plasma12,15 and in ECM.i8 Moreover, PAIbound to Vn previously immobilised on nitrocellulose could be dissociated from it by arginine, low pH and t-PA (Table). This behaviour is again consistent with the properties of native Vn.9,12,16 These experiments indicate that the PAIbinding properties of Vn subjected to SDS-PAGE and immobilization on nitrocellulose are similar to those of Vn in ECM and plasma. DISCUSSION PAI- is a relatively unstable molecule in solution,’ rapidly decaying into the latent form upon
secretion.However, PAIin ECM is quite stable.’ Thus, PAI- BP(s) may represent a new family of molecules that are present in blood and tissues, and that have evolved to localise and concentrate PAI- in tissues and to stabilise it in its active form.’ These considerations indicate that assays for the detection of PAI- BP(s) in complex biological samples (i.e. plasma and tissue extracts) may be important to further define and characterise this class of molecules. In this report, we describe a simple ligand blotting procedure for the detection of PAIBP(s). The assay was developed using purified Vn as a prototype PAI- BP. This assay showed that after SDS-PAGE and transfer to nitrocellulose, Vn continued to bind to PAI- in a dose-dependent manner and that binding was saturable (Figs. 1 and 2). The PAIbinding property of Vn seems to be unchanged by SDS-PAGE and immobilisation on nitrocellulose since arginine, low pH, and t-PA continue to dissociate PAI-l/Vn complexes to the same extent as observed for native Vn (Table; see a1s09,12,14,16).The specificity of PAI- binding in this system was further confirmed by the fact that only active PAI- bound to Vn (Fig. 3). Although inactive, latent PAI- binds to ECM9 and to Vn,15 its affinity for ECM and Vn is at least 15fold less than that of activated PAI-1.12,‘8 These facts may explain why no apparent binding of inactive, latent PAI- at low concentration (lOOr& ml) to Vn is revealed under the assay conditions employed. The indirect assay was relatively sensitive since as little as 50ng of Vn could be detected (Fig. 1). Although the indirect ligand blotting assay is more complex than the direct assay (i.e. it requires two additional steps), its specificity is unchanged in comparison to the direct assay (Fig. 3). More importantly, its sensitivity is approximately lo-fold greater than the direct assay suggesting that the indirect assay will be the method of choice for most analyses. Plasma is a relatively complex mixture of proteins, lipids, hormones and proteoglycans. In spite of this, only a single PAI- BP was detected when plasma was
Table Effect of various treatments
nitrocellulose-associated
on the PAI-
bound to
Vn
Treatment
Remaining PAI-
BSA (lmg/ml) in PBS (control) t-PA (1 l&ml) in PBS Arginine O.lM pH 7.4 l.OM pH 7.4 Glycine O.lM pH 7.4 O.lM pH 2.5
100 10 70 50 100 10
(%)
Non-reduced Vn (500ng) was fractionated by SDS-PAGE, transferred to nitrocellulose membranes, incubated (4”C, 16h) with guanidine-activated PAI- (lOOng/ml), and washed. The membranes were then incubated (25°C 1 h) in buffer containing the reagents listed above, and the amount of PAI- remaining on the membranes was detected by the indirect approach. The data are expressed as percentage of the control incubated with BSA alone.
Fibrinolysis
analysed by both the direct and indirect methods (Fig. 3), and it comigrated with purified Vn. This PAI- BP was not detected in Vn-depleted plasma (not shown) indicating that the primary SDS-stable PAI- BP in bovine plasma is Vn. Whether other, SDS-labile PAI- BP(s) exist in plasma and tissues remains to be determined. The specificity of this result suggests that the ligand blotting assay for PAIBP(s) may be helpful for the detection of new PAIBP(s) in other complex samples (i.e. tissues), and may aid in delineating their role in PA-dependent biological processes as diverse as fibrinolysis, tumour cell invasion, inflammation, and development.26s27
LIST OF ABBREVIATIONS BAEs: bovine aortic endothelial cells BSA: bovine serum albumin ECM: extracellular matrix PA: plasminogen activator PAI-1: type 1 PA inhibitor PAI- BP: PAI- binding protein PBS: phosphate buffered saline SDS: sodium dodecyl sulfate SDS-PAGE: SDS-polyacrylamide gel phoresis t-PA: tissue-type PA u-PA: urokinase-type PA Vn: vitronectin
electro-
ACKNOWLEDGEMENTS We wish to acknowledge Dr R. Schleef for helpful comments, N. Wagner for excellent technical assistance and P. Tayman for fine secretarial assistance.
REFERENCES 1. Loskutoff D J, Sawdey M, Mimuro J 1989 Type 1
2. 3.
4.
5. 6.
plasminogen activator inhibitor. In: Coller B S (ed) Progress in Hemostasis and Thrombosis, 9th Edn. W.B. Saunders Company, Philadelphia, pp 87-116 Sprengers E D, Kluft C 1987 Plasminogen activator inhibitors. Blood 69: 381-387 Hekman C M, Loskutoff D J 1988 Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator. Arch Biochem Biophys 262: 199-210 Kooistra T, Sprengers E D, van Hinsbergh V W M 1986 Rapid inactivation of the plasminogen-activator inhibitor upon secretion from cultured human endothelial cells. Biochem J 239: 497-593 Levin E G, Sante11 L 1987 Conversion of the active to latent plasminogen activator inhibitor from human endothelial cells. Blood 70: 1090-1098 Hekman C M, Loskutoff D J 1988 Bovine plasminogen activator inhibitor 1: Specificity determinations and comparison of the active, latent, and guanidine-activated forms. Biochemistry 27: 2911-2918
201
7. Laiho M, Saksela 0, Andreasen P A, Keski-Oja J 1986 Enhanced production and extracellular deposition of the endothelial-type plasminogen activator inhibitor in cultured human lung libroblasts by transforming growth factor-p. J Cell Biol 103: 2403-2410 8. Knudsen B S, Hapel P C, Nachman R L 1987 Plasminogen activator inhibitor is associated with the extracellular matrix of cultured bovine smooth muscle cells. J Clin Invest 80: 1082-1089 9. Mimuro J, Schleef R R, Loskutoff D J 1987 The extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor. Blood 70: 721-728 10. Rheinwald J G, Jorgensen J L, Hahn W C, Terpstra A J, O’Connell T M, Plummer K K 1987 Mesosecrin: A secreted glycoprotein produced in abundance by human mesothelial, endothelial and kidnev epithelial cells in culture. J Cell Biol . 104: 263-275 11. Levin E G, Sante11 L 1987 Association of plasminogen activator inhibitor (PAI-1) with the growth substratum and membrane of human endothelial cell. J Cell Biol105: 2543-2549 12. Declerck P J, De Mol M, Alessi M-C et al 1988 Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin). J Biol Chem 263: 15454-15461 13. Wiman B, Almquist A, Sigurdardottir 0, Lindahl T 1988 Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in plasma. FEB 242: 125-128 14. Mimuro J, Loskutoff D J 1989 Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin. J Biol Chem 264: 936-939 15. Salonen E M, Vaheri A, Pollanen J et al 1989 Interaction of plasminogen activator inhibitor (PAI-1) with vitronectin. J Biol Chem 264: 6339-6343 16. Wun T-C, Palmier M 0, Siegel N R, Smith C E 1989 Affinity purification of active plasminogen activator inhibitor-l (PAI-1) using immobilized anhydrourokinase. J Biol Chem 264: 7862-7868 17. Bolton A E, Hunter W M 1973 The labelling of proteins to high specific radioactivities by conjugation to a iZ51containing acylating agent. Application to the radioimmunoassay. Biochem J 133: 529-539 18. Mimuro J, Loskutoff D J 1989 Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells. J Biol Chem 264: 50585063 19. Hekman C M, Loskutoff D J 1985 Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem 260: 11581-11587 20. van Mourik J A, Lawrence D A, Loskutoff D J 1984 Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. J Biol Chem 259: 14914-14921 21. Schleef R R, Sinha M, Loskutoff D J 1985 Characterization of two monoclonal antibodies against human tissue-type plasminogen activator. Thromb Haemost 53: 170-175 22. Schleef R R, Wagner N V, Sinha M, Loskutoff D J 1986 A monoclonal antibody that does not recognize tissue-type plasminogen activator bound to its naturally occurring inhibitor. Thromb Haemost 56: 328-332 23. Dahlback B, Podack E R 1985 Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group. Biochem 24: 2368-2374 24. Laemmli U K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227: 68&685 25. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci 76: 435t3-4354
.302
.D~;tection of Vitronectin
by Ligand Blotting with Type l.Pla.sminogen.Activator.
26. Dano K, Andreasen J, Grondahl-Hansen J et al 1985 Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res 44: 139266
Received: 11 April 1990 Accepted: 25 May 1990 Offprint orders to: D. L. Loskutoff, Research Institute of Scripps Clinic, IMM-15, 10666 North Torrey Pines Road, La Jolla, CA 92037. USA.
Inhibitor
27. Saksela 0, Rifkin D B 1988 Cell-associated plasminogen activation: Regulation and physiological functions. Ann Rev Cell Bio14: 93126
Fibrinolysis (1990) 4,197-202 01990 Longman Group UK Ltd
Detection of Vitronectin
by Ligand Blotting with Type 1 Plasminogen
Activator Inhibitor
D. Seiffert, J. Mimuro, D. J. Loskutoff SUMMARY. Ligand blotting procedures were developed for the detection of type 1 plasminogen activator inhibitor (PAI-1) binding protein(s) (BPS) transferred to nitrocellulose sheets after sodium dodecyl sulfate polyacrylamide gel electropboresis (SDS-PAGE). Purified vitronectin (Vn), a well cbaracterised PAI- BP, was employed to optimise this assay system. After blocking with casein, the sheets were washed and then incubated with either 1251-labelled PAI- (direct assay), or with unlabelled PAI- followed by a polyclonal antiserum to PAI- (indirect assay). In the latter case, the bound antibody was detected by using an 1251-labelled second antibody. Binding was dose-dependent with respect to both Vn and PAI-1, and only active PAI- bound to Vn (i.e. latent PAI-1 and PAI-1 in complex with tissue-type plasminogen activator (t-PA), did not bind to Vn in this system). Moreover, t-PA, arginine and acidic conditions dissociated PAI- from Vn adsorbed to nitrocellulose. Analysis of bovine plasma by these techniques revealed the presence of a single PAI- BP, and this protein was recognised by antisera to Vn. These results indicate that Vn previously fractionated by SDS-PAGE and transferred to nitrocellulose, continues to bind to PAI- in a manner that resembles its behaviour in plasma and on extracellular matrix. These ligand blotting procedures may thus represent useful new approaches for the detection of other SDS-stable PAI- binding proteins in biological samples. KEYWORDS. Type 1 plasminogen Vitronectin Ligand blotting.
activator.
Type
1 plasminogen
activator
inhibitor
binding
protein.
recent demonstration that PAI- is a component of the extracellular matrix (ECM) of a variety of cells,7-11 and that the bound form is fully active,sr9,11 raises the possibility that PAl-1 binding proteins (PAl-1 BPS) also may regulate fibrinolytic activity by stabilising and localising PAI-1.l PAl-1 binds to plasma vitronectin (Vn), and interaction that may again stabilise PAl-1 against its spontaneous loss of activity,l’-I6 and Vn competes with ECM for PAZ-1 binding. These results suggest that PAI- in ECM is bound to Vn.14 Quantitative assays that reveal the presence of PAl-1 BP(s) in complex biological samples will be important for the further characterisation of these proteins. We have developed ligand blotting assays for detecting PAl-1 BP(s). These assays utilise 1251labelled PAl-1 (direct assay) or unlabelled PAl-1 in conjunction with 1251-labelled antibodies (indirect assay) to detect PAl-1 BP(s) transferred to nitrocellulose after SDS-PAGE. The characteristics of this assay and its utility for the analysis of PAI- BP(s) in plasma is described.
The initiation of vascular fibrinolysis is a complex process, regulated not only by tissue-type plasminogen activator (t-PA) and urokinase-type PA (u-PA), but also through the interaction of these PAS with cell surface receptors, with components of the cell matrix, and with plasma cofactors.’ However, the primary regulatory element of this system may well be type 1 PA inhibitor (PAI-l), a physiological inhibitor of both t-PA and u-PA.lm3 Active PAl-1 is a relatively unstable molecule .in solution where it rapidly decays into an inactive, latent form.“6 The D. Seiffert, D. J. Loskutoff, Committee on Vascular Biology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA, J. Mimuro, Division of Hemostasis and Thrombosis, Institute of Hematology, Jichi Medical School, Tochigi-Ken, 329-04, Japan. This work was supported in part by Grant HL319.50-06 to D. J. Loskutoff from the National Institutes of health, and was performed during the tenure of a fellowship to D. Seiffert from Deutsche Forschungsgemeinschaft, Bonn, West Germany and a fellowship to J. Mimuro from the American Heart Association, California Affiliate, with funds contributed by the Central Mission Trails Chapter. 197
198 Detection of Vitronectin by Ligand Blotting with Type 1 Plasminogen Activator Inhibitor
Vitronectin (ngl
1
500
0 50 lb0
Vitronectin
(ng)
Fig. 1 Ligand blotting: Dose-response of Vn. Increasing amounts of either purified Vn (non-reduced [O] and reduced [0]) or BSA (U) were fractionated by SDS-PAGE, transferred to nitrocellulose sheets, and analysed by the indirect ligand blotting assay using lOOng/ml guanidine-activated PAI- (see ‘Methods’). Autoradiograms were prepared and the portions of the nitrocellulose containing PAI- (as revealed by the presence of bound 1251-labelled antibody) were excised and the cpm in them determined by using a gamma counter. The inset shows the actual autoradiograms obtained using non-reduced (N.R.) and reduced (R.) Vn.
MATERIALS AND METHODS Proteins Bovine PAI- was purified from the conditioned medium of bovine aortic endothelial cells (BAEs) as described previously.6 Purified PAI- was labelled with 125I using the Bolton-Hunter procedure,l’ and its specific ‘radioactivity was 160000 cpm/kg.18 Radiolabelled and unlabelled PAI- were activated with guanidine-hydrochloride ‘19and titrated against the international u-PA standard (66/46).6 Antiserum against PAIwas raised in rabbits.20 t-PA was isolated from the conditioned medium of human melanoma cells ,2l and t-PA/PAI-1 complexes were isolated using lysine sepharose.22 Vn was purified from pooled bovine plasma as described.23 The final product consisted of a single peptide of M, 80000 when analysed by SDS-PAGE under reducing conditions.
Ligand Blotting Assay SDS-PAGE was performed on slab gels according to Laemmli24 with reagents obtained from Bio-Rad laboratory (Richmond, CA, USA). The upper stacking gel contained 4% acrylamide and the lower separating gel contained 9% acrylamide. After SDSPAGE, the proteins in the gel were electrophoretitally transferred to nitrocellulose sheets (Schleicher
and Schuell, Keene, NH, USA).9,25 The sheets were incubated for 1 h at 25°C in a 5% casein solution (pH 7.4) to block nonspecific protein binding sites, and then soaked (25”C, 20min) in phosphate buffered saline (PBS) containing 0.25% Triton X-100 to neutralise any remaining SDS. The nitrocellulose sheets were incubated (4”C, 16h) either with guanidine-activated 1251-labelled PAI- (direct assay) or with guanidine activated unlabelied PAI- (indirect assay) in PBS containing 1% Tween 80, and then were washed with PBS containing 1% casein and 0.1% Triton X-100 (PBS/casein/Triton). Nitrocellulose sheets to be used in the direct ligand blotting assay then were washed with 2M NaCl in PBS, dried and exposed to X-ray film (Kodak XAR5, Rochester, NY, USA) at -70°C with an intensifying screen. The sheets to be employed for the indirect ligand blotting assay were incubated (25”C, 1 h) with rabbit anti-PAI- antiserum (in PBS/casein/Triton), washed, and then incubated (25”C, 1 h) with 1251labelled donkey anti-rabbit IgG (200000 cpm/ml in PBS/casein/Triton; Amersham, Arlington Heights, IL, USA). Finally, the sheets were washed with 2M NaCl, dried and exposed to X-ray film as described above. Control sheets for the indirect assay were treated identically except that the PAI- incubation step was omitted. In both assays, the amount of PAI- bound to Vn was determined by excising the portion of the nitrocellulose sheet corresponding to the specific signal and subjecting it to analysis by y-counting.
Fibrinolysis
199
2 nl
1.0
0.5
5.0
PAL1 (nglml)
0.5
5
1
10
50
100
PAI1 (nglml) Fig. 2 Ligand blotting: Dose-response of PAI-1. Non-reduced Vn (500ng/lane) was fractionated by SDS-PAGE, transferred to nitrocellulose sheets, and then incubated in the presence of increasing concentrations of guanidine-activated PAI-I. The amount of PAI- was quantified as in Figure 1. The inset shows the actual autoradiogram from this experiment.
RESULTS PAI- Binding Properties of Vn Immobilised Nitrocellulose after SDS-PAGE
on
PAI- binds to Vn in plasma.‘2-14 Experiments were performed to determine whether this interaction still occurs after SDS-PAGE. Increasing amounts of Vn were fractionated by SDS-PAGE, transferred to nitrocellulose sheets, and then incubated with a constant amount of quanidine-activated PAI(lOOng/ml). Under these conditions, the binding of PAI- to Vn increased linearly in a dose-dependent manner (Fig. 1). Although chemically reduced Vn could also bind to PAI- under these conditions, its binding capability was reduced approximately 6-fold. The PAI- binding capacity of Vn (up to 1 pg) was totally abolished upon boiling in sample buffer (not shown), and no binding of PAI- to BSA (up to 1 Fg) was observed (Fig. 1). Experiments were performed to determine whether the extent of binding also was dependent on the PAI- concentration. A constant amount of non-reduced Vn (500ng) on nitrocellulose sheets was incubated with increasing concentrations of activated PAI-1, and the extent of binding was determined (Fig. 2). Binding again increased in a dose-dependent manner, reaching a plateau at a PAI- concentration of lO-50ng/ml (Fig. 2). The results shown in Figures 1 and 2 indicate that the interaction between PAI- and Vn is dose-dependent with respect to both Vn and PAI- concentrations, and suggest that the indirect assay may be
useful for detecting and quantitating SDS-stable PAI- BPS in various samples. Experiments were performed to compare the sensitivity and specificity of the indirect assay with that of the direct assay since the latter assay is much simpler. In these experiments purified Vn and whole plasma were fractionated by SDS-PAGE, the proteins were transferred to nitrocellulose and then analysed by both the direct and the indirect ligand blotting assays (Fig. 3). A single band was detected when Vn was analysed by both procedures (compare panel A, lane 1 and panel B, lane 1). Moreover, both techniques also revealed a single PAI- BP in plasma (panel A, lane 2 and panel B, lane 2) which comigrated with purified Vn. This PAI- BP was not observed in Vn-depleted plasma prepared by passing bovine plasma over an immunoaffinity column of anti-Vn IgG coupled to Sepharose 4Bi4 (not shown). The positive signal observed in the indirect assay was PAI- dependent since no radioactive band was observed in control experiments in which the PAI- incubation step was omitted (Fig. 3, panel B, lane 3). The high molecular weight band seen in the indirect assay (panel B, lane 2) was also apparent when the PAI- preincubation step was omitted (panel B, lane 3) and when the antibodies to PAI- were omitted (not shown). This band thus appears to represent binding of the second antibody to IgG present in plasma. The autoradiograms obtained from the direct assay (Fig. 3, panel A, lanes 1 and 2) required 160h exposure time while those from the indirect assay were obtained after only 6h.
200
Detection of Vitronectin by Ligand Blotting with Type 1 Plasminogen Activator Inhibitor
97K68K43K-
29K12
12
3
4
5
Fig. 3 Comparison of the direct and indirect ligand blotting assay for PAI- BPS. Purified Vn (500na: lane 1, nanels A and B) and plasma (0.5 pl; lane 2, panels A aid B) were subjected to ’ SDS-PAGE and analysed by direct (panel A) and indirect (panel B) ligand blotting as described in ‘Methods’. Lane 3, panel B shows a control for the indirect ligand blot. In this case, after the plasma proteins were transferred to nitrocellulose, the membrane was processed as above but without the PAI- preincubation step. The autoradiograms for the direct ligand blot (160 h exposure) and for the indirect ligand blot (6 h exposure) are shown. Panel B, lane 4 and 5 show a nitrocellulose sheet containing 500ng non-reduced Vn incubated with either latent PAI- (lOOng/ml, lane 4) or with t-PA/PAI-1 complexes (equivalent to lOOng/ml PAI-1, lane 5). The positions of the molecular weight protein standards are indicated.
Thus, the indirect assay is considerably tive than the direct assay.
more sensi-
Additional Properties of Nitrocellulose-associated Vn A series of experiments were performed to determine whether Vn subjected to SDS-PAGE and immobilised on nitrocellulose retains properties of native Vn. After electrophoresis and transfer, the nitrocellulosebound Vn was incubated in the presence of guanidine-reactivated PAI-1, latent PAI-1, or t-PA/PAI-1 complexes, and the amount of bound PAI- was determined. Only active PAI- bound to Vn (Fig. 3, panels A and B, lane l), consistent with the observation that latent PAI- (Fig. 3, panel B, lane 4) and t-PA/PAI-1 complexes (Fig. 3, panel B, lane 5) bind poorly to Vn in plasma12,15 and in ECM.i8 Moreover, PAIbound to Vn previously immobilised on nitrocellulose could be dissociated from it by arginine, low pH and t-PA (Table). This behaviour is again consistent with the properties of native Vn.9,12,16 These experiments indicate that the PAIbinding properties of Vn subjected to SDS-PAGE and immobilization on nitrocellulose are similar to those of Vn in ECM and plasma. DISCUSSION PAI- is a relatively unstable molecule in solution,’ rapidly decaying into the latent form upon
secretion.However, PAIin ECM is quite stable.’ Thus, PAI- BP(s) may represent a new family of molecules that are present in blood and tissues, and that have evolved to localise and concentrate PAI- in tissues and to stabilise it in its active form.’ These considerations indicate that assays for the detection of PAI- BP(s) in complex biological samples (i.e. plasma and tissue extracts) may be important to further define and characterise this class of molecules. In this report, we describe a simple ligand blotting procedure for the detection of PAIBP(s). The assay was developed using purified Vn as a prototype PAI- BP. This assay showed that after SDS-PAGE and transfer to nitrocellulose, Vn continued to bind to PAI- in a dose-dependent manner and that binding was saturable (Figs. 1 and 2). The PAIbinding property of Vn seems to be unchanged by SDS-PAGE and immobilisation on nitrocellulose since arginine, low pH, and t-PA continue to dissociate PAI-l/Vn complexes to the same extent as observed for native Vn (Table; see a1s09,12,14,16).The specificity of PAI- binding in this system was further confirmed by the fact that only active PAI- bound to Vn (Fig. 3). Although inactive, latent PAI- binds to ECM9 and to Vn,15 its affinity for ECM and Vn is at least 15fold less than that of activated PAI-1.12,‘8 These facts may explain why no apparent binding of inactive, latent PAI- at low concentration (lOOr& ml) to Vn is revealed under the assay conditions employed. The indirect assay was relatively sensitive since as little as 50ng of Vn could be detected (Fig. 1). Although the indirect ligand blotting assay is more complex than the direct assay (i.e. it requires two additional steps), its specificity is unchanged in comparison to the direct assay (Fig. 3). More importantly, its sensitivity is approximately lo-fold greater than the direct assay suggesting that the indirect assay will be the method of choice for most analyses. Plasma is a relatively complex mixture of proteins, lipids, hormones and proteoglycans. In spite of this, only a single PAI- BP was detected when plasma was
Table Effect of various treatments
nitrocellulose-associated
on the PAI-
bound to
Vn
Treatment
Remaining PAI-
BSA (lmg/ml) in PBS (control) t-PA (1 l&ml) in PBS Arginine O.lM pH 7.4 l.OM pH 7.4 Glycine O.lM pH 7.4 O.lM pH 2.5
100 10 70 50 100 10
(%)
Non-reduced Vn (500ng) was fractionated by SDS-PAGE, transferred to nitrocellulose membranes, incubated (4”C, 16h) with guanidine-activated PAI- (lOOng/ml), and washed. The membranes were then incubated (25°C 1 h) in buffer containing the reagents listed above, and the amount of PAI- remaining on the membranes was detected by the indirect approach. The data are expressed as percentage of the control incubated with BSA alone.
Fibrinolysis
analysed by both the direct and indirect methods (Fig. 3), and it comigrated with purified Vn. This PAI- BP was not detected in Vn-depleted plasma (not shown) indicating that the primary SDS-stable PAI- BP in bovine plasma is Vn. Whether other, SDS-labile PAI- BP(s) exist in plasma and tissues remains to be determined. The specificity of this result suggests that the ligand blotting assay for PAIBP(s) may be helpful for the detection of new PAIBP(s) in other complex samples (i.e. tissues), and may aid in delineating their role in PA-dependent biological processes as diverse as fibrinolysis, tumour cell invasion, inflammation, and development.26s27
LIST OF ABBREVIATIONS BAEs: bovine aortic endothelial cells BSA: bovine serum albumin ECM: extracellular matrix PA: plasminogen activator PAI-1: type 1 PA inhibitor PAI- BP: PAI- binding protein PBS: phosphate buffered saline SDS: sodium dodecyl sulfate SDS-PAGE: SDS-polyacrylamide gel phoresis t-PA: tissue-type PA u-PA: urokinase-type PA Vn: vitronectin
electro-
ACKNOWLEDGEMENTS We wish to acknowledge Dr R. Schleef for helpful comments, N. Wagner for excellent technical assistance and P. Tayman for fine secretarial assistance.
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.D~;tection of Vitronectin
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Received: 11 April 1990 Accepted: 25 May 1990 Offprint orders to: D. L. Loskutoff, Research Institute of Scripps Clinic, IMM-15, 10666 North Torrey Pines Road, La Jolla, CA 92037. USA.
Inhibitor
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