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J Biol Chem, Vol. 274, Issue 26, 18477-18486, June 25, 1999
From the Departments of Medicine, Pathology, and Biochemistry,
Saint Louis University School of Medicine,
St. Louis, Missouri 63104
Previous studies revealed that cleavage at
Arg-318-Ser-319 in the protease domain autolysis loop of factor IXa
results in its diminished binding to factor VIIIa. Now, we have
investigated the importance of adjacent surface-exposed helix 330-338
(162-170 in chymotrypsin numbering) of IXa in its interaction with
VIIIa. IXWT, eight point mutants mostly based on
hemophilia B patients, and a replacement mutant (IXhelixVII
in which helix 330-338 is replaced by that of factor VII) were
expressed, purified, and characterized. Each mutant was activated
normally by VIIa-tissue factor-Ca2+ or
XIa-Ca2+. However, in both the presence and absence of
phospholipid, interaction of each activated mutant with VIIIa was
impaired. The role of IXa EGF1 domain in binding to VIIIa was also
examined. Two mutants (IXQ50P and IXPCEGF1, in
which EGF1 domain is replaced by that of protein C) were used.
Strikingly, interactions of the activated EGF1 mutants with VIIIa were
impaired only in the presence of phospholipid. We conclude
that helix 330 in IXa provides a critical binding site for VIIIa and
that the EGF1 domain in this context primarily serves to correctly
position the protease domain above the phospholipid surface for optimal
interaction with VIIIa.
A common cause of abnormal bleeding is either the deficiency of
factor VIII (hemophilia A) or factor IX (hemophilia B). Factor IX is a
vitamin K-dependent protein, and during physiologic
clotting it is activated to a two-chain, disulfide-linked IXa molecule by VIIa/Ca2+/tissue factor
(TF)1 and by
XIa/Ca2+ (1). The domain organization of factor IXa is
similar to those of the other two enzymes (factors VIIa and Xa)
involved in the TF-induced coagulation and to that of an anticoagulant
enzyme termed activated protein C (APC) (1). The light chain of IXa consists of an amino-terminal Based upon the crystal structure of the Gla domain of factor VIIa (4)
and the Ca2+-binding properties of factor X (5), it would
appear that this domain in IXa possesses several low to intermediate
affinity Ca2+-binding sites. In addition, the EGF1 and the
protease domain each possess one high affinity Ca2+-binding
site (6, 7). The Ca2+-loaded conformer of the Gla domain
binds to PL vesicles (8) and the EGF1 domain of IX is required for its
activation by VIIa/Ca2+/TF (9). Further, Ca2+
binding to the EGF1 domain has been reported to promote enzyme activity
and factor VIIIa binding (10). The role of EGF2 domain is not clear but
may be involved in binding to platelets and in factor X activation
(11). Finally, the protease domain is thought to play a primary role in
binding to factor VIIIa (12-14).
Recently, we have shown that mutations in the protease domain
Ca2+-binding ligands decrease the affinity of factor IXa
for factor VIIIa by ~15-fold and that proteolysis at Arg-318-Ser-319
(150-151)2 in the autolysis
loop results in a further decrease in this interaction by ~8-fold
(16). Since residues in the protease domain Ca2+-binding
loop as well as those in the autolysis loop may not directly participate in binding to factor VIIIa (2, 17), we postulated that
Ca2+ binding to the protease domain and integrity of the
autolysis loop stabilize yet another region in this domain of factor
IXa that directly interacts with factor VIIIa (16). Based upon this, we
opted to examine the role of IXa helix 330-338 (162-170), which is
adjacent to the autolysis loop in its interaction with VIIIa. All known
sequences from different species in this surface-exposed helix of IXa
are identical (15) and point mutations in eight of the nine residues in
this region cause hemophilia B (18). Herein, we provide extensive data
which indicate that this helix (residues 330-338) in IXa constitutes a
critical binding site for VIIIa and that the reduced biologic activity
of helix 330 (162) mutants in hemophilia B patients stems from their
substantially reduced affinity for factor VIIIa. We also present data
which indicate that a role of the EGF1 domain in this context is to correctly position the protease domain above the PL surface for optimal
interaction of factor IXa with factor VIIIa. Initial accounts of this
work have been presented in abstract form (19, 20).
Proteins and
Reagents--
Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide
(S-2222) was purchased from Helena Laboratories.
Dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-ck) was obtained from
Calbiochem. Phosphatidylcholine, phosphatidylserine, recombinant
hirudin, and fatty acid-free bovine serum albumin (BSA) were obtained
from Sigma. Factor IX- and factor VIII-deficient plasmas were purchased
from George King Biomedicals, and activated partial thromboplastin time
reagent was obtained from Diagnostica Stago. Normal human plasma factor
IX (IXNP) and factor X were isolated as described (15), and
factor Xa was prepared as outlined (21). Purified human factor XIa,
protein C, APC, and Coagulation Assay of Factor IX and Factor IXa--
Factor IX and
factor IXa activities were measured in a one-stage assay with automated
activated partial thromboplastin time reagent as described (24).
SDS-Gel Electrophoresis--
SDS-gel electrophoresis was
performed using the Laemmli buffer system (25). The acrylamide
concentration was 12%, and the gels were stained with Coomassie
Brilliant Blue dye.
Amino Acid Sequencing and Gla Analysis--
Gla and amino acid
sequence analysis were performed by Commonwealth Biotechnologies, Inc.,
Richmond, VA. Automated Edman degradation of each factor IX protein
(~0.5 nmol) was performed using an Applied Biosystems gas phase
sequencer. Gla analysis of each sample was performed by alkaline
hydrolysis followed by high pressure liquid chromatography analysis.
The amount of Gla was quantitated based upon the 46 residues of Asp and
Asn present per mole of factor IX (26).
Construction, Expression, and Purification of Recombinant Factor
IX Proteins--
The pRc/CMV vector (Invitrogen) was used for
expression of wild-type and each mutant factor IX. In each case
HindIII and XbaI sites in the multiple cloning
sites of the vector were used for ligation of the DNA. Construction of
the wild-type factor IX (IXWT) and that of
IXPCEGF1, in which residues 52-85 in the EGF1 domain of
IXWT have been replaced by the residues 51-92 from the
corresponding domain of protein C, have been described (27). Point
mutations in helix 330 (162) of protease domain of factor IX were
introduced using the fragment elongation method of Nelson and Long
(28), as described previously for IXQ50P (9). In each case
the mutant primer was based upon factor IX gene sequence (26) and
corresponded to six codons (18 bases) with a mutant base at the desired
position involving the third codon. The base substitution for each
point mutant was: L330I (CTT Molecular Modeling--
A modeled structure of human factor IXa
was obtained using a homology model building approach described
previously (15). The starting template used was the structure of
porcine factor IXa (Ref. 2; Protein Data Bank code 1PFX). Inasmuch as
the model structure of human factor IXa, for all purposes as it relates to this paper, was the same as porcine factor IXa and the residues involved at the mutational sites are identical between the two proteins
(15), we have used the x-ray structure of porcine factor IXa for
analysis of our data. The model of IXaPCEGFI protein was constructed by replacing the EGFI domain of factor IXa with that of APC
(Ref. 31; code 1AUT) and is described elsewhere (32).
Preparation of DEGR-ck Inhibited Various Factor IXa
Proteins--
IXNP, IXWT, and each mutant
factor IX at 200 µg/ml was activated by factor XIa (2 µg/ml) for 90 min. The buffer used was TBS, pH 7.4 (0.05 M Tris, 0.15 M NaCl, pH 7.4), containing 5 mM
Ca2+. SDS-gel electrophoretic analysis revealed full
activation to factor IXa Activation of Factor VIII by IIa--
Activation of factor VIII
was performed as outlined previously (16). Briefly, 28 nM
(40 units/ml) of factor VIII was incubated with 0.2 nM IIa
in TBS/BSA, pH 7.4 (TBS containing 1 mg/ml BSA), containing 5 mM CaCl2. In initial experiments, we
established that the maximal increase (~10-fold) in procoagulant
activity occurred at 2 min, after which it steadily declined (16).
Thus, at 2 min we routinely added recombinant hirudin (2.2 nM, final concentration) to inhibit IIa and the preparation
was used immediately.
Measurements of Factor X Activation--
Factor VIIIa
potentiation of factor X activation by IXaNP,
IXaWT, and each activated mutant was measured both in the
presence and absence of PL. The buffer used was TBS/BSA, pH 7.4, and
the reaction volume in each case was 50 µl. The activation was
carried out at 37 °C for different time periods, at which point each
reaction mixture received 1 µl of 0.5 M EDTA to stop
further generation of factor Xa. A 40-µl aliquot was then added to a
0.1-ml quartz cuvette containing S-2222 in 75 µl of TBS/BSA, pH 7.4. The final concentration of S-2222 was 100 µM. The
p-nitroaniline release was measured continuously
(
Factor X activation measurements were made under four sets of different
conditions. 1) Ca2+ and PL were present. In this system,
activation was carried out for 5-15 min and the concentration of each
IXa protein was 20 nM, PL was 25 µM, and
factor X was 100 nM. 2) Ca2+, PL, and factor
VIIIa were present. In this system, activation was carried out for
15-120 s and the concentration of each IXa protein was 0.5 nM, PL was 10 µM, VIIIa was 0.07 nM, and factor X was 15 nM. 3) Only
Ca2+ was present. In this system, activation was carried
out for 2-20 min, and concentration of each IXa protein was 400 nM and factor X was 1 µM. 4) Ca2+
and VIIIa were present. In this system, activation was carried out for
15-120 s and the concentration of each IXa protein was 2 µM, VIIIa was 14 nM, and factor X was 400 nM.
In all of the above factor X activation experiments, the incubation
times chosen are those in which factor Xa generated was always less
than 10 nM. This precautionary measure was taken to prevent
activation of factor X by the generated factor Xa (16, 34). Further, in
those incubation mixtures that contained factor VIIIa, we also
performed control experiments in which factor VIIIa was omitted. The
rates of factor X activation in these control experiments were <10%
of the experimental values in the presence of factor VIIIa and were
subtracted prior to analysis of the data. For reactions done in the
presence of Ca2+ and PL, optimal concentrations of PL were
determined. Rates of factor X activation versus PL showed a
bell-shaped curve with a broad optima between 20 and 40 µM PL.3 The
rate increased linearly from 0-20 µM, and after 40 µM it showed a gradual decrease. Therefore, PL
concentration in the absence of factor VIIIa was fixed at 25 µM. In the presence of factor VIIIa, 10 µM
PL was used based upon previous observations (16, 35). The
concentrations of factor X selected for each set of reaction conditions
are those which are below or at the Km values (35,
36).3 In this region of the Michaelis-Menten curve, the
rate of formation of factor Xa is proportional to the substrate factor
X and therefore to the affinity of the factor IXa enzyme for factor X.
Determinations of Kd(app). Values for the
Interaction of Factor VIIIa with Various Active Site-blocked Factor IXa
Proteins--
Apparent Kd values of factor
VIIIa binding to various active site-blocked factor IXa proteins were
determined from inhibition experiments as detailed previously (16).
Briefly, reaction mixtures (50 µl) in the presence of PL
contained 0.2 nM factor IXaWT, 0.48 µM factor X, 0.07 nM factor VIIIa, 5 mM Ca2+, 10 µM PL, and varying
concentrations of DEGR-IXa proteins. Reaction mixtures in the absence
of PL contained 0.1 µM factor IXaWT, 2 µM factor X, 14 nM factor VIIIa, 5 mM Ca2+, and varying concentrations of DEGR-IXa
proteins. Reactions were carried out for 2 min, and the rates of factor
X activation were measured as outlined above.
In these experimental conditions, both in the presence and absence of
PL, factor IXaWT concentrations are below the
EC50 values at the factor X concentrations used. Further,
in each case <10% of factor IXa is bound to factor VIIIa in the
absence of the competitor and no measurable rates of activation of
factor X were observed under these conditions in the absence of added
factor VIIIa. For obtaining IC50 values (concentration of
DEGR-IXa yielding 50% inhibition), the data were fitted to the
IC50 four-parameter logistic equation of Halfman (37) given
below.
Purification, Neutralization of an Anti-factor IX mAb by the Mutant
Proteins--
The ability of the IX mutant proteins to bind to a mAb
(12), which interferes with the interaction of IXa and VIIIa, was studied using a coagulant based assay as described previously (40). For
mutants that possessed Activation of Protease Domain Factor IX Mutants by VIIa/TF and by
Factor Xla/Ca2+--
The rates of activation of each
protease domain mutant performed under conditions described previously
(16) either by VIIa/TF/Ca2+ or by factor
Xla/Ca2+ were similar to that of IXNP as
analyzed by SDS-gel electrophoresis. The 90-min activation sample of
IXWT and each mutant is shown in Fig. 1B. As
compared with IXaNP, coagulant activity of Xla-activated IXaWT was ~95%, of IXaL330I was ~7%, of
IXaV331A was ~6%, of IXaD332Y was ~2%, of
IXaR333L was ~0.4%, of IXaR333Q was
~0.6%, of IXaT335A was ~35%, of IXaL337I
was 0.8%, of IXaR338Q was ~80%, and of
IXahelixVII was not measurable.
Activation of Factor X by Various IXa Protease Domain
Mutants--
In this section, we examined the ability of each
activated mutant to activate factor X in the presence and absence of PL
and factor VIIIa. The data obtained in the presence of Ca2+
and PL are shown in Fig. 2A.
One should note that the concentration of factor X used in this system
was 100 nM, which is slightly less than the
Km value (36) under these conditions. In this
system, factor IXaWT and each mutant including
IXahelixVII activated factor X at similar rates. The rate
of factor Xa generation in each case was 0.1 nM/min, which
is very close to an expected rate (0.12 nM/min) at an
enzyme concentration of 20 nM used in our system. One may
infer from these data that, in the IXa-Ca2+-PL system, each
mutant interacts with factor X normally and that the active site of
each mutant is not impaired.
Next, we studied the activation of factor X in a complete intrinsic
Tenase system (IXa, Ca2+, PL, VIIIa). When a limiting
concentration of factor VIIIa (70 pM) at 0.5 nM
IXa and 15 nM factor X was employed, the rate of activation
by each IXa mutant with the exception of IXaR338Q was significantly reduced. These data are provided in Fig. 2B
and summarized in Table I. Considering an
EC50 (functional Kd of IXa:VIIIa
interaction) value of 1.2 nM (16), a
kcat value of 300/min and a
Km of 25 nM under these conditions
(36),3 an expected rate of factor X activation would be 2.3 nM/min; this rate is close to the rate of 2.6 ± 0.2 nM /min observed in our case. Under these conditions, the
rate of factor X activation by IXaL330I was ~0.95
nM/min, by IXaV331A was ~0.55
nM/min, and by IXaT335A was ~1.9
nM/min. For IXaR338Q, it was the same as IXaWT, and for other mutants (see Table I), it could not be
measured. However, when factor VIIIa concentration was increased from
70 pM to 14 nM, the following rates of factor X
activation were obtained: IXaD332Y ~4.1
nM/min, IXaR333L ~2.7 nM/min,
IXaR333Q ~2.4 nM/min, and
IXaL337I ~3.2 nM/min; for
IXahelixVII, it was still not measurable. Note that a
calculated rate for IXaWT (or IXaNP) at 14 nM VIIIa would be 0.85 nM Xa generated/s. Thus,
each of our protease domain mutants except IXaR338Q is
impaired in its interaction with VIIIa in the
IXa-Ca2+-PL-VIIIa system.
Mutations in factor IXa can affect VIIIa binding by at least two
mechanisms: 1) by perturbation of factor IXa binding site for factor
VIIIa, and 2) by altering the spacing above the PL surface of the IXa
interactive site. To distinguish between these two possibilities, we
studied the effect of VIIIa on the potentiation of factor X activation
in the absence of PL. For these studies, we first measured the rates of
factor X activation by the IXa mutants in the presence of
Ca2+ only. These data are presented in Fig.
3A. As predicted from the
Ca2+/PL system, each IXa mutant in the presence of
Ca2+ only activated factor X at a rate (0.46 ± 0.05 nM/min) comparable to IXaWT. However, as was
the case with the Ca2+/PL/VIIIa system, the IXa mutants
also activated factor X in the Ca2+/VIIIa system at rates
that were slower than those obtained with IXaWT (or
IXaNP). These data are presented in Fig. 3B and
summarized in Table I. Considering a kcat value
of 1.1/min, a Km of 380 nM3, and an EC50 (functional
Kd of IXa:VIIIa interaction) value of 2.2 µM (16) at the 400 nM factor X concentration
used in our system, the expected rate of Xa formation using 14 nM VIIIa and 2 µM IXa would be 3.3 nM/min. The experimental rate obtained with
IXaWT (or IXaNP) was ~2.75
nM/min, a value close to the expected value. The rate of
activation by IXaL330I was ~0.94 nM/min, by IXaV331A was ~0.54 nM/min, by
IXaT335A was ~1.83 nM/min, and by IXaR338Q was ~2.22 nM/min. Under these
conditions, i.e. in the presence of limiting concentrations
of VIIIa, the rates of factor X activation by other mutants could not
be measured. Cumulatively, our data indicate that the mutations in
helix 330 (162) residues lead to an impaired interaction of IXa with
VIIIa, which does not appear to be due to spatial misalignment of the
IXa contact site above the PL surface. Our data also indicate that IXa
residue Arg-338 (170) does not play a significant role in binding to
factor X or to factor VIIIa.
Measurement of Kd(app) Values for the Interaction of
Protease Domain IXa Mutants with Factor VIIIa in the Tenase
Complex--
In the previous section, we established that helix 330 mutants of IXa are impaired in their interactions with factor VIIIa. To
further these observations, we evaluated the Kd of interaction of these mutants with VIIIa. These data are presented in
Fig. 4. The steady-state inhibition
curves (41) obtained in the presence (Fig. 4A) and absence
(Fig. 4B) of PL were analyzed as outlined under
"Experimental Procedures." The Kd(app) values
for the interaction of active-site blocked mutants with factor VIIIa
are listed in Table I. The binding of factor VIIIa to each active-site
blocked mutant (except for IXaR338Q) was considerably weaker both in the presence and absence of PL. Compared with
DEGR-IXaWT, L330I and T335A mutants had similarly (~4-
and ~2.5-fold, respectively) reduced affinity for factor VIIIa in the
presence or absence of PL. However, other mutants (Table I) had
2-3-fold further reduction in affinity in the presence of PL
versus in its absence. The n-fold reductions in
affinity for these mutants in the presence and absence of PL were:
V331A, 11 and 7; D332Y, 52 and 27; R333L, 149 and 67; R333Q, 130 and
41; and L337I, 91 and 32. Conceivably, in these mutants, in addition to
the perturbation of factor VIIIa binding site, a further reduced
affinity in the presence of PL could in part be due to the misalignment
of the factor VIIIa contact site. It is noteworthy that
IXaVIIhelix mutant failed to bind to factor VIIIa at
concentrations ~140,000-fold greater than the
Kd(app) for IXaWT in the presence of PL
and at concentrations ~150-fold greater than the
Kd(app) in the absence of PL. Thus, both the kinetic
and the binding data provide strong evidence that helix 330 in the
protease domain of factor IXa provides a critical binding site for
factor VIIIa.
Role of EGF1 Domain of Factor IXa in Binding to Factor
VIIIa--
In the previous section, we provide compelling evidence
that the IXa helix 330 constitutes a critical binding site for factor VIIIa. However, data also exist to support the theory that the EGF1
domain is involved in binding to factor VIIIa (10, 42, 43). Notably,
essentially all of the studies related to the EGF1 domain have been
conducted in the presence of PL. Thus, one cannot differentiate whether
the EGF1 domain is directly involved in binding to factor VIIIa or
whether alterations in this region result in a misalignment above the
PL surface of the region(s) in the protease domain, which is the direct
contact site in interacting with factor VIIIa. To address this, we
conducted studies both in the presence and absence of PL using two EGF1
mutants generated in our laboratory (9). The two mutants employed for
these studies were factor IXQ50P, which lacks the EGF1
domain Ca2+ binding site, and IXPCEGF1, in
which the EGF1 domain of factor IX has been replaced by that of protein
C (9). Both mutants had the same NH2-terminal sequence and
Gla content as normal IX and could be readily activated to IXa-like
molecules by factor Xla (9). In the present study, we have found that
these mutants also bind to the mAb (that interferes with the IXa:VIIIa
interaction) with the same affinity (kd ~15
nM) as normal IX.
In the Ca2+/PL system (same conditions as in Fig.
2A, i.e. 20 nM IXa and 100 nM X), IXaQ50P activated factor X at 0.07 nM/min and IXaPCEGF1 at 0.06 nM/min. These rates are slightly slower than the rate (0.1 nM/min) obtained with IXaWT. These results could be attributed to a slight shift in the active site of the variant
proteins relative to the EGF1 domain (32). When limiting concentrations
of factor VIIIa in the complete Tenase system (VIIIa, 70 pM; IXa, 0.5 nM; and X, 15 nM) were
employed, both the EGF1 domain mutants failed to activate factor X at
measurable rates (Fig. 5A and
Table I). As expected, both EGF1 domain mutants activated factor X in
the presence of only Ca2+ at rates similar to that of
IXaWT. Thus, at 1 µM X and 400 nM IXa, the rates of activation by IXaWT, IXaQ50P,
and IXaPCEGF1 were ~0.46 nM/min, ~0.39
nM/min, and ~0.48 nM/min, respectively. Surprisingly, however, both mutants also activated factor X in the
Ca2+/VIIIa system at rates similar to that of
IXaWT (Fig. 5B and Table I). Consistent with
these observations, Kd(app) of IXa:VIIIa interaction
was only impaired in the presence of PL (Fig.
6A) and not in its absence
(Fig. 6B). Kd(app) values both in the
presence and absence of PL are provided in Table I. In control
experiments, neither protein C nor DEGR-APC competed with IXa in
binding to VIIIa (Fig. 6, A and B). From these
data, we conclude that the EGF1 domain of factor IXa in the absence of PL does not play a significant role in its interaction with VIIIa and
that in the presence of PL it may primarily function to correctly position the protease domain for optimal binding to VIIIa.
Role of Factor IXa Protease Domain in Binding to Factor
VIIIa--
Initial evidence that led to the proposal that the protease
domain of factor IXa is involved in binding to factor VIIIa came from
the observations that a mAb to the protease domain inhibited factor
IXa:factor VIIIa interaction (12). Additional biochemical studies
supported this concept (13, 14). Further studies mapped this antibody
to residues 180-310 of protease domain (44), and a part of the epitope
was found to be located in the calcium binding loop (7). Moreover, a
hemophilia B patient in which Glu-245 in factor IX, a ligand for
Ca2+-binding in the protease domain, was replaced by valine
has been identified (45). Based upon these studies, it was thought that the protease domain Ca2+-binding loop may constitute a part
of the factor VIIIa binding site (45). However, site-specific mutations
adjoining this loop did not lead to impairment in the clotting activity
of factor IX, indicating that the Ca2+-binding loop does
not directly contribute to factor VIIIa binding (17). Currently, it is
believed that the binding of Ca2+ to the protease domain
indirectly affects binding of factor VIIIa to this domain (16).
In order to identify the region in the protease domain for IXa:VIIIa
interaction, we studied the role of surface-exposed helix 330. The
position of this helix is shown in Fig.
7A. The sequence in this helix
is identical in factor IX from all species (15) and is different from
all other homologous blood coagulation serine proteases (Table II).
Further, helix 330 is located 12 residues away from the autolysis loop
cleavage site to which it is connected via a single
The nature and orientation of the side chains along with the point
mutants constructed in the present study are shown in Fig. 7B. All point mutants (except for R338Q) constructed in
helix 330 residues primarily based upon hemophilia B patients had
reduced affinity for factor VIIIa both in the presence and absence of PL (Table I). Residue Leu-330 (162) is located in a hydrophobic pocket
(Fig. 8A) and makes van der
Waals contacts with residues Leu-300 (131) and Phe-349 (181). In the
mutant L330I, the hydrophobic contacts with Phe-349 (181) may be
weakened (32), which could destabilize the helix resulting in a reduced
affinity for factor VIIIa. Residues Val-331 (163), Asp-332 (164), and
Thr-335 (167) are on the same side of the helix. The van der Waals and
hydrogen bond interactions involving these residues are shown in Fig.
8B. When Val-331 (163) is changed to Ala-331 (163), the
hydrophobic contacts involving this residue with Thr-335 (167), His-354
(185), and Tyr-395 (225) will be weakened. This again will destabilize the helix resulting in an impaired interaction with factor VIIIa (Table
I). A change in Asp-332 (164) to Tyr-332 (164) will disrupt essentially
all contacts in this pocket since tyrosine is a much bulkier residue
than aspartic acid, which is expected to shift the helix away from the
this pocket. Finally, a change of Thr-335 (167) to Ala-335 will result
in disruption of its contacts with Asp-332, Val-331, and His-354.
Residue Arg-333 (165) is solvent-exposed, and abolishing the positive
charge by mutational change may abolish the direct interaction of this
amino acid with factor VIIIa. As depicted in Fig. 8C,
changing Leu-337 (169) to Ile may result in the disruption of another
hydrophobic pocket and a slight shift of the helix (32). Notably,
changing Arg-338 (170) to Gln (170) resulted in a minimal loss of
biologic activity and affinity for factor VIIIa (Table I). This is
consistent with a slightly increased activity of R338A mutant without
an impairment in factor VIIIa binding described by Chang and co-workers
(46). Note that all of the helix 330 point mutants have impaired
interaction with factor VIIIa both in the presence and
absence of PL. Furthermore, IXahelixVII mutant failed to
interact with factor VIIIa even at very high concentrations, both in
the presence or absence of PL. These data strongly support the
conclusion that helix 330 in IXa represents a critical binding site for
VIIIa.
Hemophilia B mutants (18) not expressed in the present study in helix
330 may have impaired interactions with factor VIIIa due to the
following reasons. Changing Leu-330 (162) to proline may result in a
turn accompanying a subtle directional change in the propagation of the
helix, and changing Val-331 (163) to aspartic acid is expected to
disrupt the hydrophobic interactions depicted in Fig. 8B.
Replacement of Arg-333 (165) by glycine, and of Ala-334 (166) by
aspartic acid or threonine could disrupt the direct binding of factor
IXa to factor VIIIa. A change of Cys-336 to any other residue will
disrupt the disulfide bond and a possible change in the local tertiary
structure. Replacement of Leu-337 (168) to phenylalanine or proline is
expected to disrupt the putative hydrophobic pocket (Fig.
8C), and, in the case of the proline mutant, it may also
change the direction of the polypeptide. Similarly, replacement of
Arg-338 by proline could result in the change of the direction of the
polypeptide and disruption of the local tertiary structure.
Role of Factor IXa EGF1 Domain in Binding to Factor VIIIa--
The
first EGF-like domain of factor IXa has also been implicated in binding
to factor VIIIa (10, 42, 43). In the present study, two EGF1 mutants
were examined for their abilities to bind to factor VIIIa both in the
presence and absence of PL. In a system containing PL,
IXaQ50P and IXaPCEGF1 interacted with factor
VIIIa with ~20- and ~100-fold reduced affinity, respectively.
However, in the absence of PL, both activated mutants interacted with
factor VIIIa with Kd(app) indistinguishable from
that of normal IXa (Table I). Since alterations of the EGF1 domain
affect VIIIa binding only in the presence of PL but not in its absence,
whereas mutations in helix 330 affect VIIIa binding in both the
presence and absence of PL, it can be concluded that helix 330 in IXa
provides a crucial binding site for VIIIa and that the EGF1 domain in
this context primarily serves to correctly position the protease domain above the PL surface for optimal interaction with VIIIa. In a preliminary report (47), it has also been concluded that the residues
330-338 in IXa are involved in binding to factor X. However, data
presented in Figs. 2A and 3A do not support this
concept. Further studies will be required to fully resolve this issue.
Modeling studies support the concept that helix residues 330-338 may
be shifted in the IXaPCEGF1 mutant (32). In this model, in
which the membrane binding Gla domain coordinates are unchanged from
normal IXa, the EGF2/protease domain coordinates including helix 330 are shifted ~1.5 Å. Further, the eight-residue insertion in the EGF1
domain lies on the same side as helix 330. As a consequence of these
two deviations from normal IXa, it is possible that the interactions of
helix 330 residues with factor VIIIa are weakened. In other studies in
which EGF1 domain of factor IX was replaced by that of factor X (48) or
factor VII (49), the resulting molecule either had normal
activity (IXaXEGF1) or 4-fold increased activity
(IXaVIIEGF1). The activity in the IXaVIIEGF1
was attributed to the increased affinity of this mutant for factor
VIIIa (49). When EGF1 domain of factor VII was modeled into the factor
IX molecule, the EGF2/protease domain coordinates including those of
helix 330 were only shifted ~0.5 Å (32). Further, this shift in
IXaVIIEGF1 was in the opposite direction to that observed
in the IXaPCEGF1 molecule. Since subtle changes can lead to
increase in the bond strengths, it is possible that helix 330 residues in IXaVIIEGF1 molecule are positioned more favorably to
interact with factor VIIIa binding site. Furthermore, it is quite
feasible that in other EGF1 mutants (10, 24, 42, 43, 50, 51) and in a
Gla domain mutant (52), the alignment of helix 330 above the PL surface
is altered, which results in its impaired interactions with factor
VIIIa. These conclusions are consistent with the observation of Lenting
et al. (10) and of Christophe et al. (51), in
which the light chain mutants of IXa, in the absence of PL, have
minimal alterations (2-4-fold) in direct binding to factor VIIIa,
whereas in the presence of PL interactions of these IXa variants with
VIIIa were impaired 50-200-fold. As is the case with
IXaQ50P and IXaPCEGF1 (Table I), we anticipate that interactions of the above mutants with factor VIIIa in the absence
of PL may be minimally impaired.
Concluding Remarks--
Essential to hemostasis is the interaction
of factor IXa with factor VIIIa. The present studies indicate that
helix 330 in the protease domain of factor IXa provides a critical
binding site for factor VIIIa. Although weaker interactions (10, 51) and/or exposure of neoepitopes in IXa or VIIIa cannot be ruled out, a
role of the EGF1 domain of factor IXa in this context may be to
primarily serve as a spacer in properly positioning the IXa protease
domain above the PL surface for optimal interaction with factor VIIIa.
However, whether or not Gla and EGF2 domains are involved in direct
binding to factor VIIIa is not known. Since the anti-factor IX mAb
binds normally to helix 330 mutants including IXahelixVII,
it probably interferes with IXa:VIIIa interaction primarily by steric
hindrance or by locking the conformation of IXa heavy chain unfavorable
for binding to factor VIIIa. Ca2+ binding (loop 1 in Fig.
7A) to the protease domain is known to enhance IXa:VIIIa
interaction (16). It most likely occurs via interactions through the
autolysis loop (loop 2 in Fig. 7A) which directly connects
to helix 330 (162). Furthermore, helix 330 (162) interacts with another
surface-exposed helix, 293-301 (126-132) (Fig. 8A), which
is also conserved in factor IX from all species sequenced thus far
(15); this possibly could represent another binding site for factor
VIIIa; studies are under way to examine this concept. The region in
factor VIIIa that interacts with the protease domain of IXa is quite
possibly located in the A2 domain (36). Binding of A2 domain to IXa has
been reported to increase the kcat of factor X hydrolysis (36). This
could occur by modulation and/or stabilization of helix 330, which
links directly to the extended substrate binding site residues Ile-397
(227) (53) and Trp-385 (215) through hydrophobic contacts (Fig.
8C). Further, it should be noted (Table
II) that the corresponding helix residues in factor VIIa bind to TF (4) and in factor Xa, they are involved in
binding to factor Va.4 Thus,
a common function of this helix (162 in chymotrypsin numbering) in
several blood coagulation proteases may be to serve as a binding site
for the respective cofactor. Function of this helix, if any, in other
serine proteases including chymotrypsin, trypsin, and APC remains to be
elucidated.
We thank Sulekha Verma for help in
preparation of the mutants, and Degang Zhong for useful discussions. We
also thank Ravi Kurumbail and Roderick Stegeman of Monsanto and K. Padmanabhan of Michigan State University for help in using the
programs InsightII and O.
*
This work was supported by National Institutes of Health
Grant HL36365. An initial account of this work (which A. M. received the Finn Wold Travel Award) was presented at the Twelfth
Symposium of the Protein Society, July 25-29, 1998, San Diego, CA.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: Saint Louis University
Health Sciences Center, 3635 Vista Ave. at Grand Blvd., P.O. Box 15250, St. Louis, MO 63110-0250. Tel.: 314-577-8499/8854; Fax: 314-773-1167;
E-mail: bajajps{at}wpogate.slu.edu.
2
For comparison, factor IX amino acid numbering
system has been used. The number in parentheses refers to the
chymotrypsin equivalents for the protease domain of factor IXa (2,
15).
3
A. Mathur, D. Zhong, and S. P. Bajaj,
unpublished data.
4
S. P. Bajaj and J. P. Miletich,
unpublished data.
The abbreviations used are:
TF, tissue factor;
Gla,
Protease and EGF1 Domains of Factor IXa Play Distinct Roles in
Binding to Factor VIIIa
IMPORTANCE OF HELIX 330 (HELIX 162 IN CHYMOTRYPSIN) OF PROTEASE
DOMAIN OF FACTOR IXa IN ITS INTERACTION WITH FACTOR VIIIa*
and
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EXPERIMENTAL PROCEDURES
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-carboxyglutamic acid (Gla) domain (residues 1-40 out of which 12 are Gla residues), a short hydrophobic segment (residues 41-46), and two epidermal growth factor (EGF)-like domains (EGF1 residues 47-85 and EGF2 residues 86-127), whereas the
heavy chain contains the carboxyl-terminal serine protease domain with
trypsin-like specificity (1, 2). Activation peptide of residues
145-180, which is released upon conversion of factor IX to IXa, is
rich in carbohydrate and is the least conserved region in IX from
different species (3). Factor IXa hence formed converts factor X to Xa
in the coagulation cascade; for a biologically significant rate, this
reaction requires Ca2+, phospholipid (PL), and factor VIIIa
(1). Thus, to this extent essential to physiologic hemostasis is the
interaction of Ca2+-bound factor IXa (activated hemophilia
B protein) with factor VIIIa (activated hemophilia A protein) on the PL
surface provided by the platelets.
EXPERIMENTAL PROCEDURES
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INTRODUCTION
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DISCUSSION
REFERENCES
-thrombin (IIa) were purchased from Enzyme
Research Laboratories (South Bend, IN). Recombinant human TF of amino
acids 1-243 containing the transmembrane domain was generously
provided by Genentech Inc. (South San Francisco, CA) and reconstituted
as described (22). Phosphatidylcholine-phosphatidylserine vesicles
(75% phosphatidylcholine, 25% phosphatidylserine) were prepared by
the method of Husten et al. (23) as outlined (22).
Recombinant human factor VIIa was a generous gift of Novo-Nordisk
(Copenhagen, Denmark). Purified human factor VIII was obtained from Dr.
Leon Hoyer (American Red Cross, Rockville, MD). The preparation was
free of all other coagulation factors and contained human albumin as a
stabilizing agent. Purification of a mouse monoclonal antibody (mAb)
that inhibits the interaction of factor IXa with factor VIIIa has been
described (12).
ATT), V331A (GTT GCT), D332Y (GAC
TAC), R333L (CGA CTA), R333Q (CGA CAA), T335A (ACA GCA), L337I (CTT ATT), and R338Q (CGA CAA). Factor IXhelix
VII, in which 330-338 (162-170) residues of factor IX were
replaced by the corresponding residues of factor VII, was constructed
using a 63-base primer. The first 18 bases of this primer corresponded to factor IX gene sequence coding for residues 324-329 (156-161), followed by 27 bases from the factor VII gene sequence (29) coding for
residues (162-170), and the final 18 bases corresponded to the factor
IX gene sequence coding for residues 339-344 (171-177). The PCR was
performed in the same fashion as described previously for point mutants
(9, 28). All inserts were sequenced (30) to confirm the mutations and
to rule out any PCR errors. Expression of each factor IX recombinant
protein and its purification was achieved exactly as described (9).
without further degradation.
DEGR-IXaNP, DEGR-IXaWT, and various DEGR-IXa
mutant proteins were prepared as described previously (16), and free
DEGR-ck was removed as described previously (22, 33). DEGR-APC was
prepared similarly.
A405/min) for up to 20 min (5, 22). Factor
Xa generated was calculated from a standard curve constructed using
factor Xa prepared by insolubilized Russell's viper venom.
y is the rate of Xa formation in the presence of a
given concentration of DEGR-IXa protein represented by x,
a is the maximum rate of Xa formation in the absence of
DEGR-IXa, and s is the slope factor. Each point was weighted
equally, and the data were fitted to Equation 1 using the nonlinear
regression analysis program obtained from Erithacus Software (GraFit).
The values of the slope factors were 0.9 ± 0.1 in all
experiments, indicating competition for a single binding site. The
background value represented <5% of the maximum rate of Xa formation
in the absence of DEGR-IXa. To obtain the
Kd(app) values for the interaction of DEGR-IXa proteins with factor VIIIa, we used the following equation as described
by Cheng and Prusoff (38) and further discussed by Craig (39).
(Eq. 1)
A is the concentration of IXaWT, and
EC50 is the concentration of factor IXaWT that
gives a 50% maximum response in the absence of the competitor at a
specified concentration of factor X used in the experiment.
(Eq. 2)
RESULTS
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-Carboxyglutamic Acid Content,
NH2-terminal Sequence, and Activity of Protease Domain
Factor IX Mutants--
SDS- electrophoretic analysis of factor IX
proteins using the Laemmli system (25) is shown in Fig.
1A. Each protein is
effectively homogenous in this system. Further, plasma factor IX and
each recombinant protein had 11.5-12.5 Gla residues/mol. The
NH2-terminal sequence of each protein was also determined.
All recombinant proteins revealed a major and a minor sequence. The
major sequence in each case was Tyr-Asn-Ser-Gly-Lys, and the minor
sequence in each case was Thr-Val-Phe. The major sequence corresponds
to the sequence of mature protein in plasma, and the minor sequence
corresponds to the protein in which the prosequence has not been
cleaved (26). The minor sequence was not detected in plasma factor IX,
and it amounted to less than 5% in each recombinant protein. The
relative coagulant activity of each protein was: IXNP,
100% (180 ± 10 units/mg); IXWT, ~90%;
IXL330I, ~8%; IXV331A, ~6%;
IXD332Y, ~2%; IXR333L, ~0.3%;
IXR333Q, ~0.5%; IXT335A, ~40%;
IXL337I, ~1%; IXR338Q, ~65%; and
IXhelixVII, not measurable.
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Fig. 1.
SDS-gel electrophoretic analysis of various
protease domain mutants of factor IX before (A) and
after (B) activation with factor Xla and
Ca2+. Approximately 2.3 µg of protein was applied to
each lane. Activation for each gel in B was performed at
37 °C for 90 min in TBS, 5 mM Ca2+, pH 7.4, and the reaction mixture contained 3.5 µM (200 µg/ml)
of factor IX and 25 nM (2.0 µg/ml) of active site-factor
Xla. IX, single chain IX of residues 1-415; H , the heavy
chain of factor IX activation intermediate comprised of residues
146-415; H, the heavy chain of factor IXa comprised of
residues 181-415; L, the light of factor IXa comprised of
residues 1-145. Activation peptide (AP), which is comprised
of residues 146-180, stains poorly and was not observed on these gels.
Molecular weight markers are indicated, and each lane is identified by
the mutant protein applied.
8% coagulant activity, a competition-based assay in which the mutant protein competed with the normal IX in
binding to the mAb was used (40). Each mutant protein bound to the mAb
with an equal affinity (Kd ~15 ± 8 nM).
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Fig. 2.
Factor VIIIa-mediated potentiation of factor
X activation by each protease domain IXa mutant in a system containing
Ca2+ and PL. A, the time course of
activation of factor X in IXa-Ca2+-PL system. Activation
mixture contained 100 nM factor X, 25 µM PL,
5 mM Ca2+, and 20 nM various IXa
proteins. B, the time course of activation of factor X in
IXa-Ca2+-PL-VIIIa system. Activation mixture contained 15 nM factor X, 10 µM PL, 5 mM
Ca2+, 70 pM VIIIa, and 0.5 nM
various factor IXa proteins. Each reaction was stopped at a given time
by the addition of EDTA and Xa generated was measured by S-2222
hydrolysis. Note that the optimal concentration of PL was found to be
25 µM in the absence and 10 µM in the
presence of factor VIIIa (see "Experimental Procedures" and
Footnote 3). Also note that the effective concentrations of enzyme and
substrate are different in A and B (see
"Experimental Procedures" for details). Factor IXa proteins, in
both A and B, are: WT ( 
), L3301 (*), V331A
(
), D332Y (
), R333L (
), R333Q (
), T335A (
), L337I (
),
R338Q (
), and IXahelixVII (×). The data presented are
the average of two experiments.
Effect of factor VIIIa on the rates of factor X activation and
Kd(app) values for the interaction of various factor IXa
proteins with factor VIIIa
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Fig. 3.
Factor VIIIa-mediated potentiation of factor
X activation by each protease domain IXa mutant in a system containing
Ca2+ only. A, the time course of activation
of factor X in IXa-Ca2+ system. Activation mixture
contained 1 µM factor X, 5 mM
Ca2+, and 400 nM various IXa proteins.
B, the time course of activation of factor X in
IXa-Ca2+-VIIIa system. Activation mixture contained 400 nM factor X, 5 mM Ca2+, 14 nM factor VIIIa, and 2 µM various factor IXa
proteins. Each reaction was stopped at a given time by the addition of
EDTA and Xa generated was measured by S-2222 hydrolysis. Note that the
effective concentrations of enzyme and substrate are different in
A and B. Further, the data plotted in
B represent the data which were obtained after subtraction
of the background rates ( 10%) in the absence of VIIIa (see
"Experimental Procedures" for details). Factor IXa proteins in both
panels in A and B are: WT (), L330I (*), V331A
(), D332Y (), R333L (), R333Q (), T335A (), L337I
(), R338Q (), and IXahelixVII (×). The data
presented are average of two experiments.
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Fig. 4.
Inhibition of factor Xa generation by the
active site-blocked protease domain IXa mutants. A,
abilities of activated mutants to inhibit binding of IXaWT
to VIIIa in a system containing PL. Each reaction mixture
contained: IXaWT, 0.2 nM; VIIIa, 0.07 nM; factor X, 480 nM; PL, 10 µM;
Ca2+, 5 mM; and varying amounts of active
site-blocked IXa mutant. B, abilities of activated mutants
to inhibit binding of IXaWT to VIIIa in a system
without PL. Each reaction mixture contained:
IXaWT, 100 nM; VIIIa, 14 nM; factor
X, 2 µM; Ca2+, 5 mM; and varying
amounts of active site-blocked IXa mutants. For both A and
B, the activation was stopped at 2 min by the addition of
EDTA and Xa generated was measured by S-2222 hydrolysis (see
"Experimental Procedures"). Competitors (DEGR-IXa proteins) in both
A and B are: WT ( ), L330I (*), V331A (),
D332Y (), R333L (), R333Q (), T335A (), L337I (), R338Q
(), and IXahelixVII (×). The data presented are average
of two experiments.
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Fig. 5.
Factor VIIIa-mediated potentiation of factor
X activation by the EGF1 domain IXa mutants. A, the
time course of activation of factor X in IXa-Ca2+-PL-VIIIa
system. Activation mixture contained 15 nM factor X, 10 µM PL, 5 mM Ca2+, 70 pM VIIIa, and 0.5 nM various IXa proteins.
B, the time course of activation of factor X in
IXa-Ca2+-VIIIa system. Activation mixture contained 400 nM factor X, 5 mM Ca2+, 14 nM factor VIIIa, and 2 µM various factor IXa
proteins. Factor IXa proteins in both A and B
are: WT ( ), Q50P (), and PCEGF1 (). Explanation of reaction
conditions and other details are provided in the legend to Figs. 2 and
3. The data provided are the average of two experiments.
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Fig. 6.
Inhibition of factor Xa generation by the
active-site blocked EGF1 domain IXa mutants. The data in
A were generated in the presence of PL, and data
in B were generated in the absence of PL. The
reaction conditions are the same as in Fig. 4 except the competitors
were EGF1 domain IXa mutants. DEGR-IXa proteins in both A
and B are: WT ( ), Q50P (), and PCEGF1 (). Control
proteins are protein C zymogen () and DEGR-APC (). The data
presented are average of two experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strand, marked
2 in Fig. 7A. Moreover, point mutations in eight
of the nine residues in this helix are reported to cause hemophilia B
(18).
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Fig. 7.
Position of helix 330 (162) in factor IXa and
the point mutants investigated. A, schematic
representation of the structure of porcine factor IXa. Coordinates
(Protein Data Bank code 1PFX) used are that of Brandstetter et
al. (2), and the location of each domain is indicated.
Yellow arrows represent -strands,
red cylinders represent -helixes, and
green with blue arrows represent
directions of the loops. The COOH-terminal helix of the Gla domain is
shown in blue. Protease domain Ca2+-binding loop
marked 1 and the autolysis loop marked 2 are
shown in white. Note that the autolysis loop leads to helix
330. B, orientation of the amino acid side chains of helix
330 (162) of protease domain of factor IXa. The point mutants
investigated in the present study are given in parentheses.
The direction of the helix, shown in red, is from bottom to
top.
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Fig. 8.
Schematic representation of the nature of
interactions of helix 330 residues. A, location of
factor IXa residue Leu-330 (162) in a hydrophobic pocket surrounded by
Phe-349 (181) and Leu-300 (131). The van der Waals contact distances
(Å) are shown by dashed lines. B,
nature of interactions of residues Val-331 (163), Asp-332 (164), and
Thr-335 (167) of factor IXa protease domain. Both the hydrogen bonds
and the van der Waals contacts are shown by dashed
lines. C, location of residue Leu-337 (169) in a
hydrophobic pocket. The van der Waals distances of residues located in
this hydrophobic pocket are shown by dashed
lines. Chymotrypsin numbering for the residues shown is as
follows: Ile-344 (176), Met-348 (180), Trp-385 (215), and Ile-397
(227).
Sequence of helix 330 (162) in vitamin K-dependent four
coagulant and one anticoagulant serine proteases
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by a predoctoral fellowship from the American Heart
Association, Missouri Affiliate. Portions of this study were taken from
a thesis to be submitted to the Graduate School of Saint Louis
University in partial fulfillment of the requirements for the Ph.D. degree.
ABBREVIATIONS
-carboxyglutamic acid;
EGF, epidermal growth factor;
IIa, -thrombin;
PL, phospholipid;
S-2222, benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide;
DEGR-ck, dansyl-Glu-Gly-Arg-chloromethylketone;
BSA, fatty acid-free bovine
serum albumin;
DEGR-IXa, IXa inactivated with DEGR-ck;
WT, wild type;
NP, normal plasma;
APC, activated protein C;
mAb, monoclonal antibody;
TBS, Tris-buffered saline;
PCR, polymerase chain reaction;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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S. P. Bajaj, A. E. Schmidt, A. Mathur, K. Padmanabhan, D. Zhong, M. Mastri, and P. J. Fay Factor IXa:Factor VIIIa Interaction. HELIX 330-338 OF FACTOR IXa INTERACTS WITH RESIDUES 558-565 AND SPATIALLY ADJACENT REGIONS OF THE A2 SUBUNIT OF FACTOR VIIIa J. Biol. Chem., May 4, 2001; 276(19): 16302 - 16309. [Abstract] [Full Text] [PDF]
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F. H. Wilkinson, F. S. London, and P. N. Walsh Residues 88-109 of Factor IXa Are Important for Assembly of the Factor X Activating Complex J. Biol. Chem., February 15, 2002; 277(8): 5725 - 5733. [Abstract] [Full Text] [PDF]
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F. H. Wilkinson, S. S. Ahmad, and P. N. Walsh The Factor IXa Second Epidermal Growth Factor (EGF2) Domain Mediates Platelet Binding and Assembly of the Factor X Activating Complex J. Biol. Chem., February 15, 2002; 277(8): 5734 - 5741. [Abstract] [Full Text] [PDF]
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