Factor VIIa Modified in the 170 Loop Shows Enhanced
Catalytic Activity but Does Not Change the Zymogen-like Property*
Kenji
Soejima
,
Jun
Mizuguchi§¶,
Masato
Yuguchi§,
Tomohiro
Nakagaki§,
Shouichi
Higashi
, and
Sadaaki
Iwanaga§**
From the
First Research Department and the
§ Blood Products Research Department, The
Chemo-Sero-Therapeutic Research Institute, Kumamoto 869-1298, the
Division of Cell Biology, Kihara Institute for Biological
Research, Yokohama City University, Yokohama 244-0813, and ** Institute
for Comprehensive Medical Science, School of Medicine, Fujita Health
University, Nagoya 470-1192, Japan
Received for publication, October 9, 2000, and in revised form, January 12, 2001
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ABSTRACT |
Factor VIIa (VIIa) is an unusual trypsin-type
serine proteinase that appears to exist in an equilibrium between minor
active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the
equilibrium into the active state. The proteinase domain of VIIa
contains a unique structure: a loop formed by a disulfide bond between Cys310 and Cys329, which is five residues
longer than those of other trypsin types. To examine the functional
role of the loop region, we prepared two mutants of VIIa. One of the
mutants, named VII-11, had five extra corresponding residues 316-320
of VII deleted. The other mutant, VII-31, had all of the residues in
its loop replaced with those of trypsin. Functional analysis of the two
mutants showed that VIIa-11 (Kd = 41 nM) and VIIa-31 (Kd = 160 nM) had lower affinities for soluble tissue factor as
compared with the wild-type VIIa (Kd = 11 nM). The magnitude of tissue factor-mediated acceleration
of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31
(2-fold) were also smaller than that of wild-type VIIa (30-fold). In
the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced
activity; the catalytic efficiencies of VIIa-31 toward various
chromogenic substrates were 2-18-fold greater than those of the
wild-type VIIa. Susceptibility of the
-amino group of Ile-153 of
VIIa-31 to carbamylation was almost the same as that of wild-type VIIa,
suggesting that VIIa-31 as well as wild-type VIIa exist predominantly
in the zymogen-like state. Therefore, the tested modifications in the
loop region had adverse effects on affinity for tissue factor,
disturbed the tissue factor-induced conformational transition, and
changed the catalytic efficiency of VIIa, but they did not affect the
equilibrium between active and zymogen-like conformational states.
 |
INTRODUCTION |
Factor VIIa (VIIa)1 is a
plasma serine proteinase that is essential for the initiation of
extrinsic blood coagulation (1). When VIIa forms a complex with tissue
factor (TF) in the presence of Ca2+ and phospholipids, the
proteinase activity of VIIa toward its natural substrates, factors IX
and X, is enhanced by several orders of magnitude, and the coagulation
cascade is triggered (2). In vitro, the formation of the
active complex can be evidenced by measuring the esterolytic and
amidolytic activities of VIIa (3-6); this activity is also enhanced in
the presence of soluble TF (sTF) and Ca2+ (4). Human
zymogen VII is a single-chain enzyme precursor with an
NH2-terminal Gla domain (residues 1-39), followed by two EGF-like domains, EGF 1 (residues 50-81), and EGF 2 (residues 91-127), and a COOH-terminal serine proteinase domain (residues 153-406). Through the limited proteolysis of the
Arg152-Ile153 peptide bond, zymogen VII is
converted to a two-chain form enzyme, activated VII (VIIa), bridged by
a disulfide bond (Cys135-Cys262), which is
composed of a light chain (residues 1-152) with Gla, EGF 1, and EGF 2 domains, and a heavy chain with a serine proteinase domain (residues
153-406) (7).
On the other hand, the TF molecule consists of two immunoglobulin-like
extracellular domains, a single membrane-spanning region, and a
COOH-terminal cytoplasmic tail (8). Solution of the crystal structure
of the human VIIa-sTF complex revealed that there are several amino
acid residues in the proteinase domain of VIIa that come into direct
contact with TF, in addition to a number of interaction sites in the
NH2-terminal portion (9). Six of these,
Phe275[c129F], Arg277[c134],
Arg304[c162], Met306[c164],
Gln308[c166], and Asp309[c167]
(chymotrypsinogen numbering in brackets), are located in an almost
cluster-like form (9, 10). The predicted sites of interaction between
VIIa and TF on mutants from patients, and the results of alanine
scanning analysis are consistent with the results obtained from
crystallography (11-15). Furthermore, studies of the chemical
modification of bovine VIIa provide a model of the mechanism of
TF-mediated acceleration of VIIa activity. In this model, the
proteinase domain of VIIa exists in equilibrium between the minor
active and dominant zymogen-like inactive conformational states, and
preferential binding of TF to the active state leads to a shift in
equilibrium, thereby accelerating VIIa activity (16-18). However, the
structural elements of VIIa required for catalytic site formation are
still not clear. The proteinase domain of VIIa contains a unique
primary structure; the 170 loop (chymotrypsinogen number, 168th to
182nd) has five extra amino acid residues compared with those of other
trypsin-types. In this study, we made two mutants of VIIa and studied
the functional role of the 170 loop in formation of the catalytic site.
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EXPERIMENTAL PROCEDURES |
Materials--
The materials used were as follows: S-2288
(H-D-Ile-Pro-Arg-pNA·2HCl), S-2366
(pyroGlu-Pro-Arg-pNA·HCl), S-2238
(H-D-Phe-Pip-Arg-pNA·2HCl), S-2302
(H-D-Pro-Phe-Arg-pNA·2HCl), S-2765
(Z-D-Arg-Gly-Arg-pNA·2HCl), S-2444
(pyroGlu-Gly-Arg-pNA·HCl), S-2222
(Bz-IIe-Glu(Glu
OMe)-Gly-Arg-pNA·HCl), and S-2403
(pyroGlu-Phe-Lys-pNA·HCl), from Chromogenix AB, Stockholm, Sweden; Chromozym® t-PA
(MeSO2-D-Phe-Gly-Arg-pNA), and Chromozym® X
(MeO-CO-D-Nle-Gly-Arg-pNA), from Roche Molecular
Biochemicals; p-amidinophenyl methanesulfonyl fluoride
hydrochloride (APMSF) and butyric acid, from Wako Pure Chemical
Industries, Ltd., Osaka, Japan; benzamidine-HCl, from Tokyo Chemical
Industry Co., Ltd.; LIPOFECTACETM Reagent, GENETICIN®
(antibiotics G418), and
-minimum essential medium, from Life
Technologies, Inc.; ASF-104 medium, from Ajinomoto Co., Inc., Tokyo,
Japan; fetal bovine serum, from HyClone Co., Ltd.; penicillin G
potassium, from Banyu Pharmaceutical Co., Ltd., Tokyo, Japan;
streptomycin sulfate, from Meiji Seika Kaisha, Ltd., Tokyo, Japan;
vitamin K, polyethylene glycol (PEG) 8000, bovine serum albumin (fatty
acid free), phosphatidylcholine, and phosphatidylserine, from
Sigma-Aldrich; human trypsin, from Athens Research & Technology, Inc.;
fluorescein-Phe-Pro-Arg-chloromethyl ketone (FPR-ck), from Hematologic
Technologies, Inc.; Asserachrom® VII: Ag (enzyme-linked immunosorbent
assay kit), from Diagnostica Stago, Asnieres, France; Citrated control
plasma (Ci-trol®), immunoabsorbed factor VII depleted plasma, and
Dade® thromboplastin·C plus, from DADE International Inc., Miami,
FL; and Platelin®, from Organon Teknika Corporation. Synthetic Xa
inhibitor (DX-9065a) was a gift from Dr. T. Hara, Daiichi
Pharmaceutical Co., Ltd., Tokyo, Japan (19). All custom oligo-DNA
primers were provided by Nippon Flour Mills Co., Ltd. All other
chemicals were of analytical grade or of the highest quality
commercially available.
Proteins--
Recombinant human soluble TF (residues 1-218;
sTF) was prepared as described (20). Relipidated human placental
full-length TF and plasma-derived human clotting factors VII, VIIa, X,
and Xa were prepared as described (21).
Gene Construction of VII Mutants--
cDNA of human VII was
a gift from Drs. T. Matsushita and H. Saito, Nagoya University.
Mutagenesis by polymerase chain reaction-based methods was performed
for construction of mutant VII under standard conditions as described
(22). Sequences of specific primers for cloning VII cDNA
were as follows: 5'-GGGGTCGACATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTG-3' (sense primer containing a SalI site),
5'-CCCGGATCCCTAGGGAAATGGGGCTCGCAGGAGGACTCCTGGGCG-3' (antisense primer
containing a BamHI site). Sequences of primers for
mutagenetic polymerase chain reaction were as follows:
5'-CAGCAGTCACGGCCAAATATCACGGAGTACATGTTCTGTGCCGGC-3' (sense),
5'-GCCGGCACAGAACATGTACTCCGTGATATTTGGCCGTGACTGCTG-3' (antisense) for
VII-11; 5'-ATGACCCAGGACTGCGAAGCCTCCTACCCTGGAAAGATCACGGAGTACATG-3' (sense), 5'-CATGTACTCCGTGATCTTTCCAGGGTAGGAGGCTTCGCAGTCCTGGGTCAT-3' (antisense) for VII-31. Amplified mutant VII DNA fragments were digested with SalI and BamHI and then cloned into
the mammalian expression vector pCAG-neo (23). The vectors were
confirmed to contain the entire sequences of the wild-type and mutant
VII DNA fragments by sequencing on an automated DNA sequencer.
Expression, Cell Culture, and Purification of VII
Mutants--
Each of the mutant expression vectors was transfected
into the Chinese hamster ovary cell line CHO-K1 with Lipofectin reagent (LIPOFECTACETM). Two days after transfection, the medium
was changed to
-minimum essential medium containing 10% fetal
bovine serum, 1 µg/ml G418, 10 units/ml penicillin G, 100 µg/ml
streptomycin, and 10 µg/ml vitamin K. After 2 weeks, cells were
cloned by the limiting dilution method. Expression of each clone was
confirmed by enzyme-linked immunosorbent assay (Asserachrom® VII:
Ag). The highly expressed clones were selected, cultured, and expanded.
48 h before harvesting, culture media were replaced with
serum-free media (ASF-104) supplemented with 1 mM butyric
acid and 20 µg/ml vitamin K. After harvesting, each conditioned
medium was mixed with 0.1% bovine serum albumin and 50 mM
benzamidine-HCl, and centrifuged at 5000 rpm for 20 min at 4 °C. The
supernatants (1.5-3 liters) were stored at
80 °C. Subsequently,
the frozen media were thawed and filtrated through a 0.45 µm membrane
filter (Corning Costar), mixed with 2 mM CaCl2, and subjected to the Ca2+-dependent anti-factor
VII monoclonal antibody-conjugated column chromatography (21) at
4 °C. The column had been equilibrated in 50 mM
Tris-HCl, pH 7.2, containing 0.1 M NaCl, 50 mM
benzamidine-HCl, and 2 mM CaCl2. After the
medium was loaded, the column was washed with equilibration buffer.
Mutant factor VII was eluted with 50 mM Tris-HCl, pH 7.2, containing 0.1 M NaCl, 50 mM benzamidine-HCl, and a 0-10 mM EDTA gradient, and eluted peak fractions
were pooled. The pooled fractions were analyzed by SDS-PAGE, and
clotting activity was determined in a Behring Fibrintimer® using
VII-depleted plasma. The protein concentrations were determined by the
Bradford method and/or absorbance at 280 nm
(A280 = 13.9 for 1% VII) after dialysis to
remove benzamidine-HCl. Purified VII derived from human plasma was used
as a standard to estimate the protein concentration.
Preparation of VIIa Mutants--
Activation of VII mutants was
achieved at 37 °C for 15-60 min by addition of a 1:100 molar ratio
of plasma-derived Xa in 50 mM Tris-HCl, pH 7.45, containing
0.1 M NaCl, 0.1% PEG 8000, phospholipids (Platelin®),
and 10 mM CaCl2. This reaction was terminated
by the addition of 50 mM benzamidine-HCl. Mutant VIIa was
purified by Ca2+-dependent anti-VII monoclonal
column chromatography, as described above. The eluted VIIa fractions
were pooled and dialyzed against 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl. PEG 8000 was added to aliquots at a
final concentration of 0.1%, and the samples were stored at
80 °C
until use. Any potentially contaminating Xa was inactivated with a
synthetic Xa-specific inhibitor (DX-9065a). Amidolytic activities of
wild-type VIIa and mutants of VIIa were not affected by DX-9065a (data
not shown).
Active Site Titration of VIIa Mutants--
Active site titration
of VIIa was performed by calculating the ratio of the concentrations of
fluorescein and the protein in the fluorescein-labeled VIIa as
described (18, 24) with some modifications. Fluorescein-FPR-ck and VIIa
were mixed (molar ratio of 50:1) and incubated for 18 h until VIIa
activity was no longer detectable by clotting assay. Free fluorescein
inhibitor was removed by gel filtration (Sephadex G-25) and extensive
dialysis. The concentrations of fluorescein and the protein in the
complex were determined by spectrofluorometry and the Bradford method, respectively. Fluorescein-labeled VIIa was excited at 485 nm, and
emission was detected at 535 nm in FluoroNunc® C8-white
96-well microwell plates (NalgeNunc International) with a microplate
spectrofluorometer (SPECTRAFLUOR, TECAN Austria GmbH.).
Amidolysis of Synthetic Chromogenic Substrates--
Unless
otherwise noted, all kinetic experiments were carried out under the
following conditions: 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl, 10 mM CaCl2, and 0.1%
PEG 8000; 200 µl of total volume of sample in 96-well microplates
(F96 Polysorp NuncTM-Immuno Plate; NalgeNunc International
Denmark); measuring at 30 °C with a temperature controlled
microplate spectrophotometer SPECTRAmax plus® (Molecular Devices
Inc.). Kinetics were measured based on initial rates. All experiments
were performed in at least two independent trials.
TF Dependence of VIIa Mutants on Amidolysis of S-2288--
A
mixture of 2 nM VIIa and various concentrations (0-400
nM) of sTF was preincubated for 5 min at 30 °C. In one
series of experiments, the initial rate (
mOD/min) was measured as
amidolytic activity under the above conditions on the additional of a
final concentration of 1 mM S-2288. The data were subjected
to Hanes-Woolf plot analysis to determine the apparent dissociation constants.
Measurement of Kinetic Parameters for Mutant VIIa-catalyzed
Amidolysis of S-2288--
For assays in the absence of sTF, the mutant
VIIa concentration was 100 nM, and substrate concentrations
ranged from 0.2 to 1.2 mM. In the presence of sTF, a
mixture of 1 µM enzyme and 5 µM sTF was
preincubated for 5 min. 20 µl of this mixtures was diluted 10-fold, and the initial rate of hydrolysis was
measured under the above conditions with substrate concentrations
ranging from 0.2 to 1.2 mM.
APMSF Incorporation into VIIa Mutants--
5 µl each of a
given concentration of APMSF was added to 45 µl of 1 µM
VIIa with or without 5 µM sTF. The mixture was incubated for 5 min to incorporate the APMSF molecule. To measure residual VIIa
amidolytic activities, 20 µl of each of the mixtures was diluted
9-fold, and kinetic analyses were carried out by adding 20 µl of 10 mM S-2288. For measurement of residual human trypsin amidolytic activity, the sample mixture was diluted 90-fold.
Carbamylation of the
-Amino Group in VIIa Mutants--
One
hundred µl of each VIIa mutant (1 µM) with or without
sTF (5 µM), preincubated at 30 °C for 5 min, was mixed
with 25 µl of 1 M KNCO. The mixture was incubated at
30 °C for 0, 10, 20, 40, 80, and 160 min. After each incubation,
aliquots of 20 µl of each sample taken from the reaction mixture were
diluted 9-fold, and kinetic analyses were carried out by addition
of 20 µl of 10 mM S-2288.
Substrate Specificities of VIIa Mutants--
Amidolytic
activities of VIIa toward various substrates were measured by kinetic
analysis as described above. The enzyme and substrate concentrations
were 100 nM and 1 mM, respectively.
Zymogen X Activation by VIIa Mutants--
The mutant
VIIa-mediated activation of zymogen X in the presence or absence of
relipidated TF was performed as described by Komiyama et al.
(2).
 |
RESULTS |
Purification and Activation of VII Mutants--
To investigate the
function of the 170 loop, we prepared two mutants: VII-11, with
deletion of the five extra residues in the 170 loop, and
VII-31, in which the 170 loop was
replaced with that of trypsin (Figs. 1 and
2). Both mutants and wild-type VII (VII-W) were purified from 1.5-3 liters of transfected CHO-K1 cell
culture conditioned medium using a monoclonal antibody column. The
specific activity (clotting activity/protein concentration) of purified
recombinant VII-W was equivalent to that of plasma-derived VII (about
2500 units/mg). The activation of the wild-type zymogen VII to VIIa
mediated by Xa was completed within 15 min under the conditions
described under "Experimental Procedures." Under these conditions,
the activation of VII-31 was similar to that of VII-W, whereas that of
VII-11 required a farther incubation for 15-45 min. On SDS-PAGE, the
band of the heavy chain of VIIa-31 showed further mobility than those
of VIIa-W and VIIa-11 (Fig. 3). This was
probably because a carbohydrate attaching site in the VIIa-heavy chain,
Asn322[c175] (25), was lost in the creation of VIIa-31.
The carbohydrate chain in the VIIa-W-heavy chain, but not in the
VIIa-31-heavy chain, was detected by lectin blotting analysis (data not
shown).

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Fig. 1.
Unique amino acid sequence and location of
the 170 loop in the proteinase domain of VIIa. Sequence
comparisons of the 170 loop derived from serine proteinases of various
species are indicated in the box. IX,
factor IX; PC, protein C; IIa, thrombin;
Tryp, trypsin; -CT,
-chymotrypsin. The unique five extra amino acid (KVGDS) residues
located in the 170 loop are shown in orange. Locations of
various surface loops are indicated on the right.
There is no direct contact between the 170 loop and the
NH2-terminal domain of soluble tissue factor
(left). The crystal structure is cited from Protein Data
Bank code 1DAN (9). The NH2-terminal domain of sTF is
colored green, and activation domains are light
blue.
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Fig. 2.
Circumferential structures of the 170 loop
and design of VII mutants. The unique five extra amino acid
(KVGDS) residues located in the 170 loop are shown in
orange. A cluster of amino acid residues that are in direct
contact with sTF are shown in pink. The
NH2-terminal domain of sTF is colored green.
Other circumferential structures of the 170 loop, which are involved in
catalytic-site formation, are indicated as follows: S1 pocket,
red circle; activation domains, light blue;
NH2-terminal insertion of Ile153[c16],
yellow dot. Sequences of the wild-type and both VII mutants
are indicated in the orange box.
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Fig. 3.
SDS-PAGE of the time course of activation of
each mutant VII by Xa. SDS-PAGE was performed under reducing
conditions followed by Coomassie Brilliant Blue staining. Incubation
time of activation is indicated above lanes. Bz indicates
purified mutant VII-31 with 50 mM benzamidine-HCl. After
remove benzamidine-HCl by dialysis, VII-31 showed some auto
activation.
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TF Dependence of Amidolytic Activities of VIIa
Mutants--
Amidolytic activities of plasma-derived VIIa, VIIa-W,
VIIa-11, and VIIa-31 toward S-2288, as well as the effects of TF on these activities, were examined in the absence and presence of various
concentrations of sTF. In the absence of sTF, the amidolytic activity
of VIIa-11 and that of VIIa-31 were about 50 and 700% of that of
VIIa-W, respectively. As shown in Fig.
4A, sTF potentiated the
amidolytic activities of both mutants and VIIa-W in a
dose-dependent manner. The data shown in Fig. 4A
were subjected to Hanes-Woolf plot analysis to determine the apparent
dissociation constants (Kd app) for the
plasma-derived VIIa-sTF, VIIa-W-sTF, VIIa-11-sTF, and VIIa-31-sTF
complexes (Fig. 4B). We found that both VIIa-11
(Kd app = 41 nM) and
VIIa-31 (Kd app = 160 nM)
had reduced affinities for sTF as compared with VIIa-W (Kd app = 11 nM) and
plasma-derived VIIa (Kd app = 10 nM). We also determined the kinetic parameters for the
hydrolysis of S-2288 catalyzed by mutants or wild-type VIIa (Table
I). In the absence of sTF, the
kcat/Km value of VIIa-31 was about 7-fold higher than that of VIIa-W, whereas the
kcat/Km value of VIIa-11 was
about 50% of that of VIIa-W. In the presence of saturating
concentration of sTF, the
kcat/Km value of VIIa-W was
enhanced by 30-fold, whereas those of mutants VIIa-11 and VIIa-31 were
enhanced by 7-fold and 2-fold, respectively. The
kcat/Km values of VIIa-11-sTF
and VIIa-31-sTF complexes were 9.6 and 46% of that of VIIa-W-sTF,
respectively.

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Fig. 4.
TF dependence of VIIa mutants. In
A, the hydrolysis of chromogenic substrate S-2288 (1 mM) by VIIa-W (circle; 2 nM),
VIIa-11 (square; 2 nM), and VIIa-31
(triangle; 2 nM) in the presence of various
concentrations of sTF was analyzed at 30 °C. In B, the
apparent dissociation constants of VIIa-W (circle), VIIa-11
(square), and VIIa-31 (triangle) with sTF were
calculated by Hanes-Woolf plot analysis. In both panels, data of
plasma-derived VIIa (diamond) is equivalent to that of
VIIa-W.
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Activation of the Zymogen X by VIIa Mutants--
Plasma-derived
VIIa-, VIIa-W-, VIIa-11-, and VIIa-31-mediated activation of zymogen X
with phospholipids (PCPS) were examined in the presence or absence of
full-length TF (Fig. 5). In the absence
of TF, VIIa-31 showed the highest Xa generation as compared with VIIa-W
and VIIa-11. This is consistent with data of the amidolytic activity
toward S-2288. In the presence of TF, VIIa-W-mediated Xa generation was
strongly enhanced and showed the highest activity as compared with
VIIa-11 and VIIa-31 in this condition.

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Fig. 5.
Zymogen X activation by VIIa mutants. In
A, the activations of zymogen X (FX) by VIIa-W
(open circle; 50 nM), VIIa-11 (open
square; 200 nM), and VIIa-31 (open
triangle; 50 nM) were analyzed at 30 °C. In
B, the activation of zymogen X by the complex of
VIIa-W-TF/PCPS (closed circle; 20 pM),
VIIa-11-TF/PCPS (closed square; 20 pM), or
VIIa-31-TF/PCPS (closed triangle; 20 pM) was
also analyzed at 30 °C. In both panels, data of plasma-derived VIIa
(diamond) is equivalent to that of VIIa-W.
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Incorporation of APMSF into VIIa Mutants--
APMSF is a synthetic
serine proteinase inhibitor in which an amidino group was designed to
interact with the Asp-189 (chymotrypsinogen numbering) in the subsite 1 (S1) pocket of trypsin-type serine proteinases. The interaction brings
the sulfofluoride moiety of APMSF into proximity with the active serine
residue of the enzyme, thus facilitating modification of the active
site serine. Because APMSF does not interact with the S2 or S3 sites of
serine proteinases, the inhibitor is a useful probe to examine the
active site and S1 site of VIIa mutants without considering the
contribution of extended subsite-peptidyl site interactions. We
compared the susceptibilities of VIIa mutants to APMSF inhibition with
those of wild-type and plasma-derived VIIa. In the absence of sTF,
VIIa-31 showed the highest susceptibility to APMSF, and the
concentration of APMSF required for 50% inhibition was 120 µM (Fig. 6). In contrast, 2000 µM APMSF was needed for 50% inhibition of
plasma-derived VIIa, VIIa-W, and VIIa-11 activities. Therefore, it is
likely that VIIa-31 but not VIIa-11 gained enhanced catalytic
efficiency after modification in the 170 loop. In the presence of sTF,
the concentrations of APMSF required for 50% inhibition of
plasma-derived VIIa, VIIa-W, VIIa-11, and VIIa-31 were shifted to 60 µM, 40 µM, 60 µM and 20 µM, respectively. On the other hand, the susceptibility of human trypsin was much higher than those of VIIa derivatives, and
the APMSF concentration required for 50% inhibition of trypsin activity was ~1 µM.

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Fig. 6.
Susceptibility of VIIa mutants to APMSF.
In A, VIIa-W (open circle), VIIa-11 (open
square), and VIIa-31 (open triangle) were incubated
with various concentrations of APMSF for 5 min at 30 °C, followed by
measurement of the remaining amidolytic activity with 1 mM
S-2288. In B, the complexes of VIIa-W-sTF (closed
circle), VIIa-11-sTF (closed square), VIIa-31-sTF
(closed triangle), and human trypsin (asterisk)
were incubated with various concentrations of APMSF for 5 min at
30 °C, followed by measurement of the remaining amidolytic activity
with 1 mM S-2288. In both panels, data of plasma-derived
VIIa (diamond) is equivalent to that of VIIa-W.
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Carbamylation of VIIa Mutants--
It has been reported that
treatment of VIIa with cyanate ions results in a loss of activity
caused by carbamylation of the
-amino group of
Ile153[c16] (16-18). This carbamylation reaction is
inhibited by TF, hence the critical ion pair between
Ile153[c16] and Asp343[c194] in VIIa
appears to be formed only after complex formation with TF. This
suggests that carbamylation might be useful as a tool to examine the
microenvironment of the NH2-terminal
Ile153[c16] and/or formation of the critical ion pair in
VIIa mutants. In the presence of KNCO, the rates of inactivation of the
mutants VIIa-11 and VIIa-31 and of VIIa-W were measured under the same conditions as those described previously (18). In the absence of sTF,
the inactivation rates of both mutants VIIa-11 an VIIa-31 were almost
the same as that of VIIa-W, suggesting that
-amino groups of
Ile153[c16] of the mutants as well as that of VIIa-W are
accessible to cyanate ions (Fig.
7A). After complex formation
with sTF (Fig. 7B), the inactivation rate of VIIa-W declined
significantly. In contrast, binding of sTF partly reduced the
inactivation rate of VIIa-11 but did not change the rate of VIIa-31,
suggesting that the allosteric effect of TF is not transmitted
sufficiently to the NH2-terminal region of these
mutants.

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Fig. 7.
Effect of carbamylation on VIIa mutants.
In A, VIIa-W (open circle), VIIa-11 (open
square), and VIIa-31 (open triangle) were each
incubated with 0.2 M KNCO at 30 °C for the periods
indicated, and the remaining amidolytic activity toward S-2288 was
measured. In B, VIIa-W-sTF (closed circle),
VIIa-11-sTF (closed square), and VIIa-31-sTF (closed
triangle) were each incubated with 0.2 M KNCO at
30 °C for the periods indicated, and the remaining amidolytic
activity toward S-2288 was measured. The concentration used for the
wild-type and the VIIa mutants was 1 µM, and the
concentration of sTF was 5 µM. In both panels, data of
plasma-derived VIIa (diamond) is equivalent to that of
VIIa-W.
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Substrate Specificity of VIIa Mutants--
To examine the
substrate specificities of the two mutant and wild-type VIIa, we tested
various chromogenic substrates that are used to measure the activities
of various clotting serine proteinases. As shown in Table
II, amidolytic activities of VIIa-31 toward the tested substrate were always higher than those of VIIa-W. It
seems likely that VIIa-31 especially may have some favor
pyroGlu structure at their P3 site.
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DISCUSSION |
The catalytic domain of the coagulation proteinases
has an active site and internal core that are similar to those of the typical serine proteinase, trypsin. Although these proteinases have a
highly homologous three-dimensional structure, they display significant
differences in specificity and catalytic activity. The serine
proteinase domain consists of 12
-strands and the loops between
them. The
-strands form a scaffold of the catalytic domain, and
their C
traces are very similar despite differences in amino acid
residues. The individual proteinases may be distinguished by surface
loops of varying lengths and compositions (26). These surface loops are
thought to participate closely in the mechanism of the enzymatic
activity. In the case of
-tryptase, the 60 loop, 99 loop, and 170 loop have a major interface for making a tetramer, and this
tetramerization is important for the expressing activity (27). The 170 loop of factor IX has some function in the interaction with factor
VIIIa (28, 29). It was also reported that replacement of some residues
in the 99 loop of factor IXa allows the enzyme to gain enhanced
catalytic activity toward synthetic peptidyl substrates (30). The 170 loop consists of 15 amino acid residues in trypsin and many vitamin
K-dependent coagulation proteinases but 20 residues in VIIa
(16, 31-35). The 170 loop in VIIa is not in direct contact with TF but
is located close to the region in which amino acid residues are
clustered for binding with TF (Figs. 1 and 2). To clarify the role of
the 170 loop in the catalytic activity and/or TF-induced conformational
transition of VIIa, we prepared two mutants of VIIa in which the 170 loops were modified.
We found that both VIIa-11 and VIIa-31 had lower affinities for sTF
(Fig. 4B), suggesting that not only the surface making contact with TF but also that the loop region of VIIa play important roles in the high affinity interaction between VIIa and TF. Because the
170 loop of VIIa is close to the TF-contacting surface, modification of
this loop may change the conformation or the orientation of the binding
site. It has been reported that reduction and alkylation of the
disulfide bond between Cys310[c168] and
Cys329[c182] of VIIa lead to a loss of binding ability
with sTF (16). Substitution of Phe328[c181] to Ser in the
170 loop of VIIa was reported to have an adverse effect on affinity for
TF (36). Alanine scanning study in the 170 loop was reported (15).
These studies also suggested the importance of certain residues in the
170 loop for the interaction with TF. As shown in Table I, the
magnitudes of the TF-mediated acceleration of amidolytic activity of
VIIa-11 (7-fold) and of VIIa-31 (2-fold) were smaller than that of
wild-type VIIa (30-fold). We speculated, therefore, that the allosteric
effect of TF is not transmitted sufficiently to the active site of
these mutants. After complex formation with TF, the
-amino group of
Ile153[c16] of wild-type VIIa was protected from
carbamylation. However, the Ile153[c16] of VIIa-11 was
only partially protected from carbamylation, and almost no protection
of the
-amino group was observed in the case of VIIa-31 (Fig. 7).
This suggested that the allosteric effect of TF is not transmitted
sufficiently to the NH2-terminal region of these mutants
either. The 170 loop of VIIa would play an important role in the
TF-induced conformational transition of VIIa by transmitting the effect
of TF to the activation domain that contains both the catalytic center
and the ion pair between Ile153[c16] and
Asp343[c194].
Modification of the 170 loop also seems to affect the catalytic
efficiency of VIIa. Although the 170 loop size of VIIa-11 is equal to
that of VIIa-31 (Fig. 2), catalytic efficiencies are different from
each other (Tables I and II). As compared with wild-type VIIa, VIIa-31
had markedly higher catalytic efficiency, whereas VIIa-11 showed
reduced activity in the absence of sTF. On the other hand, because VIIa
is assumed to exist in equilibrium between minor active and dominant
zymogen-like inactive conformational states, the observed enhancement
of the activity of VIIa after modification can be explained in two
alternative ways. One explanation is that the modification leads to a
shift of the unusual equilibrium into the active state, thus enhancing
activity. The other explanation is that the modification converts VIIa
into a trypsin-like highly efficient enzyme, without shifting the
equilibrium. Based on the results shown in Fig. 7A, the
latter explanation is more likely, because VIIa-31 continued to exist
predominantly in a zymogen-like state after modification. Although both
trypsin and VIIa-TF complex are assumed to exist in an active state,
trypsin showed 40-fold higher susceptibility to APMSF inhibition as
compared with the VIIa-W-sTF complex (Fig. 6B). Inactivation
of trypsin by lower concentrations of APMSF may reflect higher affinity
interaction between S1 subsite of trypsin and APMSF. Considering that
APMSF is a transition state analog of substrate (37) and that high affinity interaction between enzyme and substrate at transition state
provides an energy required for enzyme catalysis, the susceptibility to
APMSF inhibition probably reflects catalytic efficiency of proteinase.
Therefore, the result in Fig. 6B suggests that trypsin has
much higher catalytic efficiency than the active state of VIIa.
Susceptibility of VIIa-31 to APMSF inhibition was 17-fold higher than
that of VIIa-W (Fig. 6A). The difference in the
susceptibility between VIIa-31 and free VIIa-W was similar to that
between trypsin and VIIa-W-sTF complex. Therefore, it is possible that
the catalytic efficiency of VIIa-31 becomes close to that of trypsin if
the zymogen-like state of the mutant VIIa is converted to the active state. However, we could not examine this possibility because TF failed
to convert the zymogen-like state of VIIa-31 into its active state
(Fig. 7B). Because replacement of the 170 loop of VIIa with
that of trypsin conferred enhanced activity on VIIa, the loop region in
trypsin may be important for its high catalytic efficiency.
Although a relationship between the flexible structure of the 170 loop
of VIIa and the unusual zymogenicity of this enzyme has been suggested
(16), the results of the present study showed that modification of this
loop did not change the zymogenicity of VIIa. It is likely that
structural elements related to the zymogenicity of VIIa exist in some
regions other than the 170 loop. We are currently exploring the
structural elements to clarify the mechanism responsible for
TF-mediated acceleration of VIIa activity.
 |
ACKNOWLEDGEMENTS |
We thank Noriko Mimura, Yuki
Miyagawa, Mari Kawamoto, and Yasue Sakaguchi for excellent technical
assistance in the preparations of recombinant proteins. We also thank
Kazuhiko Tomokiyo for providing plasma-derived factor VII and VIIa. We
also thank Drs. T. Matsushita and H. Saito (Nagoya University) for
providing cDNA of human VII. We also thank to Dr. T. Hara (Daiichi
Pharmaceutical Co., Ltd., Tokyo) for providing Synthetic Xa
inhibitor (DX-9065a).
 |
FOOTNOTES |
*
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: Blood Products
Research Dept., The Chemo-Sero-Therapeutic Research Inst., Kyokushi, Kikuchi, Kumamoto 869-1298, Japan. Tel.: 81-968-37-4052; Fax: 81-968-37-3616; E-mail: mizuguchi@kaketsuken.or.jp.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M009206200
 |
ABBREVIATIONS |
The abbreviations used are:
VIIa, activated
coagulation factor VII;
VII, coagulation factor VII;
VII-W, wild-type
coagulation factor VII;
VIIa-W, activated wild-type coagulation factor
VII;
VIIa-11, activated coagulation factor VII-11;
VIIa-31, activated
coagulation factor VII-31;
X, coagulation factor X;
Xa, activated
coagulation factor X;
TF, tissue factor (full-length;
residues 1-263), sTF, soluble tissue factor (extracellular domain of TF;
residues
1-218), APMSF, p-amidinophenyl methanesulfonyl
fluoride-hydrochloride;
PCPS, mixture of phosphatidylcholine and
phosphatidylserine;
PAGE, polyacrylamide gel electrophoresis;
EGF, epidermal growth factor;
pNA, p-nitroanilide;
PEG, polyethylene glycol.
 |
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