(Received for publication, December 18, 1996, and in revised form, May 19, 1997)
From the Departments of Medicine,
Pathology, and Biochemistry, Saint Louis University School of Medicine,
St. Louis, Missouri 63104, the ¶ Department of Chemistry, Michigan
State University, East Lansing, Michigan 48824, and the
Department of Biochemistry and Molecular Biophysics, Washington
University School of Medicine, St. Louis, Missouri 63110
Human factor X is a two-chain, 58-kDa, vitamin
K-dependent blood coagulation zymogen. The light chain of
factor X consists of an NH2-terminal
-carboxyglutamic acid (Gla) domain, followed by a few helical
hydrophobic residues and the two epidermal growth factor-like domains,
whereas the heavy chain contains the serine protease domain. In this
study, native factor X was found to contain three classes of
Ca2+-binding sites: two high affinity
(Kd 100 ± 30 µM), four intermediate affinity (Kd 450 ± 70 µM), and five to six low affinity (Kd
2 ± 0.2 mM). Decarboxylated factor X in which the Gla
residues were converted to Glu retained the two high affinity sites
(Kd 140 ± 20 µM). In contrast,
factor X lacking the Gla domain as well as a part of the helical
hydrophobic residues (des-44-X) retained only one high affinity
Ca2+-binding site (Kd 130 ± 20 µM). Moreover, a synthetic peptide composed of residues
238-277 (58-97 in chymotrypsinogen numbering) from the protease
domain of factor X bound one Ca2+ with high affinity
(Kd 150 ± 20 µM). From
competitive inhibition assays for binding of active site-blocked factor
Xa to factor Va in the prothrombinase complex, the
Kd for peptide-Va interaction was calculated to be
~10 µM as compared with 30 pM for factor Xa
and ~1.5 µM for decarboxylated factor Xa. A peptide
containing residues 238-262(58-82) bound Ca2+ with
reduced affinity (Kd ~600 µM) and
did not inhibit Xa:Va interaction. In contrast, a peptide containing
residues 253-277(73-97) inhibited Xa:Va interaction
(Kd ~10 µM) but did not bind
Ca2+. In additional studies, Ca2+ increased the
amidolytic activity of native and des-44-Xa toward a tetrapeptide
substrate (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide) by
approximately 1.6-fold. The half-maximal increase was observed at
~150 µM Ca2+ and the effect was primarily
on the kcat. Ca2+ also
significantly protected cleavage at Arg-332-Gln-333(150-151) in the
protease domain autolysis loop. Des-44-Xa in which the autolysis loop
was cleaved possessed
5% of the amidolytic activity of the
noncleaved form; however, the S1 binding site was not affected, as
determined by the p-aminobenzamidine binding. Additionally, autolysis loop-cleaved, active site-blocked native factor Xa was calculated to have ~10-fold reduced affinity for factor Va as compared with that of the noncleaved form.
Factor X is a vitamin K-dependent
multidomain protein that participates in the middle phase of blood
coagulation (1). Factor X is essential for hemostasis since a reduction
in its functional activity results in a rare autosomal recessive
bleeding disorder known as Stuart-Prower factor deficiency (2). The
human protein is synthesized in the liver as a precursor molecule of
488 amino acids (3). The amino-terminal 40 amino acids constitute the prepro leader sequence, which is removed prior to secretion of the
molecule. Additionally, during biosynthesis, the protein undergoes several posttranslational modifications including glycosylation, -carboxylation (of the first 11 glutamic acid residues),
-hydroxylation (of Asp-63), and removal of a tripeptide
(Arg-Lys-Arg) between Arg-139 and Ser-143 (3). The resulting mature
protein is a zymogen of serine protease factor Xa and consists of a
light chain (amino acids 1-139) and a heavy chain (amino acids
143-448) held together by a single disulfide bond between Cys-132 and
Cys-302.
Gene arrangement, amino acid sequence, and modular structure
of factor X strongly suggest that the protein is organized into several
distinct domains (3, 4). The amino terminus of the light chain contains
11 -carboxyglutamic acid
(Gla)1 residues and
represents the Gla domain (residues 1-39) of factor X (3). The Gla
domain is followed by a short hydrophobic stack (residues 40-45) and
two epidermal growth factor (EGF)-like domains (residues 46-84 (EGF1)
and residues 85-128 (EGF2)). The amino terminus of the heavy chain of
factor X contains the activation peptide region of 52 amino acids
(residues 143-194) followed by the serine protease domain of 254 amino
acids (residues 195-448), which features the active site triad of
His-236(57),2 Asp-282(102),
and Ser-379(195) (3).
During physiologic hemostasis, factor X can be activated by factor IXa,
requiring Ca2+, phospholipid (PL), and factor VIIIa or
factor VIIa requiring Ca2+ and tissue factor (1, 5). A
potent nonphysiologic activator of factor X is the coagulant protein
from Russell's viper venom (6). In all cases, the activation results
from the cleavage of the Arg-194-Ile-195(15-16) bond in the heavy
chain of factor X and release of a 52-residue activation peptide; the
light chain remains unaltered during this process (3, 7). Factors X and
Xa3 can also be converted
to their respective
-forms where ~4 kDa peptide is cleaved off
from the COOH terminus of the heavy chain; this, however, does not
result in a loss of coagulant activity (6). Factor Xa converts
prothrombin to thrombin in the coagulation cascade; for a
physiologically significant rate, this reaction requires
Ca2+, PL, and factor Va (8). Thus, Ca2+ plays
an important role both in the activation of factor X and in the
conversion of prothrombin to thrombin by factor Xa.
Ca2+ binding to human factor X has been studied by Monroe
et al. (9). The authors reported that the protein contains
one high affinity Gla-independent and 19 weak Gla-dependent
Ca2+-binding sites (9). In the present report, we have
extensively investigated the Ca2+-binding properties of
native, decarboxylated, and Gla domainless (des-44-X) human factor X. The data strongly indicate that the Gla domain contains four
intermediate and five to six low affinity Ca2+-binding
sites, whereas the EGF1 and the protease domains each contains one high
affinity Ca2+-binding site. Further, proteolysis in the
autolysis loop of the catalytic domain results in a virtual loss of
amidolytic activity without affecting the S1 binding site. Autolysis
loop-cleaved factor Xa (Xa) also has ~10-fold reduced affinity for
factor Va. Importantly, Ca2+ protects the proteolytic
cleavage in the autolysis loop, thereby stabilizing this domain for
maximal biologic activity. An initial account of this work has been
presented in abstract form (10).
45CaCl2 (7.4 Ci/g of calcium) and 3H2O (5 Ci/g) were
obtained from ICN. Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide
(S-2222) and H-D-Phe-Pip-Arg-p-nitroanilide (S-2238) were purchased from Helena Laboratories.
Dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CK) was obtained from
Calbiochem.
N-p-Tosyl-L-lysine
chloromethyl ketone-treated
-chymotrypsin, diisopropyl
fluorophosphate, p-aminobenzamidine (p-AB),
polyethylene glycol 8000, bovine serum albumin (BSA), bovine brain
phosphatidylcholine (PC), and phosphatidylserine (PS) were obtained
from Sigma. Human factor X and prothrombin were purified as described
earlier (11). A total of ~100 mg of factor X obtained from six
isolations, each starting with 8 liters of plasma, was used for these
investigations. Decarboxylated factor X was prepared as outlined
previously (12). Five preparations of 10 mg each were made and pooled.
Small amounts of higher molecular weight materials (~10% of the
total protein) in decarboxylated factor X were removed by gel
filtration utilizing a Sephadex G-100 column (2.5 × 150 cm)
equilibrated in TBS, pH 7.4 (0.05 M Tris, 0.15 M NaCl, pH 7.4). Des-44-X was prepared and purified by
QAE-Sephadex A-50 column chromatography as described by Morita and
Jackson (13). The NH2-terminal sequence analysis of
des-44-X revealed two sequences of approximately equimolar amounts, one
corresponding to the heavy chain (Ser-Val-Ala-Gln-Ala) and the other
corresponding to the modified light chain, des-Gla L
(Lys-Asp-Gly-Asp-Gln). Decarboxylated factor X and des-44-X each
contained <0.5 Gla/mol as compared with factor X, which contained 10.7 ± 0.6 Gla/mol as measured by the specific 3H
incorporation (14). Factor Xa
, des-44-Xa
, and decarboxylated Xa
were prepared using insolubilized Russell's viper venom as outlined previously (11, 13). PCPS vesicles (75% PC, 25% PS) were
prepared as earlier (15). Human factor Va was obtained from Hematologic
Technologies, Inc., and
-thrombin (IIa) was purchased from Enzyme
Research Laboratories, Inc.
Des-44-Xa was prepared by incubation of 10 mg (2 mg/ml) of
des-44-Xa
in TBS, pH 7.4, containing 1 mM EDTA for
30 h at 37 °C. SDS-gel electrophoretic analysis revealed that
after a 30-h incubation period, all of the H
had been converted to
H
N and H
C (see "Results and Discussion"). In our efforts to
prepare native factor Xa
, we found that in addition to proteolysis
in the autolysis loop, the Gla domain was also cleaved off, albeit
slowly.4 Therefore, to
investigate the properties of factor Xa
, we prepared a mixture of
factors Xa
and Xa
(factor Xa
) as follows. We incubated
factor Xa
in TBS, pH 7.4, in the presence of 1 mM EDTA for 5 h at 37 °C. This sample, as analyzed
by reduced SDS gels, contained both factor Xa
and factor Xa
as
well as des-44-Xa
and des-44-Xa
. Des-44-Xa
was removed from
factor Xa
utilizing Mono Q fast protein liquid chromatography as
outlined for separating Gla-domainless factor VIIa from native
factor VIIa (17). The purified factor Xa
was free of
Gla-domainless factor Xa
(see "Results and
Discussion").
DEGR-Xa, DEGR-des-44-Xa
, and decarboxylated DEGR-Xa
were
prepared by incubating the enzymes (~500 µg/ml) in TBS, pH 7.4, with 20-fold molar excess of DEGR-CK for 2 h at 37 °C, at which time an additional 20-fold excess of the inhibitor was added to each
tube and the pH adjusted to 7.4. The samples were then allowed to sit
overnight at 4 °C, and the excess inhibitor in each sample was
removed as described earlier (15). The preparations had no measurable
S-2222 hydrolytic activity. DEGR-des-44-Xa
and DEGR-Xa
were
prepared as above, except three successive additions of the DEGR-CK
were made instead of the two earlier; after the second addition of the
inhibitor, each tube was incubated for 2 h at 37 °C prior to
the last addition and incubation overnight. When a known extinction
coefficient (3940 M
1 at 340 nm) of the dansyl
probe (18) was used, we obtained stoichiometric (1.1 ± 0.05)
incorporation of the inhibitor into each factor Xa protein.
Binding of Ca2+ to factor X, decarboxylated factor X, and des-44-X was investigated by the technique of equilibrium dialysis using 45Ca, as described in detail previously (19). The only change from the described method was the use of microcells of 0.1 ml volume. At the end of each experiment, the proteins were analyzed by SDS-gel electrophoresis and were stable for at least 48 h at room temperature. Ca2+ binding to the peptides was determined using a Ca2+-specific electrode and a model 601A digital ion analyzer. Titrations of peptides in 4 ml of buffer were performed by adding small increments (4-8 µl) of 10 or 100 mM CaCl2 at room temperature. In these titrations, bound Ca2+ was taken as the difference between measured free Ca2+ concentration and total added (15). Data were analyzed using the nonlinear, least-squares, curve fitting program LIGAND (20).
Peptide SynthesisA total of three peptides were
synthesized using Merrifield's solid phase method (21) on an Applied
Biosystems model 430A peptide synthesizer. The peptides were
deprotected and cleaved from the resin with anhydrous hydrogen
fluoride/anisole/dimethyl sulfide (10:1:1) (v/v/v) for 50 min at
0 °C. The cleaved peptides were washed with diethyl ether and
extracted from the resin with 30% acetic acid. After removing the
acetic acid by rotary evaporation, the remaining aqueous solution was
diluted 4-fold with water, shell frozen, and lyophilized. Peptides were
further purified (90%) by reverse phase high performance liquid
chromatography on a Vydac C-18 (22 × 250 mm) column using
standard trifluoroacetic acid/acetonitrile conditions (22). The
sequence of peptide 1 was:
Leu-Tyr-Gln-Ala-Lys-Arg-Phe-Lys-Val-Arg-Val-Gly-Asp-Arg-Asn-Thr-Glu-Gln-Glu-Glu-Gly-Gly-Glu-Ala-Val-His-Glu-Val-Glu-Val-Val-Ile-Lys-His-Asn-Arg-Phe-Thr-Lys-Glu. The sequence of peptide 1 corresponds to residues
238-277(58-97) of human factor X (3, 4). The sequence of peptide 2 corresponds to residues 238-262(58-82), and the sequence of peptide 3 corresponds to residues 253-277(73-97) of factor X. Tyrosine was
added at the NH2 terminus of peptide 3 to facilitate
determination of its concentration in solution. Peptide concentrations
were determined using the molar extinction coefficient of 2390 at 293 nm for tyrosine in 0.1 M NaOH (23).
SDS-gel electrophoresis was performed using the Laemmli buffer system (24). The acrylamide concentration was 15%, and the gels were stained with Commassie Brilliant Blue dye. All proteins used in the present study were ~98% pure.
Molecular ModelingThe putative model of the Gla domain of
factor X was constructed using a homology model building approach
described earlier (25). Crystallographic structures of the Gla domain
of prothrombin in the presence of Ca2+ (26) and
Sr2+ (27) were used as the starting templates. EGF1 domain
was modeled using the NMR coordinates of EGF1 domain of bovine factor X
in the presence of Ca2+ (28). Structures of the EGF2 and
protease domains of human factor Xa were from Padmanabhan et
al. (4). In this structure, Glu-250(80) side chain was disordered
and there was no electron density beyond the -carbon; the side chain
of this glutamic acid was introduced during the modeling of
Ca2+-binding site in the 250-260(70-80) loop. A single
Ca2+-binding site in trypsin was first identified by Bode
and Schwager (29); coordinates of the Ca2+-binding loop
were taken from the Protein Data Bank (Brookhaven National Laboratory,
code 4PTP) for modeling of the Ca2+-binding site in factor
Xa. In modeling of the whole factor Xa molecule, individual domains
with respect to each other were positioned based upon the crystal
structure of porcine factor IXa (Ref. 30, code 1PFX), a protein whose
domain organization and modular structure is similar to that of factor
Xa.
Automated Edman degradation of each protein component was performed using an Applied Biosystems model 477A gas phase sequencer. Approximately 0.1-0.5 nmol of protein was loaded on the filter cartridge. The proteins from SDS gels were transferred to polyvinylidene difluoride membranes as described by Rosenberg (31).
Measurements of S-2222 Amidolytic Activity of Factor Xa ProteinsThe concentration of factor Xa and des-44-Xa
used
was 1 nM each and the concentration of des-44-Xa
was 20 nM. The S-2222 concentration ranged from 20 µM to 1 mM. The buffer used was TBS, 0.1 mg/ml BSA, pH 7.4, containing 1 mM Ca2+ or 1 mM EDTA. The p-nitroaniline release was measured
continuously (
A405/min) for up to 30 min
using a Beckman DU65 spectrophotometer equipped with a Soft-Pac
kinetics module. An extinction coefficient of 9.9 mM
1·cm
1 at 405 nM
was used in calculating the amount of p-nitroaniline released (32). All reactions were performed in triplicate. The Km and kcat values were
obtained using the Enzyme Kinetics program from Erithacus Software. In
Ca2+ titration experiments, the Ca2+
concentrations ranged from 0-500 µM (0, 20, 50, 100, 200, 300, 400, and 500 µM), and the substrate
concentration was 170 µM.
Binding of p-AB was measured by increase in its intrinsic fluorescence upon binding to the active site of each factor Xa protein using Perkin-Elmer 650-10S Fluorescence Spectrophotometer. The concentration of each factor Xa protein used was 100 µg/ml (1.7 µM) in TBS, pH 7.4 containing 0.3% polyethylene glycol 8000, and the excitation wavelength was 336 nm (33). A titration of the protein solution (700 µl) in the presence of EDTA (1 mM) or Ca2+ (5 mM) was performed by adding small increments (2-4 µl) of 1.8 mM stock solution of p-AB, and the resulting fluorescence at 376 nm was measured at each point after the attainment of equilibrium conditions (usually 1 min). Both excitation bandwidth and emission bandwidth were 5 nm.
Measurements of Kd Values for the Interaction of Each Factor Xa Species with Factor Va in the Prothrombinase ComplexFor these experiments, we first determined the functional
Kd (EC50) of interaction of factor Xa
with factor Va in our system. Reaction mixtures (50 µl final volume)
were prepared in Eppendorf tubes containing 5 pM factor Va,
10 µM PCPS, 5 mM Ca2+, and
varying concentrations (5, 12, 19, 25, 37, 50, 75, and 100 pM) of factor Xa
. The buffer used was TBS/BSA, pH 7.4 (TBS containing 1 mg/ml BSA). After 5 min at 37 °C prothrombin (700 nM, final concentration) was added to each tube, and the
mixture was incubated for a variable time period ranging from 2 to 10 min. A 20-µl aliquot of the reaction mixture was removed at a given
time and added to 10 µl of 30 mM EDTA to stop further
generation of thrombin. Twenty µl of each chelated sample was then
transferred to a 0.1-ml quartz cuvette containing 100 µl of S-2238
(final concentration, 125 µM). The
p-nitroaniline release was measured continuously (
A405/min) for up to 30 min. All reactions
were performed in triplicate. Thrombin generated was calculated from a
standard curve constructed using purified thrombin. Functional
Kd was obtained form a plot of factor Xa
versus rate of thrombin generation using the Enzyme Kinetics
program from Erithacus Software. The functional Kd
of factor Xa
:Va interaction at several additional concentrations
(15, 50, 150, 300, 500, 1200, and 1500 nM) of prothrombin
was also determined as outlined above using 750 nM
prothrombin. Kd values of factor Va binding to DEGR-Xa
, of decarboxylated DEGR-Xa
, and of the three peptides were determined from their abilities to inhibit prothrombin activation in a system containing 5 pM factor Va, 15 pM
factor Xa
, 10 µM PCPS, and 750 nM
prothrombin. In each case factor Xa
was mixed with the competitor
prior to incubation with factor Va. The steady state inhibition curves
were analyzed using the program LIGAND (20).
SDS-gel electrophoretic
analysis of the proteins used in Ca2+-binding experiments
is presented in Fig. 1A. Under
nonreducing conditions, each of the three proteins (native factor X,
decarboxylated factor X, and des-44-X) used for
Ca2+-binding experiments revealed a single Commassie
Brilliant Blue dye staining band. Under reducing conditions, native and
decarboxylated factor X revealed the typical heavy and light chains,
whereas the des-44-X protein revealed a smaller light chain indicative of removal of the Gla domain. The NH2-terminal sequence
analysis of des-44-X is given under "Experimental Procedures." In
addition, the disulfide reduced native factor X contained a small
amount of high molecular weight component that could represent an
incompletely processed protein in which the tripeptide (residues
140-142) has not been removed (3, 7). An apparent absence of this high molecular weight component in decarboxylated factor X may indicate its
removal during the gel filtration step (see "Experimental Procedures"). The purity of the factor X preparations is shown here,
since it is important to the conclusions drawn from the data presented
in this paper.
The Scatchard plots of the Ca2+-binding data for native factor X and decarboxylated factor X as measured by the equilibrium dialysis technique are shown in Fig. 1B. The data on native factor X fit to a model in which there are three classes of Ca2+-binding sites: two high affinity (Kd ~100 µM), four intermediate affinity (Kd ~450 µM), and five to six low affinity (Kd ~2 mM). An analysis by the LIGAND program (20) gave a p value (significance of fit) of <0.01 between the high and intermediate affinity sites and of <0.05 between the intermediate and low affinity sites. The two high affinity sites are present in the non-Gla region of the molecule since decarboxylated factor X retains these sites (Fig. 1B, inset). In contrast, des-44-X contained only one high affinity site (data not shown). Ca2+-binding data are summarized in Table I. From the structural similarities and identical domain organizations between factors VII, IX, and X, it is clear that out of the two non-Gla high affinity sites in factor X, one is located in the EGF1 domain (15, 34, 35) and the other in the protease domain (10, 36-39). Because des-44-X has only one high affinity Ca2+-binding site (Ref. 9 and the present study) as compared with the two in decarboxylated factor X, it is likely that residues Asp-46 to Gln-49 are flexibly disordered in des-44-X and cannot effectively participate in forming the high affinity site in the EGF1 domain (40).
|
Since decarboxylated factor X retained the two high affinity Ca2+-binding sites, it is reasonable to conclude that the intermediate and low affinity sites in native factor X are located in the Gla region of the molecule. Bovine factor X (41) and prothrombin (42-44) have been reported to bind Ca2+ with positive cooperativity and two or three sites may be involved in this interaction; furthermore, the possibility of four cooperative sites may not be ruled out (45). In light of these observations, we explored whether or not the intermediate affinity sites in the Gla domain of human factor X exhibited positive cooperativity in binding to Ca2+. As is evident from Fig. 1 (B-D), we were unable to demonstrate cooperative binding of Ca2+ to native (Hill coefficient 0.87) or decarboxylated (Hill coefficient 0.91) factor X from the direct measurement data. However, cooperativity in Ca2+ interaction became evident when we obtained a hypothetical curve of binding to the Gla domain by subtracting the binding curve of the decarboxylated factor X from that of the native factor X (Fig. 1C). The Hill coefficient, at midpoint of 0.1 mM Ca2+ concentration was 1.57 and at midpoint of 2 mM Ca2+ concentration was 0.89 (Fig. 1D). Since in these calculations we have used nine as the total (intermediate and low affinity) number of binding sites in the Gla domain, the real value of the Hill coefficient at lower concentrations of Ca2+ may be higher than 1.57. These data therefore suggest that there are at least two and possibly more cooperative Ca2+-binding sites in the Gla domain of human factor X. This analysis is consistent with the data obtained earlier with bovine factor X and prothrombin (41-44). However, one should note that our analysis is based upon a hypothetical curve, and additional data will be needed to fully establish the number of sites that exhibit cooperativity in binding of Ca2+ to the Gla domain of factor X.
To further investigate the structural requirements for the protease
domain binding site, we studied the Ca2+-binding properties
of three peptides derived from the catalytic domain of factor X. Peptide 1 containing residues 238-277(58-97) was found to contain a
single Ca2+-binding site with a Kd
~150 µM (Fig. 2).
Ca2+ binding to peptide 2 containing residues
238-262(58-82) was weaker (Kd ~600
µM), and Ca2+ binding to peptide 3 containing
residues 253-277(73-97) was not observed. In peptide 2, the two
residues Asp-250(Glu-70) and Glu-260(80) are included, which
participate in the Ca2+ binding in the known serine
proteases (25, 29, 39). The reason that peptide 2 binds
Ca2+ with a slightly lower affinity (as compared with
peptide 1 and decarboxylated factor X) may be that the -sheet (
4
and
5 strands) that holds these residues together in a loop is not
fully formed and is less stable; out of the potential five H-bonds,
only three would be possible (Table II).
As a result, a significant population of the peptide may not exist in a
conformation favorable for Ca2+ binding. The binding of
Ca2+ to the peptide in the favorable conformation will
continuously shift the equilibrium and generate additional conformers
suitable for Ca2+ binding. This could explain the reduced
affinity of this peptide for Ca2+. Peptide 3, which lacked
Asp-250(Glu-70), did not bind Ca2+ out to 5 mM.
This is consistent with the observation that mutation of Asp-250(70) to
lysine abolishes this Ca2+-binding site in factor X
(46).
|
Ca2+-binding sites in the Gla domain of factor
X were modeled based upon the structure of the Gla domain of
prothrombin in the presence of Ca2+ (26) and
Sr2+ (27). The folding of the Gla domain in this model
(Fig. 3) is very similar to the Gla
domain of factor VIIa determined by x-ray crystallography (39) and to
the modeled structure of protein C (47). Both of these proteins have
one residue deletion corresponding to residue 4 in bovine prothrombin.
No difficulties were encountered in incorporating four intermediate
affinity and four low affinity Ca2+-binding sites in the
modeled Gla domain of factor X (Fig. 3). Compared with bovine
prothrombin, two additional low affinity sites could be formed
involving Gla-32 and Gla-39 in factor X. Ca2+ or
Sr2+ was not found coordinated to Gla-32 (Gla-33 in
prothrombin), which is flexibly disordered in the crystal structure of
prothrombin fragment 1 (26, 27), and Gla-39 is Ala-40 in bovine
prothrombin. In our modeled structure of the Gla domain of human factor
X, three hydrophobic residues (Phe-4, Leu-5, and Met-8) point out into
the solvent. Based upon the work on other vitamin
K-dependent proteins (26, 39, 48-51), these residues could
insert into the membrane during prothrombinase assembly. Calcium ions
bound to the low affinity sites may also participate in binding to the membrane via phosphate head groups. Quenching of the intrinsic fluorescence observed on Ca2+ binding to the Gla domain
(52) would appear to be due to the perturbation of the environment of
Trp-41 (Fig. 3). Thus, all of the known properties of Gla domain of
factor X, including the intermediate and low affinity
Ca2+-binding sites determined experimentally (Fig. 1) could
be accounted for in the modeled structure (Fig. 3).
High affinity Ca2+-binding site in the protease domain of
factor X is analogous to the site first observed in trypsin (29). In
trypsin, Ca2+-binding site is contained in a surface loop
that is formed by residues 249(69)-260(80). In trypsin as well as in
factor VIIa and elastase, all of the calcium ligands are provided by
the residues in this loop (29, 39, 53). These are the side chain
carboxyl oxygens of Asp-250(Glu-70) and Glu-260(80) and the main chain carbonyl oxygens of residues 252(72) and 255(75). Additional ligands may be water molecules or other side chains contained in this so-called
calcium binding loop (29, 39, 53). Additionally, this surface loop is
stabilized by hydrogen bonds between the carboxyl group of residue
260(80) and the main chain amide groups of residues 258(78) and
259(79). The crystal structure of factor Xa is in the absence of
Ca2+, and Glu-260(80) residue is disordered in this
structure (4). However, Glu-260(80) could be easily modeled, and all of
the above features observed in the calcium binding loop of trypsin (29) could be readily incorporated into the factor Xa structure. This Ca2+-binding site in the protease domain of factor Xa
is shown in Fig. 4.
Functional Significance of the Protease Domain Ca2+-binding Site in Factor Xa
Ca2+
potentiated the S-2222 hydrolytic activity of factor Xa and
des-44-Xa
by approximately ~1.6-fold, and the effect was primarily
on the kcat (Table
III). In three separate experiments, the
half-maximal increase was observed at 150 ± 20 µM
Ca2+. Since des-44-Xa
does not contain the high affinity
site in the EGF1 domain, the Ca2+ effects are due to the
occupancy of the protease domain Ca2+-binding site. Rezaie
and Esmon (46) reported that Ca2+ increases
(Kd ~250 µM) the amidolytic activity
of a recombinant factor Xa mutant (lacking the Gla and EGF1 domains) toward a synthetic substrate
(methoxycarbonyl-D-cyclohexylglycin-Gly-Arg-p-nitroanilide) by 35%. Similarly, Sherrill et al. (54) reported that
Ca2+ (Kd ~200 µM)
enhances the amidolytic activity of native and des-44-Xa by 25-35%.
Our data on S-2222 hydrolysis are qualitatively similar to the
observations of these authors (46, 54). Importantly, we show that the
effect of Ca2+ is primarily on the
kcat and that the Km is
essentially unaffected (Table III).
|
In further experiments, we tested whether or not occupancy of
Ca2+-binding site in the protease domain prevents
proteolytic cleavages in the autolysis loop. Des-44-Xa was incubated at
37 °C in the presence of 1 mM Ca2+ or 1 mM EDTA. Samples were removed at different times for SDS gel analysis and for S-2222 activity in the presence or absence of 1 mM Ca2+. These data are presented in Fig.
5. In the presence of Ca2+,
the rate of proteolysis in the autolysis loop is ~50% (apparent first order rate constant 0.04/h) of that observed in the presence of
EDTA (apparent first order rate constant 0.08/h) as analyzed by the
disappearance of H and appearance of H
N and H
C. Additionally, the cleavage(s) in the autolysis loop results in a virtual loss of
amidolytic activity. The loss of activity was exponential (Fig. 5),
probably as a result of depletion of the substrate and the activator
simultaneously. A 30-h preparation of des-44-Xa
that appeared to be
free of des-44-Xa
(as analyzed by SDS gels) retained ~5% of the
amidolytic activity of des-44-Xa
. However, our des-44-Xa
preparation may contain small amounts of des-44-Xa
, which could partly contribute to the observed amidolytic activity. Proteolytic cleavage in our des-44-Xa preparation had occurred at
Arg-332(150)-Gln-333(151) (see legend to Fig. 5). Thus proteolysis at
the Arg-332(150)-Gln-333(151) peptide bond leads to a loss of
catalytic efficiency in factor Xa. Furthermore, Ca2+
significantly protects the protease domain from proteolysis in this
loop. It should be noted that while our studies were in progress, Pryzdial and Kessler (16) reported that factor Xa cleaved at Lys-330(147)-Gly-331(148) by plasmin is devoid of amidolytic and clotting activity. Moreover, thrombin cleaved in the autolysis loop is
less stable, and within hours at 37 °C loses its catalytic efficiency (55). Thus, an overall conclusion to be reached from these
observations is that proteolysis in the autolysis loop in factor Xa and
thrombin leads to an unstable enzyme with concomitant loss of catalytic
efficiency.
We next examined whether or not des-44-Xa has the ability to bind to
p-AB. These experiments were designed to test the
availability of S1 site in the enzyme. Consistent with earlier
observations (54), factor Xa
, decarboxylated Xa
, and des-44-Xa
each were found to interact with p-AB with
Kd 30 ± 3 µM in both the
presence and absence of Ca2+. These observations are also
consistent with our earlier findings that the Km of
S-2222 hydrolysis by factor Xa is not affected by Ca2+
(Table III). Des-44-Xa
also interacted with p-AB,
although with slightly reduced affinity (Kd 40 ± 5 µM); however, the enhancement in the intrinsic
fluorescence of p-AB upon binding at the active site was
only ~25% of that observed with des-44-Xa
. It should be noted
that an enhancement of the intrinsic fluorescence of p-AB
was not observed when DEGR-des-44-Xa
(or DEGR-des-44-Xa
) was used
in the p-AB titration experiments; this strongly indicates that the increase in intrinsic fluorescence observed with des-44-Xa
is due to the binding of p-AB at the active site. Since a
reduced fluorescence increase was observed with des-44-Xa
, it would
indicate that the environment of the p-AB bound to the S1
site in des-44-Xa
is less nonpolar as compared with that in the
des-44-Xa
molecule. Based upon the work with other serine proteases
(4, 56), it would appear that Trp-399(215) in factor Xa contributes to the nonpolar environment and therefore enhancement of p-AB
intrinsic fluorescence upon binding at the active site; if so, then
this region in factor Xa
is slightly perturbed without the loss of S1 binding site.
In the following experiments, we wished to investigate the effect of
proteolysis in the autolysis loop of factor Xa in its binding to
factor Va. The rationale for these experiments is based upon the
knowledge that the protease domain of factor Xa may be involved in
binding to factor Va (38, 57). For these studies, we prepared
DEGR-Xa
, decarboxylated DEGR-Xa
, and DEGR-Xa
(a mixture
containing 50% of DEGR-Xa
and 50% of DEGR-Xa
, see Experimental Procedures and legend to Fig. 6), and
evaluated their abilities to compete with active factor Xa
in
binding to factor Va in the prothrombinase complex. We also evaluated
the abilities of the three peptides to compete for factor Va in the
prothrombinase complex. First, we determined the functional
Kd (EC50) of interaction of factor Xa
with factor Va in our system. A Kd value of 30 ± 5 pM was obtained for this interaction and its value was
not dependent upon the concentration of prothrombin utilized in the
assay (see "Experimental Procedures"). Next, using this value of
functional Kd and steady state inhibition curves, the Kd values for interaction of factor Va with
various competitors were calculated. These data are presented in Fig. 6. Using the data in this figure, a Kd value of
26 ± 4 pM for the interaction of DEGR-Xa
and
factor Va was calculated. This agrees well with the functional
Kd value of 30 ± 5 pM calculated
from the initial rates of prothrombin activation assays using different
concentrations of factor Xa
and a fixed (5 pM)
concentration of factor Va (see above). When DEGR-Xa
(50% each
of
and
species; see legend to Fig. 6) was used as a competitor,
the inhibition curve best fitted (p < 0.01) to a two-site model, with a Kd value of 19 ± 3 pM and 231 ± 15 pM. The value of 19 pM was taken as the Kd for the interaction of factor Va and DEGR-Xa
, and the value of 231 pM was taken as the Kd for the
interaction of factor Va and DEGR-Xa
. Decarboxylated DEGR-Xa
interacted with factor Va with Kd ~1.5 ± 0.5 µM, and peptides 1 and 3 interacted with factor Va with
Kd ~10 ± 2 µM, whereas peptide
2 did not bind to factor
Va.5
Our observation that the interaction of factor Va and factor Xa on the
PCPS vesicles is not influenced by the substrate concentration in the
prothrombinase assay system is in agreement with the earlier data of
Nesheim et al. (60). Our Kd value of 30 pM for the association of factor Xa and factor Va on the
phospholipid vesicles is also in agreement with the value of 25 pM recently obtained by Ye and Esmon (61). Furthermore, our
Kd value of 1.5 µM for the interaction
of decarboxylated DEGR-Xa
(which cannot bind to PL vesicles) and
factor Va also agrees well with previous determinations on the
Kd of interaction of factors Va and Xa in the
absence of phospholipid using physical methods (62). Finally, our
peptide inhibition data are also in general agreement with the data of
Chattopadhyay et al. (63), who observed that a peptide
containing the sequence 263(81)-274(94) of factor Xa inhibited
prothrombin activation with an IC50 (50% inhibition) value
of 20 µM. The above comparisons attest to the validity of
our system in studying Xa:Va interactions. Using this system, we show
that as compared with factor Xa
, factor Xa
binds to factor Va
with ~10-fold reduced affinity. Overall, our data reveal that factor
Xa
is essentially devoid of catalytic activity and has a reduced
affinity for factor Va; however, the S1 binding site is retained in
this molecule. Similarly, in another study (65), we show that factor
IXa cleaved in the autolysis loop retains the S1 site, has reduced
affinity for factor VIIIa and is devoid of enzymatic activity.
Collectively, our data combined with
previous observations support the conclusion that factor X contains one
high affinity Ca2+-binding site in the protease domain and
one in the EGF1 domain. The formation of the high affinity site in the
EGF1 domain requires the presence of the Gla domain, which could be in
the decarboxylated form. The Gla domain contains four intermediate and
five to six low affinity Ca2+-binding sites, which could be
successfully modeled based upon the crystallographically determined
structure of the Gla domain of prothrombin (26, 27). The protease
domain Ca2+-binding site could also be easily modeled into
the x-ray structure of factor Xa (4). Using the x-ray structure of EGF2
and protease domains of factor Xa (4), the NMR coordinates of bovine
factor X EGF1 domain (28), and x-ray coordinates of the Gla domain of
bovine prothrombin (26, 27), we were able to model the entire factor Xa
molecule. This is shown in Fig. 7. The
model of the whole factor Xa molecule is shown here for clarity in
pointing out the location of the Ca2+-binding and membrane
insertion sites in the protein. However, it should be borne in mind
that it is a modeled view of the protein, and it is depicted here
simply to illustrate the biologic features. Biochemical data support
the conclusion that the protease domain of factor Xa is important in
its interaction with factor Va and that the Gla domain is needed for
insertion into the PL membrane. Based upon peptide inhibition data, the
region of the protease domain containing the peptide sequence
263(81)-274(94)(63) may be important for Xa:Va interaction. However,
one should note that a peptide may adopt a different conformation than
that occurring in the protein. Thus, additional data will be required
to confirm whether or not this segment of the protease domain interacts
with factor Va. The precise functions of the EGF1 and EGF2 domains are
not as yet clear, but may be involved in protein·protein or protein·cofactor interactions. Finally, Ca2+ binding to
the protease domain protects proteolysis in the autolysis loop, and
stabilizes this domain for optimal catalytic activity and factor Va
binding.