- Email: [email protected]
Immunological detection of conformational changes of type 1 plasminogen activator inhibitor associated with activation
Fibnnolysu (1991) 5,225231 01991 Longman Group UK Lfd
Immunological Plasminogen
D. Seiffert**,
Detection of Conformational
Changes of Type 1
Activator Inhibitor Associated with Activation
T. J. Podor”
SUMMARY. The effect of activation, solid-phase surfaces, tissue-type plasminogen activator (t-PA) and vitronectin (Vn) on the epitope expression of monoclonal antibody (Mab) to bovine type 1 plasminogen activator inhibitor (PAI-1) was analysed. The epitope for Mab 12C2 was optimally expressed on PAI- in solution (active, free, complexed or cleaved by t-PA) and bound to Vn. Activation of PAI- by guanidine hydrochloride increased the apparent PAI- antigen concentration in a Mab 12C2-based immunoassay by about 300%, whereas sodium dodecyl sulfate (SDS) decreased the antigen concentration to 44%. Both treatments increased PAI- activity to 1300%. Thus, activation by denaturants may lead to differences in specific activity (molar units of urinary type PA inhibitory activity/molar PAI- antigen) of about 3000%. These results thus suggest different conformations of SDS vs guanidine-activated PAI-1. KEYWORDS. Extracellular
Type 1 plasminogen matrix. Vitronectin.
activator
inhibitor.
Monoclonal
Plasminogen
activator.
as sodium dodecyl sulfate (SDS),’ guanidine hydrochloride’.879 or negatively charged phospholipids’O and heat.” Conversion of latent PAl-1 to its active form by denaturants is believed to be associated with unfolding of the protein’s tertiary structure as suggested by an apparent change in M, which was determined by gel filtration chromatography” and equilibrium centrifugation.’ However, little is known about changes in PAl-1 antigenicity associated with activation by denaturants or following its interaction with PAS and vitronectin (Vn). In order to investigate the conformational changes associated with latent, activated and vitronectin (Vn)- or PA-modified PAI-1, we have characterised a monoclonal antibody (Mab) to bovine PAl-1 and compared expression of its distinct epitope on various forms of solution- and surface-phase PAI-1. These studies demonstrate that PAI-I antigenic conformation was differentially altered by SDS- or guanidine-hydrochloride activation.
The initiation of vascular fibrinolysis is a complex process, regulated not only by tissue-type plasminogen activator (t-PA) and urinary-type PA (u-PA), but also through the interactions of plasminogen and PAS with plasma-cofactors and solid-phase supports such as cell surface receptors and extracellular matrices (ECM) (for review, see ref. 1). The primary regulator of this system appears to be type 1 plasminogen activator inhibitor (PAI-l), a member of the serine proteinase inhibitor superfamily of proteins.2-5 PAl-1 is one of the most highly regulated of the fibrinolytic components (for review, see ref.5). Changes in its activity, whether naturally occurring or experimentally induced, may disrupi normal fibrinolytic balance and result in dramatic increases or decreases in net fibrinolytic activity (for review, see ref.5). PAl-1 is a relatively unstable molecule that exists in vivo and in vitro, in both an active and latent form.68 Activation of latent PAl-1 activity in plasma or conditioned medium (CM) from cultured cells can be achieved following treatment with denaturants such
MATERIALS D. Seiffert, T. J. Podor, Committee on Vascular Biology. CVB-3. Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037. USA. *Present address: Department of Pathology, McMaster University, Hamilton, Ontario, Canada. “*Clinical Experimental Physiology Department of Medical Physiology. Schwarzspanierstrasse 17. A-1090, Vienna, Austria.
Chemicals,
AND METHODS
Proteins,
Reagents
All chemicals were the highest analytic grade commercially available. Tissue culture plasticware was obtained from Corning (Corning, NY); minimal 225
R
antibodies.
226
Immunological Detection of Conformational Changes of Type 1 Plasminogen Activator Inhibitor Associated with Activation
essential medium (MEM) and 96 well ELISA plates from Flow Laboratories (McLean, VA); methioninefree media from Irvine Scientific (Irvine, CA); calf serum, trypsin, penicillin, streptomycin, Freund’s adjuvant from GIBCO (Grand Island, NY); ProteinSepharose A Sepharose beads, CNBr-activated beads from Pharmacia Fine Chemical (Piscataway, NJ); prestained high molecular weight protein standards from BRL Life Tech Inc (Gaithersburg, MD); goat anti-mouse IgG Sepharose beads, low endotoxin bovine serum albumin (BSA), Triton X-100, Tris base, casein, ethylenediamine tetraacetate, and caprylic acid from Sigma Chemical Co (St. reagent Louis, MO); ‘251-labelled Bolton-Hunter from New England Nuclear (Boston, MA); i2’Ilabelled donkey anti-rabbit and sheep anti-mouse from IgG, and 35S-methionine (llOOCi-mmol) Amersham Radiochemicals (Irvine, CA); Kodak XARS X-ray film from Eastman Kodak (Rochester, NY); monoclonal antibody (Mab) isotyping and chain-class determining kit, biotinylated or unconjugated goat anti-mouse IgG F(ab’)* fragments, alkaline phosphate conjugated streptavidin, alkaline phosphate substrate buffer and p-nitrophenyl phosphate (pNPP) substrate from Zymed Laboratories (San Francisco, CA); reagents for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) from BioRad Laboratories (Richmond, CA); nitrocellulose paper from Schliecher and Schuell (Keene, NH). PAI- was purified from serum-free bovine aortic endothelial cell (BAE) CM as described8 and radioiodination by the Bolton-Hunter method to a specific activity of 5000-20000 cpm/ng protein. Human t-PA was purified from melanoma cell-conditioned medium. l2 Bovine Vn was purified from bovine plasma as described.i3 Hybridoma cells were obtained by the fusion of x-63-Ag 8.653 myeloma cells with spleen cells from Balb/cByJ mice injected with purified bovine PAI- using standard hybridoma techniques. l4 Mabs were purified from ascites fluid by the caprylic acid method.” Antiserum to bovine PAI- was raised in rabbits as described,16 and the IgG fraction was further purified by affinity chromatography on immobilised PAI- using standard procedures. l4 Biotinylation of specific PAI- IgG with amino-hexanoyl-biotin-N-hydroxysuccinimide ester was performed according to the manufacturer (Zymed Laboratories Inc, San Francisco, CA). Cell Culture BAEs were isolated from bovine aorta, cloned from a single Factor VIII positive cell, and cultured in MEM containing 10% calf serum.16 Cultures were grown to confluency in 48-well culture plates and maintained at confluency for 3-5 days before use. Cells were serumstarved (24 h) and then incubated with 10% serum or serum free MEM (0.5ml/well) in the presence or absence of transforming growth factor-beta (TGF-P, courtesy of Dr. M. Sporn, NCI). The CM was
harvested and the cell lysates and ECM prepared as described.i7 BAEs also were metabolically labelled with 35S-methionine for 24h in the presence of purified bacterial lipopolysaccharide and in the presence or absence of 2 pg/ml of t-PA as described.” Screening
Assays for Anti-PAL1
IgG
Binding of Mabs to bovine PAI- was determined by four different screening methods: (a) solid-phase PAI- (plastic-adsorbed); (b) solution-phase, (radiolabelled PAI-1); (c) BAE ECM-associated PAI-1; and (d) Vn-associated PAI-1. Solid Phase PAl-1
Purified PAI- (50kl/well, l.Oug/ml in PBS) was adsorbed (4°C 16h) to EIA plates. The wells were blocked (3% BSA in PBS) and the washed wells were incubated with CM from subcloned hybridomas in IRMA buffer (PBS containing 3% BSA, 0.1% Tween 80, 5 m EDTA and 20U/ml aprotinin). After washing, the bound IgG was detected with biotinylated goat anti-mouse IgG F(ab’)2 fragments followed by streptavidin alkaline phosphatase and p-nitrophenyl phosphate (pNPP). Solution Phase PAZ-1
Purified goat anti-mouse IgG F(ab’);! fragments (50uVwell; lOpg/ml in PBS) were coated onto EIA plates. The blocked plates were incubated with CM from subcloned hybridomas in IRMA buffer. After washing, each well was incubated with 50000 cpm of ‘251-labelled PAI- and the radioactivity bound to each well determined by y-counting. ECM-associated
PAI-
BAEs were grown in 96-well plates to confluence and the ECM was prepared as described.17 ECM containing plates were incubated with various dilutions of hybridoma CM in IRMA buffer. PAI- specific IgG was detected with ‘251-sheep anti-mouse IgG. The bound radioactivity was extracted with 1% SDS and quantified by y-counting. Vn-associated PAI-
Vn/PAI-1 complexes were preformed on microtitre wells as described.” The wells then were incubated with purified Mab IgG in PBS containing 0.1% BSA and 0.1% Tween 80 and bound IgG was detected by incubation with ‘*‘I-sheep anti-mouse IgG. PAI-1 EIA PAIantigen concentration in the CM or cell extracts was determined by a sandwich EIA specific for bovine PAI-1. Purified Mab 12C2 IgG (lOO@
Fibrinolysis
well, 5 Fg/rnl in PBS) was coated on microtitre plates by incubation at 4°C for 16h. After washing (PBS), the plates were incubated (37°C 1 h) with 3% BSA in PBS, washed (PBS) and samples diluted in IRMA buffer incubated in the antibody-coated wells at 4°C for 16h. After washing with PBS, bound PAI- was quantitated with affinity-purified, biotinylated rabbit anti-bovine PAI- antibody (37”C, 1 h), followed by streptavidin-alkaline phosphatase conjugate/pNPP. Results of duplicate wells were averaged and corrected for absorbance at 405nm from antibody coated wells incubated with secondary antibody and substrate in the absence of PAI-1. Direct-Binding Radioimmunoassay Associated PAI-
(RIA) for ECM-
ECM-associated PAI- was quantitated by directbinding of Mab 12C2 IgG and compared to PAIstandards diluted in distilled water and adsorbed to the surface of equivalent culture dishes. The wells containing PAI- standards were incubated with 3% BSA in PBS and washed with PBS buffer, then the ECM containing wells and PAI- standards were incubated for 1 h at 4°C with 200 ~1 of purified Mab 12C2 IgG (0.5 l&ml) in PBS containing 0.1% BSA. The washed wells were then incubated for 1 h at 4°C with 200~1 of 12”1-sheep anti-mouse IgG (100000 cpm/well). The bound radioactivity was determined by extracting each washed well with 1.0% SDS for 15 min at 37°C and y-counting. The radioactivity of duplicate wells were averaged and corrected for non-specific binding of Mab 12C2 and secondary antibodies to BSA coated wells. Activation Inhibitory
of PAIActivity
and Determination
227
precipitation analysis using Mab ascites (0.1% VOY vol) was essentially performed as described.‘*
RESULTS Initial Characterisation
of Mah 12C2
Hybridoma secreting Mabs specific for bovine PAIwere prepared by immunising mice with purified PAI-1. From the resulting hybridomas, 17 supernatants contained antibodies reactive with purified PAI- immobilised on microtitre plates. The clone 12C2 was further analysed, and had a dissociation constant for PAI- of 8.0 X 10-s mol/L and secreted IgG of the IgGkl subtypes (not shown). Screening assays were designed to determine the binding of Mabs to either solution- or surface-phase forms of PAI-I (Figs 1 & 2). The clone 12C2 detected PAIimmobilised on plastic (Fig. 1, panel A), in solution (Fig. 1, panel B), and bound to ECM (Fig. 1, panel C) or Vn (Fig. 2). The reactivity of Mab 12C2 with SDS-denatured and immobilised bovine PAI- in an immunoblot was considerably weaker in comparison
0.5
.5 & g
0.4
i
0.2
0.3
0.1
of PA
Purified PAI- (33kg/ml) was activated with either SDS or guanidine hydrochloride as described.6 Briefly, PAI- was incubated with 0.1% SDS for 30 min at 37°C and then dialysed for 18h at 4°C into PBS containing 0.01% Tween 80. Alternatively, PAIwas dialysed for 2h at 37°C in 4M guanidine hydrochloride (pH 7.4), and then dialysed for 18h at 4°C into PBS containing 0.01% Tween 80. Trace amounts of 12”I-PAI-1 were added to the above samples of PAI- to correct for volume changes associated with dialysis. Following dialysis, samples were diluted 1:50 with PBS/O.l% Triton-X 100; subsequent dilutions for EIA analysis were in IRMA buffer, or for activity analysis in PBS containing 0.01% Tween 80. The activity of PAI- was determined by its ability to inhibit purified u-PA as described.s Miscellaneous
SDS-PAGE was performed in slab gels20 The upper stacking gel contained 4% acrylamide while the lower separating gel contained 9% acrylamide. Immuno-
*-
c
5t
5
DILUTION Fig. 1 Screening assays to quantitate the binding of Mab 12C2 various forms of PAI-1. The binding of Mab 12C2 (0) to solid-phase PAI- (panel A), solution-phase PAI- (panel B), BAE-ECM associated PAI- (panel C) was analysed and compared with normal mouse IgG (0). PAI- absorbed to microtitre plates (panel A), or PAI- in BAE-ECM (panel C) was detected by incubating the wells with logarithmic dilutions 12C2 IgG (stock @ 5 mg/ml) followed by ‘zSI-labelled sheep anti-mouse IgG. In panel B, the binding of ‘Z”I-labelled PAImicrotitre wells coated with dilutions of 12C2 IgG was determined.
to or
of to
228
Immunological
0.1
Detection
1
of Conformational
10
Changes
100
of Type
1,000
IktGl (ngimt) Fig. 2 Binding of Mab 12C2 to PAI-IIVn complexes. PAI-l/Vn complexes were prepared by incubating Vn coated wells with activated PAI-1. The complexes were detected by the indicated concentration of purified Mah 12C2 IgG (0) or normal mouse IgG (0), followed by ‘~iI-labelled sheep anti-mouse IgG.
to a polyclonal antiserum and no cross-reactivity with human PAI- was detectable (not shown). The effect of t-PA on the epitope expression of Mab 12C2 in solution was analysed. BAEs were metabolically labelled with “‘S-methionine in the presence or absence of exogenous, purified t-PA and the CM subjected to immunoprecipitation analysis (Fig. 3). Mab 12C2 detected a single protein of Mr 50000 in the absence of t-PA (Fig. 3, panel A, lane 2). If BAEs were labelled in the presence of t-PA, Mab 12C2 immunoprecipitated Mr 50000 ‘native’ PAI- as well as additional polypeptides of Mr 47000 and 116000 (Fig. 3, panel B, lane 2, A & C). A Mab to t-PA, shown to detect t-PA in complex with PAI-lZJ was used to identify these additional polypeptides. This antibody did not detect any radiolabelled protein in BAEs labelled in the absence of t-PA (Fig. 3, panel A, lane 3). However. upon addition of t-PA. both peptides and Mr 47000 (representing dissociated t-PA/PAI-1 complexes) andMr 116000 (representing intact t-PA/PAI-1 complexes) were detected (Fig. 3, panelB,lane4,A&C).
Effect of Activation on the Epitope Expression Mab 12C2 and Development of lmmunoassays Quantitate PAI-
of to
To quantitate total PAIantigen in biological fluids such as CM or cell lysates, we developed an EIA utilising purified Mab 12C2 as a catching antibody and affinity-purified, biotinylated rabbit anti-PAIas an detecting antibody. PAIconcentrations between 1 .O-lOOng/ml could accurately be detected (not shown). When purified PAIwas added to complete media containing 10% bovine serum or cell lysates, recoveries were essentially 100% (not shown). The assay variability was assessed using 4 different CM standards with a wide range of PAIconcentrations (0.02-4,Oug/ml); each assayed 10
I Plasminogen
Activator
Inhibitor
Associated
with Activation
times on 4 occasions. Intraassay and interassay coefficients of variation were 11% and 14%) respectively. Attempts to quantitate ECM-associated PAIin unstimulated or stimulated (TGF beta) BAEs by the EIA after extraction of ECM-associated PAIwith 0.5% SDS significantly underestimated total PAIantigen in comparison to immunoblotting analysis using a rabbit polyclonal antiserum to bovine PAI(Table 1). One possible explanation for these differences may be that the protein denaturant SDS, used for the extraction of ECM associated proteins, changed the epitope expression for Mab 12C2 in PAI-1. We therefore evaluated the effects of denaturants on the Mab 12C2 epitope expression (EIA) and PAIactivity levels and compared to PAIstored at -20°C without multiple freeze/thawing cycles prior to analysis (Table 2). SDS-treatment of PAIincreases its PA inhibitory activity 13-fold, but decreased the Mab 12C2 detectable antigen levels, leading to a 2900% increase in PAI- specific activity (SA) (molar units of u-PA inhibitory activity/molar PAIantigen) (Table 2). Conversely, treatment of PAIwith guanidine hydrochloride increases activity and antigen levels 13- and 3-fold respectively, increasing the SA by only 430%. However, an additional incubation with SDS decreased both activity and antigen levels and SA to 250% of controls
a
b
zoo-
zoo-
97-
97-
68-
68-
43-
43-
29-
2912
3
Fig. 3 Immunoprecipitation of PAI-I and t-PA1 complexes by Mab 1X2. BAEs were metabolically labeled with “Smethionine in the absence (panel A) or presence (panel B) of purified t-PA (2&ml). The samples were fractionated by SDSPAGE, and the gels were dried and analyscd by fluorography (panels A and B. lane I). The radiolabelled PAI- or t-PAIPAI-1 complexes were collected by immunoprecipitation using goat anti-mouse IgG Sepharose, and analysed by SDS-PAGE and Buorography: Mab 12C2 (panels A and B. lane 2); pre-immune mouse IgG (panel B. lane 3); and Mab HI-2 directed against human t-PA (panel A, lane 3 and panel B. lane 4). Arrow in panel A indicates M, 47000 PAI- and arrows in panel B indicate: A. undisscociated t-PA/PAI-I complexes; B. native Mr SOlJO PAI-1; C, t-PA cleaved Mr 47000 PAI-1.
Fibrinolysis
Table 1 Quantitative determination of BAE ECM-associated PAI- antigen content by various immunological methods
Control TGFB
PAI- EIA (Mab 12C2)
Immunoblot (Rb anti-PAI-1)
ECM RIA (Mab 12C2)
5.0 35
50-80 4OfL600
80 700
BAEs were cultured in the presence or (Sng/ml) for 24h. The ECM associated (ng/l@ cells) by EIA, immunoblotting 1, and ECM RIA using purified PAI-I Methods).
absence of TGFP PAI- was estimated utilising rabbit anti-PAIas a standard (see
(Table 2). Incubation of PAIat 37°C for 24h decreased the relative SA to 20% of controls by reducing activity to 21% and not significantly altering antigen levels. Moreover, heat-treatment (56°C) decreased PAI- activity to 29% but increased PAIantigen levels approximately 12-fold, reducing the SA to 2% of controls (Table 2). These results indicate that quantitating PAIantigen using the Mab 12C2 EIA may lead to estimations for SA of purified PAIpreparations that may vary greater than lOOO-fold (Table 2, compare SA of SDS- vs heat-treated [56”C]). Furthermore, the underestimation of ECMassociated PAIusing the solution-phase EIA appeared to be due to SDS-induced reduction in the expression of the Mab 12C2 PAIepitope. To more accurately quantitate total ECM-associated PAIantigen, we designed a direct-binding, solid-phase RIA based on the ability of Mab 12C2 to recognise solid-phase PAI- associated with the ECM and Vn (Figs 1 & 2). For a standard curve, various doses of purified PAI-I were absorbed to tissue culture wells. The coating efficiency was determined by adding trace quantities of 12”I-labelled PAIto unlabelled PAIstandards and was approximately 40%. The PAIRIA dose response curve had therefore been corrected for the actual PAI- bound and was linear over a range of 0.02-2.0)&m* (not shown). The assay variability was assessed by incubating confluent BAEs for 3 days in 6 individual wells of 6 different 48-well plates and then analysing the individual wells in each plate for ECM-associated PAIon 6 different occasions. Intraassay and interassay coefficient of variation were 20% and 18%, respectively. Results of the RIA were essentially in agreement with that of the semi-quantitative immunoblotting (compare Table 1) and suggest that the direct-binding RIA was a simple and sensitive method to determine ECM-associated PAI- concentrations.
DISCUSSION PAIis found in plasma, serum, and platelets releasates, and is synthesized by a variety of cultured cells (for review, see ‘) and deposited into their ECM bound to serum-derived vitronectin. 18,22 Active
229
PAI- in solution is an unstable molecule and rapidly decays into the latent form.‘,’ In contrast, PAIassociated with the ECM of cultured cells or Vn is functionally active and more stable.” PAIantigen in plasma or CM may be present in several distinct forms: first, in a complex with endogenous PAS; second, in a free active form; third, in an inactive latent form; fourth, in an irreversibly inactive, and/or proteolytically cleaved form; and fifth, in complex with plasma or serum-derived vitronectin (for references, see ‘). In contrast, ECM-associated PAIantigen is primarily found in an active form bound to and apparently stabilised by vitronectin.17,18,22,23 This broad range of PAIfunctional heterogeneity suggests the possibility of an equally broad range of PAIantigenic heterogeneity. The data in this report indicate that the antigenic conformation of purified PAIwas very labile and that expression of the PAIepitope for Mab 12C2 were significantly dependent on whether the protein was in solution, immobilised on solid-phase supports, or treated with denaturants. Optimal expression of the Mab 12C2 PAI- epitope was associated with native (M, 50kDA) and t-PA modified PAI(cleaved and/or complexed) in solution, and ECMor vitronectin-associated PAI-1; in contrast, immunoblot analysis indicated that the Mab 12C2 epitope was virtually absent on the immobilised M, 50000 PAI-1. The regulation of PAIgene and protein expression, in cultured BAEs and various other cell types, is modulated by a variety of growth factors, hormones and inflammatory mediators.2”26Thus, development of simple, sensitive immunoassays which quantitate total BAE PAI- antigen in the CM, cell lysates and ECM may aid studies designed to elucidate mechanisms regulating PAI- synthesis function and distribution.*’ Since Mab 12C2 could immunoprecipitate native PAIfrom biosynthetically labelled BAE CM, it was selected as the capture antibody for a two-site, sandwich EIA. However, efforts to quantitate ECM-associated PAIby the EIA proved
Table 2 Effect of chemical and thermal denaturation on the quantitation of PAI-I antigen and plasminogen activator (PA) inhibitory activity PAItreatment
% PAIactivity
% PAIantigen
% Relative specific activity
Control SDS Gn-HCI Gn-HCl + SDS 37°C (24 h) 56°C (30 min)
100 1292 1300 321 21 29
100 44 302 130 110 1180
100 2940 430 250 20 2
Purified bovine PAI-I was either activated with SDS and/or guanidine hydrochloride or incubated at various temperatures for the indicated time interval. PAI- antigen concentrations were determined by the PAI-I EIA and PA inhibitory activity by a synthetic plasmin substrate assay (see Methods). The data are expressed as percentage of controls stored at -20°C.
230
Immunological
Detection
of Conformational
Changes
of Type 1 Plasminogen
unsuccessful due to the significant underestimation of PAIin the SDS-extracted ECM samples. Since denaturants, such as SDS or guanidine hydrochloride, are used routinely in conjunction with PAIactivity assays to estimate the levels of latent and activatable PAIin CM or blood samples,6s*9 we further investigated the effects of denaturants on the antigenicity of PAIusing the Mab 12C2 EIA. Treatment of latent PAIwith SDS or guanidine hydrochloride both stimulated PA inhibitory activity as expected;6 however, SDS decreases, and guanidine increased the apparent PAI- antigen concentrations, suggesting different conformation of SDS vs guanidine activated PAI-1. The inhibitory effects of SDS on the PAIbinding to Mab 12C2 might be a direct result of residual SDS interfering with the epitope expression or antibody-antigen interaction. However, this seems unlikely since neutralising excess SDS with Triton X-100, and a 500 to 5000-fold dilution in IRMA buffer should have sufficiently negated direct SDS effects on antibody binding. In addition, not all of the Mabs produced from the same fusion displayed sensitivity to SDS-treatment of PAI-1; in fact, several Mabs demonstrated a relative increased binding to SDS-treated PAI(data not shown).
List of Abbreviations BAEs BSA CM ECM EIA IgG IRMA
Mab MEM PAGE PAIPAS PBS pNPP RIA SA SDS TGFB t-PA u-PA
buffer
:
bovine aortic endothelial cells bovine serum albumin conditioned media extracellular matrix enzyme immunoassay immunoglobulin G . PBS containine 3% BSA. 0.1% Tween 80. 5mM EDTA,,-20U/ml aprotinin, 0.05% sodium azide monoclonal antibody minimal essential media polyacrylamide gel electrophoresis type 1 plasminogen activator inhibitor plasminogen activator phosphate buffered saline para-nitrophenyl phosphate radioimmunoassay specific activity sodium dodecyl sulfate transforming growth factor beta tissue-type PA urinary-type PA
ACKNOWLEDGEMENTS We thank Dr D. J. Loskutoff for his helpful discussions, Dr Carla Hekman for assistance with the PAIactivity assays, D. Lawrence, S. Curriden, and N. N. Wagner for excellent technical assistance, and P. Tayman for secretarial assistance. This work was supported by grants #HL16411 and HL82229 to David J. Loskutoff from the National Institute of Health and was conducted during the tenure of a research fellowship to T J P from the Canadian Heart Foundation and to D S from the American Heart Association, California Affiliate, with funds contributed by the Orange County Chapter.
Activator
Inhibitor
Associated
with Activation
REFERENCES P A, Grondahl-Hansen J, Kristensen P, 1. Dano K, Andreasen Nielsen L S, Skriver L 1985 Plasminogen activators, tissue degradation and cancer. Adv Can Res 44: 139239 and the serpins: 2. Carrel1 R, Travis J 1985 Alpha-1-antitrypsin Variation and countervariation. Trends Biochem Sci 10: 20 3. Ny T, Sawdey M, Lawrence D A, Millan J L, Loskutoff D J 1986 Cloning and sequence of a cDNA coding for the human B-migrating endothelial-cell-type plasminogen activator inhibitor. Proc Nat1 Acad Sci USA 83: 67766780 H, Veerman H, Lambers H et al 1986 4. Pannekoek Endothelial plasminogen activator inhibitor (PAI): A new member of Serpin gene family. EMBO J 5: 2539-2544 5. Loskutoff D J, Sawdey M, Mimuro J 1989 Type 1 plasminogen activator inhibitor. In: Coller B (ed): Progress in Hemostasis and Thrombosis. Vol9, W B Saunders cells produce 6. Hekman C M, Loskutoff D J 1985 Endothelial a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem 260: 11581 of the active to latent 7. Levin E G, Sante11 L 1987 Conversion plasminogen activator inhibitor from human endothelial cells. Blood 70: 109&1098 8. Hekman C M, Loskutoff D J 1988 Bovine plasminogen activator 1: Specificity determinations and comparison of the active, latent and guanidine-activated forms. Biochem 27: 2911 9. Erickson L A, Heckman C M, Loskutoff D J 1986 Denaturant induced stimulation of the B-migrating plasminogen activator inhibitor in endothelial cells and serum. Blood 68: 1298 10. Lambers J W J, Cammenga M, Konig B, Pannekoek H, van Mourik J A 1987 Activation of human endothelial cell type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids. J Biol Chem 262: 17492 11. Katagiri K, Okada K, Hattori H, Yano M 1988 Bovine endothelial cell plasminogen activator inhibitor: Purification and heat activation. Eur J Biochem 176: 81-87 12. Schleef R R, Sinha, M., Loskutoff D J 1985 Characterization of two monoclonal antibodies to human tissue-type plasminogen activator. Thromb Haemost 53: 170 13. Yatohgo T, Izumi M, Kashiwagi H, Hayashi M 1988 Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Structure and Function 13: 281-292 14. Harlow E, Lane D 1988 In: Antibodies: A laboratory manual. Cold Spring Harbor Laboratory, New York 15. Steinbuch M, Audrin A 1969 The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch Biochem Biophys 134: 279 16. 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 2.59: 14914 17. Mimuro J, Schleef R R, Loskutoff D J 1987 Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor. Blood 70: 721 18. Seiffert D, Wagner N N, Loskutoff D J 1990 Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor. J Cell Biol 111: 1283-1291 19. Seiffert D, Loskutoff D J 1991 Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin. J Biol Chem 266: 2824-2830 20. Laemmli U K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680 21. 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 occurringinhibitor. Thromb Haem 56: 328-332 22. Preissner K T, Gulich-Henn J, Ehrlich H J et al 1990 Structural requirements for the extracellular interaction of plasminogen activator inhibitor 1 with endothelial cell matrix-associated vitronectin. J Biol Chem 265: 1849&18498
Fibrinolysis
23. 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. J Biol Chem 263: 15454 24. Schleef R R, Bevilacqua M P, Sawdey M, Gimbrone M A, Loskutoff D J 1988 Cytokine activation of vascular endothelium: effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor. J Biol Chem 263: 5797 25. Sawdey M, Podor T J, Loskutoff D J 1989 Regulation of
Received: 3 December 1990 Accepted after revision: 13 April 1991 Offprint orders to: T. Podor, Hamilton Civic Hospital Research Centers, Henderson General Divison, 711 Concession Street, Hamilton, Ontario, Canada, L8V IC3.
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type 1 plasminogen activator inhibitor gene expression in cultured bovine aortic endothelial cells: Induction by transforming growth factor-beta, lipopolysaccharide. and tumor necrosis factor. J Biol Chem 264: 10396 26. Sawdey M, Ny T, Loskutoff D J 1986 Messenger RNA for plasminogen activator inhibitor. Thromb Res 41: 151-160 27. Schleef R R, Loskutoff D J, Podor T J 1991 Immunelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells. J cell Biol 113: 14lS1423
Immunological Plasminogen
D. Seiffert**,
Detection of Conformational
Changes of Type 1
Activator Inhibitor Associated with Activation
T. J. Podor”
SUMMARY. The effect of activation, solid-phase surfaces, tissue-type plasminogen activator (t-PA) and vitronectin (Vn) on the epitope expression of monoclonal antibody (Mab) to bovine type 1 plasminogen activator inhibitor (PAI-1) was analysed. The epitope for Mab 12C2 was optimally expressed on PAI- in solution (active, free, complexed or cleaved by t-PA) and bound to Vn. Activation of PAI- by guanidine hydrochloride increased the apparent PAI- antigen concentration in a Mab 12C2-based immunoassay by about 300%, whereas sodium dodecyl sulfate (SDS) decreased the antigen concentration to 44%. Both treatments increased PAI- activity to 1300%. Thus, activation by denaturants may lead to differences in specific activity (molar units of urinary type PA inhibitory activity/molar PAI- antigen) of about 3000%. These results thus suggest different conformations of SDS vs guanidine-activated PAI-1. KEYWORDS. Extracellular
Type 1 plasminogen matrix. Vitronectin.
activator
inhibitor.
Monoclonal
Plasminogen
activator.
as sodium dodecyl sulfate (SDS),’ guanidine hydrochloride’.879 or negatively charged phospholipids’O and heat.” Conversion of latent PAl-1 to its active form by denaturants is believed to be associated with unfolding of the protein’s tertiary structure as suggested by an apparent change in M, which was determined by gel filtration chromatography” and equilibrium centrifugation.’ However, little is known about changes in PAl-1 antigenicity associated with activation by denaturants or following its interaction with PAS and vitronectin (Vn). In order to investigate the conformational changes associated with latent, activated and vitronectin (Vn)- or PA-modified PAI-1, we have characterised a monoclonal antibody (Mab) to bovine PAl-1 and compared expression of its distinct epitope on various forms of solution- and surface-phase PAI-1. These studies demonstrate that PAI-I antigenic conformation was differentially altered by SDS- or guanidine-hydrochloride activation.
The initiation of vascular fibrinolysis is a complex process, regulated not only by tissue-type plasminogen activator (t-PA) and urinary-type PA (u-PA), but also through the interactions of plasminogen and PAS with plasma-cofactors and solid-phase supports such as cell surface receptors and extracellular matrices (ECM) (for review, see ref. 1). The primary regulator of this system appears to be type 1 plasminogen activator inhibitor (PAI-l), a member of the serine proteinase inhibitor superfamily of proteins.2-5 PAl-1 is one of the most highly regulated of the fibrinolytic components (for review, see ref.5). Changes in its activity, whether naturally occurring or experimentally induced, may disrupi normal fibrinolytic balance and result in dramatic increases or decreases in net fibrinolytic activity (for review, see ref.5). PAl-1 is a relatively unstable molecule that exists in vivo and in vitro, in both an active and latent form.68 Activation of latent PAl-1 activity in plasma or conditioned medium (CM) from cultured cells can be achieved following treatment with denaturants such
MATERIALS D. Seiffert, T. J. Podor, Committee on Vascular Biology. CVB-3. Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037. USA. *Present address: Department of Pathology, McMaster University, Hamilton, Ontario, Canada. “*Clinical Experimental Physiology Department of Medical Physiology. Schwarzspanierstrasse 17. A-1090, Vienna, Austria.
Chemicals,
AND METHODS
Proteins,
Reagents
All chemicals were the highest analytic grade commercially available. Tissue culture plasticware was obtained from Corning (Corning, NY); minimal 225
R
antibodies.
226
Immunological Detection of Conformational Changes of Type 1 Plasminogen Activator Inhibitor Associated with Activation
essential medium (MEM) and 96 well ELISA plates from Flow Laboratories (McLean, VA); methioninefree media from Irvine Scientific (Irvine, CA); calf serum, trypsin, penicillin, streptomycin, Freund’s adjuvant from GIBCO (Grand Island, NY); ProteinSepharose A Sepharose beads, CNBr-activated beads from Pharmacia Fine Chemical (Piscataway, NJ); prestained high molecular weight protein standards from BRL Life Tech Inc (Gaithersburg, MD); goat anti-mouse IgG Sepharose beads, low endotoxin bovine serum albumin (BSA), Triton X-100, Tris base, casein, ethylenediamine tetraacetate, and caprylic acid from Sigma Chemical Co (St. reagent Louis, MO); ‘251-labelled Bolton-Hunter from New England Nuclear (Boston, MA); i2’Ilabelled donkey anti-rabbit and sheep anti-mouse from IgG, and 35S-methionine (llOOCi-mmol) Amersham Radiochemicals (Irvine, CA); Kodak XARS X-ray film from Eastman Kodak (Rochester, NY); monoclonal antibody (Mab) isotyping and chain-class determining kit, biotinylated or unconjugated goat anti-mouse IgG F(ab’)* fragments, alkaline phosphate conjugated streptavidin, alkaline phosphate substrate buffer and p-nitrophenyl phosphate (pNPP) substrate from Zymed Laboratories (San Francisco, CA); reagents for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) from BioRad Laboratories (Richmond, CA); nitrocellulose paper from Schliecher and Schuell (Keene, NH). PAI- was purified from serum-free bovine aortic endothelial cell (BAE) CM as described8 and radioiodination by the Bolton-Hunter method to a specific activity of 5000-20000 cpm/ng protein. Human t-PA was purified from melanoma cell-conditioned medium. l2 Bovine Vn was purified from bovine plasma as described.i3 Hybridoma cells were obtained by the fusion of x-63-Ag 8.653 myeloma cells with spleen cells from Balb/cByJ mice injected with purified bovine PAI- using standard hybridoma techniques. l4 Mabs were purified from ascites fluid by the caprylic acid method.” Antiserum to bovine PAI- was raised in rabbits as described,16 and the IgG fraction was further purified by affinity chromatography on immobilised PAI- using standard procedures. l4 Biotinylation of specific PAI- IgG with amino-hexanoyl-biotin-N-hydroxysuccinimide ester was performed according to the manufacturer (Zymed Laboratories Inc, San Francisco, CA). Cell Culture BAEs were isolated from bovine aorta, cloned from a single Factor VIII positive cell, and cultured in MEM containing 10% calf serum.16 Cultures were grown to confluency in 48-well culture plates and maintained at confluency for 3-5 days before use. Cells were serumstarved (24 h) and then incubated with 10% serum or serum free MEM (0.5ml/well) in the presence or absence of transforming growth factor-beta (TGF-P, courtesy of Dr. M. Sporn, NCI). The CM was
harvested and the cell lysates and ECM prepared as described.i7 BAEs also were metabolically labelled with 35S-methionine for 24h in the presence of purified bacterial lipopolysaccharide and in the presence or absence of 2 pg/ml of t-PA as described.” Screening
Assays for Anti-PAL1
IgG
Binding of Mabs to bovine PAI- was determined by four different screening methods: (a) solid-phase PAI- (plastic-adsorbed); (b) solution-phase, (radiolabelled PAI-1); (c) BAE ECM-associated PAI-1; and (d) Vn-associated PAI-1. Solid Phase PAl-1
Purified PAI- (50kl/well, l.Oug/ml in PBS) was adsorbed (4°C 16h) to EIA plates. The wells were blocked (3% BSA in PBS) and the washed wells were incubated with CM from subcloned hybridomas in IRMA buffer (PBS containing 3% BSA, 0.1% Tween 80, 5 m EDTA and 20U/ml aprotinin). After washing, the bound IgG was detected with biotinylated goat anti-mouse IgG F(ab’)2 fragments followed by streptavidin alkaline phosphatase and p-nitrophenyl phosphate (pNPP). Solution Phase PAZ-1
Purified goat anti-mouse IgG F(ab’);! fragments (50uVwell; lOpg/ml in PBS) were coated onto EIA plates. The blocked plates were incubated with CM from subcloned hybridomas in IRMA buffer. After washing, each well was incubated with 50000 cpm of ‘251-labelled PAI- and the radioactivity bound to each well determined by y-counting. ECM-associated
PAI-
BAEs were grown in 96-well plates to confluence and the ECM was prepared as described.17 ECM containing plates were incubated with various dilutions of hybridoma CM in IRMA buffer. PAI- specific IgG was detected with ‘251-sheep anti-mouse IgG. The bound radioactivity was extracted with 1% SDS and quantified by y-counting. Vn-associated PAI-
Vn/PAI-1 complexes were preformed on microtitre wells as described.” The wells then were incubated with purified Mab IgG in PBS containing 0.1% BSA and 0.1% Tween 80 and bound IgG was detected by incubation with ‘*‘I-sheep anti-mouse IgG. PAI-1 EIA PAIantigen concentration in the CM or cell extracts was determined by a sandwich EIA specific for bovine PAI-1. Purified Mab 12C2 IgG (lOO@
Fibrinolysis
well, 5 Fg/rnl in PBS) was coated on microtitre plates by incubation at 4°C for 16h. After washing (PBS), the plates were incubated (37°C 1 h) with 3% BSA in PBS, washed (PBS) and samples diluted in IRMA buffer incubated in the antibody-coated wells at 4°C for 16h. After washing with PBS, bound PAI- was quantitated with affinity-purified, biotinylated rabbit anti-bovine PAI- antibody (37”C, 1 h), followed by streptavidin-alkaline phosphatase conjugate/pNPP. Results of duplicate wells were averaged and corrected for absorbance at 405nm from antibody coated wells incubated with secondary antibody and substrate in the absence of PAI-1. Direct-Binding Radioimmunoassay Associated PAI-
(RIA) for ECM-
ECM-associated PAI- was quantitated by directbinding of Mab 12C2 IgG and compared to PAIstandards diluted in distilled water and adsorbed to the surface of equivalent culture dishes. The wells containing PAI- standards were incubated with 3% BSA in PBS and washed with PBS buffer, then the ECM containing wells and PAI- standards were incubated for 1 h at 4°C with 200 ~1 of purified Mab 12C2 IgG (0.5 l&ml) in PBS containing 0.1% BSA. The washed wells were then incubated for 1 h at 4°C with 200~1 of 12”1-sheep anti-mouse IgG (100000 cpm/well). The bound radioactivity was determined by extracting each washed well with 1.0% SDS for 15 min at 37°C and y-counting. The radioactivity of duplicate wells were averaged and corrected for non-specific binding of Mab 12C2 and secondary antibodies to BSA coated wells. Activation Inhibitory
of PAIActivity
and Determination
227
precipitation analysis using Mab ascites (0.1% VOY vol) was essentially performed as described.‘*
RESULTS Initial Characterisation
of Mah 12C2
Hybridoma secreting Mabs specific for bovine PAIwere prepared by immunising mice with purified PAI-1. From the resulting hybridomas, 17 supernatants contained antibodies reactive with purified PAI- immobilised on microtitre plates. The clone 12C2 was further analysed, and had a dissociation constant for PAI- of 8.0 X 10-s mol/L and secreted IgG of the IgGkl subtypes (not shown). Screening assays were designed to determine the binding of Mabs to either solution- or surface-phase forms of PAI-I (Figs 1 & 2). The clone 12C2 detected PAIimmobilised on plastic (Fig. 1, panel A), in solution (Fig. 1, panel B), and bound to ECM (Fig. 1, panel C) or Vn (Fig. 2). The reactivity of Mab 12C2 with SDS-denatured and immobilised bovine PAI- in an immunoblot was considerably weaker in comparison
0.5
.5 & g
0.4
i
0.2
0.3
0.1
of PA
Purified PAI- (33kg/ml) was activated with either SDS or guanidine hydrochloride as described.6 Briefly, PAI- was incubated with 0.1% SDS for 30 min at 37°C and then dialysed for 18h at 4°C into PBS containing 0.01% Tween 80. Alternatively, PAIwas dialysed for 2h at 37°C in 4M guanidine hydrochloride (pH 7.4), and then dialysed for 18h at 4°C into PBS containing 0.01% Tween 80. Trace amounts of 12”I-PAI-1 were added to the above samples of PAI- to correct for volume changes associated with dialysis. Following dialysis, samples were diluted 1:50 with PBS/O.l% Triton-X 100; subsequent dilutions for EIA analysis were in IRMA buffer, or for activity analysis in PBS containing 0.01% Tween 80. The activity of PAI- was determined by its ability to inhibit purified u-PA as described.s Miscellaneous
SDS-PAGE was performed in slab gels20 The upper stacking gel contained 4% acrylamide while the lower separating gel contained 9% acrylamide. Immuno-
*-
c
5t
5
DILUTION Fig. 1 Screening assays to quantitate the binding of Mab 12C2 various forms of PAI-1. The binding of Mab 12C2 (0) to solid-phase PAI- (panel A), solution-phase PAI- (panel B), BAE-ECM associated PAI- (panel C) was analysed and compared with normal mouse IgG (0). PAI- absorbed to microtitre plates (panel A), or PAI- in BAE-ECM (panel C) was detected by incubating the wells with logarithmic dilutions 12C2 IgG (stock @ 5 mg/ml) followed by ‘zSI-labelled sheep anti-mouse IgG. In panel B, the binding of ‘Z”I-labelled PAImicrotitre wells coated with dilutions of 12C2 IgG was determined.
to or
of to
228
Immunological
0.1
Detection
1
of Conformational
10
Changes
100
of Type
1,000
IktGl (ngimt) Fig. 2 Binding of Mab 12C2 to PAI-IIVn complexes. PAI-l/Vn complexes were prepared by incubating Vn coated wells with activated PAI-1. The complexes were detected by the indicated concentration of purified Mah 12C2 IgG (0) or normal mouse IgG (0), followed by ‘~iI-labelled sheep anti-mouse IgG.
to a polyclonal antiserum and no cross-reactivity with human PAI- was detectable (not shown). The effect of t-PA on the epitope expression of Mab 12C2 in solution was analysed. BAEs were metabolically labelled with “‘S-methionine in the presence or absence of exogenous, purified t-PA and the CM subjected to immunoprecipitation analysis (Fig. 3). Mab 12C2 detected a single protein of Mr 50000 in the absence of t-PA (Fig. 3, panel A, lane 2). If BAEs were labelled in the presence of t-PA, Mab 12C2 immunoprecipitated Mr 50000 ‘native’ PAI- as well as additional polypeptides of Mr 47000 and 116000 (Fig. 3, panel B, lane 2, A & C). A Mab to t-PA, shown to detect t-PA in complex with PAI-lZJ was used to identify these additional polypeptides. This antibody did not detect any radiolabelled protein in BAEs labelled in the absence of t-PA (Fig. 3, panel A, lane 3). However. upon addition of t-PA. both peptides and Mr 47000 (representing dissociated t-PA/PAI-1 complexes) andMr 116000 (representing intact t-PA/PAI-1 complexes) were detected (Fig. 3, panelB,lane4,A&C).
Effect of Activation on the Epitope Expression Mab 12C2 and Development of lmmunoassays Quantitate PAI-
of to
To quantitate total PAIantigen in biological fluids such as CM or cell lysates, we developed an EIA utilising purified Mab 12C2 as a catching antibody and affinity-purified, biotinylated rabbit anti-PAIas an detecting antibody. PAIconcentrations between 1 .O-lOOng/ml could accurately be detected (not shown). When purified PAIwas added to complete media containing 10% bovine serum or cell lysates, recoveries were essentially 100% (not shown). The assay variability was assessed using 4 different CM standards with a wide range of PAIconcentrations (0.02-4,Oug/ml); each assayed 10
I Plasminogen
Activator
Inhibitor
Associated
with Activation
times on 4 occasions. Intraassay and interassay coefficients of variation were 11% and 14%) respectively. Attempts to quantitate ECM-associated PAIin unstimulated or stimulated (TGF beta) BAEs by the EIA after extraction of ECM-associated PAIwith 0.5% SDS significantly underestimated total PAIantigen in comparison to immunoblotting analysis using a rabbit polyclonal antiserum to bovine PAI(Table 1). One possible explanation for these differences may be that the protein denaturant SDS, used for the extraction of ECM associated proteins, changed the epitope expression for Mab 12C2 in PAI-1. We therefore evaluated the effects of denaturants on the Mab 12C2 epitope expression (EIA) and PAIactivity levels and compared to PAIstored at -20°C without multiple freeze/thawing cycles prior to analysis (Table 2). SDS-treatment of PAIincreases its PA inhibitory activity 13-fold, but decreased the Mab 12C2 detectable antigen levels, leading to a 2900% increase in PAI- specific activity (SA) (molar units of u-PA inhibitory activity/molar PAIantigen) (Table 2). Conversely, treatment of PAIwith guanidine hydrochloride increases activity and antigen levels 13- and 3-fold respectively, increasing the SA by only 430%. However, an additional incubation with SDS decreased both activity and antigen levels and SA to 250% of controls
a
b
zoo-
zoo-
97-
97-
68-
68-
43-
43-
29-
2912
3
Fig. 3 Immunoprecipitation of PAI-I and t-PA1 complexes by Mab 1X2. BAEs were metabolically labeled with “Smethionine in the absence (panel A) or presence (panel B) of purified t-PA (2&ml). The samples were fractionated by SDSPAGE, and the gels were dried and analyscd by fluorography (panels A and B. lane I). The radiolabelled PAI- or t-PAIPAI-1 complexes were collected by immunoprecipitation using goat anti-mouse IgG Sepharose, and analysed by SDS-PAGE and Buorography: Mab 12C2 (panels A and B. lane 2); pre-immune mouse IgG (panel B. lane 3); and Mab HI-2 directed against human t-PA (panel A, lane 3 and panel B. lane 4). Arrow in panel A indicates M, 47000 PAI- and arrows in panel B indicate: A. undisscociated t-PA/PAI-I complexes; B. native Mr SOlJO PAI-1; C, t-PA cleaved Mr 47000 PAI-1.
Fibrinolysis
Table 1 Quantitative determination of BAE ECM-associated PAI- antigen content by various immunological methods
Control TGFB
PAI- EIA (Mab 12C2)
Immunoblot (Rb anti-PAI-1)
ECM RIA (Mab 12C2)
5.0 35
50-80 4OfL600
80 700
BAEs were cultured in the presence or (Sng/ml) for 24h. The ECM associated (ng/l@ cells) by EIA, immunoblotting 1, and ECM RIA using purified PAI-I Methods).
absence of TGFP PAI- was estimated utilising rabbit anti-PAIas a standard (see
(Table 2). Incubation of PAIat 37°C for 24h decreased the relative SA to 20% of controls by reducing activity to 21% and not significantly altering antigen levels. Moreover, heat-treatment (56°C) decreased PAI- activity to 29% but increased PAIantigen levels approximately 12-fold, reducing the SA to 2% of controls (Table 2). These results indicate that quantitating PAIantigen using the Mab 12C2 EIA may lead to estimations for SA of purified PAIpreparations that may vary greater than lOOO-fold (Table 2, compare SA of SDS- vs heat-treated [56”C]). Furthermore, the underestimation of ECMassociated PAIusing the solution-phase EIA appeared to be due to SDS-induced reduction in the expression of the Mab 12C2 PAIepitope. To more accurately quantitate total ECM-associated PAIantigen, we designed a direct-binding, solid-phase RIA based on the ability of Mab 12C2 to recognise solid-phase PAI- associated with the ECM and Vn (Figs 1 & 2). For a standard curve, various doses of purified PAI-I were absorbed to tissue culture wells. The coating efficiency was determined by adding trace quantities of 12”I-labelled PAIto unlabelled PAIstandards and was approximately 40%. The PAIRIA dose response curve had therefore been corrected for the actual PAI- bound and was linear over a range of 0.02-2.0)&m* (not shown). The assay variability was assessed by incubating confluent BAEs for 3 days in 6 individual wells of 6 different 48-well plates and then analysing the individual wells in each plate for ECM-associated PAIon 6 different occasions. Intraassay and interassay coefficient of variation were 20% and 18%, respectively. Results of the RIA were essentially in agreement with that of the semi-quantitative immunoblotting (compare Table 1) and suggest that the direct-binding RIA was a simple and sensitive method to determine ECM-associated PAI- concentrations.
DISCUSSION PAIis found in plasma, serum, and platelets releasates, and is synthesized by a variety of cultured cells (for review, see ‘) and deposited into their ECM bound to serum-derived vitronectin. 18,22 Active
229
PAI- in solution is an unstable molecule and rapidly decays into the latent form.‘,’ In contrast, PAIassociated with the ECM of cultured cells or Vn is functionally active and more stable.” PAIantigen in plasma or CM may be present in several distinct forms: first, in a complex with endogenous PAS; second, in a free active form; third, in an inactive latent form; fourth, in an irreversibly inactive, and/or proteolytically cleaved form; and fifth, in complex with plasma or serum-derived vitronectin (for references, see ‘). In contrast, ECM-associated PAIantigen is primarily found in an active form bound to and apparently stabilised by vitronectin.17,18,22,23 This broad range of PAIfunctional heterogeneity suggests the possibility of an equally broad range of PAIantigenic heterogeneity. The data in this report indicate that the antigenic conformation of purified PAIwas very labile and that expression of the PAIepitope for Mab 12C2 were significantly dependent on whether the protein was in solution, immobilised on solid-phase supports, or treated with denaturants. Optimal expression of the Mab 12C2 PAI- epitope was associated with native (M, 50kDA) and t-PA modified PAI(cleaved and/or complexed) in solution, and ECMor vitronectin-associated PAI-1; in contrast, immunoblot analysis indicated that the Mab 12C2 epitope was virtually absent on the immobilised M, 50000 PAI-1. The regulation of PAIgene and protein expression, in cultured BAEs and various other cell types, is modulated by a variety of growth factors, hormones and inflammatory mediators.2”26Thus, development of simple, sensitive immunoassays which quantitate total BAE PAI- antigen in the CM, cell lysates and ECM may aid studies designed to elucidate mechanisms regulating PAI- synthesis function and distribution.*’ Since Mab 12C2 could immunoprecipitate native PAIfrom biosynthetically labelled BAE CM, it was selected as the capture antibody for a two-site, sandwich EIA. However, efforts to quantitate ECM-associated PAIby the EIA proved
Table 2 Effect of chemical and thermal denaturation on the quantitation of PAI-I antigen and plasminogen activator (PA) inhibitory activity PAItreatment
% PAIactivity
% PAIantigen
% Relative specific activity
Control SDS Gn-HCI Gn-HCl + SDS 37°C (24 h) 56°C (30 min)
100 1292 1300 321 21 29
100 44 302 130 110 1180
100 2940 430 250 20 2
Purified bovine PAI-I was either activated with SDS and/or guanidine hydrochloride or incubated at various temperatures for the indicated time interval. PAI- antigen concentrations were determined by the PAI-I EIA and PA inhibitory activity by a synthetic plasmin substrate assay (see Methods). The data are expressed as percentage of controls stored at -20°C.
230
Immunological
Detection
of Conformational
Changes
of Type 1 Plasminogen
unsuccessful due to the significant underestimation of PAIin the SDS-extracted ECM samples. Since denaturants, such as SDS or guanidine hydrochloride, are used routinely in conjunction with PAIactivity assays to estimate the levels of latent and activatable PAIin CM or blood samples,6s*9 we further investigated the effects of denaturants on the antigenicity of PAIusing the Mab 12C2 EIA. Treatment of latent PAIwith SDS or guanidine hydrochloride both stimulated PA inhibitory activity as expected;6 however, SDS decreases, and guanidine increased the apparent PAI- antigen concentrations, suggesting different conformation of SDS vs guanidine activated PAI-1. The inhibitory effects of SDS on the PAIbinding to Mab 12C2 might be a direct result of residual SDS interfering with the epitope expression or antibody-antigen interaction. However, this seems unlikely since neutralising excess SDS with Triton X-100, and a 500 to 5000-fold dilution in IRMA buffer should have sufficiently negated direct SDS effects on antibody binding. In addition, not all of the Mabs produced from the same fusion displayed sensitivity to SDS-treatment of PAI-1; in fact, several Mabs demonstrated a relative increased binding to SDS-treated PAI(data not shown).
List of Abbreviations BAEs BSA CM ECM EIA IgG IRMA
Mab MEM PAGE PAIPAS PBS pNPP RIA SA SDS TGFB t-PA u-PA
buffer
:
bovine aortic endothelial cells bovine serum albumin conditioned media extracellular matrix enzyme immunoassay immunoglobulin G . PBS containine 3% BSA. 0.1% Tween 80. 5mM EDTA,,-20U/ml aprotinin, 0.05% sodium azide monoclonal antibody minimal essential media polyacrylamide gel electrophoresis type 1 plasminogen activator inhibitor plasminogen activator phosphate buffered saline para-nitrophenyl phosphate radioimmunoassay specific activity sodium dodecyl sulfate transforming growth factor beta tissue-type PA urinary-type PA
ACKNOWLEDGEMENTS We thank Dr D. J. Loskutoff for his helpful discussions, Dr Carla Hekman for assistance with the PAIactivity assays, D. Lawrence, S. Curriden, and N. N. Wagner for excellent technical assistance, and P. Tayman for secretarial assistance. This work was supported by grants #HL16411 and HL82229 to David J. Loskutoff from the National Institute of Health and was conducted during the tenure of a research fellowship to T J P from the Canadian Heart Foundation and to D S from the American Heart Association, California Affiliate, with funds contributed by the Orange County Chapter.
Activator
Inhibitor
Associated
with Activation
REFERENCES P A, Grondahl-Hansen J, Kristensen P, 1. Dano K, Andreasen Nielsen L S, Skriver L 1985 Plasminogen activators, tissue degradation and cancer. Adv Can Res 44: 139239 and the serpins: 2. Carrel1 R, Travis J 1985 Alpha-1-antitrypsin Variation and countervariation. Trends Biochem Sci 10: 20 3. Ny T, Sawdey M, Lawrence D A, Millan J L, Loskutoff D J 1986 Cloning and sequence of a cDNA coding for the human B-migrating endothelial-cell-type plasminogen activator inhibitor. Proc Nat1 Acad Sci USA 83: 67766780 H, Veerman H, Lambers H et al 1986 4. Pannekoek Endothelial plasminogen activator inhibitor (PAI): A new member of Serpin gene family. EMBO J 5: 2539-2544 5. Loskutoff D J, Sawdey M, Mimuro J 1989 Type 1 plasminogen activator inhibitor. In: Coller B (ed): Progress in Hemostasis and Thrombosis. Vol9, W B Saunders cells produce 6. Hekman C M, Loskutoff D J 1985 Endothelial a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem 260: 11581 of the active to latent 7. Levin E G, Sante11 L 1987 Conversion plasminogen activator inhibitor from human endothelial cells. Blood 70: 109&1098 8. Hekman C M, Loskutoff D J 1988 Bovine plasminogen activator 1: Specificity determinations and comparison of the active, latent and guanidine-activated forms. Biochem 27: 2911 9. Erickson L A, Heckman C M, Loskutoff D J 1986 Denaturant induced stimulation of the B-migrating plasminogen activator inhibitor in endothelial cells and serum. Blood 68: 1298 10. Lambers J W J, Cammenga M, Konig B, Pannekoek H, van Mourik J A 1987 Activation of human endothelial cell type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids. J Biol Chem 262: 17492 11. Katagiri K, Okada K, Hattori H, Yano M 1988 Bovine endothelial cell plasminogen activator inhibitor: Purification and heat activation. Eur J Biochem 176: 81-87 12. Schleef R R, Sinha, M., Loskutoff D J 1985 Characterization of two monoclonal antibodies to human tissue-type plasminogen activator. Thromb Haemost 53: 170 13. Yatohgo T, Izumi M, Kashiwagi H, Hayashi M 1988 Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Structure and Function 13: 281-292 14. Harlow E, Lane D 1988 In: Antibodies: A laboratory manual. Cold Spring Harbor Laboratory, New York 15. Steinbuch M, Audrin A 1969 The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch Biochem Biophys 134: 279 16. 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 2.59: 14914 17. Mimuro J, Schleef R R, Loskutoff D J 1987 Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor. Blood 70: 721 18. Seiffert D, Wagner N N, Loskutoff D J 1990 Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor. J Cell Biol 111: 1283-1291 19. Seiffert D, Loskutoff D J 1991 Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin. J Biol Chem 266: 2824-2830 20. Laemmli U K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680 21. 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 occurringinhibitor. Thromb Haem 56: 328-332 22. Preissner K T, Gulich-Henn J, Ehrlich H J et al 1990 Structural requirements for the extracellular interaction of plasminogen activator inhibitor 1 with endothelial cell matrix-associated vitronectin. J Biol Chem 265: 1849&18498
Fibrinolysis
23. 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. J Biol Chem 263: 15454 24. Schleef R R, Bevilacqua M P, Sawdey M, Gimbrone M A, Loskutoff D J 1988 Cytokine activation of vascular endothelium: effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor. J Biol Chem 263: 5797 25. Sawdey M, Podor T J, Loskutoff D J 1989 Regulation of
Received: 3 December 1990 Accepted after revision: 13 April 1991 Offprint orders to: T. Podor, Hamilton Civic Hospital Research Centers, Henderson General Divison, 711 Concession Street, Hamilton, Ontario, Canada, L8V IC3.
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type 1 plasminogen activator inhibitor gene expression in cultured bovine aortic endothelial cells: Induction by transforming growth factor-beta, lipopolysaccharide. and tumor necrosis factor. J Biol Chem 264: 10396 26. Sawdey M, Ny T, Loskutoff D J 1986 Messenger RNA for plasminogen activator inhibitor. Thromb Res 41: 151-160 27. Schleef R R, Loskutoff D J, Podor T J 1991 Immunelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells. J cell Biol 113: 14lS1423