Báo cáo khoa học: Probing plasma clearance of the thrombin–antithrombin complex with a monoclonal antibody against the putative serpin–enzyme complex receptor-binding site docx

Eur. J. Biochem. 270, 4059–4069 (2003) Ó FEBS 2003

doi:10.1046/j.1432-1033.2003.03793.x

Probing plasma clearance of the thrombin–antithrombin complex
with a monoclonal antibody against the putative serpin–enzyme
complex receptor-binding site
George L. Long1,*, Margareta Kjellberg2, Bruno O. Villoutreix3 and Johan Stenflo2
1

Department of Biochemistry, University of Vermont, Burlington, VT, USA; 2Department of Clinical Chemistry, Lund University,
University Hospital Malmoă, Malmoă, Sweden; 3INSERM U428, University of Paris V, France

A high-anity monoclonal antibody (M27), raised against
the human thrombin–antithrombin complex, has been
identified and characterized. The epitope recognized by M27
was located to the linear sequence FIREVP (residues 411–
416), located in the C-terminal cleavage peptide of antithrombin. This region overlaps, by two residues, the putative
binding site of antithrombin for the serpin–enzyme complex
receptor. Studies in rats and with HepG2 cells in culture
indicated that the Fab fragment of M27 does not block
binding and uptake of the thrombin–antithrombin complex,

suggesting that this region does not play a major role in the
recognition and clearance of the thrombin–antithrombin
complex. M27 blocked the ability of antithrombin to inhibit
thrombin as well as antithrombin cleavage, both in the
presence and absence of heparin.

Antithrombin (AT), a member of the serine protease
inhibitor family (serpin), is a 58-kDa molecular mass

glycoprotein that circulates in human plasma at a concentration of  5 lM [1–5]. AT modulates blood coagulation
by inhibiting thrombin, active factor X (factor Xa) and
active factor IX (factor IXa), and thereby prevents
inappropriate clot formation and thrombosis. The rate of
AT-mediated inhibition of thrombin and factor Xa is
increased several thousand-fold by binding of the sulfated
polysaccharide heparin or heparin-like molecules. Individuals with AT deficiency are at a significantly increased risk
of venous thrombosis [6,7].
AT and other serpins inhibit their serine protease
cognates by the formation of a long-lived, covalent acyl
intermediate upon specific protease cleavage [1–5]. In AT,
cleavage is at Arg393 in a so-called reactive center loop
(RCL) with the formation of a C-terminal, 39 amino-acid
residues long, disulfide-bonded (Cys247 to Cys430) peptide
[8]. Prompt insertion of residues P1–P17 of the RCL, with

attached protease, as an additional strand into b-sheet A of
the inhibitor, causes a dramatic conformational change in
the serpin [9]. Recent X-ray crystallographic diffraction
analysis of the trypsin–antitrypsin covalent complex has
shown that insertion of the RCL also leads to a critical
distortion of the structure of the protease [10]. As a result,
the canonical active site Ser in position 195 is reoriented at a
˚
distance of more than 6 A from His57, which is too far to
form the critical hydrogen bond of the catalytic triad – a
bond that is a prerequisite for cleavage of the acyl
intermediate that links the protease to the serpin. Moreover,
the distortion of the complexed thrombin molecule renders
it susceptible to proteolytic degradation. A further consequence of the complexation-induced conformational change

in the serpin is exposure of structure(s) that are recognized
by serpin–enzyme complex (SEC) receptors on the surface
of hepatocytes. Receptor binding followed by endocytosis
results in rapid clearance of the protease–serpin complexes
from the circulatory system [11].
In addition to native and complexed AT, two forms of
AT have been characterized: a cleaved uncomplexed form
with the RCL inserted into b-sheet A, and a so-called
latent uncleaved, loop-inserted form. The cleaved, loopinserted inhibitor is formed if loop insertion, with the
acyl-linked protease, is not sufficiently rapid to compete
with deacylation, which leads to release of the active
protease. Human elastase cleaves AT at Ile390 without
complex formation [12]. The elastase-cleaved form has
properties that are indistinguishable from those of the
thrombin-cleaved inhibitor. The latent form is a conformational isomer of the native form in which the RCL has
been inserted into b-sheet A without prior complex
formation/cleavage. Neither the cleaved uncomplexed
nor the uncleaved latent form exhibit inhibitory activity

Correspondence to J. Stenflo, Department of Clinical Chemistry,
Lund University, University Hospital, Malmo, S-205 02 Malmo,
ă
ă
Sweden. Fax: + 46 40 929023, Tel.: + 46 40 331421,
E-mail: johan.stenfl
Abbreviations: AT, antithrombin; LRP, low-density lipoprotein
receptor-related protein; PVDF, poly(vinylidene difluoride); RCL,
reactive center loop; SEC, serpin–enzyme complex; SECR, serpin–
enzyme complex receptor; T–AT, covalent thrombin-antithrombin
complex.

*This work was carried out during the sabbatical of G. L. Long
to the Department of Clinical Chemistry, Lund University,
University Hospital Malmo, S-20502 Malmo, Sweden.
ă
ă
(Received 3 July 2003, revised 30 July 2003, accepted 15 August 2003)

Keywords: antithrombin; thrombin; thrombin–antithrombin complex; monoclonal antibody; serpin–enzyme complex
receptor.


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4060 G. L. Long et al. (Eur. J. Biochem. 270)

[9,13,14]. Latent AT forms spontaneously under mild
conditions [13,14], including storage of plasma at 37 °C
[15]. A naturally occurring genetic variant, AT Rouen-VI
(Asn187 fi Asp), readily forms the latent form and is
associated with fever-induced thromboembolic disease
[16]. In commercial concentrates of AT, up to 40% exists
as the latent form [17]. Recently, it was reported that AT
has potent antiangiogenic activity and inhibits tumor
growth [18,19]. Native AT has little effect, cleaved AT has
an intermediate effect, and latent AT is the most potent
antiangiogenic agent reported to date [19].
In this communication we report the characterization of
a murine mAb, M27, against human AT that binds to the
native, complexed, cleaved, and latent forms of AT. M27
blocks the thrombin-inhibiting capacity of AT and

protects it from cleavage by thrombin. M27 binds to a
linear epitope (residues 411–416; FIREVP) that partly
overlaps a region (residues 408–412; FLVFI) implicated in
the recognition of certain serpin–protease complexes by
the SEC receptor (SECR), first identified on the surface of
human hepatoma HepG2 cells [20]. M27 binds the
thrombin–AT (T–AT) complex with a picomolar dissociation constant. The rate of clearance of the ternary
T–AT–Fab complex in rats was only slightly slower than
the rate of clearance of T–AT. Studies with cultured
HepG2 cells indicated that M27 does not block the
binding of T–AT in an epitope-dependent manner. These
findings are consistent with recent results that cast doubt
on the notion that the sequence FLVFI in AT, and
homologous regions in certain other serpins, is crucial for
receptor-mediated elimination of protease–serpin complexes from blood plasma [21,22].

Materials and methods
Preparation of proteins
Native AT was purified from human, bovine, rabbit, and
mouse plasma, as previously described [23], and stored at
)20 °C. Cleaved human AT was produced essentially as
described previously [24]. Native AT was incubated with
porcine elastase (Sigma-Aldrich, Stockholm, Sweden), at a
100 : 1 molar ratio, in 50 mM Tris/HCl, 0.15 M NaCl and
0.1% (w/v) PEG 8000 for 4 h at 37 °C. After addition of
phenylmethanesulfonyl fluoride to a final concentration of
1 mM, the solution was dialyzed against 50 mM Tris/HCl,
0.1 M NaCl (pH 7.5) and purified by heparin–Sepharose
chromatography. Elution with a NaCl gradient (0.1–1.0 M)
gave one peak at 0.3 M NaCl. Sequence analyses revealed

cleavage at positions 389, 390 and 393. The material was
aliquoted and frozen at )70 °C.
Latent human AT was prepared as described previously [13]. Native AT was incubated in 10 mM Tris/HCl,
0.25 M sodium citrate, pH 7.4, for 70 h at 60 °C and
purified on a heparin–Sepharose column, as described
above. A portion of the AT did not bind to the heparin–
Sepharose, and a second major peak, which eluted at
0.3 M NaCl, showed a sequence corresponding to native
AT, but migration by SDS/PAGE corresponding to that
of latent AT. The material eluting at 0.3 M NaCl was
concentrated, stored at )70 °C, and used in further
experiments.

Human prothrombin was purified by slight modification
of a standard procedure, including precipitation with
barium chloride and ammonium sulfate, followed by
DEAE Sephacel chromatography [25]. Prothrombin was
activated with venom from Oxyuranus scutellatus (ICN
Biomedicals Inc., Irvine, CA) and purified employing
Q-Sepharose followed by SP–Sepharose column chromatography [26].
Production of mAb M27
Murine mAbs were produced as described previously [27].
The mice were immunized with human T–AT complexes.
These complexes were also used to test the clones in an
ELISA. Conditioned media were collected from cloned
mouse myeloma cells cultured in a TECNOMOUSE
(Integra Biosciences, Wallisellen, Switzerland) hollow fiber
chamber, and stored at )20 °C until used. Thawed media
were centrifuged at low speed to remove cellular debris and
the supernatant was diluted with an equal volume of

column equilibration buffer (1 M glycine, 150 mM NaCl,
pH 8.0) and purified by Protein A–Sepharose chromatography [28]. mAb M27 was stored in aliquots at )20 °C.
Its concentration was estimated from the absorbance at
280 nm, assuming an absorbance of 1.34 for a 1 mgỈmL)1
solution [28]. M27 was determined to be of the IgG2b
isotype by a standard procedure using a commercial kit
(Miles Laboratories, Elkhart, IN).
Production of the M27 Fab fragment
The general procedure for generating Fab was that
described by Parham [29]. Purified M27 was dialyzed
against 100 mM sodium acetate, pH 5.5, and D,L-cysteine
and EDTA were added to final concentrations of 45 and
0.9 mM, respectively. After 5 min of preincubation at 37 °C,
digestion was performed for 30 min with 0.5% (w/w) fresh
papain (Sigma). After incubation with iodoacetamide (final
concentration 70 mM) for 30 min at room temperature, the
digest was dialyzed at 4 °C against 1 M glycine, 150 mM
NaCl, pH 8.0. The digest was then purified by protein A–
Sepharose chromatography to remove traces of undigested
IgG and Fc fragment. The material in the flow-through
peak (unbound Fab fragment) was dialyzed against 20 mM
Tris/HCl, pH 8.5, and subjected to chromatography on a
Q-Sepharose Fast Flow column that was eluted with a
linear NaCl gradient (0–250 mM). The Fab fragment eluted
at  70 mM NaCl. It was homogenous, as judged by SDS/
PAGE, and was able to bind Eu3+ chelate-labeled native
antithrombin in a DELPHIA assay [30]. The Fab fragment
was dialyzed against NaCl/Tris buffer, pH 7.5, aliquoted
and stored at )20 °C. About 20% of the material eluted at
 160 mM NaCl; it possessed no ability to bind AT and

presumAbly consisted of Fab fragments with an aberrant j
chain [31].
SDS/PAGE and Western blotting
SDS/PAGE and Western blotting were performed using
standard methods. For Western blotting, poly(vinylidene
difluoride) (PVDF; Immobilon P, 0.45-lm pore size)
membranes (Millipore, Bedford, MA) were used. Proteins


Ó FEBS 2003

T–AT complexes and the SEC receptor (Eur. J. Biochem. 270) 4061

were stained with GelCode Blue Stain Reagent (Pierce,
Rockford, IL) according to the supplier’s instructions.
Peptide synthesis and epitope mapping
Peptides were synthesized on a Milligen 9050 Plus instrument (Perkin-Elmer, Stockholm, Sweden), using Fmoc
chemistry, and purified by reverse-phase HPLC followed by
lyophilization and storage at )20 °C until required for use.
All peptides were readily dissolved in deionized water, to a
nominal concentration of 0.5 mM, and stored frozen. The
exact concentrations were determined by amino acid
analysis after acid hydrolysis.
Binding of the synthetic peptides to M27 was studied
by an ELISA-based method. The peptides and native AT,
1.5 nmol and 15 pmol, respectively, in 100 lL of coating
buffer (100 mM sodium bicarbonate, pH 9.6), were delivered to wells of high-binding, polystyrene microtitre plates
(Costar, Corning, NY). After incubation for 15 h at 4 °C,
the wells were rinsed several times with 10 mM sodium
phosphate buffer containing 0.5 M NaCl and 0.1% Tween

20, pH 8.0 (NaCl/Pi/Tween), followed by blocking for
15 min with 1% (w/v) BSA (Sigma Fraction V) in NaCl/
Pi/Tween. Wells were rinsed again, and different amounts
of M27, diluted in NaCl/Pi/Tween containing 0.1% BSA,
were added to the wells followed by incubation on a
platform shaker for 1 h at room temperature. Wells were
rinsed again and then incubated, as described above, with
a horseradish peroxidase-conjugated rabbit anti-mouse
IgG (DAKOPATTS AB, Alvsjo, Sweden) diluted 1 : 1000
in NaCl/Pi. After washing, the chromogenic enzyme
substrate
2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic
acid) (ABTS) was added and the absorbance at 405 nm
was recorded as a function of time. Binding of M27 to the
peptides (all containing a single cysteine residue, see
Fig. 3A) was also determined after coating the peptides,
dissolved in NaCl/Pi, pH 7.4, to a maleimide-activated
microtitre plate (Pierce) for 15 h at room temperature.
Following coating and subsequent rinsing with NaCl/Pi,
pH 7.4, wells were blocked by incubation for 90 min with
150 lL of D,L-cysteine in 10 lgỈmL)1 NaCl/Pi, pH 7.4.
Wells were then rinsed with NaCl/Pi/Tween, followed by
antibody binding and enzymatic color development, as
described above.
Effect of AT and M27 on thrombin activity
A two-stage assay was used to determine the effect of M27
on the inhibition of thrombin by AT. The first stage consists
of a-thrombin incubation with native AT at 37 °C for
different lengths of time in the presence or absence of M27.
Freshly diluted thrombin (0.5 lg in buffer comprising

50 lL of 50 mM Tris/HCl, 150 mM NaCl, 0.1% BSA,
pH 7.5; Tris/HCl/NaCl/BSA) was delivered into wells of a
low-affinity microtitre plate (Bibby Sterilin, Ltd, Staffs.,
UK) and allowed to equilibrate for 5 min at 37 °C. Native
AT (0.8 lg in 10 lL of Tris/HCl/NaCl/BSA), M27 (4 lg in
10 lL of Tris/HCl/NaCl/BSA) or AT preincubated with
M27 (same concentrations and volume) were added to the
thrombin solution, mixed and incubated at 37 °C. At
different time-points, 10-lL aliquots were removed for
SDS/PAGE or mixed into 90 lL of Tris/HCl/NaCl/BSA

(at room temperature). Duplicate aliquots (10 lL) of the
latter were immediately transferred into clean wells containing 190 lL of freshly prepared thrombin substrate
(400 lM S-2238; Chromogenix, Gothenburg, Sweden) in
50 mM Tris/HCl, 0.1% BSA, pH 8.4. The samples were
briefly mixed, and the increase in absorbance at 405 nm was
recorded as a function of time.
A similar procedure was used to measure the effect of
heparin on the above system. Native AT (90 lgỈmL)1),
M27 (435 lgỈmL)1), or AT plus M27 (same concentrations) were preincubated (for 1 h at 37 °C) in Tris/HCl/
NaCl/BSA containing 100 mL)1 heparin (average MW,
15 kDa; Lovens Kemiske Fabrik, Ballerup, Denmark).
ă
Aliquots (10 lL) were then added to an equal volume
of Tris/HCl/NaCl/BSA containing 20 lgỈmL)1 thrombin,
followed by brief mixing and incubation at 25 °C for
5 min. The interaction with thrombin was stopped by the
addition of 180 lL of ÔquenchÕ solution: 110 lg of
protamine sulfate (Lovens Kemiske Fabrik) per ml of
ă

Tris/HCl/NaCl/BSA. Duplicate 10-lL quenched aliquots
were then used in the measurement of thrombin amidolytic activity, as described above. The experiments, with
and without heparin, were performed on three separate
occasions, with essentially identical results. They were
also performed once with a molar equivalent (antigen-binding sites) of purified M27 Fab fragment, and
gave results identical to those obtained with the intact
mAb.
Surface plasmon resonance spectroscopy
Binding of M27 IgG and the Fab fragment to different
forms of AT was studied using a BIACORE 2000
biosensor (Biacore AB, Uppsala, Sweden). Purified M27
or Fab fragments were diluted into 10 mM Hepes,
150 mM NaCl, 3 mM EDTA, 0.005% Polysorbate 20,
pH 7.4 (Hepes/NaCl/EDTA/Polysorbate 20). They were
immobilized with NH2-coupling to a CM5 sensor chip
(Biacore) to levels of 1700 and 740 response units (RU)
for the intact mAb and the corresponding Fab fragment,
respectively. Analytes were diluted into Hepes/NaCl/
EDTA/Polysorbate 20 and used to measure binding to
the immobilized IgG and Fab using a programmed
protocol with 20 s preinjection delay, 180 s association
time and 600 s dissociation time The experiments were
performed at room temperature and the proteins were
pumped at 30 lLỈmin)1. The chip was regenerated with
two pulses of 5 lL 0.1 M glycine/HCl, 0.5 M NaCl,
pH 2.75, at a flow rate of 5 lLỈmin)1. The analyte
concentrations ranged from 0.1 to 100 nM (based on
amino acid analysis). Runs were performed three times
and with five different concentrations for each analyte.
Data were analyzed using the BIAEVALUATION 3.0 software package (Biacore), assuming noninteracting antibody-binding sites and a 1 : 1 stoichiometry of binding.

In vivo clearance of the T–AT complex in rats
Thrombin, native AT, and elastase-cleaved AT were labeled
with 125I (Amersham Pharmacia AB, Uppsala, Sweden)
using the chloramine T method, according to the supplier’s
instructions. 125I-labeled thrombin was covalently


Ó FEBS 2003

4062 G. L. Long et al. (Eur. J. Biochem. 270)

complexed with unlabeled AT at a 1 : 2 molar ratio for 1 h
at room temperature, then applied to a heparin–Sepharose
column and eluted. The concentrations of two fractions
containing the T–AT complex were estimated by comparing
the absorbance at 280 nm with a nonradioactive T–AT
complex, for which the concentration had been determined
by amino acid analysis. The proteins were stored in aliquots
at )70 °C.
Sprague-Dawley rats (350 g) were anesthesized
(2 mLỈkg)1) with a 1 : 1 : 2 (v/v/v) mixture of Hypnorm
(JANSSEN-CILAG Ltd, High Wycombe, UK), Dormicum (Pharma hameln GmbH, Hamelm, Germany) and
deionized water. 125I-labeled T–AT (73 pmol in 500 lL),
with and without 2.9 nmol M27 Fab, were injected in a tail
vein, and blood samples (200 lL) were drawn from a
jugular vein into tubes containing 10 lL of 0.5 M EDTA
after 1, 3, 5, 10, 15 and 20 min. Radioactivity was measured
in 50 lL aliquots of the plasma. Blood samples drawn after
1 min were considered to represent the amount of injected

radioactivity after equilibration in the circulation. 125Ilabeled thrombin, native AT, and cleaved AT alone were
also injected into control animals.
Binding of

125

I-labeled T–AT complex to HepG2 cells

The binding studies were performed, as previously
described, with minor modifications [21]. HepG2 cells
were cultured at 37 °C, 5% CO2 in 75-cm2 flasks (Nunc,
Roskilde, Denmark) containing Dulbecco’s modified
Eagle’s medium (DMEM) (Invitrogen Corp.) supplemented with 10% (v/v) fetal bovine serum (HyCloneÒ),
2 mM L-glutamine (Invitrogen Corp.), and penicillin/
streptomycin (100 unitsỈmL)1/100 lgỈmL)1) (Invitrogen
Corp.). Cells were transferred to 24-well plates, at a
concentration of 2–3 · 105 cells per well, and grown for
2 days before the experiments. The cells were washed
twice with DMEM containing 0.2% BSA, 0.5 lM
PPACK and 10 mM Hepes, pH 7.4 (binding buffer).
Radiolabeled T–AT complex (20 nM) in binding buffer,
with or without unlabelled T–AT (2 lM) or M27
(0.2 lM) or M38 (0.2 lM), or cleaved AT (1 lM), were
each added to four wells. After incubation at 4 °C for
2 h, the wells were washed three times with 10 mM
Hepes, pH 7.4, containing 0.15 M NaCl, 1 mM CaCl2,
2 mM MgCl2 and 0.2% BSA. Cells were lysed in 0.4 mL
of 2 M NaOH overnight at room temperature and the
radioactivity connected to the cells was measured. The
results are expressed as the mean of triplicate samples.

Results
Identification of the M27 epitope
Western blotting was used in the initial characterization of
the epitope of mAb M27 (Fig. 1). Bands corresponding to
all forms of nonreduced AT were rapidly visible. However,
when the inhibitors were analyzed after reduction of the
disulfide bonds, only the native and latent forms of AT (i.e.
not cleaved at Arg393) were observed. The 39-residue
C-terminal peptide (residues 394–432), formed upon complex formation with thrombin, is linked by means of Cys430
to Cys247 in the main body of the inhibitor. The results
suggest that this peptide, which is not visible owing to its
small size, harbors the eptiope that is recognized by M27.
Binding of the mAb to reduced and nonreduced native and
latent AT indicates that the mAb recognizes a linear
sequence.
These results warranted a test of the reactivity of mAb
M27 in Western blotting with AT from mouse, rabbit and
bovine blood plasma. ATs from these species all differ from
their human counterpart at three positions in the C-terminal
peptide: residues 411, 416 and 432 (Fig. 2A) [37]. M27 did
not react with AT from any of the three species (Fig. 2B),
whereas a control rabbit polyclonal antiserum against
human AT gave positive results. As mAb M27 is not
sensitive to reduction of the Cys residue at position 430 of
AT, it is unlikely that the epitope is at the very C-terminus of
the peptide. The results therefore suggested that residues 411
and 416 are part of the epitope of M27.
Synthetic peptides were used to localize the epitope of
M27 more precisely (Fig. 3A). Wells of microtitre plates

were coated with the peptides for ELISA-binding studies. A
peptide including residues 404–420 (P-74) was found to bind
M27 (Fig. 3B). In contrast, a corresponding peptide, with
substitutions at positions 411 (P-80), 416 (P-81), or both
(P-79), showed a very weak reaction with M27. Native AT
competed less well with the synthetic peptides than with
native immobilized AT for binding to M27. A control
peptide with the same composition as the 404–420 peptide,
but with the sequence scrambled, did not react with M27.
Identical results were obtained when the peptides were
covalently linked to the wells of microtitre plates by a
maleimide reaction with C-terminal Cys residues (data not

Molecular modeling
X-ray structures of human native AT [32], latent AT [9],
heparin–AT complex [33], cleaved bovine AT [34], and
trypsin–antichymotrypsin covalent complex [10], were
analyzed using the ACCELRYS molecular modeling package
(San Diego, CA, USA), running on a Silicon Graphics
workstation 02 or Fuel. Solvent accessibility was computed
using the method of Lee & Richards [35]. The packing
density was calculated with the method of Kurochking &
Privalov [36], with the MOLE molecular graphics software
package kindly provided by R. Tarr (Applied Thermodynamics, Inc., Hunt Valley, MD, USA).

Fig. 1. Western blotting of AT with mAb M27. Different forms of AT
were electrophoresed by SDS/PAGE (12% gels), transferred to
poly(vinylidene difluoride) (PVDF) membrane, and detected with
M27. Nonreduced native, latent, elastase-cleaved, or thrombin–AT
complex (1.7 pmol each) were electrophoresed in lanes 2–5, respectively. The same amounts of dithiothreitol-reduced proteins were electrophoresed in lanes 6–9. The arrow points to the position of the

62 kDa molecular mass protein marker in lane 1.


Ó FEBS 2003

T–AT complexes and the SEC receptor (Eur. J. Biochem. 270) 4063

Fig. 2. Western blotting of AT from different species with M27. (A)
C-terminal sequences of AT from different species are compared
(human AT numbering). Amino acids identical to that of the human
are shown with dashes (–). The arrow indicates the thrombin cleavage
site in AT that forms the C-terminal 39 residue peptide. Cys430, which
is disulfide bonded to Cys247, is underlined. (B) Nonreduced native
human AT (0.34 pmol) was included in lanes 2 and 6. Equal amounts
of purified, nonreduced native mouse, rabbit or bovine AT were
included in lanes 3–5 and 7–9, respectively. Protein markers were run
in lane 1. Membrane-bound proteins from lanes 1–5 were incubated
with M27, followed by incubation with rabbit anti-mouse IgG alkaline
phosphatase conjugate and enzyme color development. Membranebound proteins from lanes 6–9 were incubated in the same manner,
except with a primary polyclonal rabbit anti-(human AT) Ig followed
by secondary goat anti-rabbit IgG alkaline phosphatase conjugate
(DAKOPATTS).

shown). The results of the ELISA-binding studies are
consistent with the results of the Western blotting experiments and establish that the sequence including FIREVP
(residues 411–416) constitutes a critical part of the linear
epitope of mAb M27.

Fig. 3. Binding of M27 to immobilized synthetic peptides. (A) Synthetic
peptide sequences. The vertical arrow designates the thrombin-cleavage site in antithrombin (AT). Peptide P-78 corresponds to the 42 Cterminal residues of human AT. Bold, underlined residues are changes

from the wild-type sequence. Peptide P-82 is a ÔscrambledÕ sequence,
whose composition is the same as that of the wild-type plus a Cterminal cysteine residue. (B) Peptides were immobilized on the surface
of polystyrene microtitre plate wells. Binding of M27 was determined
using an ELISA, with a final development of 405 nm absorption vs.
time. Bars represent average values of duplicates at the 10-min timepoint. Error bars indicate the range. Color development in the assay is
still linear, with respect to time, at the 10-min time-point. Key: black
bars, 200 ng of M27 added; white bars, 1000 ng of M27 added; stippled bars, 200 ng of M27 + 2 lg of native AT added.

Effect of M27 on inhibition of thrombin by AT
The effect of mAb M27 on the formation of covalent
complexes between thrombin and AT was studied in a twostage assay. First, thrombin and AT were incubated in
microtitre plate wells, with and without M27. Aliquots were
then removed at different time-points for SDS/PAGE and
for measurements of the amidolytic activity. M27 inhibited
AT both in the presence and absence of heparin (Table 1).
In the absence of heparin the M27-mediated inhibition was
complete, whereas in the presence of heparin it was partial.
The inhibition in the presence of heparin was not influenced
by increasing the mAb concentration from a four- to a
40-fold molar excess over the AT concentration, suggesting
that the heparin and mAb binding sites on AT are
independent of one another (data not presented). Solution
binding studies with Eu3+ chelate-labeled native AT have
also indicated that there is no competition between heparin
and M27 for binding to AT (data not presented). As shown

in Fig. 4, Western blotting of aliquots removed after the first
stage of the assay also revealed that the presence of M27
blocked the T–AT formation. Moreover, at most, a minute
amount of AT could have been cleaved by thrombin when

M27 was bound to AT. A very weak band above the bands
of native AT in lanes 2 and 4 is probably a small amount of
cleaved AT, which is an impurity of our native AT. The
bands correspond to the mobility of the cleaved inhibitor
during SDS/PAGE.
Measurement of AT binding to M27 by surface
plasmon resonance
The binding of different forms of AT to immobilized intact
IgG and the Fab fragment of mAb M27 was studied in real
time by surface plasmon resonance using a BIACORE
biosensor. Representative binding curves and binding


Ó FEBS 2003

4064 G. L. Long et al. (Eur. J. Biochem. 270)

Table 1. Effect of M27 on the inhibition of thrombin by AT. Concentration and molar ratios of proteins in the second stage amidolytic assay are
shown in parentheses. Vmax is the rate of substrate hydrolysis, as measured by the change in absorbance at 405 nm over a period of 20 min. In all
cases the change was linear during this time-period. Values represent the average and range of duplicate measurements. In part A, first-stage
components were combined and incubated for 60 min at 37 °C prior to addition to the second stage. In part B, first-stage components were
combined and incubated for 5 min at 25 °C prior to addition to the second stage. Other details of the assay are described in the Materials and
methods.
Additions

Vmax (mODỈmin)1)

Part A
None (TBSB buffer alone)
Thrombin (50 ngỈmL)1)

Thrombin (50 ngỈmL)1) +
Thrombin (50 ngỈmL)1) +
Thrombin (50 ngỈmL)1) +
Thrombin (50 ngỈmL)1) +
Thrombin (50 ngỈmL)1) +

AT (1 : 1.04)
AT (1 : 1.04) + M27 (1 : 1.04 : 3.7)
M27 (1 : 3.7)
AT (1 : 1.04) + Fab (1 : 1.04 : 3.7)
Fab (1 : 3.7)

0.02
36.42
3.52
35.88
35.19
33.30
35.22

±
±
±
±
±
±
±

0.01
1.50

0.12
1.30
1.53
1.47
1.36

Part B
Thrombin
Thrombin
Thrombin
Thrombin
Thrombin
Thrombin
Thrombin

Heparin
Heparin
Heparin
Heparin
Heparin
Heparin

33.60
31.28
0.13
19.90
32.04
19.44
32.19

±
±
±
±
±
±
±

0.90
1.20
0.00
0.77
1.28
0.83
0.86

(50
(50
(50
(50
(50
(50
(50

ngỈmL)1)
ngỈmL)1)
ngỈmL)1)
ngỈmL)1)
ngỈmL)1)
ngỈmL)1)

ngỈmL)1)

+
+
+
+
+
+

(0.25
(0.25
(0.25
(0.25
(0.25
(0.25

unitsỈmL)1)
unitsỈmL)1)
unitsỈmL)1)
unitsỈmL)1)
unitsỈmL)1)
unitsỈmL)1)

+
+
+
+
+

AT (1 : 2.6)

AT (1 : 2.6) + M27 (1 : 2.6 : 9.8)
M27 (1 : 9.8)
AT (1 : 2.6) + Fab (1 : 2.6 : 9.8)
Fab (1 : 9.8)

Clearance of the T–AT complex in rats

Fig. 4. M27 protection of antithrombin (AT) from thrombin cleavage.
Samples of native AT incubated for 40 min in the presence of
thrombin, with or without M27, were submitted to SDS/PAGE and
Western blotting with M27, as described in the legend to Fig. 1. Lanes
1–5 contain nonreduced samples, and lanes 6–9 contain reduced
samples. Lane 1, size markers; lane 2, 80 ng of AT before incubation;
lane 3, 60 ng of AT + thrombin; lane 4, 60 ng of AT + thrombin +
M27; lane 5, thrombin + M27; lane 6, 64 ng of AT before incubation;
lane 7, 48 ng of AT + thrombin; lane 8, 48 ng of AT + thrombin +
M27; lane 9, thrombin + M27. The intense bands at the top of lanes 4
and 5 are caused by reaction of M27 in the loaded samples with the
secondary antibody–enzyme conjugate. The arrow denotes the position of the 62 kDa molecular mass marker protein.

constants are presented in Fig. 5 and Table 2. Results
obtained with immobilized intact mAb and the corresponding Fab fragment were identical within experimental error.
The derived dissociation constants ranged from 20 pM to
8 nM, i.e. indicating that M27 has a high affinity for all
forms of AT. The Kd for native and elastase-cleaved AT
were almost identical. The highest Kd, for latent AT, was the
result of both a decrease in the association rate constant (ka)
and an increase in the dissociation rate constant (kd) relative
to native AT, each about one order of magnitude. M27 had
the highest affinity for the T–AT complex (KD

 2 · 10)11 M). This can be attributed almost totally to a
very slow dissociation rate (Fig. 5C).

SECs are removed from the circulation by cellular receptors
that recognize receptor-binding site(s) on the complex [11].
A pentapeptide (residues 408–412; FLVFI in AT) in the
C-terminal fragment of the cleaved inhibitor has been
implicated in receptor binding and internalization [38]. As
the M27-binding site on complexed AT (residues 411–416;
FIREVP) partially overlaps the proposed SECR-binding
site, a clearance study in rats was performed to determine
whether the antibody would inhibit removal of the complex
from the circulation. The results obtained for the complex,
in the absence of the Fab fragment, and for the native and
cleaved AT, were similar to that reported previously [39].
Although the Fab fragment of M27 caused a small, but
significant (P < 0.0001, two-factorial ANOVA), reduction in
the rate of clearance of the complex, from a half-life (t½) of
 7 min in the absence of the Fab fragment to a t½ of
 9 min in its presence, it did not block the uptake to the
liver (Fig. 6). Considering the very high affinity of the M27
Fab fragment for the T–AT complex, this result is not
consistent with a model where the putative SECR-binding
site on the T–AT complexed AT is important for complex
clearance [11].
To determine whether the M27 could inhibit the binding
of T–AT to the SEC receptor in a less complex system,
HepG2 cells were incubated with radiolabeled T–AT in the
presence and absence of the antibody. The binding of
radiolabeled T–AT was reduced to 33 and 16% when 50fold (not shown) and 100-fold molar excess of unlabeled T–

AT complex was added, respectively, which is in accordance
with a previous study [38]. The binding was 69 and 79%
in the presence of a 10-fold molar excess of M27 and Fab
M27, respectively. The high affinity of M27 to T–AT


Ó FEBS 2003

T–AT complexes and the SEC receptor (Eur. J. Biochem. 270) 4065

Discussion

Fig. 5. BIACORE binding curves for M27 Fab interaction with AT.
Data obtained by surface plasmon resonance experiments were analyzed using the BIAEVALUATION 3.0 software package. Symbols represent actual data points and solid lines are simulation curves, assuming
univalent, noninteracting binding sites. (A) Concentrations of latent
AT are 24.1, 40.2, 56.2, 64.3, and 80.3 nM. (B) Concentrations of
elastase-cleaved AT are 0.9, 2.3, 4.6, 9.2 and 18.4 nM. (C) Concentrations of the T–AT complex are 0.9, 2.3, 4.3, 8.7 and 17.4 nM.
(D) Concentrations of native AT are 0.8, 2.0, 4.0, 8.0 and 16.0 nM.

(KD 3.7 · 10)11 M), ensured that > 99.9% of the T–AT was
complexed with M27 in these experiments. Similar results
were obtained with a 50-fold molar excess of the antibodies
(not shown). As a control, HepG2 cells were incubated with
labeled T–AT in the presence of M38, which is an anti-AT
mAb that does not compete with M27 for binding to AT.
The binding of labeled T–AT was decreased to 54%. A
50-fold molar excess of cleaved AT decreased the binding to
79%. These results also argue against a major role for the
FIREVP sequence in receptor-mediated complex binding.

We have generated and characterized a murine mAb, M27,
possessing high affinity for human AT. This antibody reacts
with all naturally occurring forms of AT, with KD values
ranging from 8 to 0.02 nM. The epitope for M27 was
identified by comparison of Western blotting results for
reduced with nonreduced cleaved and noncleaved forms of
AT. The results indicate that the epitope resides in the
C-terminal 39-amino acid residue peptide generated by
T–AT complex formation and cleavage. Synthetic peptides
helped define the epitope to the linear sequence, FIREVP
(residues 411–416), although native AT could not compete
with the synthetic immobilized peptides for the binding to
M27, as well as with the immobilized AT. One explanation
could be that the antibody only binds with of one of its
binding sites to AT and therefore could bind to a small
immobilized peptide with the other. It is clear that the
binding of M27 to immobilized peptides is not saturated
[100-fold more peptides (1.5 nmol) than AT (15 pmol) were
added to the wells]. Yet, the same amount of antibody gives
only  50% of the signal in peptide-coated wells compared
with AT-coated wells. We consider that only a small
fraction of the peptides immobilized on the plastic surface
(without spacer arm) bind the mAb. Another possibility is
that our antibody is, to some extent, conformation
dependent (Fig. 1), but with its crucial binding pointing at
the hexapeptide FIREVP.
Several mAbs against human AT have been reported that
map to the C-terminal region of the molecule. Asakura
et al. described a murine mAb (JITAT-16) raised to human
AT that recognizes the T–AT complex as well as cleaved

AT, but not native AT [40]. The epitope of JITAT-16
(AAAST; residues 382–386) is just upstream of the Arg393
cleavage site [41]. JITAT-16 destroyed the ability of AT to
inhibit thrombin, as does M27. However, the mechanism of
inhibition is different for the two antibodies. JITAT-16 acts
by enhancing the hydrolysis of the T–AT acyl intermediate
to free, cleaved AT and active thrombin (normally a slow
process) relative to the formation of a stable covalent
complex. Presumably, this results from delayed insertion of
the RCL into b-sheet A. In contrast, M27 seems to inhibit
formation of the acyl intermediate quantitatively and hence
complex formation and subsequent hydrolysis to the
cleaved form of AT (Fig. 4). Picard et al. described a
mAb (12A5) recognizing the linear sequence, DAFHK
(residues 366–370), in the C-terminal region of AT [24].
Antibody 12A5 also differs from M27 in that the former
recognizes AT when it exists as a binary complex with
thrombin, factor Xa and, to some exent, the P14-P9 synthetic
peptide, but not native, latent or cleaved forms of AT.
Dawes et al. have reported a conformationally sensitive
mAb (ESAH 1) that recognizes native, thrombin-complexed, and cleaved AT [42]. The authors also observed that
binding of heparin to AT counteracts the ability of ESAH 1
to neutralize AT inhibition of thrombin, but heparin
binding had no effect on ESAH 1 binding to AT. These
observations are different from those (seen by us) for M27,
where the effects of heparin and antibody on AT inhibition
of thrombin are independent of one another. The epitope
recognized by ESAH 1, involves residues 402–407 and 429
(FKANRP/P), all in the C-terminal cleavage peptide, but

Ó FEBS 2003

4066 G. L. Long et al. (Eur. J. Biochem. 270)

Table 2. Binding constants for M27 IgG and Fab interaction with AT. Measurements were made with surface plasmon resonance on a BIAcore
instrument and analyzed using the software package BIAEVALUATION 3.0. See the Materials and methods for details. Values represent the mean (±
standard deviation) of three independent determinations, except for AT native/Fab where only two determinations were made.
Analyte/ligand
AT native/IgG
AT native/Fab
AT latent/IgG
AT latent/Fab
AT cleaved/IgG
AT cleaved/Fab
T–AT complex/IgG
T–AT complex/Fab

ka (M)1Ỉs)1)
3.85
4.64
4.20
3.81
3.14
3.45
1.80
2.28

·
·

·
·
·
·
·
·

5

10
105
104
104
105
105
105
105

(±
(±
(±
(±
(±
(±
(±
(±

kDa (s)1)
0.04)
0.04)

0.36)
0.46)
0.00)
0.02)
0.11)
0.14)

5.64
6.28
2.87
3.01
5.18
5.23
6.53
4.37

·
·
·
·
·
·
·
·

KD (M)
)5

10
10)5

10)4
10)4
10)5
10)5
10)6
10)6

(±
(±
(±
(±
(±
(±
(±
(±

0.20)
0.03)
0.11)
0.10)
0.16)
0.17)
4.37)
3.51)

1.46
1.36
6.83
8.02
1.65

1.51
3.73
1.98

·
·
·
·
·
·
·
·

10)10 (± 0.07)
10)10 (± 0.02)
10)9 (± 0.85)
10)9 (± 1.06)
10)10 (± 0.05)
10)10 (± 0.06)
10)11 (± 2.57)
10)11 (± 1.66)

Fig. 6. Effect of mAb M27 on T–AT clearance in vivo and in vitro. (A) 125I-T–AT complex (73 pmol in 500 lL) was injected into the tail vein of rats
in the absence (h) and presence (d) of M27 Fab fragment (2.9 nmol). Blood samples were collected at the time-points indicated and the
radioactivity was measured. As controls, 125I-labeled diisopropylphosphoryl (DIP)-thrombin (j), native AT (n) and elastase-cleaved AT (s) were
injected in the same manner as the complex. The complexes with and without M27 Fab fragment were injected into two rats each. The error bars
represent the range. (B) HepG2 cells were incubated with 200 lL of 20 nM 125I-T–AT in the absence or presence of 2 lM unlabeled T–AT, 0.2 lM
Fab M27, 0.2 lM mAb M27, 0.2 lM mAb M38, and 1 lM cleaved AT. Each bar represents the percentage of binding ± 2SD. Binding of
radiolabeled T–AT without competitor was set to 100%.

distinct from the region recognized by M27 (residues 411–
416).Three-dimensional molecular models for native AT are
presented in Fig. 7. Examination of the model reveals that
the epitope recognized by M27 resides in strand 4 of b-sheet
B of AT, on the face opposite b-sheet A into which the
reactive-center loop inserts. Analyses of X-ray-derived
structures for the latent and cleaved forms of AT suggest
that the epitope is in the same general location for all forms
of AT and at least partially surface exposed (Fig. 7). The
model derived from the crystal structures indicates that only
the side-chains of residues 413–416 are exposed to the
surface, in agreement with residue 416 playing an important
role in M27 binding. However, X-ray structures showing
that residue 411 is buried is in apparent contradiction with
our peptide epitope mapping that implicates residue 411 in
M27 binding. Examination of molecular models for native,

cleaved, and latent AT, based upon X-ray crystallography,
does not lead to a clear explanation of the differences in the
equilibrium-binding constants of M27 for the different
forms.
The antibody-binding site shown in Fig. 7 is sufficiently
˚
close to the RCL of AT (40–50 A) to allow antibodymediated blocking of the formation of the Michaelis-like
complex with thrombin, even in the case of the Fab
fragment, which typically has a length from the antigen˚
binding site to the papain cleavage site of  45 A. However,
this explanation for the neutralization of AT by M27 would
require that the bound antibody has very little free
movement relative to the RCL, and is always in an

orientation that blocks the binding of thrombin to AT.
An alternative explanation is that binding of M27 distorts
the conformation of the RCL in a manner that prevents


Ó FEBS 2003

T–AT complexes and the SEC receptor (Eur. J. Biochem. 270) 4067

Fig. 7. Molecular models of the M27 epitope on antithrombin.

recognition by thrombin. A precedent for such a subtle, yet
significant, conformational change is offered by heparin
binding to AT, resulting in a conformational change in the
RCL [33]. Also consistent with this proposal is the ease with
which AT can adopt alternative conformations (e.g. the
latent form). This explanation may also, in part, explain the
only partial neutralization by M27 observed in the presence
of heparin.

In the presence of high-molecular-weight heparin, the
M27-mediated block of AT inhibition was not complete and
was not influenced by a 10-fold increase in antibody
concentration (data not presented). This suggests that the
binding sites on AT for antibody and heparin are
independent of one another. Second-order association rate
constants of 8.9 · 10)3 and 3.7 · 10)7ỈM)1Ỉs)1 have been
reported for T–AT in the absence and presence of heparin,

Ó FEBS 2003

4068 G. L. Long et al. (Eur. J. Biochem. 270)

respectively [43]. Dissociation of M27 from AT (the Kd
value for the M27AT complex is 5.6 Ã 10)5ặs)1; tẵ, 3.4 h)
cannot account for the T–AT complex formation in the
presence of heparin. We propose that heparin induces a
conformational change in the RCL of AT that allows slow
complex formation with thrombin, even with M27 bound.
Lollar & Owen were the first to demonstrate that the
AT–125I-thrombin complex is rapidly cleared by the liver in
rabbits [44]. Subsequent studies by others, involving competitive clearance studies, indicated that a common pathway
exists for several SECs, including AT–thrombin, heparin
cofactor II–thrombin, a1-antitrypsin–trypsin, and a1-antitrypsin–elastase [45,46]. Mast et al. demonstrated that
the rate of SEC clearance is 10–50 times faster than that
of the corresponding free inhibitor [47]. In the case of T–AT,
the t½ for the elimination of the complex from the
circulation is in the order of several minutes [45,47]. The
first receptor identified as being involved in SEC binding
and clearance, termed SECR, was implicated by Perlmutter
and co-workers based on in vitro studies of HepG2 and
monocyte stimulation of a1-antitrypsin biosynthesis [20].
Subsequently, SECR was reported to recognize a minimal
pentapeptide sequence (FVFLM) in a1-antitrypsin, based
upon synthetic peptide competitive-binding studies [38]. The
authors also proposed that the corresponding pentapeptide
(FLVFI in antithrombin) in the homologous serpin portion
of SEC complexes, is similarly and competitively recognized
by SECR. In contrast to Perlmutter and co-workers,

Maekawa et al. showed that five recombinant heparin
cofactor II–thrombin complexes, each with different single
mutations in the proposed receptor-binding pentapeptide,
were not prevented from binding and uptake to HepG2 cells
[22]. In recent years, conjugates of poly Lys-peptides,
containing the FVFLM sequence, have been used to
effectively transfer DNA into hepatic cells both in vitro
and in vivo [48–50]. Kounnas et al. demonstrated the
importance of the low-density lipoprotein receptor-related
protein (LRP) in the clearance of SEC complexes in vivo and
in vitro [39]. Their conclusions were based on the ability of
the receptor-associated protein, an inhibitor of LRP
activity, to prevent uptake of SEC complexes to liver in
rats and to HepG2 cells.
We have demonstrated that mAb M27 recognizes a
hexapeptide segment (FIREVP), which overlaps the last
two residues of the pentapeptide (FLVFI) reported to be
recognized by the SECR. We found that clearance of 125IT–AT from the blood circulation of rats was only slightly
reduced by bound M27 Fab fragment. Furthermore, the
Fab fragment only marginally reduced binding of the
complex to HepG2 cells. The effect was similar to that
obtained with a control antibody against AT. The limited
effect obtained with Fab M27 bound to T–AT is presumably nonspecific and caused by to an increase in size and/or
change of charge. Our interpretation is that the pentapeptide site is, at most, marginally involved in complex
clearance.

Acknowledgements
We acknowledge the technical assistance of Bjorn Hambe in purifying
antithrombin from bovine, rabbit and mouse plasma, and of Ulla
Persson in production of conditioned media containing the mAb, M27.

Financial support to G. L. L., while on sabbatical stay at the
Department of Clinical Chemistry, Lund University Hospital, Malmo,
ă
was provided, in part, by the Wenner-Gren Foundation. This work was
supported by grants from the Swedish Medical Research Council (B9603X-04487-22B and B96-03X-10825-03A), the Swedish Foundation of
˚
Strategic Research, the Kock Foundation, the Pahlsson Foundations,
and the Foundation of University Hospital, Malmo.
ă

References
1. Carrell, R.W. & Stein, P.E. (1996) The biostructural pathology of
the serpins: critical function of sheet opening mechanism. Biol.
Chem. Hoppe–Seyler. 377, 1–17.
2. Gettins, P.G.W., Patston, P.A. & Olsen, S.T. (1996) Serpins:
Structure, Function and Biology. R. G. Landes, Austin, TX.
3. Gils, A. & Declerck, P.J. (1998) Structure–function relationships
in serpins: current concepts and controversies. Thromb. Haemost.
80, 531–541.
4. Schulze, A.J., Huber, R., Bode, W. & Engh, R.A. (1994) Structural aspects of serpin inhibition. FEBS Lett. 334, 117–124.
5. Gettins, P.G.W. (2002) Serpin structure, mechanism, and function. Chem. Rev. 102, 4751–4803.
6. Miletich, J.P., Prescott, S.M., White, R., Majerus, P.W. & Bovill,
E.G. (1993) Inherited predisposition to thrombosis. Cell 72,
477–480.
7. Florell, S.R. & Rodgers, G.M. (1997) Inherited thrombotic disorders: an update. Am. J. Hematol. 54, 53–60.
8. Fish, W.W., Orre, K. & Bjork, I. (1979) The production of an
ă
inactive form of antithrombin through limited proteolysis by
thrombin. FEBS Lett. 98, 103–106.

9. Carrell, R.W., Stein, P.E., Fermi, G. & Wardell, M.R. (1994)
˚
Biological implications of a 3A structure for dimeric antithrombin.
Structure 2, 257–270.
10. Huntington, J.A., Read, R.J. & Carrell, R.W. (2000) Structure of a
serpin–protease complex shows inhibition by deformation. Nature
407, 923–926.
11. Wells, M.J., Sheffield, W.P. & Blajckman, M.A. (1999) The
clearance of thrombin–antithrombin and related serpin–enzyme
complexes from the circulation: role of various hepatocyte receptors. Thromb. Haemost. 81, 325–337.
12. Chang, W.-S.W., Wardell, M.R., Lomas, D.A. & Carrell, R.W.
(1996) Probing serpin reactive-loop conformations by proteolytic
cleavage. Biochem. J. 314, 647–653.
13. Wardell, M.R., Chang, M.-S.W., Bruce, D., Skinner, R., Lesk,
A.M. & Carrell, R.W. (1997) Preparative induction and
chracterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1. Biochemistry 36, 13133–
13142.
14. Carrell, R.W., Huntington, J.A., Mushunje, A. & Zhou, A. (2001)
The conformational basis of thrombosis. Thromb. Haemost. 86,
14–22.
15. Zhou, A., Huntington, J.A. & Carrell, R.W. (1999) Formation of
antithrombin heterodimer in vivo and the onset of thrombosis.
Blood 94, 3388–3396.
16. Bruce, D., Perry, D.J., Borg, J.-Y., Carrell, R.W. & Wardell, M.R.
(1994) Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn fi Asp).
J. Clin. Invest. 94, 2265–2274.
17. Chang, W.-S.W. & Harper, P.L. (1997) Commercial antithrombin
concentrate contains inactive L-forms of antithrombin. Thromb.
Haemost. 77, 323–328.
18. O’Reilly, M.S., Pirie-Shepherd, S., Lane, W.S. & Folkman, J.

(1999) Antiangiogenic activity of the cleaved conformation of the
serpin antithrombin. Science 285, 19261928.
ă
19. Larsson, H., Sjoblom, T., Dixelius, J., Ostman, A., Ylinenjarvi,
ă
ă
K., Bjork, I. & Claesson-Welsh, L. (2000) Antiangiogenic eects
ă


Ó FEBS 2003

20.

21.

22.

23.

24.

25.

26.

27.

28.
29.

30.

31.

32.

33.

34.

35.

T–AT complexes and the SEC receptor (Eur. J. Biochem. 270) 4069

of latent antithrombin through perturbed cell–matrix interactions
and apoptosis of endothelial cells. Cancer Res. 60, 6723–6729.
Perlmutter, D.H., Glover, G.I., Rivetna, M., Schasteen, C.S. &
Fallon, R.J. (1990) Identification of a serpin–enzyme complex
receptor on human hepatoma cells and human monocytes. Proc.
Natl Acad. Sci. USA 87, 3753–3757.
Wells, M., Hatton, M.W.C., Hewlett, B., Podar, T.J., Scheffield,
W.P. & Blajchman, M.A. (1997) Cytokeratin 18 is expressed on
the hepatocyte plasma membrane surface and interacts with
thrombin–antithrombin complexes. J. Biol. Chem. 272, 28574–
28581.
Maekawa, H. & Tollefsen, D.M. (1996) Role of the proposed
serpin–enzyme complex receptor recognition site in binding and
internalization of thrombin–heparin cofactor II complexes by
hepatocytes. J. Biol. Chem. 271, 18604–18609.

McKay, E.J. (1981) A simple two-step procedure for the isolation
of antithrombin III from biological fluids. Thromb. Res. 21,
375–382.
Picard, V., Marque, P.-E., Paolucci, F., Aiach, M. & Le Bonniec,
B.F. (1999) Topology of the stable serpin–protease complexes
revealed by an autoantibody that fails to react with the monomeric
conformers of antithrombin. J. Biol. Chem. 274, 4586–4593.
Bajaj, S.P. & Mann, K.G. (1973) Simultaneous purification of
bovine prothrombin and factor X. Activation of prothrombin by
trypsin-activated factor X. J. Biol. Chem. 248, 7729–7741.
Speijer, H., Govers-Reimslag, J.W.P., Zwall, R.F.A. & Rosing, J.
(1986) Prothrombin activation by an activator from the venom
of Oxyuranus scutellatus (Taipan Snake). J. Biol. Chem. 261,
13258–13267.
Strandberg, K., Kjellberg, M., Erb, E.-M., Persson, U., Mosher,
D.F., Villoutreix, B.O. & Stenflo, J. (2000) Activated protein
C–protein C inhibitor complex formation: characterization of a
neoepitope provides evidence for extensive insertion of the reactive
center loop. Biochemistry 39, 15713–15720.
Harlow, E. & Lane, D. (1988) Antibodies – A Laboratory Manual.
CSH Laboratory, Cold Spring Harbor, NY.
Parham, P. (1986) Preparation and purification of active fragments from mouse monoclonal antibodies. In: Cellular Immunology, (Weir, D.M., ed), 4th edn, Vol. 1, pp. 14.1–14.23. Blackwell
Scientific Publications, Malden, MA.
Brown, M.A., Stenberg, L.M., Perrson, U. & Stenflo, J. (2000)
Identification and purification of vitamin K-dependent proteins
and peptides with monoclonal antibodies specific for c-carboxyglutamyl (Gla) residues. J. Biol. Chem. 275, 19795–19802.
Cowan, N.J., Secher, D.S. & Milstein, C.J. (1974) Intracellular
immunoglobulin chain synthesis in non-secreting variants of a
mouse myeloma: detection of inactive light chain messenger RNA.
J. Mol. Biol. 90, 691–701.

Schreuder, H.A., De Boer, B., Dijkema, R., Mulders, J., Theunisson, H.J.M., Grootenhuis, P.D.J. & Hol, W.G.J. (1994) The
intact and cleaved human antithrombin III complex as a model for
serpin–proteinase interactions. Nat. Struct. Biol. 1, 48–54.
Skinner, R., Abrahams, J.P., Whisslock, J.C., Lesk, A.M., Carrell,
˚
R.W. & Wardell, M.R. (1997) The 2.6 A structure of antithrombin
indicates a conformational change at the heparin binding site.
J. Mol. Biol. 266, 601–609.
Mourny, L., Samama, J.P., Delarue, M., Petitou, M., Choay, J. &
Moras, D. (1993) Crystal structure of cleaved bovine antithrombin
˚
III at 3.2 A resolution. J. Mol. Biol. 232, 223–241.
Lee, B. & Richards, F.M. (1971) The interpretation of protein
structures: estimation of static accessibility. J. Mol. Biol. 55,
379–400.

36. Kurochking, N. & Privalov, G. (1998) Heterogeneity of packing:
Structural approach. Protein Sci. 7, 897–905.
37. Wu, J.K., Sheffield, W.P. & Blajchman, M.A. (1992) Molecular
cloning and cell-free expression of mouse antithrombin III.
Thromb. Haemost. 68, 291–296.
38. Joslin, G., Fallon, R.J., Bullock, J., Adams, S.P. & Perlmutter,
D.H. (1991) The SEC receptor recognizes a pentapeptide neodomain of a1-antitrypsin–proteinase complex. J. Biol. Chem. 266,
11282–11288.
39. Kounnas, M.Z., Church, F.C., Argraves, W.S. & Strickland,
D.K. (1996) Cellular internalization and degradation of antithrombin III–thrombin, heparin cofactor II–thrombin, and a1antitrypsin–trypsin complexes is mediated by the low density
lipoprotein receptor-related protein. J. Biol. Chem. 271, 6523–
6529.
40. Asakura, S., Matsuda, M., Yoshida, N., Terukina, S. & Kihara,
H. (1989) A monoclonal antibody that triggers deacylation of an

intermediate thrombin–antithrombin III complex. J. Biol. Chem.
264, 13736–13739.
41. Asakura, S., Hirata, H., Okazaki, H., Hashimoto-Gotoh, T. &
Matsuda, M. (1990) Hydrophobic residues 382–386 of antithrombin III, Ala-Ala-Ala-Ser-Thr, serve as the epitope for an
antibody which facilitates hydrolysis of the inhibitor by thrombin.
J. Biol. Chem. 265, 5135–5138.
42. Dawes, J., James, K. & Lane, D.A. (1994) Conformational
change in antithrombin induced by heparin, probed with a
monoclonal antibody against the 1C/4B region. Biochemistry 33,
4375–4383.
43. Olson, S.T. & Bjork, I. (1991) Predominant contribution of sură
face approximation to the mechanism of heparin acceleration of
the antithrombin–thrombin reaction. Elucidation from salt concentration effects. J. Biol. Chem. 266, 6353–6364.
44. Lollar, P. & Owen, W.G. (1980) Clearance of thrombin from
circulation in rabbits by high-affinity binding sites on endothelium.
Possible roles in the inactivation of thrombin by antithrombin III.
J. Clin. Invest. 66, 1222–1230.
45. Fuchs, H.E., Shifman, M.A. & Pizzo, S.V. (1982) In vivo
catabolism of a1-proteinase inhibitor–trypsin, antithrombin III–
thrombin and a2-macroglobulin–methylamine. Biochim. Biophys.
Acta 716, 151–157.
46. Pratt, C.W., Church, F.C. & Pizzo, S.V. (1988) In vivo catabolism of heparin cofactor II and its complex with thrombin:
evidence for a common receptor-mediated clearance pathway for
three serine proteinase inhibitors. Arch. Biochem. Biophys. 262,
111–117.
47. Mast, A.E., Enghild, J.J., Pizzo, S.V. & Salveson, G. (1991)
Analysis of the plasma elimination kinetics and conformational
stabilities of native, proteinase-complexed, and reactive site
cleaved serpins: comparison of a1-proteinase inhibitor, a1-antichymotrypsin, antithrombin III, a2-antiplasmin, angiotensinogen,
and ovalbumin. Biochemistry 30, 1723–1730.

48. Ziady, A.G., Perales, J.C., Ferkol, T., Gerken, T., Beegen, H.,
Perlmutter, D.H. & Davis, P.B. (1997) Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor. Am. J.
Physiol. 273, G545–G552.
49. Patel, S., Zhang, X., Collins, L. & Fabre, J.W. (2001) A small
synthetic peptide for gene delivery via the serpin–enzyme complex
receptor. J. Gene Med. 3, 271–279.
50. Zaidy, A.G., Ferkol, T., Dawson, D.V., Perlmutter, D.H. &
Davis, P.B. (1999) Chain length of the polylysine in receptortargeted transfer complexes affects duration of reporter gene
expression both in vitro and in vivo. J. Biol. Chem. 274, 4908–4916.