Definition of a Factor Va Binding Site in Factor Xa*
Amy E.
Rudolph
,
Rhonda
Porche-Sorbet, and
Joseph P.
Miletich§
From the Departments of Pathology and Medicine, Division of
Laboratory Medicine, Washington University School of Medicine,
St. Louis, Missouri 63110
Received for publication, August 2, 2000, and in revised form, October 11, 2000
 |
ABSTRACT |
We reported previously that residue 347 in
activated fX (fXa) contributes to binding of the cofactor, factor Va
(fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861-2867). Four additional
residues that participate in fVa binding have now been identified by
mutagenesis. All five resulting fX species, fXR306A,
fXE310N, fXR347N, fXK351A, and
fXK414A, are activated and inhibited normally. However, the
rate of inhibition by antithrombin III in the presence of submaximal
concentrations of heparin is reduced for all the enzymes. In the
absence of fVa, all of the enzymes bind and activate prothrombin
similarly except fXaE310N, which has a reduced apparent
affinity (~3-fold) for prothrombin compared with wild type fXa
(fXaWT). In the absence of phospholipid, fVa enhances the
catalytic activity of fXaWT significantly, but the response
of the variant enzymes was greatly diminished. On addition of 100 nm
PC:PS (3:1) vesicles, fVa enhanced fXaWT,
fXaR306A, and fXaE310N similarly, whereas
fXaR347N, fXaK351A, and fXaK414A
demonstrated near-normal catalytic activity but reduced apparent
affinity for fVa under these conditions. All enzymes function similarly
to fXaWT on activated platelets, which provide saturating
fVa on an ideal surface. Loss of binding affinity for fVa as a result
of the substitutions in residues Arg-347, Lys-351, and Lys-414 was
verified by a competition binding assay. Thus, Arg-347, Lys-351, and
Lys-414 are likely part of a core fVa binding site, whereas Arg-306 and
Glu-310 serve a less critical role.
 |
INTRODUCTION |
The vitamin K-dependent plasma serine protease,
activated factor X (fXa)1
serves as the physiological activator of prothrombin. The zymogen precursor, fX, is activated to fXa by complexes in the intrinsic (factor IXa/factor VIIIa) and extrinsic (factor VIIa/TF) coagulation pathways and by RVV-X in the presence of calcium (1-6). Once activated, fXa is inhibited by antithrombin III (ATIII) and tissue factor pathway inhibitor (TFPI, Refs. 7-10). The light chain of fX
contains 11
-carboxylated glutamic acid residues that compose the
Gla domain and two epidermal growth factor-like domains (EGF-1 and
EGF-2). The activation peptide and the serine protease domain form the
heavy chain of fX, which is bound to the light chain via a single
disulfide bond.
Factor Xa interacts synergistically with cofactor (fVa), substrate
(prothrombin), and a phospholipid surface to form the prothrombinase complex which supports maximally efficient prothrombin activation (11).
Several of these interactions were independently evaluated for a
variant recombinant fX, fXaR347N (residue 165 in
chymotrypsin numbering), and it was found that substitution of Arg-347
selectively reduces fVa affinity (12). The current study describes
further delineation of the fVa binding site of fXa through targeted
mutagenesis of additional residues in the surface epitope that includes
Arg-347.
Arg-306, Lys-351, and Lys-414 (125, 169, and 230 in chymotrypsin
numbering, respectively) have been substituted by alanine. Glutamate
310 (129 in chymotrypsin numbering) was substituted by asparagine. The
fVa affinity of the resulting enzymes was probed using functional and
binding studies. It was determined that Arg-347, Lys-351, and Lys-414
compose a core cofactor binding epitope and Glu-310 and Arg-306 form an
extended region of the epitope.
 |
EXPERIMENTAL PROCEDURES |
Reagents and Chemicals--
Crude snake venoms,
L-
-phosphatidylcholine, and
L-
-phosphatidylserine were purchased from Sigma.
Spectrozyme FXaTM and Spectrozyme THTM were purchased from American
Diagnostica Inc. (Greenwich, CT). Full-length heparin was obtained from
Elkins-Sinn Inc. (Cherry Hill, NJ). Heparin pentasaccharide was a
generous gift of J. C. Lormeau of Sanofi Recherché (Gentilly
Cedex, France). 100 nm phosphatidylcholine:phosphatidylserine (PC:PS,
3:1) vesicles were prepared as described (12-14).
Proteins--
Prothrombin (13, 14) and fVII (15) were purified
from human plasma as described. Prothrombin was immunodepleted of
residual fX (<1 nM) using a anti-fX monoclonal antibody.
ATIII was purchased from Kabi Pharmacia Diagnostics (Piscataway, NJ),
and recombinant tissue factor pathway inhibitor was kindly provided by
Monsanto/Searle Co. (St. Louis, MO). Human fVa and fIXa were purchased
from Hematologic Technologies (Essex Junction, VT). Thrombin was
prepared as described (13, 14). Porcine fVIII was purchased from Porton
Products (Agoura Hill, CA) and purified as described (13, 14).
Recombinant, lipidated tissue factor, Innovin, was purchased from
Baxter Diagnostics Inc. (Deerfield, IL). RVV-X was purified from
Russell's viper venom (16). SDS-polyacrylamide gel electrophoresis was
performed by the method of Laemmli (17). All recombinant fX species
were purified as described (14).
Immunoglobulins--
Anti-fX murine monoclonal antibodies
utilized in the study were developed by standard methods. 3698.1A8.10
reacts with the Gla domain in the presence of calcium, 3514.5H12.10
reacts with the Gla domain independent of calcium, and 3448.1D7.20
binds to EGF-2. Activation of fX was monitored by a two-site
immunofluorescent assay utilizing 3448.1D7.20 and 3514.5H12.10 as
described (13). Murine anti-fX monoclonal antibody directed against the
heavy chain was purchased from Enzyme Research Laboratories, Inc.
(South Bend, IN). For immunoblotting, an antibody mixture of
3448.1D7.20, 3514.5H12.10, and the anti-fX heavy chain monoclonal were utilized.
Mutant Construction and Cell Culture--
The fXWT
cDNA was cloned and modified as described previously (13, 14).
Sequence changes for the variants include: fXR306A, CCGAGCGTG
CCGAAGCTG; fXE310N,
GCCGAGTCC
GCAAATTCC;
fXR347N, GACCGCAAC
GACAACAAC; fXK351A, TGCAAGCTG
TGTGCACTG);
fXK414A, ACCAAGGTC
ACCGCGGTC. All
sequence changes and the entire fXWT sequence were verified
by sequence analysis. Mutant and fXWT sequences were
shuttled into the expression vector (ZMB3) kindly provided by Dr. Don
Foster. All constructs were transfected into human kidney cells (293)
using calcium phosphate precipitation (18). 293 cells were cultured as
described (13, 14). Isolated colonies were screened for fX production
by immunoassay and expanded.
Activation--
The assay buffer used was 10 mM
HEPES, pH 7.0, 100 mM NaCl, 5 mM
CaCl2, 1 mg/ml bovine serum albumin, 1 mg/ml polyethylene glycol 8000. All variant zymogens were activated as described previously for fXR347N (12). Briefly, variants or
fXWT (100 nM) were activated with 2 nM RVV-X for end point activation or 50 pM
RVV-X for initial rate measurements. Each fX was activated by fVIIa (40 pM) in the presence of 0.5 nM lipidated tissue
factor, and by fIXa (2 nM) in the presence of fVIIIa (4 units/ml) on PC:PS vesicles (20 µM). All concentrations
were subsaturating for the activators and initial rates of activation
(<10% of substrate utilized) were determined. Reactions were
monitored over time by quenching aliquots of the reaction in EDTA
buffer (10 mM HEPES, pH 7.0, 100 mM NaCl, 5 mM EDTA, 1 mg/ml bovine serum albumin, 1 mg/ml polyethylene
glycol 8000) and measuring the rate of hydrolysis of Spectrozyme FXaTM
(100 µM).
Inhibition--
Inhibition of all variants was measured using
established methodologies (12, 13, 19, 20). All zymogens (100 nM) were activated by RVV-X (2 nM). Each fXa
(0.5 nM) was incubated with Spectrozyme FXaTM (100 µM) in the presence of inhibitor: ATIII alone (0-4
µM), ATIII (0-50 nM) in the presence of
heparin pentasaccharide (0.5 µM), ATIII (0-16
nM) in the presence of 2.5 milliunits/ml full-length
heparin, or TFPI (0-50 nM). Concentrations of heparin pentasaccharide and full length heparin were empirically determined to
support half-maximal acceleration of inhibition. Residual fXa activity
was quantitated from Spectrozyme FXaTM hydrolysis. First and second
order rate constants were derived as described (12).
Thrombin Formation--
Assays were performed in assay buffer as
described (12). Kinetic values were calculated from the least squares
fit of the data to Equation 1,
|
(Eq. 1)
|
where v = the observed initial rate of thrombin
formation, Vmax = the maximal initial rate of
thrombin formation, [Z]= the concentration of the
component being varied in the experiment, and Km=
the apparent concentration of the variable component required to reach
half-maximal thrombin formation under the conditions specified. Data
are expressed as moles of thrombin (IIa)/s/mol of Xa. Samples were
removed from each reaction at various times and quenched into EDTA
buffer. Thrombin formed was quantitated by incubating quenched samples
with 500 µM Spectrozyme THTM, a thrombin chromogenic
substrate. The conditions for the experiments have been described in
detail (12). In summary, 10 nM each fXa was incubated with
prothrombin alone (0-450 µM). In the absence of
phospholipid, varying concentrations of fVa (0-250 nM)
were incubated with 10 µM prothrombin and fXa (0.5 nM). In the presence of PC:PS vesicles (3:1, 20 µM), 20 pM fXa was incubated with fVa (0-1
nM) and the reaction was initiated with 1 µM
prothrombin. Thrombin-activated platelets (108/ml) were
incubated with fXa (0-1 nM), and thrombin formation was
initiated by the addition of 1 µM prothrombin.
Competition Binding Assay--
Reagents for the latex bead-based
binding assay were prepared as described (12). Briefly,
fXS379A was labeled with 125I using
Bolton-Hunter reagent (Amersham Pharmacia Biotech). Radioactivity not
incorporated into protein was removed using a Bio-Spin 6 column (Bio-Rad). The specific activity of the labeled protein was typically 2000 cpm/ng. Labeled FXS379A was activated using RVV-X as
described above.
Latex beads (1.0 µm; Interfacial Dynamics Corp., Portland, OR) were
coated with PC:PS (3:1) according to published methods (12). Beads
coated with PC only were also prepared and used in pilot experiments to
demonstrate specificity; i.e. no fVa-dependent binding without PS. Nonspecific binding accounted for <10% of binding, and ~50% of 125I-fXaS379A binding
was prevented by the addition of 1 nM unlabeled fXaWT.
The concentrations of all assay components were empirically determined
as described (12). In the assay, 1 nM fVa was incubated with 0.2% (v/v) PC:PS beads and 0.8% (v/v) uncoated beads for 10 min.
125I-fXaS379A (final concentration 1.0 nM) was mixed with various concentrations of either wild
type or mutant fXa, added to the fVa/bead mixture, and incubated with
mixing for an additional 10 min. 125I-fXaS379A
was utilized as the labeled fXa species for greater stability over
time, i.e. to minimize fXa-dependent
proteolysis. The beads were collected by centrifugation, and the
pellets and supernatants were counted separately. Nonspecific binding
was determined from reactions not containing fVa and subtracted from counts bound in the pellets. The percentage of bound fXa was
quantitated relative to the amount bound in reactions with no unlabeled
fXa. The apparent affinity (Kd(app)) represents
the concentration of unlabeled fXa required to displace 50% of
125I-fXaS379A from the fVa-bound beads and is
calculated from the following equation.
|
(Eq. 2)
|
In the equation, y = % bound
125I-fXaS379A, x = concentration of added, unlabeled fXa, and s = a slope factor.
fXa Model of the fVa Binding Site--
The model of fXa was
generated from the published coordinates (21) using RIBBONS version
3.0, developed by M. Carson at the University of Alabama (Birmingham,
AL). In the structure, the Gla domain is missing and EGF-1 is disordered.
 |
RESULTS |
Mutagenesis--
We demonstrated previously that the employed
expression system is efficacious for the production of fully functional
recombinant wild-type and variant fX (12-14). Construction of the
variant molecules in the current study was based on two mutational
strategies. Residues Arg-306, Lys-351, and Lys-414 were substituted
with alanine, whereas Glu-310 and Arg-347 were substituted with
asparagine, resulting in the creation of a potential
N-linked glycosylation site, NX(C/S/T), (Table
I). The apparent molecular weight of
fXE310N was elevated, indicating the addition of a
carbohydrate group at this residue (Fig.
1). However, as previously described,
asparagine substitution of Arg-347 did not result in an added
carbohydrate, as the electrophoretic mobility of fXR347N
was not altered as compared with fXWT (Fig. 1; Ref.
12).

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 1.
Gel electrophoresis of fX species. Each
lane represents 2.5 ng of fX. Proteins were analyzed by 12.5%
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and immunoblotted using monoclonal antibodies directed against the
light chain and heavy chain. Molecular weight standards are as
indicated. Lane 1, fXWT; lane
2, fXK414A; lane 3,
fXR347N; lane 4, fXE310N;
lane 5, fXR306A; lane 6,
fXK351A.
|
|
Activation--
All variants hydrolyzed a peptide substrate,
Spectrozyme FXaTM, normally (data not shown). Initial rates of
activation by RVV-X and the extrinsic (fVIIa/TF) and intrinsic
(fIXa/fVIIIa) activation complexes were quantitated by monitoring
Spectrozyme FXaTM hydrolysis. Activation of all variant zymogens by
all activators was very similar to the activation of
fXWT.
Inhibition--
The impact of the substitutions on the active site
of fXa was probed by studying the interaction between the enzymes and
the physiologic inhibitors, ATIII and TFPI. All variant enzymes were inhibited normally by these inhibitors as compared with
fXaWT and second order rate constants for inhibition of
fXaWT were consistent with reported values (Table
II; Ref. 22). Therefore, the active site
of fXa was not compromised by these mutations. Inhibition of each
enzyme by ATIII was also examined in the presence of heparin pentasaccharide and full-length heparin. The second order rate constants for ATIII inhibition in the presence of full-length heparin
were reduced for all variants as compared fXaWT (Table II).
Rate constants for inhibition of fXaR306A and
fXaE310N were modestly reduced (66% and 64% of
fXaWT, respectively), whereas those for
fXaR347N (12.6% of fXaWT, 12),
fXaK351A (20.1%), and fXaK414A (21.1%) were
markedly reduced. In contrast to these effects with full-length
heparin, inhibition of all variants in the presence of the
pentasaccharide was equivalent to that of fXaWT (Table II).
Thus, the basic residues at positions 347, 351, and 414 likely contribute to the heparin binding site of fXa.
View this table:
[in this window]
[in a new window]
|
Table II
Inhibition of variant and wild type fXa
Second order rate constants were determined as described under
"Experimental Procedures."
|
|
Prothrombin Activation in the Absence of fVa and
Phospholipid--
The catalytic function of the enzymes was evaluated
using the physiological substrate, prothrombin (Fig.
2). Multiple concentrations of
prothrombin were incubated with the mutant enzymes and the apparent
affinity for prothrombin and catalytic turnover were compared with that
of fXaWT. In the absence of cofactor and phospholipid, all
variant enzymes, with the exception of fXaE310N, interact similarly with prothrombin as indicated by similar
Km(app) values (range
Km(app) = 88.4-96.4 µM).
fXaE310N demonstrated a 3-fold reduction in the apparent
affinity for prothrombin (fXaWT Km(app) = 91.9 µM
versus fXaE310N Km(app) = 299 µM). The variation in the range of maximum
prothrombin turnover rates for all enzymes is less than 2-fold
(0.026-0.047 s
1), indicating that all
variant enzymes have similar catalytic activity compared with
fXaWT.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
Prothrombin activation in the absence of fVa
and phospholipid. 10 nM fXaWT ( ),
fXaR306A ( ), fXaE310N ( ),
fXaR347N ( ), fXaK351A ( ), or
fXaK414A ( ) was incubated with varying concentrations of
prothrombin (0-450 µM). Aliquots were removed from the
reactions for initial rate measurements, diluted 10-fold in EDTA
buffer, and incubated with 500 µM Spectrozyme THTM. The
initial rate of hydrolysis was monitored at 405 nm. The concentration
of thrombin was determined from a standard curve prepared from
dilutions of maximally activated prothrombin. The rate of thrombin
formation was calculated for the indicated prothrombin concentrations
and expressed as moles of thrombin (IIa)/mole of Xa/s. The maximal rate
of thrombin formation is expressed as kcat
(s 1).
|
|
Prothrombin Activation in the Presence of fVa ± Phospholipid--
To examine the interaction between the variant
enzymes and cofactor, thrombin formation was first quantitated in the
presence of fVa, but in the absence of phospholipid. All enzymes
demonstrated markedly reduced function as compared with
fXaWT (Fig. 3). fVa enhanced
the catalytic function of fXaWT, e.g. at 10 µM prothrombin, fXaWT was catalytically more
than 1000-fold faster in the presence of 250 nM fVa. The
catalytic activity of the variants was also enhanced by the addition of
cofactor, although not nearly to the same extent. In an effort to
increase the concentration of cofactor in the presence of the enzymes,
prothrombin turnover was evaluated on 100 nm PC:PS (3:1) vesicles which
bind fXa and fVa and colocalize the enzyme with cofactor. Under these
conditions, all variant enzymes displayed measurable function (Fig.
4). Here, the apparent fVa affinity for
fXaR306A and fXaE310N were only slightly
reduced relative to fXaWT (fXaWT
Kd(app) = 106 pM,
fXaR306A = 203 pM, and fXaE310N = 157 pM), whereas fXaK351A and
fXaK414A demonstrated a greater reduction in cofactor
affinity (fXaK351A Kd(app) = 366 pM, and fXaK414A = 556 pM). As
previously reported, the relative fVa affinity of fXaR347N
was markedly reduced under these conditions (fXaR347N
Kd(app) = 2299 pM). Maximal turnover
of prothrombin by all variant enzymes was found to be within 2-fold
that of fXaWT (range, 18.9-28.9
s
1) in the presence of cofactor and
phospholipid vesicles. These data are most easily explained by the
hypothesis that the major effect of substitution of Arg-347, Lys-351,
or Lys-414 is the reduction in fVa affinity in the presence of PC:PS
vesicles with little or no impact on catalytic activity of the fVa-fXa
complex.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Prothrombin activation in the presence of fVa
without phospholipid. 10 µM prothrombin was
pre-incubated with varying concentrations of fVa (0-250
nM). Reactions were initiated by the addition of 0.5 nM fXaWT ( ), fXaR306A ( ),
fXaE310N ( ), fXaR347N ( ),
fXaK351A ( ), or fXaK414A ( ). Samples were
removed from the reactions for initial rate measurements, quenched in
EDTA buffer, and incubated with 500 µM Spectrozyme THTM.
Initial rates of Spectrozyme THTM hydrolysis were determined at 405 nm, and thrombin formation was quantitated as described in the legend
to Fig. 2 for the fVa concentrations indicated.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Prothrombin activation in the presence of fVa
and PC:PS vesicles. Reactions contained 20 µM PC:PS
vesicles (3:1), fVa (0-1 nM), and 20 pM
fXaWT ( ), fXaR306A ( ),
fXaE310N ( ), fXaR347N ( ),
fXaK351A ( ), or fXaK414A ( ). Thrombin
formation was initiated by the addition of 1 µM
prothrombin and quantitated by incubating quenched reaction aliquots
with 500 µM Spectrozyme THTM. Thrombin formation was
quantitated as described in the legend to Fig. 2 and expressed as moles
of thrombin (IIa)/mole of Xa/s, and the maximal turnover rate in the
presence of saturating fVa is represented as
kcat(Va) (s 1).
|
|
If the mutations only impact cofactor affinity, the full catalytic
potential should be realized under ideal conditions. This hypothesis
was evaluated using activated platelets, which provide an ideal
phospholipid surface and saturating fVa. Indeed, under these
conditions, all fXa species are very similar to fXaWT (Fig. 5).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Prothrombin activation on an activated
platelet surface. Washed platelets (108/ml) were
incubated with 0.5 units/ml thrombin in the presence of varying
concentrations (0-1 nM) of fXaWT ( ),
fXaR306A ( ), fXaE310N ( ),
fXaR347N ( ), fXaK351A ( ), or
fXaK414A ( ). Thrombin generation was initiated by the
addition of 1 µM prothrombin. Initial rates of thrombin
formation were determined by incubating quenched aliquots of the
reactions with 500 µM Spectrozyme THTM. Thrombin formed
was quantitated as described in the legend to Fig. 2 and expressed as
thrombin (nM)/s, and the maximal turnover rate is expressed
as Vmax (nM/s).
|
|
Competition Binding Assay--
To confirm the results of the
functional studies and more directly probe the role of these residues
in fVa binding, we employed a previously described binding assay (12).
In the assay, active-site modified, radiolabeled fXa
(125I-fXaS379A) was mixed with varying
concentrations of unlabeled, mutant enzyme. Both enzymes were then
allowed to compete for fVa binding on a PC:PS-coated latex bead. Factor
Va binding is expressed for the variant enzymes in Fig.
6 as the percentage of
125I-fXaS379A bound to the bead in the presence
of increasing concentrations of added, unlabeled fXa.
fXaR306A and fXaE310N demonstrated modest reductions in fVa affinity in the assay (fXaWT
Kd(app) = 1.83 nM,
fXaR306A = 2.96 nM, fXaE310N = 4.03 nM), whereas the relative fVa affinity of
fXaK351A, fXaK414A, and fXaR347N
was markedly reduced (Kd(app) = 8.81, 12.17, and
19.45 nM, respectively). Consistent with the functional
studies on phospholipid vesicles, substitution of residues Lys-351,
Lys-414, and Arg-347 resulted in a selective loss of fVa affinity in
the absence of prothrombin, whereas mutation of Arg-306 and Glu-310
reduced fVa binding to a much lower extent.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 6.
Factor Va binding of fXa variants.
Factor Va (1 nM) was incubated with PC:PS-coated latex
beads (0.2%, v/v) for 10 min. 125I-fXaS379A (1 nM) was added in the presence of increasing concentrations
of each fXa species: fXaWT ( ), fXaR306A
( ), fXaE310N ( ), fXaR347N ( ),
fXaK351A ( ), or fXaK414A ( ), and the
reaction was incubated for an additional 10 min. The beads were
collected by centrifugation, and the supernatant and pellet were
counted separately. The fVa-specific binding (calcium-,
phosphatidylserine-, and fVa-dependent) was quantitated as
the percentage of bound 125I-fXaS379A. The
concentration of unlabeled fXa required to displace 50% of bound,
125I-fXaS379A is represented as the apparent
affinity (Kd(app)). Nonspecific binding has been
subtracted from all values shown.
|
|
fVa Binding Site of fXa--
The juxtaposition of the targeted
residues is apparent from the fXa structure (Fig.
7; Ref. 21). Residues found to contribute to the fVa binding site are clustered in a defined region on the fXa
surface in the serine protease domain. The impact of surrounding residues (Arg-273, Ile-357, Arg-406, Lys-406, and Lys-420) on fVa
affinity was evaluated using the fVa-sensitive functional assays
described above, i.e. in the presence and absence of PC:PS vesicles. Substitution of these residues had no impact on fVa affinity
in these assays (data not shown), thereby delineating the fVa binding
epitope.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 7.
A fVa binding site of fXa. The model was
generated using RIBBONS version 3.0. Arg-347, Lys-351, and
Lys-414 are shown in green and were found to contribute to
fVa binding. Arg-306 and Glu-310 are on the periphery of the binding
site and are highlighted in dark blue. Residues shown in
red (Arg-273, Ile-357, Arg-406, Lys-406, and Lys-420) were
found not to contribute to the fVa binding site of fXa. The active site
residues (Ser-379, Asp-282, and His-236) are shown in
gold.
|
|
 |
DISCUSSION |
The current study describes substitutions in the surface epitope
of fX that includes Arg-306, Glu-310, Arg-347, Lys-351, and Lys-414.
All mutants were synthesized and secreted in the expression system in a
manner equivalent to that of fXWT. Activation of these zymogens by fVIIa/TF, fIXa/fVIIIa, and RVV-X, hydrolysis of a small,
peptide substrate, and inhibition by TFPI and ATIII all occurred at a
near-normal rates. Based on these studies, substitution of these
residues does not compromise the active site structure of fXa.
Heparin can accelerate the rate of fXa inhibition by ATIII by mediating
conformational changes in the inhibitor and by acting as a template
that binds both inhibitor and enzyme (23). The individual contributions
of structural alterations in ATIII and binding to fXa were evaluated
using heparin pentasaccharide, which binds ATIII, but does not act as a
template. All variant enzymes were inhibited normally by ATIII in the
presence of heparin pentasaccharide. However, the rate of inhibition by
ATIII in the presence of full-length heparin was reduced for all
variants, indicating that these residues contribute to the heparin
binding capacity of fXa. Substitution of the glutamic acid at position
310 results in the addition of a carbohydrate group, which could
sterically hinder the binding of heparin to the fXa surface.
Substitution of the basic residues at positions 347, 351, 414, and 306 likely attenuates ionic interactions between heparin and fXa. These
data are corroborated by structural analysis of thrombin and fXa.
Lys-351 and Lys-414 are adjacent to the analogous epitope defined in
thrombin as the heparin binding exosite (24). Moreover, Padmanabhan and
co-workers (25) have described the heparin binding domain of fX as a
large basic region, which either includes or is adjacent to these
residues. Delineation of the entire heparin binding domain of fXa will
require additional mutagenesis.
All variant enzymes bound and activated prothrombin in a manner similar
to fXaWT with one exception, fXaE310N. The
apparent affinity of fXaE310N for prothrombin was 3-fold
lower than fXaWT. It is conceivable that the added
carbohydrate sterically hinders substrate binding. Alternatively, the
added sugar may restrict conformational mobility of fXa and prevent the
enzyme from adapting a favorable conformation for substrate binding in
the absence of a lipid surface.
This study examined the independent interactions between fXa and other
prothrombinase complex constituents. It was found that the sensitivity
to aberrant fVa binding is heightened under suboptimal conditions for
prothrombin turnover, i.e. in the presence of fVa ± PC:PS vesicles, allowing clear delineation of the residues that compose
the fVa binding site. As expected, substitutions of residues that
comprise the core binding site result in a more severe phenotype as
compared with mutations in the residues that form the extended regions
of the site. The power of this methodology is exemplified by
fXaR306A and fXaE310N, which displayed a
reduced function in the absence of phospholipid. On addition of PC:PS
vesicles, which increase the local concentration of prothrombinase
complex components and maximize productive interactions, these enzymes
bind fVa with a similar apparent affinity to fXaWT. In
contrast, the phenotype of fXaR347N, fXaK351A,
and fXaK414A was only overcome under the ideal conditions
of activated platelets, suggesting a more critical role of these
residues in fVa binding. Therefore, Arg-306 and Glu-310 may form part
of the binding site that extends beyond the core epitope whereas
Arg-347, Lys-351, and Lys-414 likely contribute to the core of the
binding epitope.
It has been proposed that fVa mediates conformational changes in fXa
that contribute to the cofactor-driven enhancement of prothrombin
activation (11, 26-28). In the absence of a phospholipid surface, cofactor enhanced the activity of fXaWT, but had
little effect on the variant enzymes. There are at least two possible explanations for these data. Either the mutations compromised the
functional capacity of fXa by attenuating these proposed
cofactor-mediated structural rearrangements, or fVa binding is impaired
by the mutations. If these structural rearrangements are attenuated by
the substitutions, the aberrant phenotype would not likely be overcome
even under optimal conditions for substrate turnover. It was
demonstrated that mutant enzymes with less severe phenotypes
(fXaE310N and fXaR306A) can function nearly
normally on a PC:PS vesicle surface in the presence of fVa and the
function of all mutant enzymes was similar to the wild-type enzyme
under the conditions provided by activated platelets. Moreover, the
competition binding assay, which directly probes cofactor binding,
demonstrated a more marked loss of fVa affinity for variant enzymes
with more severe phenotypes in the functional assays. The data do not
support the postulate that substitution of these residues prevented
fVa-mediated changes in the fXa structure, but rather that the fVa
affinity is selectively attenuated by the mutations.
Analysis of the tertiary structure of fXa provides compelling
structural support for the existence of a fVa binding epitope in the
serine protease domain of fXa. Using peptide mapping studies, Chattopadhyay et al. (30) identified three regions of fXa
that were found to contribute to fVa binding: 211-222, 254-269, and 263-274 (30). Each of these regions was evaluated by mutagenesis using
functional assays and found not to contribute to fVa binding (data not
shown). The discrepancy between the studies may reflect differences in
methodologies. The region identified by the current study has also been
described as a cofactor binding site for other coagulation enzymes. For
example, the analogous surface loop of fIXa, which corresponds to
residues 344-352 in fXa, was recently identified to form a binding
epitope for fVIIIa (31, 32). Moreover, Banner et al. (29)
identified multiple regions of the fVIIa surface that contribute to TF
binding including one region analogous to fX residues 346-349.
Targeted mutagenesis of the residues that surround the proposed
cofactor binding epitope was also conducted as part of this study. It
was found that residues Arg-273, Ile-357, Arg-406, Lys-406, and Lys-420
do not contribute to fVa binding, but rather outline the fVa binding
epitope. Based on this survey of adjacent epitopes, structural
examination, and comparisons to other cofactor binding epitopes, it is
likely that Arg-347, Lys-351, and Lys-414 form the core of a fVa
binding epitope of fXa, which is extended to include Arg-306 and
Glu-310.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Tom Girard and Kevin Conricode
for critical review of the manuscript and Dr. Ravi Kurumbail for
assistance with the RIBBONS program.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant NHLBI HL14147 and a grant from the Monsanto/Searle Co.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 and reprint requests should be addressed.
Present address: Pharmacia Corp., 800 N. Lindbergh Blvd., St. Louis, MO
63167. Tel.: 314-694-9017; Fax: 314-694-8153; E-mail: amy.e.rudolph@monsanto.com.
§
Present address: Merck & Co., Inc., West Point, PA 19486.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M006961200
 |
ABBREVIATIONS |
The abbreviations used are:
f, factor (as in fXa);
TF, human tissue factor;
TFPI, Tissue Factor Pathway
Inhibitor;
RVV-X, X activating protein from Russell's viper
venom;
ATIII, antithrombin III;
PC, phosphatidylcholine;
PS, phosphatidylserine;
EGF, epidermal growth factor.
 |
REFERENCES |
1.
|
Jesty, J.,
Spencer, A. K.,
and Nemerson, Y.
(1974)
J. Biol. Chem.
249,
5614-5622[Abstract/Free Full Text]
|
2.
|
DiScipio, R. G.,
Hermodson, M. A.,
and Davie, E. W.
(1977)
Biochemistry
16,
5253-5260[Medline]
[Order article via Infotrieve]
|
3.
|
Pfeiffer, R. A.,
Ott, R.,
Gilgenkrantz, S.,
and Alexandre, P.
(1982)
Hum. Genet.
62,
358-360[Medline]
[Order article via Infotrieve]
|
4.
|
Nemerson, Y.,
and Bach, R.
(1982)
Prog. Hemostasis Thromb.
6,
237-261[Medline]
[Order article via Infotrieve]
|
5.
|
Nemerson, Y.
(1986)
Blood
71,
1-8[Medline]
[Order article via Infotrieve]
|
6.
|
Mann, K. G.,
Jenny, R. J.,
and Krishnaswamy, S.
(1988)
Annu. Rev. Biochem.
57,
915-956[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Rosenberg, R. D.,
and Damus, P. S.
(1973)
J. Biol. Chem.
248,
6490-6505[Abstract/Free Full Text]
|
8.
|
Owen, B. A.,
and Owen, W. G.
(1990)
Biochemistry
29,
9412-9417[Medline]
[Order article via Infotrieve]
|
9.
|
Broze, G. J., Jr.,
Warren, L. A.,
Novotny, W. F.,
Higuchi, D. A.,
Girard, T. J.,
and Miletich, J. P.
(1988)
Blood
71,
335-343[Abstract]
|
10.
|
Griard, T. J.,
Warren, L. A.,
Novotny, W. F.,
Likert, K. M.,
Brown, S. G.,
Miletich, J. P.,
and Broze, G. J., Jr.
(1989)
Nature
338,
518-520[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Krishnaswamy, S.
(1990)
J. Biol. Chem.
265,
3708-3718[Abstract/Free Full Text]
|
12.
|
Rudolph, A. E.,
Porche-Sorbet, R.,
and Miletich, J. P.
(2000)
Biochemistry
39,
2861-2867[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Rudolph, A. E.,
Mullane, M. P.,
Porche-Sorbet, R.,
Tsuda, S.,
and Miletich, J. P.
(1996)
J. Biol. Chem.
271,
28601-28606[Abstract/Free Full Text]
|
14.
|
Rudolph, A. E.,
Mullane, M. P.,
Porche-Sorbet, R.,
and Miletich, J. P.
(1997)
Protein Exp. Purif.
10,
373-378[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Broze, G. J., Jr.,
and Miletich, J. P.
(1984)
J. Clin. Invest.
73,
933-938[Medline]
[Order article via Infotrieve]
|
16.
|
Kisiel, W.,
Hermodson, M. A.,
and Davie, E. W.
(1976)
Biochemistry
15,
4901-4906[Medline]
[Order article via Infotrieve]
|
17.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
|
18.
|
Graham, F. L.,
and Eb, A. J.
(1973)
Virology
52,
456-467[Medline]
[Order article via Infotrieve]
|
19.
|
Huang, Z.,
Wun, T.,
and Broze, G. J., Jr.
(1993)
J. Biol. Chem.
268,
26950-26955[Abstract/Free Full Text]
|
20.
|
Olson, S. T.,
Bjork, I.,
and Shore, J. D.
(1993)
Methods Enzymol.
222,
525-559[Medline]
[Order article via Infotrieve]
|
21.
|
Brandstetter, H.,
Kuhne, A.,
Bode, W.,
Huber, R.,
von der Saal, W.,
Wirthensohn, K.,
and Engh, R. A.
(1996)
J. Biol. Chem.
271,
29988-29992[Abstract/Free Full Text]
|
22.
|
Craig, P. A.,
Olson, S. T.,
and Shore, J. D.
(1989)
J. Biol. Chem.
264,
5452-5461[Abstract/Free Full Text]
|
23.
|
Olson, S. T.,
Bjork, I.,
Sheffer, R.,
Craig, P. A.,
Shore, J. D.,
and Choay, J.
(1992)
J. Biol. Chem.
267,
12528-12538[Abstract/Free Full Text]
|
24.
|
Sheehan, J. P.,
and Sadler, J. E.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
5518-5522[Abstract]
|
25.
|
Padmanabhan, K.,
Padmanabha, K. P.,
Tulinsky, A.,
Park, C. H.,
Bode, W.,
Huber, R.,
Blankenship, D. T.,
Cardin, A. D.,
and Kisiel, W.
(1993)
J. Mol. Biol.
232,
947-966[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Husten, J. E.,
Esmon, C. T.,
and Johnson, A. E.
(1987)
J. Biol. Chem.
262,
12953-12961[Abstract/Free Full Text]
|
27.
|
Walker, R. K.,
and Krishnaswamy, S.
(1993)
J. Biol. Chem.
268,
13920-13929[Abstract/Free Full Text]
|
28.
|
Boskovic, D. S.,
Giles, A. R.,
and Nesheim, M. E.
(1990)
J. Biol. Chem.
265,
10497-10505[Abstract/Free Full Text]
|
29.
|
Banner, D. W.,
D'Arcy, A.,
Chene, C.,
Winkler, F. K.,
Guha, A.,
Konigsberg, W. H.,
Nemerson, Y.,
and Kirchhofer, D.
(1996)
Nature
380,
41-46[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Chattopadhyay, A.,
James, H. L.,
and Fair, D. S.
(1992)
J. Biol. Chem.
267,
12323-12329[Abstract/Free Full Text]
|
31.
|
Mathur, A.,
and Bajaj, S. P.
(1999)
J. Biol. Chem.
274,
18477-18486[Abstract/Free Full Text]
|
32.
|
Bajaj, S. P.
(1999)
Thromb. Haemostasis
82,
218-225[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.