From the
Mutation of residue 192 (chymotrypsin numbering) from Glu to Gln
in thrombin and activated protein C has been shown to dramatically
alter substrate and inhibitor specificity, in large part by allowing
these enzymes to accept substrates with acidic residues in the P3
and/or P3` positions. In factor Xa, residue 192 is already a Gln. We
now compare the properties of a Q192E mutant of Gla-domainless factor X
(GDFX). Kinetic analysis of prothrombin activation indicates similar
affinity of factor Va for GDFXa and GDFXa Q192E (K
Factor X is a vitamin K-dependent plasma zymogen(1) .
After activation to generate factor Xa, this enzyme can form a
Ca
The mechanism by which
factor Va increases the k
Prothrombin activation requires two proteolytic cleavages:
one to release the activation fragments, which correspond to roughly
half of the mass of prothrombin, and the other to generate the
two-chain enzyme thrombin(2) . Depending on the order of bond
cleavage, two intermediates can be formed, prethrombin 2 or
meizothrombin. With factor Xa alone, the reaction intermediate is
prethrombin 2 and a large activation fragment 1.2(9) . The order
of bond cleavage is altered by the presence of factor Va and
membranes(7, 10) . In the presence of factor Va, the
majority of the thrombin is derived from the meizothrombin
intermediate(7, 10) . Meizothrombin hydrolyzes small
peptide chromogenic substrates well, but does not clot fibrinogen
effectively(11) . Meizothrombin is also inhibited by
antithrombin, but the inhibition is not accelerated by
heparin(12, 13) .
Our previous studies (24, 28) have shown that residue 192 in thrombin and
activated protein C plays an important role in enzyme specificity.
Mutation of Glu-192 to Gln in both thrombin and activated protein C
resulted in mutant enzymes that cleaved substrates with the acidic P3
residues better than the wild-type enzymes, indicating that Glu-192 in
both thrombin and activated protein C may be responsible for the slow
cleavage of substrates with acidic residues at the P3 positions.
Residue 192 in factor Xa is Gln. Interestingly, the sequence near the
cleavage sites in human and bovine prothrombins contains acidic
residues at both the P3 and P3` positions in both cleavage sites
(except for a Thr at the P3` position of the first cleavage site in
human prothrombin). We hypothesized that if residue 192 in factor Xa
interacts with the P3 position of the substrate, then the Q192E mutant
would activate prothrombin at a slower rate. Furthermore, if factor Va
changes the conformation of the active-site pocket in factor Xa and/or
residues near the scissile bonds in prothrombin, then factor Va may
overcome the inhibitory interactions in a manner analogous to
thrombomodulin with thrombin.
Overall, the results of this study
indicate that residue 192 contributes significantly to the substrate
and inhibitor specificity of factor Xa and that it may be one of the
residues sensitive to conformational changes induced by factor Va
binding.
Since the activity of meizothrombin des-fragment 1
toward fibrinogen is considerably less than that of thrombin, the
initial rate of meizothrombin des-fragment 1 activation was measured in
a fibrinogen clotting assay using an ST4 Bio coagulometer
(Diagnostica/Stago, Asnieres, France). In this case, meizothrombin
des-fragment 1 (2 µM) was incubated with GDFXa or GDFXa
Q192E (15 nM) at 37 °C in TBS containing 5 mM Ca
Factor Va can accelerate GDFXa
activation of prothrombin at least 1000-fold(14) . To determine
the affinity of factor Va for the Q192E mutant of GDFXa, the rates of
human prothrombin activation by GDFXa and GDFXa Q192E were compared as
a function of factor Va concentration (Fig. 1). Analysis of the
data in Fig. 1indicated that factor Va interacted with both
enzymes with a similar affinity (K
In
meizothrombin des-fragment 1, the Arg-323 bond is already cleaved, and
cleavage of Arg-274 bond leads to thrombin generation. Meizothrombin
des-fragment 1 activation by GDFXa and GDFXa Q192E in the presence and
absence of factor Va is shown in Fig. 3. In the absence of factor
Va, GDFXa activated meizothrombin des-fragment 1 10-fold faster than
GDFXa Q192E (Fig. 3A). In the presence of factor Va,
however, the activation by GDFXa was also
This study indicates that residue 192 in factor Xa plays a
key role in determining the macromolecular substrate and inhibitor
specificity of factor Xa. Particularly interesting is that in
comparison with GDFXa, GDFXa Q192E by itself activates prothrombin
poorly. In the presence of factor Va, however, the activation rates
with both derivatives of factor Xa are similar. Inspection of residues
surrounding the scissile bonds may reveal an explanation for the slower
rate of activation of prothrombin by GDFXa Q192E alone. All but one of
the P3 and P3` residues in human prothrombin and all of these residues
in bovine prothrombin are acidic. It is our hypothesis that charge
repulsion between Glu-192 in the factor Xa mutant and any one of these
P3 and/or P3` acidic residues present on prothrombin activation
peptides prevents optimal activation by GDFXa Q192E. This is consistent
with the relative specificities of the two enzymes toward chromogenic
substrates. It appears that factor Va alleviates this inhibitory
interaction.
The mechanism by which factor Va influences the k
It should be noted, however, that factor Va
binding to the substrate also occurs and could change the conformation
near the scissile bond and overcome inhibitory interactions in a
similar way(5, 31) . Previous studies demonstrated that
prothrombin fragment 2 was necessary for factor Va to accelerate
prethrombin 2 activation effectively(8) . In principle, at least
two mechanisms could account for this requirement. Fragment 2 could
alter the conformation near the scissile bond so that it was recognized
better by the factor Xa-Va complex, or it could only provide a binding
site for factor Va. The results of the present study suggest that
fragment 2 alters the conformation near the scissile bond since it
selectively enhances prethrombin 2 activation by GDFXa Q192E. It is
therefore likely that fragment 2 binding to prethrombin 2 (or these
interactions within prothrombin) contributes to an altered conformation
near Arg-323 that is complementary to the active site of factor Xa
within the factor Xa-Va complex. These results indicate that a function
for fragment 2 in prethrombin 2 activation may involve substrate
presentation.
It is known that clotting proteases such as activated
protein C and thrombin, which have Glu at position 192, are resistant
to inhibition by the Kunitz inhibitors TFPI and BPTI, but factor Xa
with Gln at this position is inhibited effectively by TFPI. Our
previous studies with activated protein C (24) and thrombin (32) indicated that substitution of Glu-192 with Gln increased
the reactivity of these enzymes toward Kunitz inhibitors. Consistent
with those observations, substitution of Gln-192 with Glu in factor Xa
reduces TFPI reactivity by 2 orders of magnitude and completely
abolishes BPTI reactivity up to 300 µM inhibitor
concentration.
In contrast to Kunitz inhibitors, GDFXa and the Q192E
mutant had nearly identical second-order association rate constants
with antithrombin in the presence or absence of heparin. This may
reflect the strong preference of factor Xa for Gly at the P2 position
since antithrombin contains Gly at this site. In support of this
hypothesis, S2765 and S2222, two of the best factor Xa synthetic
substrates, both contain Gly at the P2 positions, but contain Arg and
Glu, respectively, at the P3 positions. The tolerance of two oppositely
charged residues at the P3 site may indicate that factor Xa specificity
is largely dependent on the S2 specificity pocket.
The studies
presented here were performed with GDFXa and the Q192E mutant.
Truncation of the Gla domain does alter some of the properties of
factor Xa, but the deletion mutants share many properties in common
with the wild-type enzyme. The Gla domain of factor X is critical for
membrane interaction, partially modulates substrate specificity when
associated with factor Va on the membrane surface(2) , and
enhances the affinity for factor Va even in the absence of
membranes(33) . Factor Va still enhances prothrombin activation
by GDFXa and factor Xa to nearly the same extent, however; and as seen
in this study, like intact factor Xa, GDFXa requires the presence of
prothrombin fragment 2 for maximum acceleration of prethrombin 2
activation. Thus, these forms of factor Xa are useful models for
examining enzyme specificity and cofactor function in solution. The
deletion mutants avoid the potential for Ca
Based on the analysis of thrombin, activated
protein C, and factor Xa, it is now apparent that residue 192 plays a
central role in determining the substrate specificity of the
coagulation serine proteases. It also appears that the conformation of
this region in the coagulation proteases is altered by binding to the
respective cofactors. The combination of enzyme and substrate
mutagenesis with x-ray crystallographic studies promises to provide
future insights into the mechanisms by which the cofactors accelerate
coagulation reactions.
The K
We thank Gary Ferrell and Bronson Sievers for help
with cell cultures; Barbara Carpenter and Clendon Brown for isolation
of recombinant and plasma proteins used in this study; Naomi L. Esmon
for useful discussions; and Jeff Box, Karen Deatherage, and Julie
Wiseman for assistance with preparation of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 3.6 and 3.7
µM, respectively). Prothrombin activation rates are
similar for both enzymes with factor Va, but are
10-fold slower
for the Q192E mutant without factor Va. This defect is in the
activation of prethrombin 2 and is corrected by factor Va only in the
presence of fragment 2. Without factor Va, fragment 2 has no influence
on bovine prethrombin 2 activation by GDFXa, but fragment 2 enhances
prethrombin 2 activation by the Q192E mutant at least 10-fold. These
results indicate that the fragment 2 domain of prothrombin probably
alters the conformation of the prethrombin 2 domain, selectively
improving its presentation to GDFXa Q192E. With respect to inhibition,
tissue factor pathway inhibitor and bovine pancreatic trypsin inhibitor
are
30 times poorer inhibitors of GDFXa Q192E than of GDFXa, but
these enzymes are inhibited at comparable rates by antithrombin. These
results indicate that Gln-192 in factor Xa is a key determinant of
substrate/inhibitor specificity.
-dependent complex with factor Va on membrane
surfaces (prothrombinase complex) that rapidly activates prothrombin to
thrombin(1, 2) . In comparison to factor Xa alone,
formation of the complete complex enhances the rate of prothrombin
activation
10
-fold(2) . The dramatic increase
in catalytic efficiency of prothrombinase is thought to arise from a
100-fold decrease in apparent K
and a
3000-fold increase in the k
for the
reaction(2) . The decrease in K
is believed to be the
result of negatively charged phospholipid-protein interaction, which
raises the local concentration of prothrombin near the enzyme complex,
and the increase in k
is attributed to the
cofactor effect of factor Va(2) .
of prothrombin
activation may involve conformational changes in the enzyme, substrate,
or both(3, 4, 5, 6) . In support of the
first hypothesis, Husten et al.(4) , using
fluorescently labeled active site-specific probes, demonstrated that
upon association with factor Va, the environment of the probe in factor
Xa is altered, suggesting a change in the conformation of the active
site of the enzyme. Also consistent with this hypothesis, Krishnaswamy et al.(7) demonstrated that factor Va complex
formation with factor Xa enhanced the binding affinity of the tick
anticoagulant protease inhibitor. Support for the second hypothesis is
provided by the observation that optimal cofactor effect of factor Va
requires the presence of the fragment 2 domain of prothrombin in either
covalent or noncovalent association with prethrombin
2(8, 9) . The simplest interpretation of the fragment 2
requirement in prothrombin activation is that it provides a binding
site for factor Va. Alternatively, fragment 2 may change the
conformation of residues near the scissile bonds to resemble the
transition state (5) and be complementary to the factor Xa-Va
complex.
Mutagenesis and Expression of Recombinant
Proteins
Construction and expression of GDFX(
)in the RSV-PL4 vector were described
previously(14, 15) . Mutagenesis of the GDFX cDNA
fragment for preparing the Q192E mutant was performed by the polymerase
chain reaction. The mutant codon and the native ApaI
restriction enzyme site were included in the 3`-antisense primer. The
sense primer started from the native StuI restriction enzyme
site and was designed to contain a silent mutation to eliminate the
second ApaI restriction enzyme site. The sense mutagenesis
primer was 5`-CCTGCATTCCCACAGGGCCTTACCCCTGT-3`, and the antisense
primer was 5`-CGTGCGGGCCCCCGCTGTCCCCCTCGCAGGCATCCT-3`. After
amplification of factor X cDNA with these two primers, the resulting
DNA fragment was ligated into the StuI and ApaI sites
of the GDFX DNA fragment in the RSV-PL4 expression
vector(14, 15) . After confirmation of the mutation by
DNA sequencing(16) , the expression vector was transfected into
293 cells, and the mutant protein was isolated from cell culture
supernatants by immunoaffinity chromatography as described
previously(14, 15) .
Protein Preparation
Human factor X(17) ,
human thrombin (9), human meizothrombin(3) , human
prothrombin(18) , bovine prothrombin and prethrombin
2(9) , bovine prothrombin fragment 2(9, 19) ,
bovine factor Va(20) , bovine antithrombin(21) , and the
factor X-activating enzyme from Russell's viper venom (22) were isolated as described. Recombinant full-length tissue
factor pathway inhibitor was expressed and isolated from Escherichia coli and was a generous gift of Dr. Gerald Gallupi
(Monsanto). Bovine pancreatic trypsin inhibitor was purchased from
Sigma.
Activation of GDFX and GDFX Q192E and Analysis of
Active-site Concentration
The relative concentrations of factor
X mutants were determined based on their relative A values assuming a molecular mass of 55,000 Da for the zymogen
forms and 45,000 Da for the active forms and an E
of 10 for both forms of mutants. Activation was
performed by incubating 20 µg of zymogens with 500 ng of the factor
X activator from Russell's viper venom in 0.1 M NaCl,
0.02 Tris-HCl, pH 7.5, 0.02% NaN
(TBS) containing 0.1%
gelatin and 1 mM CaCl
at 37 °C for 4 h, which
is sufficient to obtain complete activation as demonstrated by
SDS-polyacrylamide gel electrophoresis analysis and amidolytic activity
assays using the chromogenic substrate Spectrozyme FXa (American
Diagnostica Inc., Greenwich, CT). The active-site concentration was
determined by an active site-specific immunoassay using BioCap-EGR-ck
(Haematologic Technologies Inc.) as described(23) . This
procedure was used as an alternative to the conventional active-site
titration method using p-nitrophenyl p`-guanidinobenzoate. In contrast to the p-nitrophenyl p`-guanidinobenzoate burst, the
BioCap-EGR-ck immunoassay method is sensitive to <1 ng/ml factor Xa.
Briefly, factor Xa and the active mutant derivatives (1 µg/ml) were
incubated with a 10-fold molar excess of BioCap-EGR-ck for 15 min at 37
°C, after which no factor Xa activity was detected by an amidolytic
activity assay using Spectrozyme FXa. A 96-well microtiter plate was
coated with 2 µg/ml goat anti-factor X polyclonal antibody in TBS
and placed at 4 °C overnight. The wells were washed with TBS
containing 0.1% Tween 20 and then blocked with TBS containing 1% bovine
serum albumin at room temperature for 1 h. After removing the blocking
buffer, BioCap-EGR-ck-treated wild-type factor Xa of known
concentrations as a standard or BioCap-EGR-ck-treated GDFXa and GDFXa
Q192E as unknowns were added. After a 1-h incubation at room
temperature and washing with TBS, streptavidin-alkaline phosphatase
conjugate was added for 1 h at room temperature. The substrate (the
reduced form of NADPH) was added, and the signal was amplified
according to the manufacturer's direction using the enzyme-linked
immunosorbent assay amplification system kit (Life Technologies, Inc.).
This enzyme-linked immunosorbent assay detected active site-inhibited
factor Xa concentrations as low as 300 pg/ml.
Prothrombin Activation and Apparent Affinity for Factor
Va
The initial rates of prothrombin (5 µM)
activation by GDFXa and GDFXa Q192E (2.5 nM in the presence of
500 nM factor Va and 50 nM in the absence of factor
Va) were measured in TBS containing 5 mM Ca and 0.1% gelatin at room temperature. The initial rate was
determined by measuring the increase in amidolytic activity as a
function of time. The initial rates of prothrombin activation were also
measured as a function of enzyme concentration. In this case,
prothrombin (5 µM) was incubated with 500 nM factor Va and different concentrations of GDFXa or GDFXa Q192E
(2.5, 5, 10, 20, 30, and 50 nM) for 3 min at room temperature
in the same buffer. The rate of prothrombin activation was also used to
determine the apparent affinity of factor Va for GDFXa and GDFXa Q192E.
In this case, prothrombin (1 µM) was incubated with 5
nM enzymes and 5 mM Ca
in the same
buffer at room temperature in the presence of different concentrations
of factor Va. The rate of prothrombin activation was determined from a
standard curve in an amidolytic assay using the chromogenic substrate
S2238 (Kabi Pharmacia/Chromogenix, Franklin, OH) and plotted as a
function of the free factor Va concentration as described
previously(14) .
Activation of Prethrombin 2 and Meizothrombin
Des-fragment 1 by GDFXa and GDFXa Q192E
The initial rate of
bovine prethrombin 2 (2 µM) activation by GDFXa or GDFXa
Q192E (200 nM) was measured in the presence or absence of
bovine prothrombin fragment 2 (2 µM) at 37 °C in TBS
containing 5 mM Ca and 0.1% gelatin.
Prethrombin 2 + fragment 2 activation was also carried out in the
presence of factor Va. In this case, prethrombin 2 + fragment 2 (2
µM each) was incubated with GDFXa or GDFXa Q192E (10
nM) and factor Va (100 nM) at room temperature in TBS
containing 5 mM Ca
and 0.1% gelatin. The
initial rate of activation was measured from the rate of thrombin
generation as a function of time by an amidolytic activity assay as
described above.
and 0.1% gelatin (in the presence of factor
Va (300 nM), the concentration of GDFXa or GDFXa Q192E was 4
nM). At different time points, the activation mixture was
diluted into TBS buffer, and 100 µl of this dilution was added to
100 µl of 6 mg/ml human fibrinogen (Kabi Diagnostica, Stockholm,
Sweden) in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1
mM Ca
at 37 °C. The rate of thrombin
generation was measured from the clotting time and a clotting reference
curve made with known amounts of thrombin. At each time point, the
background clotting time of meizothrombin des-fragment 1 was subtracted
from the samples.
Amidolytic Activity
Steady-state kinetic analysis
of commercially available chromogenic substrate hydrolyses were
performed in the presence of 2.5 nM GDFXa or GDFXa Q192E in
TBS containing 5 mM Ca and 0.1% gelatin. The k
and K
values
were determined individually for S2765 and S2222 (Kabi
Pharmacia/Chromogenix), Spectrozyme PCa (American Diagnostica Inc.),
and Chromozym tPA (Boehringer Mannheim). The rates of hydrolysis were
monitored at 405 nm at room temperature in a V
kinetic plate reader (Molecular Devices, Menlo Park, CA). The
concentration of synthetic substrate ranged from 15 µM to
2 mM.
Inhibition by Kunitz Inhibitors and
Antithrombin
Bovine pancreatic trypsin inhibitor (BPTI) and
recombinant TFPI were incubated at room temperature with 2.5 nM GDFXa or GDFXa Q192E for 30 min. The inhibitor concentration
ranged from 0 to 300 µM BPTI for both GDFXa and GDFXa
Q192E, from 0 to 100 nM TFPI for GDFXa, and from 0 to 1
µM TFPI for GDFXa Q192E. Residual amidolytic activity was
determined at equilibrium, and the inhibition constant (K) for each inhibitor was estimated as
described previously(24) . The second-order rate constant for
antithrombin inhibition of GDFXa and GDFXa Q192E (2.5 nM) was
estimated from inhibition time courses performed at antithrombin
concentrations ranging from 0 to 1.6 µM as described
previously(24) .
Electrophoresis
SDS-polyacrylamide gel
electrophoresis was performed on a 10% polyacrylamide gel as described
by Laemmli (25) and stained with Coomassie Blue R-250.
Data Analysis
The apparent affinity of factor Va
for GDFXa and GDFXa Q192E and the k and K
values were determined by nonlinear
regression analysis using the one-site ligand binding and the
Michaelis-Menten equation in the ENZFITTER program (Elsevier-Biosoft,
London) with simple weighting.
Expression and Activation of Recombinant
Proteins
GDFX and GDFX Q192E were expressed in 293 cells and
purified from cell culture supernatants as described under
``Experimental Procedures.'' The SDS-polyacrylamide gel
electrophoresis analysis indicated that the isolated proteins are
essentially homogeneous (data not shown). Under nonreducing conditions,
both derivatives migrated as a single chain with a similar apparent
molecular mass of 62 kDa. When reduced, a heavy and a light chain of
50 and 25 kDa were observed in both cases, indicating that 293
cells processed both factor X mutants completely to the two-chain form
characteristic of plasma factor X. GDFX and GDFX Q192E were activated
separately with the factor X-activating enzyme from Russell's
viper venom. The concentration of active enzymes was determined by an
active site-specific immunoassay as described under ``Experimental
Procedures.'' As expected from homogeneity on SDS-polyacrylamide
gel electrophoresis, the concentration of both enzymes as determined by
this assay was in good agreement with that calculated by their
absorbance values at 280 nm (0.89 and 0.87 mol of active site/mol for
GDFXa and GDFXa Q192E, respectively).
= 3.6 ±
0.4 µM for GDFXa and 3.7 ± 0.8 µM for
GDFXa Q192E). We next compared the time course of activation of
prothrombin in the absence and presence of factor Va. As shown in Fig. 2A, the rate of human prothrombin activation by
GDFXa Q192E in the absence of factor Va was 6-10-fold slower than
that by GDFXa, but in the presence of factor Va, both derivatives of
factor Xa activated prothrombin at a similar rate (Fig. 2B). These results indicate that factor Va
essentially overcomes the catalytic deficiency in GDFXa Q192E. Similar
results were obtained with bovine prothrombin. However, GDFXa and GDFXa
Q192E activated human prothrombin
3- and 9-fold faster than bovine
prothrombin in the presence of factor Va and 3- and 8-fold faster than
bovine prothrombin in the absence of factor Va, respectively (data not
shown). This may be due to sequence differences near the cleavage
sites. The P3-P3` sequences of the two cleavage sites of human
prothrombin are 1) Glu-Gly-Arg-Thr-Ala-Thr and 2)
Asp-Gly-Arg-Ile-Val-Glu. In bovine prothrombin, the corresponding
sequences are 1) Glu-Gly-Arg-Thr-Ser-Glu and 2)
Glu-Gly-Arg-Ile-Val-Glu. Similar results were obtained when the
concentration of prothrombin was kept constant, and the initial rates
were compared as a function of different concentrations of factor Xa
derivatives (data not shown).
Figure 1:
Comparison of
the apparent affinity of GDFXa and GDFXa Q192E for factor Va. 2.5
nM GDFXa () or GDFXa Q192E (
) was incubated at room
temperature with 1 µM prothrombin in TBS containing 0.1%
gelatin, 5 mM Ca
, and the concentrations of
factor Va indicated on the xaxis. Apparent
dissociation constants for factor Va were measured from the rate of
thrombin formation as described under ``Experimental
Procedures.'' Three independent estimates of K
were obtained with GDFXa. With prothrombin as the substrate, the K
values were 3.0, 4.0, and 3.3 µM (average of 3.6 µM). With GDFXa Q192E and prothrombin
as the substrate, two independent estimates of K
were performed, and both gave K
=
3.7 µM. With prethrombin 1 as the substrate, the K
for GDFXa was 1.3 µM and for
GDFXa Q192E was 1.5 µM.
Figure 2:
Initial rates of prothrombin activation by
GDFXa and GDFXa Q192E with and without factor Va. A, 50 nM GDFXa () or GDFXa Q192E (
) was incubated at room
temperature with 5 µM human prothrombin in TBS containing
5 mM Ca
and 0.1% gelatin. At the indicated
time points, samples were removed into 20 mM EDTA on ice, and
the amidolytic activities were determined with S2238 as described under
``Experimental Procedures.'' B, in the presence of
500 nM factor Va, 2.5 nM GDFXa (
) or GDFXa
Q192E (
) was incubated at room temperature with 5 µM human prothrombin in TBS containing 5 mM Ca
and 0.1% gelatin. Similar to the conditions described for Fig.
4A, at the indicated time points, samples were removed, and
the amidolytic activities were determined with
S2238.
Thrombin generation is the result of
two peptide bond cleavages, Arg-274-Thr-275 and
Arg-323-Ile-324 (bovine prothrombin numbering). The order in
which these bonds are cleaved varies with activation
conditions(7, 10) . One pathway generates prethrombin 2
(a single-chain thrombin precursor) as the reaction intermediate; the
other generates meizothrombin, a two-chain amidolytically active
protein of the same mass as prothrombin(9) . Since, in the
absence of factor Va, prothrombin activation by the mutant was
6-10-fold slower than the wild type and, in the presence of
factor Va, the activation rates were comparable, we decided to
determine whether cleavage of the Arg-274 bond or the Arg-324 bond is
impaired by the Q192E mutation. To address this issue, the activation
of both intermediates by GDFXa and the Q192E mutant was studied
separately in the presence and absence of factor Va.
5-fold faster than that
by GDFXa Q192E (Fig. 3B), indicating that factor Va has
little effect in correcting the defect of the Q192E mutation through
cleavage of the Arg-274 bond.
Figure 3:
Initial
rate of meizothrombin des-fragment 1 activation by GDFXa and GDFXa
Q192E. A, meizothrombin des-fragment 1 (2 µM) was
incubated with GDFXa () or GDFXa Q192E (
) (15 nM)
at 37 °C in TBS containing 5 mM Ca
and
0.1% gelatin. At different time points, aliquots of the meizothrombin
des-fragment 1 activation mixture were removed, and the rate of
thrombin generation was measured from the rate of fibrinogen cleavage
as described under ``Experimental Procedures.'' B,
meizothrombin des-fragment 1 (2 µM) was incubated with
GDFXa (
) or GDFXa Q192E (
) (4 nM) in the presence
of factor Va (300 nM) in the same buffer, and the rate of
thrombin generation was measured as described for A.
In prethrombin 2, the Arg-274 bond is
cleaved, and only the cleavage of the Arg-323 bond is required for
thrombin formation. In the absence of factor Va, the Q192E mutation
decreases the prethrombin 2 activation rate at least 10-fold (Fig. 4A). It has been demonstrated in the past that the
optimal cofactor function of factor Va in prothrombin activation
requires the fragment 2 domain(8) . In the presence of
prothrombin fragment 2 and factor Va, the activation rate by the Q192E
mutant was nearly similar to that by GDFXa (Fig. 4B).
These results indicate that in the presence of fragment 2, factor Va
largely overcomes the inhibitory effect of the Q192E mutation.
Figure 4:
Initial rate of prethrombin 2 activation
by GDFXa or GDFXa Q192E. A, bovine prethrombin 2 (2
µM) was incubated with GDFXa () or GDFXa Q192E
(
) (200 nM) at 37 °C in TBS containing 0.1% gelatin.
At the indicated time points, samples were removed into 20 mM EDTA on ice, and the amidolytic activities were determined with
S2238 as described under ``Experimental Procedures.'' B, prethrombin 2 (2 µM) and an equimolar
concentration of fragment 2 were incubated with GDFXa (
) or GDFXa
Q192E (
) (10 nM) in the presence of factor Va (100
nM) at room temperature in the same buffer. The rate of
thrombin generation was measured as described for A.
To
determine whether fragment 2 by itself would change the properties of
prethrombin 2, we examined prethrombin 2 activation by GDFXa and GDFXa
Q192E in the presence and absence of the fragment 2 domain. As shown in Fig. 5A, the presence or absence of fragment 2 has no
influence on prethrombin 2 activation by GDFXa, consistent with
previous results reported for full-length factor Xa (8). Interestingly,
with GDFXa Q192E, the presence of the fragment 2 domain enhanced
prethrombin 2 activation at least 10-fold (Fig. 5B).
Figure 5:
Initial rates of prethrombin 2 activation
by GDFXa and GDFXa Q192E in the presence or absence of prothrombin
fragment 2. A, 200 nM GDFXa was incubated at 37
°C with 2 µM bovine prethrombin 2 in the absence
() or presence (
) of 2 µM bovine fragment 2 in
TBS containing 5 mM Ca
and 0.1% gelatin. At
the indicated time points, samples were removed into 20 mM EDTA, and the rate of thrombin generation was measured with the
chromogenic substrate S2238. B, all the experimental
conditions were the same as described for A, except that GDFXa
Q192E was used to activate prethrombin 2 in the absence (
) or
presence (
) of fragment 2.
Comparison of the Enzyme Specificities toward Peptide
Chromogenic Substrates
To examine if the Q192E substitution in
factor Xa influences the p-nitroanilide substrate specificity,
kinetic analysis was performed with S2765, S2222, Spectrozyme PCa, and
Chromozym tPA. Comparison of the ratio of the second-order rate
constants of GDFXa Q192E to GDFXa for each substrate indicates that
GDFXa Q192E cleaves the chromogenic substrates with basic residues at
the P3 positions (Arg in S2765 and Lys in Spectrozyme PCa) better than
GDFXa (). In contrast, GDFXa Q192E cleaves the substrates
with an acidic (Glu in S2222) or a hydrophobic (Phe in tPA substrate)
residue less effectively that GDFXa. These results are consistent with
the concept that residue 192 in factor Xa influences the P3 substrate
specificity.
Inhibition by the Kunitz-type Inhibitors and
Antithrombin
TFPI is a member of the Kunitz family of inhibitors
and is a competitive inhibitor of factor Xa(26) . TFPI inhibited
GDFXa (K = 6
10
M)
2 orders of magnitude better
than GDFXa Q192E (K
= 5
10
M) in TBS containing either 2.5 mM Ca
or EDTA (Fig. 6). BPTI inhibited GDFXa
with a K
of 30.0 µM (data
not shown). With up to 300 µM BPTI, no inhibition of GDFXa
Q192E was detected (data not shown). In contrast to these Kunitz
inhibitors, the serine protease inhibitor antithrombin inhibited both
GDFXa and GDFXa Q192E at comparable rates with second-order association
rate constants of 1.0
10
and 7.6
10
M
s
, respectively
(data not shown). The presence of 5 units/ml heparin accelerated the
antithrombin inhibition of GDFXa and GDFXa Q192E 720- and 728-fold,
respectively (data not shown).
Figure 6:
Inhibition of GDFXa and GDFXa Q192E by
TFPI. GDFXa () or GDFXa Q192E (
) (2.5 nM) was
incubated with TFPI at room temperature for 30 min at the
concentrations indicated. At equilibrium, the residual activity was
measured from the rate of hydrolysis of Spectrozyme FXa, and the K was determined as described under ``Experimental
Procedures.''
of prothrombin activation has been
hypothesized to be the result of either alteration in the active-site
pocket of factor Xa or stabilization of the structure near the scissile
bonds in prothrombin to better approximate the transition
state(4, 5) . The first mechanism of cofactor function
is reminiscent of thrombomodulin changing the active-site conformation
of thrombin(27) . Thrombomodulin-induced conformational change
appears to move Glu-192 in the catalytic pocket of thrombin to a
location that it is no longer inhibitory for activation of substrates
(such as protein C) with acidic residues at the P3 and/or P3`
sites(28) . Interaction of residue 192 in thrombin with P3 and
P3` residues is consistent with kinetic studies (28) of thrombin and
thrombin mutants with protein C and protein C mutants (15) and
with the thrombin crystal structure as analyzed by Stubbs et
al.(29) . Similar to thrombomodulin, therefore, a function
for factor Va in prothrombin activation could very well be the
alteration of the active-site conformation of factor Xa for efficient
catalysis. This hypothesis is consistent with studies employing
fluorescently labeled active-site probes that demonstrated that upon
association with factor Va, the environment of the probe in factor Xa
was altered, suggesting a change in the conformation of the active site
(or substrate-binding site) of the enzyme(4, 5) . Also
consistent with this hypothesis is the observation that factor Va
complex formation with factor Xa enhances the binding affinity of the
tick anticoagulant protease inhibitor(7) . Factor Xa assembly
into the prothrombinase complex is also shown to enhance the reactivity
with TFPI(30) .
-mediated
factor Xa dimerization through the Gla domain, which could complicate
interpretations.
Table: Steady-state kinetics of p-nitroanilide
hydrolysis by GDFXa and GDFXa Q192E
and k
for each chromogenic substrate
were determined as described under ``Experimental
Procedures.'' The numbers in the last column represent the k
/k
values for
GDFXa Q192E divided by GDFXa. Cbz, benzyloxycarbonyl; pNA,
p-nitroanilide; Bz, benzoyl.
-aminocaproyl-D-glutamic acid
glycylarginine chloromethyl ketone; BPTI, bovine pancreatic trypsin
inhibitor; TFPI, tissue factor pathway inhibitor; tPA, tissue
plasminogen activator.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.