(Received for publication, April 28, 1997)
From Coagulation factor VIIa belongs to a family of
homologous enzymes, including factors IXa and Xa and activated protein
C, composed of two epidermal growth factor-like domains located between
an N-terminal domain rich in The epidermal growth factor
(EGF)1-like domain is a
widespread building block in extracellular proteins, containing 40-50
amino acid residues with a characteristic disulfide bond pattern. This type of domain is well represented among proteins involved in blood
coagulation, and the homologous factors VII (fVII), IX, and X and
protein C contain two of them located between the membrane-proximal The interaction between factor VIIa (fVIIa) and its cell surface
receptor tissue factor (TF) is Ca2+-dependent
and involves all four domains in fVIIa (13). The importance of the
Ca2+ site in the first EGF-like domain is presently
unclear. This is in part due to the complex nature of Ca2+
binding to a total of nine sites in fVIIa (13): seven in the Gla domain
(14), one in the first EGF-like domain (7), and one in the protease
domain (15). To address this question, we have mutated Asp-46 and
Asp-63 in fVIIa to Asn, replacements that, based on studies of
Ca2+ binding to synthetic variants of the first EGF-like
domain from factor IX, should completely abolish Ca2+
binding to this site (16, 17). We present the results of the
characterization of the double mutant (D46N,D63N-fVIIa) and its TF
binding properties, as well as the functional status of its complex
with TF. Possible functions of Ca2+ binding to the first
EGF-like domain, such as structural stabilization of the hinge region
between the Gla and first EGF-like domains and proper presentation of
residues interacting with TF, are discussed.
The isolation of recombinant
fVIIa (18) and D46N,D63N-fVIIa (19) was carried out as described, and
the Gla-domainless forms were prepared by cleavage with
The wild-type fVII expression plasmid, pLN174, has
been described previously (19). For the construction of the mutant
cDNA (D46N,D63N), we used fVII cDNA inserted in the cloning
vector pBluescript II KS+ (Stratagene) as described. Mutagenesis was performed by PCR (25) with the following two primers: 5 The amidolytic activity of fVIIa and
D46N, D63N-fVIIa and their Gla-domainless counterparts (final
concentration 150 nM) was measured in the presence of 1 mM S-2288 (Chromogenix) in 20 mM Hepes, 0.1 M NaCl, pH 7.4, containing 1 mg/ml BSA and either 2 mM EDTA or 5 mM CaCl2 (26). The
total volume was 200 µl, and the absorbance at 405 nm was monitored
for 45 min.
The sTF-induced amidolytic activity enhancement was determined by
mixing 10 nM fVIIa or 10 nM D46N,D63N-fVIIa
with various concentrations of sTF (0-2 µM) at 5 mM CaCl2 and the conditions described above.
Alternatively, fVIIa or D46N,D63N-fVIIa (0-500 nM) was
added to 10 nM sTF. The absorbance at 405 nm was monitored for 30 min. The activity of fVIIa or D46N,D63N-fVIIa alone was subtracted.
To investigate differences between fVIIa and D46N,D63N-fVIIa in
Km for S-2288, we subjected 0.1-2 mM
substrate to hydrolysis by 100 nM fVIIa, 100 nM
D46N,D63N-fVIIa, 10 nM fVIIa/10 nM sTF, or 10 nM D46N,D63N-fVIIa/100 nM sTF. The hydrolysis
rate at different substrate concentrations was divided by the rate at 2 mM S-2288. The Ca2+ dependence of fVIIa and
D46N,D63N-fVIIa binding to sTF was studied at the same protein
concentrations in the Hepes buffer containing gelatin instead of
BSA.
In the factor X activation assay, fVIIa and D46N,D63N-fVIIa (final
concentration 2 pM to 5 nM) were mixed with a
fixed effective concentration of relipidated TF (2.5 pM
assuming quantitative reconstitution and that half the molecules are
oriented outwards from the vesicles) in 50 µl of 20 mM
Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM
CaCl2, and 1 mg/ml BSA. The reaction was started by adding
factor X (final concentration 175 nM), giving a final volume of 100 µl. After 5 min, the reaction was terminated by adding
50 µl buffer containing 20 mM EDTA instead of
CaCl2. The generated factor Xa was quantified by
subsequently adding 50 µl of 2 mM S-2765 (Chromogenix),
and the absorbance at 405 nm was measured after 10 min. The factor Xa
concentration was derived from a standard curve.
The conditions of
sTF immobilization and regeneration of the sTF-coated surface in the
BIAcore instrument (BIAcore AB, Uppsala, Sweden) have been described
(27). All experiments were performed at a flow rate of 5 µl/min, and
the interaction between fVIIa and sTF at 5 mM
CaCl2 was also monitored at 10 µl/min without any sign of
mass transport limitation. For other experimental details, see the
legend to Fig. 4. Association and dissociation phases lasted for 7 and
5 min, respectively. The presence of 2 µM sTF in solution
during the dissociation phase did not affect the dissociation rate,
showing that there was no rebinding of released fVIIa. Binding data was
fitted by non-linear regression to a one-site model using BIAevaluation
2.1 supplied with the instrument. In the Ca2+ dependence
study, 50 nM fVIIa or 200 nM D46N,D63N-fVIIa
was injected at different Ca2+ concentrations over 1760 resonance units of sTF and the amount of bound protein after 15 min of
association was measured.
The structures of fVIIa residues 1-85
were built using Modeler implemented in the modeling package Quanta
(Molecular Simulations, Inc., San Diego, CA). The models of the apo and
1 Ca2+ forms were based on the apo and 1 Ca2+
structures of the corresponding part of bovine factor X (entry codes
1WHE and 1WHF, respectively) (28). The model of the
Ca2+-loaded Gla domain was based on the Ca2+
structure of the prothrombin Gla domain (entry code 2PF2) (29) and used
to produce the Ca2+-loaded form of the Gla-EGF fragment.
Sequence alignment followed by a Modeler session and energy
minimization resulted in the final models. The model of fVIIa residues
1-85 in complex with sTF was made by fitting the
Ca2+-loaded structure to the known contacts between the two
proteins in the x-ray crystal structure (13), using the structure of sTF as the template (entry code 1HFT) (30).
The
single-step affinity-chromatographic purification resulted in a
homogeneous preparation of D46N,D63N-fVIIa with a yield of 1.3 mg/liter
of medium. The ability of the mutant to bind to the antibody (which
recognizes a Ca2+-dependent epitope in the Gla
domain) employed as affinity ligand indicated that it was properly
Amino acid substitutions in the isolated first EGF-like domain from
factor IX (16, 17) suggest that the two Asp
In the absence of Ca2+, D46N,D63N-fVIIa and fVIIa had
similar specific amidolytic activities and the two proteins displayed similar, low affinity binding to sTF as measured by surface plasmon resonance (data not shown). Hence, there are no
Ca2+-independent differences between them. However, the
amidolytic activity of fVIIa was stimulated by Ca2+ to a
2-3-fold higher level than that of D46N,D63N-fVIIa (Fig. 2), suggesting that a functional
Ca2+ site in the EGF-like domain is required for full
activity. Interestingly, the Gla-domainless forms of fVIIa and
D46N,D63N-fVIIa were similarly stimulated by Ca2+. The data
suggest that the presence of the Gla domain increases the activity in
the case of a functional Ca2+ site in the EGF-like
domain.
The ability of sTF to enhance the activities of fVIIa and
D46N,D63N-fVIIa allows the use of an amidolytic assay for the
estimation of their affinities for the cofactor. In our system, the
amidolytic activity of fVIIa was maximally enhanced at sTF
concentrations above 50 nM and half-maximum was observed
between 5 and 10 nM (Fig. 3).
The data yielded a Kd of 2.0 ± 0.7 nM. D46N,D63N-fVIIa displayed half-maximum around 100 nM sTF and was fully stimulated at sTF concentrations above
1 µM. A Kd of 54 ± 8 nM was calculated for the mutant, i.e. 25-fold
higher than that of fVIIa. Moreover, the mutant displayed lower
activity than fVIIa at saturating concentrations of sTF, similar to
what we observed in the absence of sTF. Almost identical results were
seen when titrating a constant amount of sTF with fVIIa or
D46N,D63N-fVIIa (Fig. 3). However, the sTF-independent activity
prevented us from going higher than 500 nM in
D46N,D63N-fVIIa. To elucidate the reason for the lower amidolytic
activity of D46N,D63N-fVIIa-sTF, substrate hydrolysis was measured at
varying concentrations of S-2288. Although the Km
for S-2288 appears to be high and could not be accurately determined,
the relative rate of hydrolysis obtained with fVIIa-sTF and
D46N,D63N-fVIIa-sTF was identical when compared with the respective rate at the highest substrate concentration (which was lower for D46N,D63N-fVIIa-sTF). This indicates that fVIIa-sTF and
D46N,D63N-fVIIa-sTF have similar Km values for
S-2288 and that the difference in catalytic efficiency appears to be
due to a lower kcat of the mutant in complex
with sTF. The Ca2+ dependence of fVIIa and D46N,D63N-fVIIa
binding to sTF was also investigated. At our protein concentrations,
low levels of Ca2+ (0.1-0.2 mM) resulted in a
larger absolute increase in fVIIa-sTF activity, due to the higher
affinity of this complex, than in D46N,D63N-fVIIa-sTF activity.
Nevertheless, these Ca2+ concentrations were sufficient to
stimulate D46N,D63N-fVIIa-sTF, but not fVIIa-sTF, half-maximally (not
shown). This suggests that the Ca2+ dependence of
D46N,D63N-fVIIa was mediated solely by the Ca2+ site in the
protease domain and that the Gla-EGF region has no influence in the
absence of the Ca2+ site in the first EGF-like domain.
The interaction between D46N,D63N-fVIIa and sTF was then compared with
that between fVIIa and sTF in a BIAcore instrument. The real-time
biosensor analysis of the fVIIa-sTF interaction yielded a
Kd of 4.6 nM, in agreement with previous
studies (19, 27, 33). The mutant D46N,D63N-fVIIa associated about 3-fold slower with sTF (1.1 × 105
M Wild-type fVIIa and D46N,D63N-fVIIa also had different affinities for
phospholipid-embedded TF. The Kd values were estimated to be 7.5 ± 0.4 pM for fVIIa, in good
agreement with earlier studies (14, 34), and 158 ± 16 pM for D46N,D63N-fVIIa (Fig.
5). In our system, with a fixed
concentration of TF, maximal factor X activation was observed above 200 pM fVIIa and 2 nM D46N,D63N-fVIIa. There was no
difference in the maximal rate of factor Xa generation between fVIIa-TF
and D46N,D63N-fVIIa-TF. Both complexes catalyzed the generation of
approximately 300 mol of factor Xa/mol of TF/min, assuming that all TF
molecules were successfully reconstituted into phospholipid vesicles
and that 50% of them were functionally available on the outside of the
vesicles.
Cleavage sites in the hydrophobic cluster in the
C-terminal part of the Gla domain have been exploited for more than a
decade to produce Gla-domainless clotting factors. Since the
Ca2+ binding site in the first EGF-like domain is located
close to the hydrophobic stack (or C-terminal helix of the Gla domain) and might affect the conformation of this hinge region between the Gla
and first EGF-like domains, we examined if the mutations had any effect
on the rate of enzymatic cleavage. fVIIa was rapidly cleaved in the
hydrophobic stack by chymotrypsin and cathepsin G in the absence of
Ca2+ as monitored by SDS-PAGE, and considerable protection
against cleavage by 1.5 mM Ca2+ (approximately
plasma concentration) was observed (data not shown). However, 0.3 mM Ca2+, which should give more than 70%
saturation of the Ca2+ site in the first EGF-like domain of
fVIIa, did not result in any protection against degradation. Identical
results of the chymotrypsin-catalyzed cleavage were obtained with
D46N,D63N-fVIIa, whereas cathepsin G was unable to cleave the mutant
under any of our conditions. Hence, no evidence for a specific
protective effect of an intact Ca2+ binding site in the
EGF-like domain was obtained.
The first EGF-like domain of fVIIa is known from biochemical
studies (35-37) as well as from the x-ray crystallographic structure of the fVIIa-sTF complex (13) to interact with TF, but the importance of the Ca2+ site in this domain is unclear. Based on
Ca2+ structures of EGF-like domains (8, 9) and their
homology and high degree of sequence identity with the corresponding
domain in fVIIa, the Asp residues in positions 46 and 63 in fVIIa were replaced by Asn to abolish and investigate the role of Ca2+
binding to the first EGF-like domain. D46N,D63N-fVIIa was shown not to
bind Ca2+ in the EGF-like domain, and no structural effects
were observed when the mutations were put into our model of fVIIa (22).
D46N,D63N-fVIIa and fVIIa displayed similar amidolytic activity and
similar, poor binding to sTF in the absence of Ca2+,
indicating that the proteins only differed when in their
Ca2+ conformations.
A 2-3-fold difference in specific amidolytic activity between fVIIa
and D46N,D63N-fVIIa was observed in the presence of Ca2+,
both in the absence of and in complex with sTF. Thus the stimulatory effect of sTF, which is known primarily to result from an increase in
kcat (31, 38), appears to be the same on both
forms of fVIIa. However, the reasons for the impaired activity in the
absence and presence of sTF might be different. The lower amidolytic
activity of D46N,D63N-fVIIa is in agreement with a study of factor IXa mutants in which the residue corresponding to Asp-63 was replaced by
Lys, Glu, or Val (39). Ca2+ binding to the first EGF-like
domain appears to be involved in the Ca2+-induced increase
in amidolytic activity, perhaps through an interaction between a
Ca2+-dependent structure in the Gla-EGF region
and the protease domain. The similar maximal factor X activation rates
by fVIIa and D46N,D63N-fVIIa in complex with TF suggest that factor X
and/or the phospholipid membrane is able to stabilize the optimally
active conformation of both complexes (40).
We find that D46N,D63N-fVIIa binds both sTF and lipidated TF with
approximately 20-fold higher Kd than does wild-type fVIIa, showing that Ca2+ binding to the first EGF-like
domain is essential for optimal cofactor binding. The affinity of
D46N,D63N-fVIIa for sTF is similar to that of the most defective Ala
mutant (I69A) in the EGF-like domain (41). In addition, the kinetics of
the interaction between D46N,D63N-fVIIa and sTF closely resemble those
of the interaction between R79Q-fVIIa and sTF (33). Arg-79 is a key
TF-interactive residue located in the C-terminal part of the first
EGF-like domain (13). However, in contrast to D46N,D63N-fVIIa, this
mutant displayed a lower rate of factor X activation than fVIIa but
similar amidolytic activity. The replacement of Asp-47 or Asp-64 in the
first EGF-like domain from factor IX (corresponding to Asp-46 and
Asp-63 in fVIIa) by Asn is known to abolish or severely impair
Ca2+ binding (16, 17). Mutations at these positions result
in very low biological activity of factor IXa causing hemophilia B (39,
42-47). The primary reason for the low activity appears to be a
defective interaction with factor VIIIa. The effects of mutating
Ca2+-coordinating residues on the fVIIa-TF and factor
IXa-factor VIIIa interactions suggest that Ca2+ binding to
the first EGF-like domain in the vitamin K-dependent clotting factors is important for optimal recognition of their cofactors.
Recently it was found, using a Gla-EGF fragment from factor X, that the
relative orientation of the Gla and first EGF-like domains was altered
upon Ca2+ binding to the latter domain (28). This
reorientation of domains most likely causes the increase in intrinsic
fluorescence at low Ca2+ concentrations measurable with
Gla-EGF fragments from factors VII, IX, X, and protein C (11, 26, 48,
49). These proteins all have a conserved Trp residue in the hydrophobic
stack hinge region (position 41 in fVIIa) between the Gla and first
EGF-like domains. This strongly suggests that the folding of the Gla
domain over the Ca2+ site in the first EGF-like domain is a
general feature of these proteins (Fig.
6). In addition, calorimetric and
chromatographic data supportive of a Ca2+-induced
interaction between the Gla and first EGF-like domains in factor IX
have been presented (50, 51) and a
Ca2+-dependent interaction stabilizing the
disulfide bonds in the two domains has been demonstrated in factor X
(52). In contrast, Gla-EGF from protein Z, which contains the Trp
residue in the hydrophobic stack but not the Ca2+ binding
site in the EGF-like domain, does not exhibit any fluorescence increase
(53). We therefore postulate that Ca2+ binding to
D46N,D63N-fVIIa, occurring outside the first EGF-like domain, has no
influence on the relative orientation of the Gla and first EGF-like
domains, whereas Ca2+ binding to fVIIa may affect the
domain orientation.
What is the exact role of the Ca2+ site in the first
EGF-like domain of fVIIa? The Ca2+-induced orientation of
the Gla and first EGF-like domains in Gla-EGF from factor X (28),
likely to be present in fVIIa but not in D46N,D63N-fVIIa, is not found
in fVIIa in complex with sTF (Fig. 6) (13). In the complex, the
orientation of these domains is closer to that seen in the apo
structure of the factor X Gla-EGF fragment. Hence, Ca2+
binding to the first EGF-like domain in fVIIa does not obviously facilitate the interaction with TF, but it cannot be ruled out that the
angle between the Gla and EGF-like domains is critical in the docking
of fVIIa with TF. Subsequently, the Gla-EGF region may be forced into
another conformation by TF, changing the structure of the hydrophobic
stack hinge region as inferred from CD experiments (22). In a
physiological environment, the membrane surface may also be involved in
this reorientation of domains through its interaction with the Gla
domain. A recent energy transfer study suggests that the distance
between the active site of fVIIa and the membrane surface decreases
upon tissue factor binding (54). Considering that the Gla-EGF part of
fVIIa appears to adopt a more upright conformation when bound to TF,
this implies a dramatic reorientation of the protease domain relative
to the membrane upon association with TF. However, rotational movement
of the protease domain could explain at least part of the energy
transfer change observed upon TF binding. Ca2+ binding to
the first EGF-like domain in fVIIa definitely decreases the flexibility
around the hydrophobic stack and thereby probably facilitates TF
binding. It is probably also pivotal for optimal positioning of
TF-interactive residues both in the first EGF-like domain and in the
Gla domain. We also speculated that Ca2+ binding to the
first EGF-like domain might be important for fVIIa activity in
vivo by protecting it from proteolytic degradation in the
hydrophobic stack (55-57), a rapid process in the absence of
Ca2+. However, our conclusion is that fVIIa and
D46N,D63N-fVIIa are equally susceptible to enzymatic removal of the Gla
domain at any given Ca2+ concentration. This supports the
idea that the Ca2+-induced protection against proteolysis
in the hydrophobic stack is mediated by Gla-dependent sites
(53).
We thank Lone Odborg, Annette Danielsen, and
Lone Walsøe Therkelsen for expert technical assistance and Ingrid
Dahlqvist, Lund University, for amino acid analyses.
Vessel Wall Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-carboxyglutamic acid residues and a
C-terminal serine protease domain. The first epidermal growth factor-like domain in factor VIIa contains a Ca2+
binding site, the function of which is largely unknown. Site-directed mutagenesis of two Ca2+-liganding Asp residues in this
domain abolished Ca2+ binding and resulted in a 2-3-fold
decrease in amidolytic activity at optimal Ca2+
concentrations. The lower amidolytic activity persisted in complex with
soluble tissue factor, apparently due to a lower
kcat of the mutant factor VIIa. Mutant and
wild-type factor VIIa bound to lipidated tissue factor were equally
efficient activators of factor X. The dissociation constants, derived
from amidolytic activity and surface plasmon resonance measurements,
were 2-5 nM and 50-60 nM for the interactions
between wild-type and mutant factor VIIa, respectively, and soluble
tissue factor. Binding to lipidated tissue factor was characterized by
dissociation constants of 7.5 pM for factor VIIa and 160 pM for the factor VIIa mutant. Hence, a functional
Ca2+ binding site in the first epidermal growth factor-like
domain added 7-8 kJ/mol to the total binding energy of the interaction with both lipidated and soluble tissue factor.
-carboxyglutamic acid (Gla)-containing domain and the serine protease domain (1, 2). The first EGF-like domain in these proteins
harbors one Ca2+ binding site (3-7). The side chains of
two Asp, one of which may be
-hydroxylated, and one Gln residue and
two backbone carbonyl oxygens have been identified as Ca2+
ligands in the first EGF-like domain of factors IX and X (8, 9) and the
ligands are conserved in fVII and protein C. The affinity of this site
in factor X is represented by a dissociation constant
(Kd) of about 0.1 mM in the intact
protein (10), in a fragment containing the Gla and first EGF-like
domains (11), and in a Gla-EGF fragment from which the N-terminal 28 amino acid residues have been deleted (12). Hence, under physiological conditions, the bound Ca2+ ion and the resulting
Ca2+-dependent structure can be considered as a
structural determinant of the protein. In contrast, Ca2+
binding to the isolated EGF-like domain has a 20-fold higher Kd (4).
Proteins and Standard Methods
-chymotrypsin (20) followed by purification on a column of Q
Sepharose (Pharmacia Biotech Inc.). The protein concentrations were
determined using a fVII enzyme-linked immunosorbent assay and by
absorbance measurements using an absorption coefficient
(A1 cm1% at 280 nm) of 13.2. Amino acid
analyses were performed as described (4). The resistance of fVIIa and
D46N,D63N-fVIIa against proteolytic cleavage was tested by incubation
with cathepsin G (ICN Biomedicals, Inc.) and
-chymotrypsin (Sigma),
using 0.1% (w/w) enzyme, in 50 mM Tris, 0.1 M
NaCl, pH 8.0, containing 2 mM EDTA, 0.3 mM
CaCl2, or 1.5 mM CaCl2. The
production and isolation of soluble TF (sTF) has been described (21,
22), and the concentration was estimated using an absorption
coefficient of 15.0. Full-length TF was purchased from American
Diagnostica Inc. and relipidated as described (23) using 75%
phosphatidylcholine, 25% phosphatidylserine (Sigma). Factors X and Xa
were from Enzyme Research Laboratories. SDS-PAGE was run in 12% gels
using ingredients from Serva and Bio-Rad (24). Agarose gel
electrophoresis (0.8% gels) was run in 75 mM Tris, 25 mM 5,5-diethylbarbital, pH 8.6, containing either
10 mM CaCl2 or 2 mM
EDTA, using agarose purchased from Litex.
-CCC ATT CTG
GCA TGG ACT TGA GGC ACA CTG GTC CCC ATT ACT GTA AGA-3
and 5
-GCC TCA
AGT CCA TGC CAG AAT GGG GGC TCC TGC AAG AAC CAG CTC CAG-3
. In brief,
the template plasmid was denatured by treatment with NaOH followed by
PCR with Pwo polymerase (Boehringer Mannheim). Escherichia
coli were transformed with the resulting PCR product, and clones
were screened for the presence of the mutations. The sequence was
verified between the BamHI site before the initiation codon
and the XbaI site (base number 534 of the fVII coding
sequence) for one clone containing the desired mutation. The
BamHI-XbaI fragment was removed from the plasmid
pLN174 and replaced with the corresponding fragment from the mutant
plasmid. The presence of the mutations in the final expression
construct was verified by sequencing. The baby hamster kidney cell line
BHK570 (ATCC CRL 1632) was used for transfection and expression of
mutant fVII protein as described earlier (19). Cell culture materials
were from Life Technologies, Inc.
Fig. 4.
Sensorgrams for the interactions between
wild-type fVIIa (top curve) and D46N,D63N-fVIIa
(bottom curve) and immobilized sTF. 50 nM
of analyte in 20 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl2, and 0.02%
Tween 80, was injected over 1620 resonance units (RU) of
sTF. The signals obtained when injecting the samples over a blank
biosensor surface have been subtracted.
[View Larger Version of this Image (14K GIF file)]
Purification and Characterization of D46N,D63N-fVIIa
-carboxylated (26), and amino acid analysis showed 9.7 mol of
Gla/mol of D46N,D63N-fVIIa (the calculated number according to sequence
data is 10). SDS-PAGE showed that the isolated mutant was in the
two-chain, activated form with the bands corresponding to the heavy and
light chains of fVIIa (data not shown). The conversion from
single-chain D46N,D63N-fVII appeared to occur during purification. The
starting material contained no amidolytic activity enhancable by the
addition of sTF, whereas the activity of the purified protein could be
stimulated by sTF (31, 32).
Asn mutations in
D46N,D63N-fVIIa should abolish Ca2+ binding to the first
EGF-like domain. However, it is known that the Ca2+ site in
the first EGF-like domain has a higher affinity in the intact protein
due to the presence of additional ligands and/or increased structural
stability. To confirm that the mutations had eliminated
Ca2+ binding, the electrophoretic mobility of
Gla-domainless mutant and wild-type fVIIa was analyzed on agarose gels
in the presence and absence of Ca2+. Mutant and wild-type
Gla-domainless fVIIa should contain one and two Ca2+
binding sites, respectively. The removal of two negative charges in the
mutant reduced its anodal migration rate in the absence of
Ca2+ compared with that of fVIIa (Fig.
1). In the presence of 10 mM Ca2+, the mobility of both proteins was further reduced.
The Ca2+-induced mobility shift was twice as big for fVIIa
compared with D46N,D63N-fVIIa, indicating that the mutagenesis had
successfully abolished Ca2+ binding to the EGF-like domain.
The extra Ca2+ bound to fVIIa compensated for its two
additional negative charges, resulting in identical mobilities of the
two fVIIa forms in the presence of Ca2+.
Fig. 1.
Agarose gel electrophoresis of Gla-domainless
fVIIa and Gla-domainless D46N,D63N-fVIIa. Des(1-44)-fVIIa (8 µg, lanes 1 and 3) and
des(1-44)-D46N,D63N-fVIIa (8 µg, lanes 2 and
4) were analyzed in the presence of 2 mM EDTA
(lanes 1 and 2) or 10 mM Ca2+ (lanes 3 and 4) with
1-antitrypsin, whose mobility is not affected by
Ca2+, as a reference. The positions of the sample
application slits (t0) and
1-antitrypsin (
1-AT) are shown to the right.
[View Larger Version of this Image (68K GIF file)]
Fig. 2.
The amidolytic activity of fVIIa and
D46N,D63N-fVIIa. The activities of the proteins before and after
removal of the Gla domain was measured in the absence (filled
bars) and presence of Ca2+ (open bars) as
described under "Experimental Procedures." The activity was first
measured in the presence of 2 mM EDTA, whereafter CaCl2 was added from a 0.5 M stock
solution to a final concentration of 5 mM and the
measurement continued. The activity is shown as the increase in
absorbance at 405 nm (milliunits) after 45 min of incubation.
[View Larger Version of this Image (10K GIF file)]
Fig. 3.
sTF-induced amidolytic activity of fVIIa and
D46N, D63N-fVIIa. 10 nM fVIIa () or 10 nM D46N,D63N-fVIIa (
) was incubated with the indicated
concentrations of sTF; alternatively, 10 nM sTF was
incubated with increasing concentrations of fVIIa (
) or
D46N,D63N-fVIIa (
) and the hydrolysis of S-2288 was measured. The
results are given as percent of the maximal activity of fVIIa-sTF. At
each condition, the substrate conversion in the absence of sTF was
subtracted. Each point represents the mean of three experiments.
[View Larger Version of this Image (22K GIF file)]
1 s
1) and dissociated about
3.5-fold faster from sTF (5.9 × 10
3
s
1) which resulted in a Kd of 54 nM (Fig. 4). The
Kd values are in agreement with those derived from
the sTF-dependent amidolytic activity assay. In addition,
the Ca2+ dependence of the binding of fVIIa and
D46N,D63N-fVIIa to sTF in the BIAcore closely resembled the
Ca2+ dependence of fVIIa-sTF and D46N,D63N-fVIIa-sTF
amidolytic activity (data not shown).
Fig. 5.
Factor X activation by fVIIa-TF and
D46N,D63N-fVIIa-TF. The indicated concentrations of fVIIa () or
D46N,D63N-fVIIa (
) were mixed with relipidated TF and factor X (see
"Experimental Procedures" for details). The amount of factor Xa
generated was quantified employing S-2765 amidolysis and given as
percent of maximal activation. Each point represents the mean of two
experiments. The data were fitted to a one-site model using GraFit 3.0 (Erithacus Software, Ltd., Staines, Middlesex, United Kingdom).
[View Larger Version of this Image (23K GIF file)]
Fig. 6.
Models of the Gla and first EGF-like domains
(residues 1-85) of fVIIa. The structures of the apo
(red), 1 Ca2+ (white), and sTF-bound
Ca2+-loaded (8 Ca2+, green) forms of
residues 1-85 are shown. The N-terminal Ala-1 and C-terminal Lys-85
are denoted. The structures are compared after optimally overlaying the
three helices in the Gla domain. The apo and 1 Ca2+
structures are based on the NMR structures of the corresponding region
from bovine factor X (28). Subsequently, the apo-Gla domain in the 1 Ca2+ structure was replaced by the Ca2+-loaded
Gla domain based on the x-ray crystallographic structure of bovine
prothrombin fragment 1 (29). The sTF-bound Ca2+-loaded
structure is obtained by fitting the Ca2+-loaded structure
to the known interactions between fVIIa residues 1-85 and sTF (13)
using the x-ray crystallographic structure of sTF as the template (30).
For comparison of the three different relative domain orientations, the
angle between one vector going from the C carbon of
residue 38 (in the third helix of the Gla domain) to the
C
carbon of residue 18 (in the first helix of the Gla
domain) and another vector going from the C
carbon of
residue 38 to the C
carbon of residue 72 (in the
C-terminal end of the second
-strand of the EGF-like domain)
was measured. It was 43°, 74°, and 99° for the 1 Ca2+, apo, and sTF-bound Ca2+-loaded
structure, respectively.
[View Larger Version of this Image (149K GIF file)]
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1
The abbreviations used are: EGF, epidermal
growth factor; fVIIa, coagulation factor VIIa; D46N,D63N-fVIIa, fVIIa
mutant where Asp-46 and Asp-63 have been replaced by Asn residues;
fVII, zymogen form of fVIIa; TF, tissue factor; sTF, soluble tissue
factor (residues 1-219); Gla, -carboxyglutamic acid; PAGE,
polyacrylamide gel electrophoresis; PCR. polymerase chain reaction;
BSA, bovine serum albumin.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.