Polymerization of Plasminogen Activator Inhibitor-1*
Aiwu
Zhou
§,
Richard
Faint¶,
Peter
Charlton¶,
Timothy R.
Dafforn
,
Robin W.
Carrell
, and
David A.
Lomas
From the
Department of Haematology and the
Respiratory Medicine Unit, Department of Medicine,
University of Cambridge, Wellcome Trust Centre for Molecular Mechanisms
in Disease, Cambridge Institute for Medical Research, Wellcome
Trust/MRC Building, Hills Road, Cambridge, CB2 2XY, United Kingdom
and ¶ Xenova Limited, Slough,
Berkshire, SL1 4EF, United Kingdom
Received for publication, November 26, 2000
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ABSTRACT |
The activity of the serine proteinase inhibitor
(serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the
intramolecular incorporation of the reactive loop into
-sheet A with
the generation of an inactive latent species. Other members of the
serpin superfamily can be pathologically inactivated by intermolecular
linkage between the reactive loop of one molecule and
-sheet A of a
second to form chains of polymers associated with diverse diseases. It
has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native
and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of
polymerization. The polymers of both the native and latent species
differ from the typical loop-A-sheet polymers of other serpins in that
they readily dissociate back to their original monomeric form. The
findings with PAI-1 are compatible with different mechanisms of
linkage, each involving
-strand addition of the reactive loop to s7A
in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and
latent PAI-1 can also form polymers under similar conditions, which may
be of in vivo importance in the low pH environment of the platelet.
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INTRODUCTION |
Plasminogen activator inhibitor type 1 (PAI-1)1 is a member of the
serine proteinase inhibitor or serpin superfamily (1, 2). Serpins play
an important role in the control of proteinases involved in blood
coagulation, complement activation, and inflammation and are
distinguished functionally from other types of protein inhibitors by
their ability to form SDS stable complexes with target proteinases.
Crystal structures have shown that members of the family share a highly
conserved tertiary fold consisting of three large
-sheets surrounded
by nine
-helices. This scaffold presents the reactive loop as a
pseudosubstrate that binds to and inhibits the target proteinase (3).
The target proteinases of PAI-1 are urokinase-type plasminogen
activator and tissue-type plasminogen activator (tPA) (4) and as
such it is an important modulator of events of extracellular
proteolysis in fibrinolysis and in the turnover of extracellular matrix
(5).
One of the most striking features of serpins is their ability to
undergo a dramatic conformational rearrangement with the N-terminal
portion of the reactive loop inserting into
-sheet A (6). This
transition with cleavage of the loop is central to the formation of a
stable inhibitory complex (7-10), but it can also occur spontaneously
in vivo, without cleavage of the loop, to form an inactive
latent conformation (11-14). Moreover, antithrombin and
1-antitrypsin can be induced to adopt a latent conformation by heating in stabilizing concentrations of sodium citrate
(15-17). Serpins are also able to link their reactive loop to a
-sheet of another molecule to form loop-sheet polymers, one form of
which (18) has recently been crystallized (19, 20). These polymers are
of considerable importance because they underlie the deficiency of
1-antitrypsin (3, 21-25), antithrombin (13, 26, 27),
1-antichymotrypsin (28), and C1-inhibitor (29, 30) in
association with liver cirrhosis, thromboembolism, emphysema, and
angioedema, respectively. The process also underlies a novel early
onset dementia characterized by inclusion bodies of neuroserpin
polymers (31).
Plasminogen activator inhibitor-2 can undergo this noncovalent
polymerization spontaneously (32), and most other inhibitory serpins
will polymerize upon heating or treatment with mild denaturants (16,
33). This conformational change has not been described in PAI-1, which
more readily adopts the inert latent configuration. However, the recent
crystal structure of recombinant active PAI-1 revealed PAI-1 as a
polymer in which the reactive loop anneals as strand 7A of
-sheet A
(34). This is in contrast to other models of serpin polymers in which
the loop anneals with either the A- or C-sheet of another molecule
(Fig. 1). We have examined the behavior
of PAI-1 under a variety of conditions and show here that both the
native and latent species can form polymers at low pH.

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Fig. 1.
Schematic representation of serpin
polymers. The loop-A-sheet interaction (left panel) is
believed to underlie the long chain polymers that result from naturally
occurring mutants of 1-antitrypsin (57), but the C-sheet
(middle panel) and s7A linkages (right panel)
have been described in crystal structures of antithrombin and PAI-1,
respectively (15, 34). In each of these models, the acceptor
molecule is shown in blue, the donor molecule is in red, and
the linking reactive loop center loop is in cyan.
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EXPERIMENTAL PROCEDURES |
Materials--
Unless otherwise stated, all reagents were
obtained from BDH Chemicals Ltd. Subtilisin Carlsberg, and
riboflavin were purchased from Sigma, and phenyl-Sepharose (low
substituted), CM-Sepharose FF, and Superdex S200 resins were from
Amersham Pharmacia Biotech. Human tPA was from Roche Molecular
Biochemicals. Active glycosylated PAI-1 (from HT1080 cells) was a
generous gift from Dr. Peter Andreasen (Department of Molecular and
Structural Biology, Aarhus University, Aarhus, Denmark), or
prepared by refolding latent glycosylated PAI-1 (from Alpha
Laboratories, Hants, UK) as described previously (35). The PAI-1
expression plasmid pET11c-PAI-1 was a kind gift from Prof. Xianxiu Xu
(Nanjing University) and the peptide Ac-TVASSSTA (encoding P14-P7 for
PAI-1) was synthesized by MWG-Biotech UK Ltd.
Preparation of Mutant PAI-1 and Purification of Recombinant
Protein--
The construction of pET11c-PAI-1 and expression of
recombinant PAI-1 have been described previously (36). The stable PAI-1 mutant (N150H, K154T, GQ19L, and M354I) (37) was prepared by polymerase
chain reaction mutagenesis and confirmed by DNA sequencing. The
purification protocol was a modification of published methods. Briefly
the cell lysate was applied to a CM-Sepharose column (2.5 × 40 cm), and then, after washing to base line with buffer A (20 mM NaOAc, 0.2 M NaCl, 0.1 mM EDTA,
pH 5.6), the bound proteins were eluted with a linear gradient of
buffer A containing 0.2-1.0 M NaCl. PAI-1 eluted at 0.6 M NaCl, and PAI-1 containing fractions were pooled and
mixed with
volume of buffer B (3 M
(NH4)2SO4, 20 mM NaOAc,
0.1 mM EDTA, pH 5.6). The solution was then loaded onto a
phenyl-Sepharose column (1.6 × 10 cm) that had been equilibrated
with buffer C (1 M
(NH4)2SO4, 20 mM NaOAc, 0.1 mM EDTA, pH 5.6), and the protein was eluted with a 400 ml gradient of 1.0-0 M
(NH4)2SO4 in 20 mM
NaOAc, 0.1 mM EDTA, pH 5.6. Native and latent PAI-1 were
collected separately, concentrated to 1 mg/ml in 5 mM
NaOAc, 0.1 mM EDTA, 0.1 M NaCl, pH 5.6, and stored at
70 °C. The purity of PAI-1 in each peak was
characterized by SDS-PAGE.
Preparation of Reactive Loop Cleaved PAI-1--
Native
PAI-1 (200 µg/ml) was incubated with subtilisin (4 µg/ml) at a
final ratio of 100:1 (w/w) in 5 mM NaOAc, 0.1 mM EDTA, 0.1 M NaCl, pH 5.6, at room
temperature for 45 min, and then the reaction was stopped by the
addition of phenylmethylsulfonyl fluoride to a final concentration of 1 mM. A
volume of buffer C was added and reactive
loop cleaved PAI-1 was separated from the native and latent form on the
phenyl-Sepharose column as described above. The cleaved form of PAI-1
was characterized by SDS-PAGE, mass spectroscopy, and N-terminal sequencing.
Nondenaturing Polyacrylamide Gel Electrophoresis with Low pH
Discontinuous Buffer System--
This system was first described by
Jovin in 1973 (38) and modified to assess the conformers of PAI-1. The
separating gel contained 12% (w/v) acrylamide, 0.4% (w/v)
bisacrylamide and 260 mM acetic acid adjusted to pH 4 with
KOH. The stacking gel contained 2.5% (w/v) acrylamide, 0.625% (w/v)
bisacrylamide, and 90 mM acetic acid adjusted to pH 5.0 with KOH. The pH of both were varied to assess conformations of PAI-1.
A mixture of riboflavin and TEMED (with final concentrations of 5 mg/liter and 1.25 ml/liter, respectively) was used to initiate
polymerization of the acrylamide. The electrode buffer contained 40 mM
-alanine adjusted to pH 4 with acetic acid, and
methyl green was used as the tracking dye. The proteins migrated toward
the negative electrode. All the gel electrophoresis was performed at
room temperature unless otherwise stated.
Transverse Urea Gradient Gel Electrophoresis--
12% (w/v)
polyacrylamide gels were cast with a double lumen tube and a
peristaltic pump to give a linear gradient from 0 to 8 M
urea using the low pH nondenaturing PAGE buffer system. The gels were
rotated through 90°, the stacking gel was poured, and the gels were
run using the same electrode buffer as above. The proteins were
visualized by staining with Coomassie Blue or by silver staining.
Fluorescence Measurements--
Fluorescence measurements were
made using a PerkinElmer Life Sciences 50B spectrofluorimeter as
detailed previously (39). Intrinsic tryptophan fluorescence of PAI-1
was measured using an excitation wavelength of 295 nm and detecting
photons with a wavelength of 340 nm emitted at 90° to the excitation
beam. Wherever possible the slits controlling the intensity of the
excitation light source were kept at the minimum machine-permissible
limit of 2.5 nm. Emission slit widths were varied between 2.5 and 15 nm
depending on the experimental conditions to give the optimal emission
signal. The experiments were performed in a 0.5-ml cuvette with a path
length of 1 cm on the excitation axis and 0.2 cm on the emission axis.
Light scattering experiments were performed under the same conditions
but at an excitation wavelength of 400 nm and an emission wavelength of
405 nm.
Circular Dichroism--
CD experiments were
undertaken in a 0.5 mm path length quartz cell using a JASCO J-810
spectropolarimeter. Samples of PAI-1 were prepared in 0.1 M
NaOAc for pH values between 4 and 5.5, 0.1 M sodium
phosphate for pH values between 6 and 8, and 100 mM Tris
for pH 8.9. The protein was examined at 0.28 mg/ml with 25 mM NaCl from pH 4 to 8.9. All buffers were filtered, and
samples were centrifuged before the experiment. Changes in secondary
structure of PAI-1 with temperature were measured by monitoring the CD
signal at 222 nm or 216 nm between 25 and 95 °C with a heating rate
of 2 °C/min. The second derivative of the resulting data was then used to calculate the inflection point of the transition and hence the
Tm (39). Measurement of the far UV CD spectra of
native and polymerized PAI-1 at pH 4 was complicated by the high
absorbance of the buffer (0.1 M NaOAc). This was reduced by
dialyzing PAI-1 against water at 4 °C and then diluting into 5 mM NaOAc, pH 4 (0.48 mg/ml). The spectra were recorded at
20 °C in 5 mM NaOAc, pH 4, and averaged for 20 runs to
give the profile for the native protein. PAI-1 was then incubated at
37 °C for a further 2 h before recording the spectrum of the
polymerized protein.
N-terminal Amino Acid Sequencing and Mass
Spectrometry--
Purified PAI-1 or PAI-1 electrotransferred to
Problot (40) was N-terminally sequenced by the Department of
Biochemistry, University of Cambridge. The subtilisin cleavage of PAI-1
was also analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Cleaved PAI-1 was exchanged with distilled
water by Microcon Ultrafiltration. Sinapinic acid was used as the
matrix. The Kratos Kompact matrix-assisted laser desorption ionization
IV (Kratos Analytical Ltd., Manchester, UK) was operated in positive
ion linear mode with an acceleration voltage of +20 kV. The results
were the averages of over 100 shots.
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RESULTS AND DISCUSSION |
Preparation of Native, Latent, and Cleaved
PAI-1--
Glycosylated recombinant PAI-1 spontaneously converts to
the latent conformation with a half-life of 3 h under
physiological conditions (4, 11), but this may be increased to 100 h if it is stored at high salt concentration, low pH, and room
temperature (41). Our purification protocol of PAI-1 was a modification of existing methods (42, 43), and all the steps were carried out at
room temperature. Following expression of PAI-1 and cell lysis, the
supernatant was directly loaded onto a CM-Sepharose column and eluted
with a NaCl gradient that gave ~90% pure PAI-1 with a small fraction
of latent protein (data not shown). The eluted PAI-1 was then salted to
1 M (NH4)2SO4 and
further purified by phenyl-Sepharose chromatography, which resulted in
complete separation of active PAI-1 from its latent conformation (Fig. 2a). The active peak
(hereafter referred to as the native component) was confirmed to be
PAI-1 by N-terminal sequencing (1VHHPP), and typically
1 g of wet Escherichia coli cell pellet yielded 5 mg of
active and 0.2 mg of latent PAI-1.

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Fig. 2.
Purification of recombinant PAI-1 from cell
pellets. a, separation of active (peak I)
from latent (peak II) PAI-1 by phenyl-Sepharose
chromatography by elution with a 1.0-0 M
(NH4)2SO4 gradient. The
dashed line shows the purification profile of cleaved PAI-1.
b, 10% w/v SDS-PAGE to show the purity and activity of native and
latent PAI-1. Each lane contains 2.5 µg of PAI-1 and, where
indicated, 5 µg of tPA. PAI-1·tPA complexes were formed by
incubating enzyme and inhibitor at room temperature for 30 min in PAI-1
storage buffer. Lane M, molecular mass markers (78, 66, 45, and 32 kDa); lane 1, native PAI-1 from peak I;
lane 2, native PAI-1 with tPA; lane 3, latent
PAI-1 from peak II; lane 4, latent PAI-1 with tPA;
lane 5, tPA. c, native and latent PAI-1 were
incubated with subtilisin in a 100:1 (w/w) ratio for different time
intervals at room temperature before being separated by 10% w/v
SDS-PAGE. Each lane contains 2.5 µg of PAI-1. Lane M,
molecular mass markers (78, 66, 45, and 32 kDa); lanes 2-6,
native PAI-1 incubated with subtilisin for 0, 5, 10, 20, and 30 min,
respectively; lanes 7-11, latent PAI-1 incubated with
subtilisin for 0, 5, 10, 20, and 30 min, respectively. d,
cleavage sites of native PAI-1 by subtilisin. Cleaved PAI-1 samples
were analyzed by N-terminal amino acid sequencing and mass
spectrometry. The molecular masses of C-terminal fragments of cleaved
PAI-1 are consistent with the calculated values.
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Native, but not latent, PAI-1 was able to form SDS stable
complexes with tPA (Fig. 2b) and could be cleaved at the
P1-P1' bond of the reactive loop by subtilisin (Fig. 2c).
Reactive loop cleaved PAI-1 was separated from native and latent
protein by phenyl-Sepharose chromatography as the cleaved protein
eluted before the other conformations (Fig. 2a); cleaved
PAI-1 eluted at 0.6 M
(NH4)2SO4, native PAI-1 at 0.4 M, and the latent species eluted at 0.2 M
(NH4)2SO4. The purity of all the
PAI-1 species was assessed by SDS-PAGE, and the cleavage sites of
subtilisin were analyzed by N-terminal sequencing and confirmed by
matrix-assisted laser desorption ionization-time of flight mass
spectrometry. The primary cleavage site was at P1-P1'
(Arg346-Met347), which gives an N-terminal
sequence of 1VHHPP and 347MAPEE, with secondary
sites at P1-P2 (Ala345-Arg346) and P3-P4
(Val343-Ser344) (Fig. 2d).
Characterization of PAI-1 on pH 4 Nondenaturing Gels--
Native
PAI-1 was easily distinguished from the reactive loop cleaved protein
by SDS-PAGE (Fig. 2c) but migrated with the same electrophoretic mobility as latent PAI-1. Native PAI-1 migrated as a
smear or remained in the sample well on a pH 7.8 nondenaturing gel.
Because native PAI-1 is more stable at low pH, it was characterized on
a pH 4 nondenaturing discontinuous buffer system (acid-PAGE). This
differentiated between native, cleaved, and latent PAI-1 (Fig.
3a), and to our surprise both
native and latent PAI-1 formed discrete high molecular mass ladders
(Fig. 3a, lanes 1 and 2) characterized
in other serpins as loop-sheet polymers (16, 18, 33). Varying the pH
confirmed that both native and latent PAI-1 formed polymers when
assessed on gels between pH 3.5 and 5, but these were most marked at
lower pH values. Reactive loop cleaved PAI-1 did not form polymers even
after assessment on gels at pH 3.5. The resistance of the reactive loop
cleaved PAI-1 to pH-induced polymerization may result from either
inaccessibility of the reactive loop to act as a donor for
polymerization or from the filling of the
-sheet A binding site by
the cleaved reactive loop. These alternatives were assessed using PAI-1
that had been stabilized by incubation with an exogenous peptide
corresponding to P14-P7 that binds to
-sheet A but leaves the
reactive loop in its external exposed position (44, 45). The binary
complex was formed by incubating PAI-1 with 100-fold molar excess of
P14-P7 reactive loop peptide at 37 °C in 10 mM NaOAc, 50 mM NaCl, pH 5.6, for 24 h. Formation of the complex
was confirmed by a loss of inhibitory activity with PAI-1 becoming a
substrate for tPA. The peptide-PAI-1 complex failed to form polymers on
an acid-PAGE with either the pattern seen with native PAI-1 or with
latent PAI-1. Structurally the significant difference between the
peptide-complexed and native PAI-1 is that of a six-stranded
versus a five-stranded A-sheet, and the significant
difference between latent PAI-1 and both the peptide-complexed and
cleaved form PAI-1 is the presence of a vacant strand 1C position in
the latent form. Thus, the results suggest that the polymerization of
native PAI-1 requires a five-stranded A-sheet compatible with s7A
linkage observed in the crystal structure (34) and that the different
polymerization observed with latent PAI-1 may require a vacated s1C
position, as in the C-sheet model (Fig. 1). Support for these
conclusions of separate mechanisms of loop linkage is provided by the
absence of either of the patterns of polymerization in reactive loop
cleaved PAI-1, which has both a six-stranded A-sheet and an intact
s1C.

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Fig. 3.
Polymerization of native PAI-1.
a, 12% (w/v) pH 4 nondenaturing PAGE. Lane N,
native PAI-1; lane L, latent PAI-1; lane C,
reactive loop cleaved PAI-1. Each lane contains 10 µg of PAI-1. The
band marked with an asterisk is a dimer of
cleaved PAI-1, which was confirmed by N-terminal amino acid sequencing
and gel filtration chromatography. b, native PAI-1 was
incubated at 0.1 mg/ml in 0.1 M NaOAc, pH 4, at 37 °C
for up to 12 h. Samples were withdrawn, snap frozen, and then
analyzed on a pH 4 acid PAGE at 4 °C. Each lane contained 10 µg of
protein. Lane L represents latent PAI-1 that was also
incubated in 0.1 M NaOAc, pH 4, for 12 h at 37 °C.
c, assessment of the polymerization of PAI-1 using light
scattering (dashed line) and intrinsic tryptophan
fluorescence (continuous line). PAI-1 was incubated at 0.1 mg/ml in 0.1 M NaOAc, pH 4, at 37 °C, and light
scattering was assessed at an excitation wavelength of 400 nm and a
detection wavelength of 405 nm. The data were fitted to a single
exponential curve to give a single rate (k1).
Intrinsic tryptophan fluorescence was assessed at excitation and
emission wavelengths of 295 and 340 nm, respectively. The data were
fitted with a double exponential decay to give a fast initial rate
(k1) and a slow second rate
(k2). The value of k2 was
similar to that obtained for the rate of increase in light scattering
(k1) and comparable with the rate of
polymerization observed in b. The rate of polymerization was
assessed at different protein concentrations using intrinsic tryptophan
fluorescence (d). The fast rate (k1)
was independent of protein concentration, but the slow rate
(k2) shown in the figure increased with
increasing protein concentration.
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Kinetics of the Polymerization of PAI-1--
To determine
the kinetics of polymerization, native PAI-1 was incubated at pH 4 prior to separation on an acid-PAGE. Native PAI-1 completely
polymerized after incubation at pH 4 and 37 °C for 3 h (Fig.
3b). The rate of polymerization of PAI-1 at pH 4 was
assessed using light scattering at 0.1 mg/ml and 37 °C (Fig. 3c). The data were fitted to a single exponential that gave
a rate of polymerization (k1) of 3.03 SD ± 0.49 × 10
5 s
1 (n = 4). This rate corresponded well to the rate of polymer formation when
assessed by acid-PAGE (Fig. 3b). The polymerization of PAI-1 was further examined using intrinsic tryptophan fluorescence. Incubation at 0.1 mg/ml, pH 4, and 37 °C resulted in a reduction in
the fluorescence signal (Fig. 3c). This was fitted to a
double exponential decay to generate an initial fast rate
(k1) and then a slow rate
(k2). The slow rate (2.93 ± 0.19 × 10
5 s
1; n = 4) was
comparable with the rate obtained from the light scattering and from
the acid-PAGE. The fast rate of polymerization was independent of
protein concentration (1.24 ± 0.09 × 10
3
s
1) over a range of concentrations of PAI-1 from 0.025 to
0.4 mg/ml. In contrast the slow rate increased with protein
concentration (Fig. 3d), indicating a bimolecular process in
keeping with polymer formation. The lack of concentration dependence of
the fast rate implies that it is unimolecular process in keeping with a
pH-induced conformational transition that preceeds polymerization.
These data show striking similarities with the polymerization of
another serine proteinase inhibitor
1-antitrypsin (39).
This too has a fast unimolecular step that represents a conformational
transition followed by a slow bimolecular process that correlates with
the generation of polymers.
Native and latent PAI-1 polymers were analyzed by gel filtration
on a Superdex S200 column in 20 mM NaOAc, pH 4, or 20 mM Tris-HCl, pH 8.0, with 0.15 M NaCl, 1 mM EDTA at room temperature. PAI-1 was incubated at
37 °C for 30 min at pH 4 and centrifuged to remove aggregates before
loading onto the column. In both cases only a monomeric peak was
apparent (data not shown). Thus, both polymers of native and latent
PAI-1 were dissociable. These data are similar to the results obtained
for the dimer of antithrombin which was crystallographically shown to
be due to reactive loop-C-sheet linkage (15). This similarly
dissociates to monomeric protein on gel filtration (46, 47). The
inhibitory activity of the pH 4 treated native PAI-1 could not be
assessed against tPA directly because tPA loses its enzymatic activity
at this pH. However, after polymerization at pH 3.5-5 for 30 min,
native PAI-1 was shown to regain almost full inhibitory activity
against tPA when the pH was raised above 5. Conversely, no inhibitory
activity was detected after treating latent PAI-1 at low pH for 2 h at 37 °C and then raising the protein to a neutral pH. Thus, in
the latent PAI-1 polymers, the reactive loop must remain at least partially inserted into
-sheet A.
Characterization of the Unfolding of PAI-1 on pH 4 Transverse Urea
Gradient Gels--
The demonstration that conformations of PAI-1 could
be distinguished on low pH nondenaturing gels prompted their
examination by transverse urea gradient gels with low pH buffers.
Active and cleaved PAI-1 exhibited similar profiles to the
corresponding species of
1-antitrypsin or antithrombin
(16, 48) but with an unfolding transition at ~3 M urea
for the native species and resistance to unfolding in 8 M
urea for reactive loop cleaved PAI-1 (Fig.
4, a and b). Latent
PAI-1 had an unfolding transition on transverse urea gradient gels at
~6 M urea (Fig. 4c), which is unlike the
latent conformations of
1-antitrypsin, antithrombin, and
1-antichymotrypsin, which are resistant to unfolding in
8 M urea (14, 16, 49). The binary complex of PAI-1 with the P14-P7 reactive loop peptide also starts to unfold at ~7
M urea (Fig. 4d), which again is different from
those of
1-antitrypsin and antithrombin. These data
demonstrate that incorporation of the reactive loop into the A-sheet of
latent PAI-1 is not as stable as in other serpins and are consistent
with the well recognized finding that latent PAI-1 can be reactivated
by treatment with denaturants (4, 11) or by heating (50).

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Fig. 4.
12% (w/v) pH 4 transverse urea gradient gels
of native (a), reactive loop cleaved
(b), latent (c), and binary complexed
(d) PAI-1. The binary complexed conformation was
prepared with a peptide corresponding to P14-P7 of the reactive loop.
The left and right sides of each gel represent 0 and 8 M urea, respectively.
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The Effect of pH on Reactive Loop Peptide Induced Inactivation of
Native PAI-1--
External peptides corresponding to the N-terminal
portion of the reactive center loop of serpins form stable binary
complexes, converting each inhibitor to a substrate for target
proteinases. Recent crystal structures of such binary complexes confirm
that as assumed previously, the peptides bind to the vacant strand 4A
position (45, 51). Thus, a measure of the readiness and accessibility
of the opening of the A-sheet at s4A is indicated both by the rate of
self-insertion to give the latent form and the rate of external peptide
insertion to give the binary complex. To assess this, native PAI-1 was
incubated with or without peptide (Ac-TVASSSTA, P14-P7) under different
pH conditions for 20 h at room temperature. The mixtures were
adjusted to neutral pH, excess tPA was added, and the samples were
analyzed by SDS-PAGE (Fig. 5). The result
indicates the proportion of active PAI-1 represented by the SDS stable
complex with tPA and the proportion of binary complex represented by
the cleaved band, as well as the proportion of the latent form
represented by the intact band. At pH 8, after 20 h of incubation,
most of the active PAI-1 was converted to the latent form when
incubated alone (Fig. 5, lane 6), and almost all PAI-1
formed binary complex in the presence of peptide (Fig. 5, lane
7). However, when incubated at pH 4 (Fig. 5, lane 2), PAI-1 remains almost fully active and comparable with that of the
starting material (Fig. 5, lane 1). Less than 5% of PAI-1 forms binary complex in the presence of peptide (Fig. 5, lane 3). Thus, at low pH the central
-sheet A of native PAI-1 is
tightly closed, which inhibits self-loop insertion to form the latent form or the insertion of an external peptide from another molecule to
form s4A loop-sheet polymers.

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Fig. 5.
Binary complex formation at different pH
values. 2 µM active PAI-1 was incubated with or
without the reactive center loop peptide corresponding to P14-P7 of
PAI-1 (Ac-TVASSSTA) at 1:100 ratio at room temperature (RT)
for 20 h. The pH was adjusted to 5.6-8, and the mixtures were
then incubated with excess tPA and analyzed by 10% (w/v) SDS-PAGE.
Each lane contains 2.5 µg of PAI-1. Lane N, active PAI-1
control (starting material); lane 1, starting material (pH
5.6) incubated with excess tPA; lanes 2, 4, and
6, PAI-1 incubated at pH 4, 5.6, and 8 alone, respectively;
lanes 3, 5, and 7, PAI-1 incubated
with the peptide at pH 4, 5.6, and 8, respectively. Lane C,
reactive loop cleaved PAI-1 control.
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Circular Dichroism Signal Changes during the Polymerization
of Native and Latent PAI-1--
The effect of pH on the stability of
native and latent PAI-1 was then assessed by monitoring the circular
dichroic signal at 222 nm with increasing temperature. The conformers
were incubated at pH 4-8.9 for 5 min at room temperature before
assessing stability (Fig. 6a).
Reducing the pH resulted in a progressive increase in stability of
native PAI-1 such that the melting point of native PAI-1 was over
90 °C at pH 4.5. The protein showed no marked increase in
ellipticity at temperatures of up to 98 °C at pH 4 (data not shown).
The melting points (Tm) of latent PAI-1 were
less sensitive to the change in pH (Fig. 6a). The CD signal
at 222 nm indicates the
-helical content of a protein; to assess
changes in
-sheet content, the CD signal of native PAI-1 was
monitored at 216 nm. Following incubation at pH 4.0 (but not at pH
8.0), there was a negative increase in ellipticity in keeping with an
increase in
-sheet structure (Fig. 6b). Because the
melting point of native PAI-1 incubated at pH 4 is much higher than
that of latent form of PAI-1 (approx. 72 °C), the stable species
formed at higher temperature cannot be ascribed to a transition to the
latent conformation. Moreover, pH 4 acid-PAGE confirmed that the
protein was in the polymeric conformation, and a direct comparison
between the far UV CD profiles of native and latent PAI-1 showed less
than 1% difference at 216 nm in keeping with the results of others
(52).

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[in this window]
[in a new window]
|
Fig. 6.
Thermal stability and circular dichroic
profile of native and latent PAI-1. a, native and
latent PAI-1 were incubated at different pH values, and their thermal
stability was assessed by increasing temperature at 2 °C/min and
monitoring ellipticity at 222 nm. All the results are the average of
three independent experiments. b, polymerization of native
PAI-1 assessed at 216 nm during incubation at 37 °C and pH 4 in 0.1 M NaOAc pH 4.0 buffer. The postincubation sample was
confirmed to contain predominantly PAI-1 polymers by acid-PAGE (data
not shown). c, CD spectra of native (bold line)
and polymerized (fine line) PAI-1. The spectra were recorded
at 20 °C in 5 mM NaOAc, pH 4, and averaged for 20 runs
to give the profile for the native protein. PAI-1 was then incubated at
37 °C for a further 2 h before recording the spectrum of the
polymerized protein. Each scan was repeated on three occasions, and the
difference between the two spectra is shown as a dashed
line.
|
|
The changes in secondary structure of native PAI-1 during
polymerization were examined in more detail by circular dichroic spectra (Fig. 6c). The spectra were deconvoluted using the
program Selcon3 (53) and a basis set containing 29 proteins with
spectra between 178 and 260 nm. Monomeric PAI-1 contained 35%
-helix, 19%
-strand, 20%
-turn, and 23% random coil. By
contrast the polymeric protein contained 29%
-helix, 19%
-strand, 24%
-turn, and 27% random coil. This indicates that
polymerization at low pH results in a conformational change that
includes an overall loss of
-helical structure but no increase in
-sheet structure. The loss of
-helix is consistent with a partial
unfolding of the protein, an event often observed at extremes of pH.
The lack of change of
-sheet content agrees well with the secondary
structural changes that occur during the polymerization of
1-antitrypsin (39).
Polymerization of Glycosylated PAI-1 and Stable PAI-1
Mutant--
Active and latent glycosylated PAI-1 were assessed on a pH
4 nondenaturing PAGE after incubation at pH 4 at 37 °C for 10 min. Both of them formed high molecular mass polymers similar to those of
the recombinant protein (Fig. 7,
lanes 6 and 7), respectively. PAI-1 can be
rendered stable by four point mutations, N150H, K154T, Q319L, and
M354I, that reduce the rate of intramolecular loop incorporation and
hence transition to a latent conformation (37). This stable variant was
prepared and assessed at low pH. The active conformation also formed
high molecular mass species that were similar to those of native PAI-1
(Fig. 7, lane 1).

View larger version (102K):
[in this window]
[in a new window]
|
Fig. 7.
10% (w/v) pH 4 nondenaturing PAGE of
recombinant and glycosylated PAI-1. Lane 1, stable
PAI-1 mutant (N150H, K154T, Q319L, and M354I); lane 2,
native PAI-1; lane 3, latent PAI-1; lane 4,
reactive loop cleaved PAI-1; lane 5, binary complex PAI-1
(small amount of polymers were from latent PAI-1, which formed during
the preparation of the binary complex); lane 6, glycosylated
native PAI-1; lane 7, glycosylated latent PAI-1. The protein
in lanes 6 and 7 was visualized by silver
staining.
|
|
Mechanism of PAI-1 Polymerization--
Native serpins are in a
metastable conformation, and under mild denaturing conditions they
adopt an intermediate species that may progress to a latent or
polymeric conformation. The pathway may be described by the following
equation (39).
|
(Eq. 1)
|
where step 1 represents the conformational change
of the serpin to a polymerogenic monomeric form (M*), step 2 represents the formation of polymers (P), and step 3 represents a side pathway that leads to the formation of the latent
conformation (L). Although little is known about the exact structure of
M*, it is obvious that the loop should be converted to or stay in
-strand conformation, which is necessary for polymerization.
Generation of the unstable intermediate M* occurs spontaneously in some
serpins (28) in association with clinically relevant point mutations
that facilitate the conformational transition. In serpins such as
PAI-1, the protein scaffold favors conversion of the intermediate
conformation to a latent species (54), whereas in others, such as
1-antitrypsin and C1-inhibitor, the serpin polymerizes
(21, 33). Clearly such transitions are dependent upon environment, and
this is best illustrated by
1-antitrypsin and
antithrombin, which can be induced to adopt a latent configuration by
heating in stabilizing concentrations of sodium citrate (15-17). It is
perhaps not surprising that under specific conditions PAI-1 can also
form the discrete high molecular mass bands on nondenaturing PAGE that
are characteristic of serpin loop-sheet polymers. The feature, however,
that differentiates these polymers from the typical loop-sheet polymers
observed with other serpins is their ready dissociability, a
dissociation that with polymers of native PAI-1 returns them to
their fully active form.
Our results strongly favor two structurally confirmed models with
linkage occurring by edge strand insertion into a
-sheet and hence
with ready dissociability. With native recombinant PAI-1 the defined
structure of s7A linkage (Fig. 1c) is compatible with all
the biochemical findings reported here. The active PAI-1 remains fully
active at pH 4, which is in keeping with the finding of others that
active PAI-1 is more stable at low pH (42, 55), explicable by
protonation of His143 to give closure of
-sheet A (44).
Such stabilization of the closure of the A-sheet would be a
prerequisite for the dissociable insertion of the loop of another
molecule to give the s7A linkage observed in the crystal structure of
intact PAI-1 (34). With latent PAI-1, however, the structural basis for
the formation of reversible polymers is less clear. The likelihood is
that
-sheet linkage occurs to the vacated s1C position in the latent
PAI-1, as has been structurally demonstrated with the dimerization of latent antithrombin (15, 47). The donor strand is likely to come from
the reactive site loop of another molecule, and such polymeric loop-s1C
linkage can be modeled with the reactive loop retained in the fully
inserted latent form. An alternative model of loop-s1C linkage is
suggested by our recent structure of a stable intermediate semi-latent
form of
1-antichymotrypsin with partial loop insertion
(28). That such loop release can occur with latent PAI-1 is supported
by the transverse urea gradient gels shown in Fig. 5, which indicate
that the reactive loop can insert into and be stripped out of the A
-sheet much more readily than in other serpins. Such an intermediate
with a partially opened A-sheet could allow the formation of
loop-A-sheet polymers, but the finding of dissociability of the formed
polymers of latent PAI-1 might favor, in this case, the alternative
loop-C-sheet (s1C) linkage (56). Our data suggest that the increase in
stability at pH 4 for native PAI-1 is due to polymerization rather than transition to the latent conformer. The mechanism of polymerization of
native PAI-1 is similar to that of
1-antitrypsin
(39) in that there is no increase in overall
-sheet structure
despite being characterized by an aberrant
-strand linkage. The
reactive loop can simply anneal as an extra strand of
-sheet A, as
s7A for PAI-1 (Fig. 1c), and as s4A for
1-antitrypsin (Fig. 1a). Similarly there is
no increase in overall
-sheet structure in the s4A or s1C
polymerization models, in which s1C release is necessary (Fig.
1b).
In conclusion, this study shows that both native and latent PAI-1 can
reversibly polymerize by mechanisms that are compatible with previously
structurally-demonstrated s7A and s1C
-linkages. Physiologically it
may be of significance that this polymerization occurs at low pH such
as that found in platelets or at foci of inflammation.
 |
ACKNOWLEDGEMENTS |
We are grateful to Prof. Randy
Read (Cambridge Institute for Medical Research, Cambridge, UK) for
helpful discussions. We thank Dr. Peter Andreasen (Department of
Molecular and Structural Biology, Aarhus University, Denmark) for
providing active glycosylated PAI-1 and Dr. Hui Hong (Department of
Chemistry, University of Cambridge, UK) for helping with mass
spectrometry analysis.
 |
FOOTNOTES |
*
This work was supported by Xenova Limited, the Wellcome
Trust, and the Medical Research Council (United Kingdom).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Cambridge Inst. for
Medical Research, Wellcome Trust/MRC Bldg., Hills Rd., Cambridge, CB2
2XY, UK. Tel.: 1223-336825; Fax: 1223-336827; E-mail:
awz20@ cus.cam.ac.uk.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M010631200
 |
ABBREVIATIONS |
The abbreviations used are:
PAI-1, plasminogen activator inhibitor type 1;
tPA, tissue-type plasminogen
activator;
PAGE, polyacrylamide gel electrophoresis;
TEMED, N,N,N',N'-tetramethylethyl-enediamine.
 |
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