Probing plasma clearance of the thrombin–antithrombin complex
with a monoclonal antibody against the putative serpin–enzyme
complex receptor-binding site
George L. Long
1,
*, Margareta Kjellberg
2
, Bruno O. Villoutreix
3
and Johan Stenflo
2
1
Department of Biochemistry, University of Vermont, Burlington, VT, USA;
2
Department of Clinical Chemistry, Lund University,
University Hospital Malmo
¨
, Malmo
¨
, Sweden;
3
INSERM U428, University of Paris V, France
A high-affinity 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 anti-
thrombin. 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

1
–P
17
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 conse-
quence 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, loop-
inserted inhibitor is formed if loop insertion, with the

(Received 3 July 2003, revised 30 July 2003, accepted 15 August 2003)
Eur. J. Biochem. 270, 4059–4069 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03793.x
[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].IncommercialconcentratesofAT,upto40%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 dissoci-
ation 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

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 previ-
ously [13]. Native AT was incubated in 10 m
M
Tris/HCl,
0.25
M
sodium citrate, pH 7.4, for 70 h at 60 °Cand
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,andusedinfurther
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 chromato-

D
,
L
-cysteine
and EDTA were added to final concentrations of 45 and
0.9 m
M
, 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 m
M
) for 30 min at room temperature, the
digest was dialyzed at 4 °C against 1
M
glycine, 150 m
M
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 m
M
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 m
M
). The Fab fragment eluted
at  70 m
M
NaCl. It was homogenous, as judged by SDS/

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 lLofcoating
buffer (100 m
M
sodium bicarbonate, pH 9.6), were deliv-
ered 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 m
M
sodium
phosphate buffer containing 0.5
M
NaCl and 0.1% Tween
20, pH 8.0 (NaCl/P
i
/Tween), followed by blocking for
15 min with 1% (w/v) BSA (Sigma Fraction V) in NaCl/
P
i
/Tween. Wells were rinsed again, and different amounts
of M27, diluted in NaCl/P
i
/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/P

of a-thrombin incubation with native AT at 37 °Cfor
different lengths of time in the presence or absence of M27.
Freshly diluted thrombin (0.5 lg in buffer comprising
50 lLof50m
M
Tris/HCl, 150 m
M
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 lgin10lL of Tris/HCl/NaCl/BSA), M27 (4 lgin
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 con-
taining 190 lL of freshly prepared thrombin substrate
(400 l
M
S-2238; Chromogenix, Gothenburg, Sweden) in
50 m
M
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 lgÆmL

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 m
M
Hepes,
150 m
M
NaCl, 3 m
M
EDTA, 0.005% Polysorbate 20,
pH 7.4 (Hepes/NaCl/EDTA/Polysorbate 20). They were
immobilized with NH
2
-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 lLÆmin
)1
. The chip was regenerated with
two pulses of 5 lL0.1
M

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 mLÆkg
)1
) with a 1 : 1 : 2 (v/v/v) mixture of Hypnorm
(JANSSEN-CILAG Ltd, High Wycombe, UK), Dormi-
cum (Pharma hameln GmbH, Hamelm, Germany) and
deionized water.
125
I-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 lLof0.5
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.
125
I-
labeled thrombin, native AT, and cleaved AT alone were
also injected into control animals.
Binding of
125
I-labeled T–AT complex to HepG2 cells

) in binding buffer,
with or without unlabelled T–AT (2 l
M
)orM27
(0.2 l
M
)orM38(0.2l
M
), or cleaved AT (1 l
M
), were
each added to four wells. After incubation at 4 °Cfor
2 h, the wells were washed three times with 10 m
M
Hepes, pH 7.4, containing 0.15
M
NaCl, 1 m
M
CaCl
2
,
2m
M
MgCl
2
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

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
Fig.1. WesternblottingofATwithmAbM27.Different forms of AT

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
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. Membrane-

for native and elastase-cleaved AT
were almost identical. The highest K
d
, for latent AT, was the
result of both a decrease in the association rate constant (k
a
)
and an increase in the dissociation rate constant (k
d
)relative
to native AT, each about one order of magnitude. M27 had
the highest affinity for the T–AT complex (K
D
 2 · 10
)11
M
). This can be attributed almost totally to a
very slow dissociation rate (Fig. 5C).
Clearance of the T–AT complex in rats
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

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 V
max
(mODÆmin
)1
)
Part A
None (TBSB buffer alone) 0.02 ± 0.01
Thrombin (50 ngÆmL
)1
) 36.42 ± 1.50
Thrombin (50 ngÆmL
)1
) + AT (1 : 1.04) 3.52 ± 0.12
Thrombin (50 ngÆmL
)1
) + AT (1 : 1.04) + M27 (1 : 1.04 : 3.7) 35.88 ± 1.30
Thrombin (50 ngÆmL
)1
) + M27 (1 : 3.7) 35.19 ± 1.53
Thrombin (50 ngÆmL
)1
) + AT (1 : 1.04) + Fab (1 : 1.04 : 3.7) 33.30 ± 1.47
Thrombin (50 ngÆmL
)1
) + Fab (1 : 3.7) 35.22 ± 1.36

)1
) + Heparin (0.25 unitsÆmL
)1
) + Fab (1 : 9.8) 32.19 ± 0.86
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 posi-
tionofthe62kDamolecularmassmarkerprotein.
4064 G. L. Long et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(K
D
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
labeledT–ATinthepresenceofM38,whichisananti-AT
mAb that does not compete with M27 for binding to AT.
The binding of labeled T–AT was decreased to 54%. A

(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 P
14
-P
9
synthetic

.
Ó FEBS 2003 T–AT complexes and the SEC receptor (Eur. J. Biochem. 270) 4065
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 antibody-
mediated 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

5
(± 0.04) 5.64 · 10
)5
(± 0.20) 1.46 · 10
)10
(± 0.07)
AT native/Fab 4.64 · 10
5
(± 0.04) 6.28 · 10
)5
(± 0.03) 1.36 · 10
)10
(± 0.02)
AT latent/IgG 4.20 · 10
4
(± 0.36) 2.87 · 10
)4
(± 0.11) 6.83 · 10
)9
(± 0.85)
AT latent/Fab 3.81 · 10
4
(± 0.46) 3.01 · 10
)4
(± 0.10) 8.02 · 10
)9
(± 1.06)
AT cleaved/IgG 3.14 · 10
5
(± 0.00) 5.18 · 10

125
I-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 lLof20n
M
125
I-T–AT in the absence or presence of 2 l
M
unlabeled T–AT, 0.2 l
M
Fab M27, 0.2 l
M
mAb M27, 0.2 l
M
mAbM38,and1l
M
cleaved AT. Each bar represents the percentage of binding ± 2SD. Binding of
radiolabeled T–AT without competitor was set to 100%.
4066 G. L. Long et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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

AT–
125
I-thrombin complex is rapidly cleared by the liver in
rabbits [44]. Subsequent studies by others, involving com-
petitive clearance studies, indicated that a common pathway
exists for several SECs, including AT–thrombin, heparin
cofactor II–thrombin, a
1
-antitrypsin–trypsin, and a
1
-anti-
trypsin–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 t
½
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 a
1
-antitrypsin biosynthesis [20].
Subsequently, SECR was reported to recognize a minimal
pentapeptide sequence (FVFLM) in a
1
-antitrypsin, based
upon synthetic peptide competitive-binding studies [38]. The
authors also proposed that the corresponding pentapeptide

change of charge. Our interpretation is that the penta-
peptide 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
¨
,
was provided, in part, by the Wenner-Gren Foundation. This work was
supported by grants from the Swedish Medical Research Council (B96-
03X-04487-22B and B96-03X-10825-03A), the Swedish Foundation of
Strategic Research, the Kock Foundation, the Pa
˚
hlsson Foundations,
and the Foundation of University Hospital, Malmo
¨
.
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