Báo cáo Y học: Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin Characterization of different PAI-1 mutants pdf

Interaction of plasminogen activator inhibitor type-1
(PAI-1) with vitronectin
Characterization of different PAI-1 mutants
Nuria Arroyo De Prada
1,
*, Florian Schroeck
1,
*, Eva-Kathrin Sinner
2
, Bernd Muehlenweg
1,3
,
Jens Twellmeyer
1
, Stefan Sperl
3
, Olaf G. Wilhelm
3
, Manfred Schmitt
1
and Viktor Magdolen
1
1
Klinische Forschergruppe der Frauenklinik der Technischen Universita
È
tMu
È
nchen, Klinikum rechts der Isar, Germany;
2
Max-Planck-Institut fu
È

r Biochemie, Martinsried, Germany;
3
Wilex AG, Mu
È
nchen, Germany
The serpin plasminogen activator inhibitor type 1 (PAI-1)
plays an important role in physiological processes such as
thrombolysis and ®brinolysis, a s w ell as pathophysiological
processes such as thrombosis, tumor invasion and metas-
tasis. In addition to inhibiting serine proteases, mainly
tissue-type (tPA) and urokinase-type (uPA) plasminogen
activators, PAI-1 interacts with d ierent components of
the extracellular matrix, i.e. ®brin, heparin (Hep) and
vitronectin (Vn). PAI-1 binding to Vn facilitates migration
and invasion of tumor cells. The most important deter-
minants of the Vn-binding site of PAI-1 appear to reside
between amino acid s 110±147, which includes a helix E
(hE, amino acids 1 09±118). Ten dierent PAI- 1 variants
(mostly h arboring m odi®cations in hE) a s w ell as w ild-type
PAI-1, the previously described PAI-1 mutant Q123K, and
another s erpin, PAI-2, were recombinantly p roduced in
Escherichia coli containing a His
6
tag and puri®ed by
anity chromatography. As shown in microtiter
plate-based binding assays, surface plasmon resonance and
thrombin inhibition experiments, all o f the newly generated
mutants which retained inhibitory activity against u PA still
bound to Vn. Mutant A114±118, in which all amino-acids
at positions 114±118 of PAI-1 were exchanged for alanine,

displayed a reduced anity to Vn as compared to wild-
type PAI-1. Mutants lacking inhibitory activity towards
uPA did not bind to Vn. Q123K, which inhibits uPA but
does not bind to Vn, s erved as a control. In contrast to
other active PAI-1 mutants, the inhibitory properties of
A114±118 towards thrombin a s well as uPA were signi®-
cantly reduced in the presence of Hep. Our results dem-
onstrate that the wild-type sequence of the region around
hE in PAI-1 is not a prerequisite for binding to Vn.
Keywords: plasminogen activator inhibito r t ype-1;
vitronectin; heparin; mutational analysis; surface plasmon
resonance.
The urokinase-type plasminogen activation system plays
an important role in tumor growth, invasion, and
metastasis. The serine protease urokinase-type plasmino-
gen activator (uPA) activates plasminogen, the zymogen
of plasmin, thus generating a p rotease with broad
substrate speci®city a nd leading t o d egradation of extra-
cellular matrix (ECM) proteins [1±3]. The activity of uPA
is focussed to the cell surface by interaction with its
speci®c receptor uPAR (CD87). T issue-type plasminogen
activator (tPA), the second type of human plasminogen
activator, in contrast to uPA does not bind to a h igh
af®nity receptor on tumor cell surfaces and therefore
does not promote tumor cell-associated pericellular pro-
teolysis.
There are two main inhibitors of uPA and tPA, the
serine protease inhibitors (serpin) plasminogen activator
inhibitor type-1 (PAI-1) and type-2 (PAI-2) [4]. For
inhibition, the surface-exposed reactive center loop

(RCL) of PA I-1 or P AI-2 interacts with the r eactive site
of the target protease. Initially, the P1±P1¢ bond of the
inhibitor is cleaved and an intermediate enzyme±inhibitor
complex is formed. This is followed by the insertion of
part of the RCL as additional b strand 4A, which leads t o
the translocation of the p rotease a cross t he p lane o f bsheet
A of PAI-1 and formation of an SDS-stable enzyme±
inhibitor complex [5]. This complex dissociates very slowly
and is cleared from circulation before d isassembly can
occur. In vitro, the inhibitor can be released from the
protease in a substrate-like manner, generating the
so-called RCL-cleaved form of the inhibitor [6].
Correspondence to V. Magdolen, Klinische Forschergruppe der
Frauenklinik der Technischen Universita
È
tMu
È
nchen, Klinikum rechts
der Isar, Ismaninger Str. 22, D-81675 Mu
È
nchen, Germany.
Fax: + 49 89 4140 7410, Tel.: + 49 89 4140 2493,
E-mail:
Abbreviations: ECM, extracellular matrix; Hep, heparin; hE, helix E;
HMW-uPA, high molecular weight urokinase-type plasminogen
activator; PAI-1, plasminogen activator inhibitor type-1; PAI-2,
plasminogen activator inhibitor type-2; RCL, reactive center loop;
RU, resonance units; SPR, surface plasmon resonance; tPA, tissue-
type plasminogen activator; uPA, urokinase-type plasminogen acti-
vator; uPAR, uP A r eceptor; Vn, vitronectin; IPTG, isop ropyl t hio-b-

D
-galactoside.
*Note: these au thors contributed equally to the work.
Note: web pages are available at h ttp://www.frauenklinik-tu-muen-
chen.de, a nd

(Received 04 July 2001, re vised 22 O ctober 2001, accepted 29 Oc tober
2001)
Eur. J. Biochem. 269, 184±192 (2002) Ó FEBS 2002
Active PAI-1 is metastable a nd spontaneously converts to
a latent form by inserting a major part of its RCL into the
central b sheet A [7]. Latent PAI-1, as well as PAI-1 in
complex with uPA or tPA, do not bind to ECM compo-
nents. Active PAI-1, however, interacts with ®brin, h eparin
(Hep), and vitronectin (Vn) [1,8,9]. Binding to Vn doubles
the physiological half-life of active PAI-1 in solution.
Moreover, b y b inding to Vn or Hep, the substrate speci®city
of PAI-1 i s a ltered, because i nteraction with Vn or Hep
enables PAI-1 to inhibit another serine protease, thrombin
[10].
High levels of PAI-1 in tumor tissu e indicate short
recurrence-free and overall survival of tumor p atients
af¯icted with a broad variety of cancers, e.g. mammary,
ovarian, cervical, colorectal, bladder, renal and lung
carcinomas [2,3]. In line with this, it was demonstrated
that PAI-1 affects t umor cell adhesion and/or tumor
angiogenesis and, as a result of this, may s upport tumor
invasion [11±13].
As the PAI-1-binding site on Vn partially overlaps with
the binding sites of cellular adhesion proteins, e.g. uPAR

and some integrins, addition of PAI-1 to Vn-bound tumor
cells leads to d etachment of t hese cells [11,14]. This effect can
be reversed by uPA, as Vn-bound PAI-1 dissociates from
Vn upon interacti on w ith uPA [9]. Interestingly, as demon-
strated b y Bajou et al. [13], the in¯uence of PAI-1 on tumor
vascularization is due to the i nhibition of proteases and not
due to its interaction with Vn. Thus, the balance of several
tumor-associated factors seems to control the modulatory
effects of PAI-1 on cell adhesion, migration, and angiogen-
esis and may play a crucial role in tumor invasion and
metastasis [13,15].
Various r esearch groups have attributed the Vn-binding
site on PAI-1 to different epitopes. However, essential
amino acids appear to be located in a region within amino
acids 1 10±147 [16±19], most of them being localized in the
a helix E (hE). The major aim of the present study was to
further analyze the structure-function relationship of PAI-1
mainly regarding its interactions with Vn, but also with
Hep. Therefore, a number of PAI-1 variants preferentially
containing modi®cations in hE of PAI-1 were generated and
characterized biochemically.
MATERIALS AND METHODS
Generation of PAI-1 variants
The coding regions of wild-type PAI-1 (amino acids 1±379
according to the numbering pr oposed by Ny et al. [20]) and
wild-type PAI-2 (amino acids 2±415; PIR protein sequence
database: A32853) have been cloned in frame with an
N-terminal His
6
tag into the E. coli expression vector pQE-30

(Qiagen, Hilden, Germany) as described previously [21].
Modi®cations in the w ild-type cDNA s equence of P AI-1
were generated by reverse long-range PCR ( ÔExpand High
Fidelity PCR System KitÕ; Roche, M annheim, Germany)
applying mutated primers (Metabion, Martinsried, Germa-
ny). PCR p roducts were ph osphorylated (T4 polynucleotide
kinase; R oche, M annheim, Germany), re-ligated (T4 DNA
ligase; Roche, Mannheim, Germany) and transformed into
the E. coli strain XL1 b lue (Stratagene, H eidelberg, Ger-
many). The mutated sequences were veri®ed b y DNA
sequencing performed by TopLab, Martinsried, Germany.
Expression and puri®cation of wild-type PAI-1,
wild-type PAI-2, and PAI-1 variants
Expression of recombinant proteins w as induced by adding
isopropyl thio-b-
D
-galactoside (IPTG; ®nal concentration:
2m
M
) to an X L1 blue (variant) PAI-1 or PAI-2 culture,
pregrown in Luria±Bertani medium supplemented with
100 lgámL
)1
ampicillin ( D
600
0.6±0.7). P rotein ex pression
occurred at 37 °C o vernight on an orbital shaker at
200 r.p.m. The bacterial culture was harvested by centrif-
ugation at 5000 g at 4 °C for 10 min. Then, the bacterial
pellet was frozen for 20 min at )80 °C and, subsequently

suspe nded in 20 m
M
Na±acetate, 1
M
NaCl, 0 .1% Tween-80
(v/v), pH 7.4 supplemented with the protease inhibitor mix
ÔComplete EDTA-freeÕ (Roche, Mannheim, Germany). In
the case of wild-type PAI-2, a slightly different buffer
[20 m
M
Na
2
HPO
4
,1
M
NaCl,0.1%Tween-80(v/v),pH 7.4
supplemented w ith C omplete E DTA-free] was used. Bacte-
ria were disrupted mechanically by addition of glass beads
(Sigma, Taufkirchen, G ermany) t o the bacterial cell sus-
pension and 10 subsequent cycles of vortexing and incuba-
tion on ice for 1 min each. The lysate was centrifuged for
15 min (12 000 g,4°C), the supernatant recovered and
subjected to Ni
2+
-nitrilotriacetic acid agarose af®nity
column puri®cation.
Ni
2+
-nitrilotriacetic acid af®nity chromatography

The Ni
2+
-nitrilotriacetic acid af®nity column was prepared
as described by the manufacturer (Qiagen, Hilden,
Germany). Initially, the af®nity column was equilibrated
with 20 m
M
Na-acetate, 1
M
NaCl, 0.1% Tween-80 (v/v),
pH 7.4. Then, t he cleared bacterial lysate was applied to the
column and, subsequently, the column washed with equili-
bration buffer followed by 20 m
M
Na-acetate, 1
M
NaCl,
0.1% Tween- 80 (v/v), 20 m
M
imidazole, pH 5.6 until the
absorption of the ef¯uent had r eturned to b aseline
(D
280
< 0.001). F inally, t he a dsorbed recombinant proteins
were eluted with 20 m
M
Na-acetate, 1
M
NaCl, 0.1%
Tween-80 (v/v), 200 m

M
imidazole, pH 5.6. The e luate
containing the recombinant proteins was dialyzed in
equilibration buffer and puri®ed by a second Ni
2+
-nitrilo-
triacetic acid af®nity chromatography as described above.
The supernatant of lysates of wild-type PAI-2 expr essing
bacterial cells (in a buffer containing 20 m
M
Na
2
HPO
4
,1
M
NaCl, 0.1% Tween-80, pH 7.4) was also applied to a Ni
2+
-
nitrilotriacetic a cid af®nity column, and washed with the
same buffer (at pH 6.5) supplemented with 20 m
M
imidaz-
ole. For elution, the same buffer (at pH 6.0) containing
200 m
M
imidazole was used.
Denaturation and refolding of the recombinant proteins
The puri®ed recombinant proteins, with exception of wild-
type PAI -2, were denatured f or 4 h in 4

M
guanidinium/HCl
at room temperature under light protection. Refolding of
the proteins was achieved by dialysis (2 h, 4 °C) in 20 m
M
Na-acetate, 1
M
NaCl, 0.01% Tween-80 (v/v), pH 5.6
followed by a second dialysis step (overnight, 4 °C). The
proteins were subsequently concentrated in Centricon
centrifugal ®lter devices (Millipo re, Eschborn, G ermany).
Wild-type PAI-2 was dialyzed against NaCl/P
i
(pH 7.4) in
order to remove residual i midazole, concentrated in
Ó FEBS 2002 Interaction of PAI-1 variants with vitronectin (Eur. J. Biochem. 269) 185
Centricon centrifugal ®lter d evices, and then stored at
)80 °C until use.
Characterization of the recombinant proteins
The protein c oncentration was determined ac cording to
Bradford using the Bio-Rad Protein Assay Dye Reagent
Concentrate (Bio-Rad, Krefeld, Germany). PAI-1 antigen
was determined u sing the Imubind Tissue P AI-1 ELISA Kit
(American Diagnostica, Pfungstadt, Germany). The iden-
tity of the puri®ed proteins was demonstrated by Western
blotting employing a polyclonal antibody (pAb) from
chicken directed against human PAI-1 (a k ind gift of N .
Grebenschikov, Institute of Ch emical Endocrinology, Uni-
versity of Nijmegen, the Netherlands), a monoclonal mouse
antibody (mAb) to human PAI-2 (#110 from American

Diagnostica, Pfungstadt, Germany), and the ECL Western
Blotting Detection R eagent (Amersham P h armacia, Frei-
burg, Germany) for detection. N-terminal amino-acid
sequence analysis p erformed by TopLab (Martinsried,
Germany) was used t o con®rm the identity of the puri®ed
recombinant wild-type PAI-1 and wild-type PAI-2.
Amidolytic assay for determination of the inhibitory
activity of the recombinant proteins against HMW-uPA
The assay was performed in 96-well microtiter plates
(Greiner, Frickenhausen, Germany). Puri®ed recombinant
proteins were diluted in a buffer containing 100 m
M
Tris/
HCl, 0.05% T ween-20 (v/v), p H 7 .5, and 100 lgámL
)1
BSA
(ICN, Aurora, Ohio, US A), incubated with 1 0 U high
molecular weight (HMW-)uPA (RheotrombÒ 500 000,
Curasan Pharma GmbH, Kleinostheim, Germany) for
15 min at r oom temperature and then 10 lL o f chromo-
genic substrate Bz-b-Ala-Gly-Arg-pNA.AcOH ( Pefa-
chromeÒ uPA, Pentapharm Ltd, Basel, Switzerland,
concentration 2 m
M
) w ere a dded (30 min, 37 °C). The
absorption was measured a t 405 nm. O ne unit PAI activity
was d e®ned as the amount, w hich completely inhibited one
unit of HMW-uPA activity.
Complex formation of the recombinant proteins
with HMW-uPA

For complex formation, 100 U ( 0.7 lg) of HMW-uPA
were incubated with t he recombinant proteins at room
temperature for 10 min. The complexes were visualized by
SDS/PAGE under nonreducing conditions and subsequent
silver staining or Western blotting with the chicken pAb
against PAI-1, mouse mAb #110 against PAI-2 (as
mentioned above), and polyclonal chicken anti-uPA Ig
(kindly provided by N. Grebenschikov, Institute of Chem-
ical Endocrinology, Nijmegen, the Netherlands). POX-
labeled chicken anti-(mouse IgG) Ig and POX-labeled goat
anti-(chicken IgY) Ig w ere purchased from Dianova,
Hamburg, Germany.
Amidolytic assay for determination of the inhibitory
activity of the recombinant proteins against thrombin
The assay was performed i n 96-well microtiter plates. F ifty
units of active recombinant PAI-1 (  35 n
M
) i n the presence
or absence o f 1 40 n
M
Vn (Promega GmbH, Mannheim,
Germany) or 1 UámL
)1
Hep (LiqueminÒ N 25 000,
Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany)
were incubated with 0.1 U of thrombin (from human
plasma; Sigma, T aufkirchen, Germany) i n a total volume of
130 lL of Tris/NaCl/Tween buffer [20 m
M
Tris/HCl,

100 m
M
NaCl, 0.1% Tween-80 (v/v), pH 8.0] a t 37 °Cfor
1 h [10]. Then, 10 lL of chromogenic substrate (Chromo-
zymÒ TH, Roche, Mannheim, Germany, concentration:
2m
M
) were a dded and the thrombin a ctivity measured
monitoring the change of D at 405 nm.
Complex formation of the recombinant proteins
PAI-1 with thrombin
Fifty units of PAI-1 ( variant) in the p resence or absence of
600 n
M
Vn or 1 UámL
)1
Hepwereincubatedwith0.5Uof
thrombin in a total volume of 30 lLTris/NaCl/Tween
buffer (1 h, 37 °C) and then subjected to nonreducing SDS/
PAGE followed by Western blotting employing chicken
pAb against PAI-1 and a POX-labeled goat anti-(chicken
IgY) Ig.
Binding of recombinant PAI-1 to Vn-coated
microtiter plates
Vn or collagen type IV (Sigma, Taufkirchen, Germany)
weredilutedtoaconcentrationof10lgámL
)1
in a buffer
containing 100 m
M

Na
2
CO
3
,pH9.6.Forcoating,50lLof
the V n or collagen type IV dilutions were poured into wells
of a NuncMaxiSorp microtiter plate ( Nunc GmbH & Co.
KG, Wiesbaden, Germany) and incubated overnight at
4 °C. After three washes with NaCl/P
i
/Tween [NaCl/P
i
containing 0.05% Tween-20 (v/v)], the wells were blocked
by addition of 200 lL p er well of NaCl/P
i
supplemented
with 2% BSA ( w/v) and incubation at room temperature for
2 h. T he wells were washed three times with NaCl/P
i
/
Tween. Afterwards 100 lL p er well of (mutant) PAI-1 in the
desired concentration were added at room temperature for
1 h. Following three additional washing steps, 200 lLper
well horseradish peroxidase labeled Ni
2+
-nitrilotriacetic
acid (Qiagen, Hilden, G ermany) at a dilution of 1 : 1000 in
NaCl/Pi/Tween containing 0.2% BSA (w/v) were added
(1 h, room temperature). After another four times of
washing, binding of PAI-1 to t he solid phase was visualized

by addition of 100 lL per well of a TMB substrate mix
(KPL, Gaithersburg, Maryland, USA). T he reaction was
stopped after color development with 50 lLperwellof
0.5
M
H
2
SO
4
. Optical density was measured at 450 nm.
Surface plasmon resonance analysis
of (mutant) PAI-1 binding to Vn
Surface plasmon resonance (SPR) studies were conducted
with a BIACORE 2000 (Biacore AB, Uppsala, Sweden).
Approximately 2000 resonance units (RU) of collagen type
IV (10 lgámL
)1
in 10 m
M
Na-acetate, pH 4 .0) (lane 1) and
Vn (10 lgámL
)1
in 10 m
M
Na-formiate, pH 4.0 [22]) (lanes
2±4) were immobilized to a CM5 sensor chip (research
grade, Biacore AB, Uppsala, Sweden) using the amino
coupling kit accordin g to t he manufacturer's recommenda-
tion. All experiments were performed in HBS-EP [10 m
M

Hepes, 150 m
M
NaCl, 3 m
M
EDTA, 0.005% Tween-20 (v/v),
pH 7.4] at a ¯ow rate of 20 lLámin
)1
. HMW-uPA was
186 N. Arroyo De Prada et al. (Eur. J. Biochem. 269) Ó FEBS 2002
used in a c oncentration o f 400 UámL
)1
. Regeneration of the
surface was achieved by injection of 1 0 m
M
HCl f or 8 min.
In order to check for reproducibility during the measure-
ment, at ®rst 80 lLofa200UámL
)1
dilution of wild-type
PAI-1 were injected fo r t wo subsequent experiments,
followed by two subsequent measurements of 80 lLofa
200 UámL
)1
dilution of a PAI-1 mutant. Then, wild-type
PAI-1 was measured a third time in duplicate, followed b y a
measurement in duplicate of the next mutant and so on.
Thus, for each PAI-1 variant at least t wo binding pro®les
were recorded. The kinetics obtained in the collagen type
IV-coated ¯ow cell were subtracted from the kinetics
derived from the Vn-coated ¯ow cell in order t o obtain

binding pro®les without bulk effects.
RESULTS
Expression and puri®cation of recombinant PAI-1,
PAI-2, and PAI-1 variants
The coding sequences for w ild-type PAI -1 a nd wild-type
PAI-2, respectively, have previously been cloned by us in
expression vector pQE-30 [21]. By reverse PCR, a series of
PAI-1 variants w as generated using pQE-30-wild-type P AI-1
as the template. Due to the fact that serpins have a very
compact tertiary s tructure (Fig. 1A), large modi®cations of
the molecule m ay r esult in misfolded, and t herefore inactive,
proteins. B ecause of this, we designed various strategies such
as introduction of point mutations, s ubstitution of few
amino a cids by alanine and glycine, substitution of selected
epitopes by the homologue PAI-2 sequence, and short
deletions ( Fig. 1B). The g enerated PAI-1 v ariants are
summarized in Table 1. IPTG-induced recombinant protein
expression in the bacterial strain XL1 blue y ielded 5±10% of
the total E. coli protein.
The recombinant proteins contained a His
6
tag at their
N-terminus, allowing puri®cation by Ni
2+
-nitrilotriacetic
acid af®nity chromatography. Although one cycle o f af®nity
chromatography substantially enriched the recombinant
proteins from other bacterial proteins, a puri®cation grade
of > 95% was only achieved after a second chromato-
graphic cycle as demonstrated by SDS/PAGE (Fig. 2 ).

Inhibitory activity of the recombinant proteins
against HMW-uPA
PAI-1 i s unique among serpins b y i ts metastability th at l eads
to a s hort half-life of  2 h under physiological conditions.
Therefore, it was not surprising that after puri®cation
(performed at room temperature) most of the recombinant
PAI-1 wild-type protein and variants were p resent in the
inhibitory inactive latent conformation. However, denatur-
ation a nd subsequent refolding by dialysis leads to the
reactivation of latent PAI-1 [23]. An up to 87% inhibitory
activity of wild-type PAI-1 and PAI-1 variants against
HMW-uPA (100 000 Uámg
)1
de®ned as t he maximum [ 24])
was reached after denaturation with 4
M
guanidinium/HCl
followed b y dialysis in a buffer of h igh ionic strength at
pH 5.6. Moreover, inhibitory activities of the protein
preparations remained stable for more than one year in
this buffer when stored a t )80 °C. The inhibitory activity of
the g enerated PAI-1 mutants is summarized in Table 1. All
inhibitory active mutants were metastable (half-life £ 2h
at 37 °C) and the half-life was roughly doubled in the
presence of Vn. Furthermore, inhibitory active variants
formed SDS-stable complexes with HMW-uPA; t he inactive
Fig. 1 . Three-dimensional struc ture of ac tive PAI-1. (A) Important structural elements of active PAI-1 (PDB 1B3K). The central b sheet A consisting
of strands 1A, 2A, 3A, 5A, and 6A is indicated in yellow, helix D in green, and helix E (hE) in cyan blue. The P1-residue of PAI-1 (R346) is also
indicated. (B) Location o f amino acid alterations i n some of t he generated PAI-1 mutants. P73A, single amino acid exch an ge of P73 to alanine ;
A114±118, exchange of the sequence 114FRLFR118 to ®ve a lanines; D109±112, deletion of the four amino acids 109MPHF112; Q123K, single

amino acid exchange of Q123 to lysine.
Ó FEBS 2002 Interaction of PAI-1 variants with vitronectin (Eur. J. Biochem. 269) 187
mutants d id not (Fig. 3). PAI-2 was stable (no loss of
inhibitory activity after incubation for 24 h at 37 °C) and
exerted an inhibitory activity against HMW-uPA of 90%
(equivalent to 90 000 Uámg
)1
) after the two-step af®nity
chromatography.
Interaction of (mutant) PAI-1 with Vn
All inhibitory active mutants but mutant Q123K [16] did
interact with Vn as veri®ed by measuring (mutant) PAI-1
binding to Vn-coated microtiter plates and by surface
plasmon resonance analysis (Table 1). This binding was
demonstrated to be highly speci®c, as binding of (mutant)
PAI-1 was completely abolished by preincubation of
recombinant PAI-1 with soluble Vn (10 lgámL
)1
)priorto
adding it to the wells or before injection in a reproducible
manner ( data not shown). Furthermore, latent PAI-1 (data
not shown) as well as heat-denatured PAI-1 (Fig. 4) did not
bind to the immobilized Vn.
The observed K
D
value for wild-type PAI-1
(K
D
 0.18 n
M

)aswellasforP73A(K
D
 0.33 n
M
)is
similar to t hat previously reported for the interaction of
human PAI-1 recombinantly expressed in Chinese hamster
ovary cells with vitronectin (K
D
 0.1 n
M
), also applying
Table 1. PAI-1 variants and binding t o V n. PAI-1 mutants with corresponding sp eci®c inhibitory activity towards uPA and their ability to interact
with Vn measured b y use of Vn-coated microtiter plates ( M P) and surface plasmon r esonanc e (SPR). + Indicates bind ing to Vn; ± indicates n o
binding to Vn. NT, not tested.
PAI-1 variant Modi®cation
a
Inhibitory activity
towards uPA (Uámg
)1
)
Binding to Vn
MP SPR
Wild-type PAI-1 87 000 + +
Wild-type PAI-2 90 000 ± NT
Mutant 1 (D109-123) D F109-Q123 Inactive ± NT
Mutant 2 (M2) F109-Q123 vs. AAGAGAA Inactive ± NT
Mutant 3 (M3) F109-Q123 vs. homologue Inactive ± ±
PAI-2 sequence
b

and E128G
Mutant 4 (M4) F109-Q123 vs. AAAA Inactive ± ±
Mutant 5 (D109-112) D F109-H112 10 000 + NT
Mutant 6 (A114-115) F114A and R115A 57 000 + NT
Mutant 7 (A114±118) F114-R118 vs. AAAAA 20 800 + +
Mutant 8 (M8) F114-R118 vs. AAAAA and D68G 7 700 + +
Mutant 9 (M9) V284-G294 vs. homologue
PAI-2 sequence
c
Inactive ± ±
Mutant 10 (P73A) P73A 56 250 + +
Mutant 11 (Q123K) Q123K 47 800 ± ±
a
Numbering of PAI-1 according to Ny et al. [20];
b
141YIRLCQKYYSSEPQA155, and
c
318YELRSILRSMG328, PAI-2 according to PIR
protein sequence database: A32853.
Fig. 2. Puri®cation o f recombinant wild-type PAI-1. Human recombi-
nant wild-type PA I-1 e quipped w ith a n N-terminal His
6
tag ( wild-type
PAI-1) was puri®ed from an E. coli cell lysate by Ni
2+
-nitrilotriacetic
acid anity chromatography and analyzed by SDS/PAGE. M,
marker; lane 1, E. coli ly sate; lane 2, euent o f the ®rst Ni
2+
-nitrilo-

triacetic a cid anity chro matography cycle; lane 3, elu ate of the ®rst
Ni
2+
-nitrilotriacetic acid anity chromatography cycle; lane 4, eluate
of the second Ni
2+
-nitrilotriacetic acid anity c hromatography c ycle.
Fig. 3. Formation of S DS stable complexes. Wild-type PAI-1 o r variants thereof i n the presen ce (+ uP A) or absenc e (± uPA) of 100 U ( 0.7 lg)
HMW-uPA were incubated for 10 min at room tem per ature and then subjected to n onred ucing SDS/PAGE. Subsequently, the gels were silver-
stained. 200 U o f PAI-1, 1 10 U o f Q123K, 50 U of P73A and of A114±118 were applied; alternatively 1.4 lg of ( inactive ) M 4 a nd 1 0 U of PAI-1
were used.
188 N. Arroyo De Prada et al. (Eur. J. Biochem. 269) Ó FEBS 2002
SPR analysis [22]. Mutant A114±118 displayed a slower
association to (on-rate: 5.35 ´ 10
5
M
)1
ás
)1
)andafaster
dissociation from Vn (off-rate: 1.0 ´ 10
)3
s
)1
) c ompared t o
wild-type PAI-1 (on-rate: 1.0 ´ 10
6
M
)1
ás

)1
; off-rate:
1.9 ´ 10
)4
s
)1
). Only a low amount of mutant Q123K
associated to V n and d issociated immediately a fter washing
with buffer , indicating an unspeci®c binding to Vn (Fig. 4).
Moreover, mutant Q123K did not show any change in
binding to solid phase Vn in the presence or a bsence of
soluble V n (data not shown). All Vn-binding mutants
dissociated immediately from Vn after uPA injection
(Fig. 4). Thus, these results clearly demonstrate that
(mutant) PAI-1, but not Q123K, s peci®cally binds t o Vn
immobilized to the dextran matrix of the CM5 chip, and
then still was able to form complexes with uPA.
Inhibition of thrombin by PAI-1 and binding to Hep
Binding to Vn provides wild-type P AI-1 with thrombin
inhibitory properties [10]. Therefore, we tested whether the
PAI-1 mutants generated were also able to inhibit thrombin
in the p resence of Vn. I n line w ith our data obtained by
SPR, the Vn-interacting mutants A114±118 and P73A, but
not Q123K, inhibited thrombin in t he presence of Vn to
about 40%. Vn alone did not reduce thrombin activity
signi®cantly (Fig. 5).
We also tested the e ffects of Hep on the inhibitory activity
of PAI-1 towards thrombin. All mutants tested but A114±
118 d isplayed s imilar properties as wild-type PAI-1 (> 90%
inhibition; Fig. 5). A114±118 inhibited thrombin in the

presence of Hep clearly less effectively (about 40%; Fig. 5).
These r esults determ ined by amidolytic assays measuring
(residual) thrombin activity were supported by the results
seen in Western blots visualizing the formation of SDS
stable c omplexes between thrombin and PAI-1 with or
without Vn o r H ep. Detection o f a higher residual thrombin
activity correlated with a lower a mount of SDS stable
complexes (data not shown).
The reduced capacity of A114±118 to inhibit thrombin
with Hep as a cofactor is not related to an altered af®nity to
Fig. 5. Inhibition of thrombin. Fifty un its of recombinant PAI-1
( 35 n
M
) in the presence or absence of 140 n
M
Vn or one UámL
)1
Hep were incubated with 0.1 U of thrombin in a total volume of
130 lL of Tris/NaCl/Tween buer a t 3 7 °C for 1 h . T hen, 10 lLof
chromogenic substrate were added and the thrombin activity measured
monitoring the change of optical density at 405 nm. The activity of
thrombin in the absence of inhibitor was se t to 100%, the othe r
activities were calculated accord ingly. Data shown are from three
independent experiments, each measured in duplicate (  SD). As a
control, the eect of 140 n
M
Vn or 1 UámL
)1
Hep witho ut inhib itor
was measured. Black bars, buer, only; hatched bars, plus Vn

(140 n
M
); grey bars, plus Hep (1 UámL
)1
).
Fig. 4. Surface plasmon resonance: binding of (mutant) PAI-1 to VN. Approximately 2000 R U of collagen type I V or V n were immobilized to a
CM5 sensor chip i nserted in the BIAcore 2 000 system. Then, wild-type PAI-1 (200 UámL
)1
) was injected and a llowed to bind to Vn, w hich was
followed by a washing step w ith buer. Finally, H MW-uPA ( 400 UámL
)1
) was i njected. Two subsequent measurements of the b inding kinetics of
wild-type PAI-1 were followed by two independent measurements of a PAI-1 variant (200 UámL
)1
). After that, wild-type PAI-1 was measured a
third time, followed by a measurement in duplicate of the next mutant and so on. Thus, for each PAI-1 variant at l east two independent binding
pro®les we re o btained. All experiments were pe rf ormed a t a ¯ow rate of 2 0 lLámin
)1
; regeneration of the surface was achieved by treatment with
10 m
M
HCl f or 8 min. The k inetics obtained in the collagen t ype IV-coated ¯ow c ell w ere subtracted f rom t he k inetics de rived f rom t he Vn -coated
¯ow cell in o rd er to obtain binding pro®les without bulk eects. Heat d enature d controls were measured to compare the binding signal w ith the
unspeci®c binding to the identical surface conditions.
Ó FEBS 2002 Interaction of PAI-1 variants with vitronectin (Eur. J. Biochem. 269) 189
Hep, as in SPR we observed that A 114±118 displayed
similar binding to biotinylated Hep immobilized on a SA-5
sensor chip as wild-type PAI-1 (data not shown). Interest-
ingly, Hep did not only provide A114±118 with thrombin
inhibitory properties less e f®ciently than t he other t ested

mutants, it also reduced the inhibitory property of A114±
118 towards uPA in a dose dependent manner. Hep had
only marginal e ffects on t he inhibition of uPA by other
inhibitory active PAI-1 proteins (wild-type P AI-1, P 73A,
Q123K, data not shown).
DISCUSSION
Puri®cation and inhibitory activity of recombinant
PAI-1, PAI-2, and PAI-1 variants
Recombinant expre ssion i n a bac terial s ystem i s a n easy a nd
quick method for the productio n of large a mounts of
human PAI-1 and PAI-2. It has been s hown p reviously,
that, although PAI-1 and extracellular PAI-2 are glycosy-
lated proteins, expression of both proteins i n a nonglycosy-
lated form in prokaryotes does not affect production and
inhibitory activity of these proteins [25]. We cloned the
cDNA sequence of both wild-type PAI-1 and wild-type
PAI-2 in an expression vector that provides an additional
histidine
6
-sequence at t he N-terminus of the r ecombinant
proteins [21], thus allowing puri®cation by Ni
2+
-nitrilotri-
acetic acid af®nity chromatography. The obtained speci®c
activities of wild-type PAI-1 and wild-type PAI-2, respec-
tively, strongly indicate that this mo di®cation does not have
any signi®cant effect on the inhibitory activity of the
recombinant in comparison to the natural proteins. This is
most likely due to the fact that the RCL of both serpins is
located close to the C-terminus.

The recombinant proteins were puri®ed under native
conditions using a modi®ed version of a b uffer t hat had
been described previously for the isolation of PAI-1 from
conditioned medium of human endothelial cells [26]. This
buffer i s characterized by a l ow pH (5.6) as well as a
relatively high ionic strength (1
M
NaCl). These conditions
resemble the inner milieu of the a-granula contained in
thrombocytes, which are the main reservoir o f PAI-1 in
blood [27]. Sancho et al. [28] reported the isolation of
signi®cant amounts of a ctive PAI-1 under native conditions
by using such a buffer. However, we did not obtain such
high amounts of a ctive PAI-1 under similar conditions, but
the inhibitory activity of PAI-1 was dramatically enhanced
by denaturation, refolding, and storage in a buffer a t p H 5.6
containing 1
M
NaCl.
The generated PAI-1 var iants did also not display any
inhibitory activity after puri®cation under native conditions.
Again, the inhibitory activity of at least some of the PAI-1
mutants could be restored by denaturation and refolding.
The achieved activities among the variants were highly
related to the number of modi®cations introduced. The
variants A114±115, P73A , a nd Q 123K, i n w hich only one or
two amino-acid were exchanged, showed the highest
inhibitory capacity against H MW-uPA among the PAI-1
variants ( 50 000 Uámg
)1

). Mutant A114±118 containing
®ve amino-acid substitutions displayed 24%, D109±112
(four deleted amino acids) and M8 (six amino-acid substi-
tutions) a bout 10% of the inhibitory activity of recombinant
wild-type PAI-1, only. All of the other PAI-1 variants
(D109-123, M2, M3, M4, M9), which c ontained more than
seven modi®ed amino-acid positions did not display a ny
inhibitory activity towards uPA, indicating that larger
modi®cations at these positions are not tolerated and lead to
a loss o f activity m ost likely due to misfolding of the
compact PAI-1 structure.
The active PAI-1 variants behaved similarly to wild-type
PAI-1 with respect to the ir metastability and the stabiliza-
tion of the active f orm b y V n. In contrast to wild-type PAI-1
and its active variants, r ecombinant P AI-2 did not show any
signi®cant loss of activity after puri®cation under native
conditions, underlining the uniqueness of the metastable
PAI-1 a mo ng se rp ins.
Interaction of (mutant) PAI-1 with Vn
Using Vn-coated microtiter plates and SPR, we a nalyzed
speci®c binding of wild-type PAI-1 and PAI-1 variants to
Vn. I n general, SPR measurements are c onsidered to be very
quantitative and association/dissociation constants are
normally easily analyzed by Langmuir binding isotherms
to obtain t he respective bin ding constants. H owever, in the
case of PAI-1 and variants thereof, this kind of measure-
ments may not be accurate for t he following reasons: PAI-1
spontaneously converts from its active to a latent form. To
determine t he p roportion o f active PAI-1, w e m easured t he
speci®c inhibitory activity against uPA with a theoretical

maximum de®ned a s 100 000 Uámg
)1
[24]. This m ethod can
be used for wild-type PAI-1, but for the PAI-1 mutants a
different maximum for the speci®c activity may exist.
Furthermore, within the time frame of determination of t he
amount of active PAI-1 in a given preparation to the time-
point when PAI-1 is u sed in the SPR analysis, anoth er a s y et
unknown part of active PAI-1 converts to the l atent form
and thus cannot bind to Vn anymore. Therefore, the
analysis of the interaction of PAI-1 and, especially, PAI-1
variants with Vn can only b e semiquantitative. It has t o be
emphasized, however, that in the present s tudy the main a im
was to analyze whether mutants with variations in hE are
still able to bind to Vn and n ot to compare the binding
af®nities of the various mutants in a quantitative manner.
As only inhibitory active PAI-1 binds to Vn [8],
conclusions about the Vn-binding site on PAI-1 can only
be drawn from the mutants with inhibitory activity. All
mutants with var iations covering the w hole area of hE
(D109-123, M2, M3, M4) yielded inactive PAI-1 variants.
However, D109-112, in which t he N-t erminal amino a cids of
hE were deleted, as well as A114±118, with changes in the
C-terminal region of hE, still bo und to Vn and inhibited
uPA. Thus, m utations within hE of PAI-1 are tolerated t o
some extent. Our results from measuring binding of wild-
type PAI-1 and its variants to Vn-coated microtiter plates
were reproduced in SPR. Furthermore, as these results are
in line with those obtained in thrombin inhibition experi-
ments, it can be c oncluded that the mutants still interact

functionally with Vn. Moreover, concerning Q123K and its
dramatically reduced af®nity to Vn, we r eproduced the
results of Lawrence et al. [16], which again underlines the
functionality of our test systems. Lawrence et al.[16]also
reported a bout another mutation (L116P) i n hE that led to
Vn-binding de®ciency. In two of our mutants (A114±118,
M8), L116, among other amino acid changes, was substi-
tuted by alanine. The resulting mutant proteins, however,
190 N. Arroyo De Prada et al. (Eur. J. Biochem. 269) Ó FEBS 2002
still displayed Vn-binding activity. This may indicate that
the rather conservative alteration of L116 to alanine ( as
compared to proline) may not be dramatic enough to
eliminate the Vn-binding capacity of these PAI-1 variants.
Padmanabhan and Sane [19] located t he Vn-binding site
on PAI-1 to amino acids 115±130 employing PAI-1/PAI-2
chimera a nd protease-digested P AI-1. This seems to b e
contradictory to our results, but one has to keep in mind
that proteolytic treatment of PAI-1 most likely leads to an
altered overall structure in the resulting fragments. Fur-
thermore, all of the chimera that did not bind to Vn were
not only modi®ed around hE but additionally in the area
around Q123 of PAI-1. Van Meijer et al. [ 17] localized the
Vn-binding region of PAI-1 to amino acids 110±145 using
epitope-mapped m onoclonal antibodies which inhibited V n/
PAI)1-interaction. The region encompassing amino acids
110±145 not only comprises hE but also the region of the
strand 1 edge o f bsheet A, where Q123 is located [29]. Cross-
linking studies reported b y D eng et al. [18] localized the
Vn-binding region of PAI-1 to the same region. However,
Sui and Wiman [30] did not report any changes in the

Vn-binding behavior of mutants w ith single amino-acid
substitutions in the region of F113 to D138, which is in line
with our results. Summarizing these data, it is much likely
that the importance of hE for Vn-binding has been
overestimated previously. Although hE still plays a role
for high a f®nity binding of PAI-1 to V n as demonstrated in
the altered association to and especially dissociation from
Vn in the case of A114±118 in SPR, there seems to exist
some cooperation of the region a round s trand 1 of b sheet A
with hE in Vn-binding.
Inhibition of thrombin by PAI-1 and binding to Hep
Ehrlich et al. [10] demonstrated th at w ild-t ype P AI-1
inhibits thrombin in the presence of Vn. This is also true
for all tested variants in the present study that did interact
with Vn. Furthermore, in line with the results from Ehrlich
et al .[31],1UámL
)1
was determined as the ideal Hep
concentration f or inhibition of thrombin by wild-type PAI-
1. In addition to wild-type PAI-1, we a lso t ested t he mutants
A114±118, Q123K, and P73A at this concentration. P73A
did not show any differences in interaction with Hep,
although this amino acid is located in helix D, which was
previously reported to contribute to the Hep binding site
[32]. As e xpected, Hep-bound Q123K inhibited thrombin
exactly like w ild-type PAI-1. A 114±118 did not display a
signi®cantly altered af®nity to Hep i n SPR, although it was
not able to inhibit t hrombin a s e f®ciently a s w ild-type P AI-1
together with Hep . This contras ts w ith the results o f Sui and
Wiman [30] who detected a change in a f®nity to Hep in their

mutant R118D and proposed that mainly ionic interactions
occur between PAI-1 and Hep. However, a change o f R118
to alanine (as in the mutant A114±118) and not to aspartate
(as in R118D) may not change the surface charge of this
region signi®cantly enough to reduce af®nity to Hep.
Surprisingly, t he i nhibitory activity of A114±118 towards
uPA was reduced in a dose dependent manner by addition
of Hep. This may suggest that upon Hep-binding to A114±
118 c onformational c hanges occur, affecting the inhibition
characteristics of this PAI-1 mutant towards both thrombin
and uPA.
CONCLUSIONS
Comparison of the m ain p rotease targets of PAI-1, uPA and
tPA, has previously shown that the proteolytic activity of
these enzymes is not exclusively the relevant feature for
cancer spread. Rath er, it seems that further interactions of
one of the proteases, uPA, w ith other molecules support
tumor i nvasion a nd metastasis. W hereas tPA, at least in
breast and ovarian cancer, does not play a major role in
tumor cell invasion, uPA is an important, multifunctional
component of the invasion m achinery most likely due to
effects excerted upon interaction with its speci®c receptor,
uPAR [3]. Similarly, the additional b inding partners of PAI-
1 (Vn, Hep, and ®brin) strongly differentiate it from PAI-2,
which extracellularly targets serine proteases only. Especial-
ly, interaction of PAI-1 with Vn is strongly related to the
modulation of cancer cell adhe sion and, thus, m ay facilitate
tumor cell invasion via a balanced interference/induction of
tumor cell attachment/migration [11,12,33,34].
Detailed knowledge of the structural region(s) of the PAI-1

molecule implicated in the P AI-1/Vn-interaction is the basis
for the rational development of site-speci®c PAI-1 modu-
lators [35]. Surface-exposed loop structures, s uch as hE in
PAI-1, represent attractive targets for the development of
such modulators because of their high ac cessibility. hE has
been implicated in the binding of PAI-1 to Vn [16±19]. An
hE-blocking compound m ay not block further PAI-1
activities (inhibition of serine proteases o r binding to ®brin)
and, thus, would not alter functions of PAI-1 important for
physiological processes such a s ®brinolysis. However, in the
present paper, we demonstrate that the region around hE in
PAI-1 is not a prerequisite for b inding to Vn and, thus, m ay
not be a target for th e d evelopment o f a therapeutically
applicable PAI-1 modulator.
ACKNOWLEDGEMENTS
The excellent technical assistance of S. Creutzburg is g ratefully
acknowledged. We thank J. Stu
È
rzebecher, J. Krol, and S. Sato for
stimulating d iscussions. Part of this work w as supported by grants of
the Graduiertenkolle g 333, the Sonderforschungsbereich 469 (A4), and
the Sonderforschungsbereich 456 (B9) of the Deutsche Forschungs-
gemeinschaft.
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