 |
INTRODUCTION |
The blood coagulation cascade consists of a series of enzymatic
conversions driven by the formation of complexes between serine proteases and cell membrane-bound cofactors. Human factor X
(FX)1 is one of the serine
protease zymogens playing a central role in coagulation processes
leading to the formation of a fibrin clot. This is illustrated by the
behavior of FX as a substrate or as an enzyme in three essential blood
coagulation complexes. First, FX is a natural substrate, as well as
factor IX (FIX), of the tissue factor-factor VIIa (TF-FVIIa) complex
(1) considered as the initial enzyme complex in the cascade following
vascular damage. FX activation by TF-FVIIa results from specific
cleavage and release of a 52-residue activation peptide. Activated FX
(FXa) can generate a tiny amount of thrombin from prothrombin in an extremely inefficient reaction (2). Tissue factor pathway inhibitor (TFPI) binds to TF-FVIIa-FXa to limit the production of FXa and FIXa by
TF-FVIIa (3, 4). Nevertheless, once produced, thrombin and the
initially formed FXa activate small quantities of factor V (FV) to FVa
and factor VIII (FVIII) to FVIIIa (5-8). The activation of these two
cofactors leads to the formation of two other essential procoagulant
complexes, both involving FX, at the surface of procoagulant phospholipids in the presence of calcium ions (9), FIXa-FVIIIa and
FXa-FVa complexes, which convert FX to FXa and prothrombin to thrombin,
respectively. These complexes are 105-106-fold
more active than the serine proteases devoid of their respective cofactors (10-12) and promote the formation of a fibrin clot.
FX circulates in blood as a two-chain molecule and has the same modular
structure as other vitamin K-dependent blood coagulation proteins such as FVII and FIX (13). The light chain has 11 amino-terminal glutamyl residues that are post-translationally modified
in a vitamin K-dependent reaction to form a
-carboxyglutamic acid-containing domain or "Gla domain" critical
for the binding of calcium ions and phospholipids (14). The Gla domain
is followed by two domains homologous to the epidermal growth factor
(EGF) precursor, considered important for protein-protein interactions
(15). The heavy chain is joined to the light chain by a single
disulfide bond and contains a 52-amino acid peptide and a trypsin-like
serine protease domain (16), which forms the carboxyl-terminal end of
the molecule. Although FXa has substantial sequence similarities and
high homologous three-dimensional structure with FVIIa and FIXa
(17-19), it displays significant differences in substrate specificity
and catalytic activity. Furthermore, each protease requires a specific
cofactor to express enhanced catalytic activity within the
procoagulant complexes of blood coagulation.
It is clear that the catalytic specificities of blood coagulation
proteases are supported by the carboxyl-terminal half of the enzymes, a
trypsin-like serine protease domain (20). The prime role of the serine
protease domain has also been demonstrated in the interaction of the
blood coagulation proteases with their cofactors. For instance,
site-directed mutagenesis revealed that the serine protease domain is
the most important part of FIXa and FXa in the FIXa-FVIIIa and FXa-FVa
interactions, respectively (21-23). However, there is also evidence
that Gla- and EGF-like domains may also mediate protein-protein
interactions and may consequently be implicated in the assembly of the
protein complexes of blood coagulation. For instance, FVIIa multiple
residues, distributed over the entire light chain, are in contact with
TF (19). The identification of the FIXa light chain residues directly
involved in the interaction with FVIIIa has to be performed, but three different approaches have suggested that the region around residues 85-90 in the linker area between EGF1 and EGF2 might contact residues 1804-1818 of FVIIIa (24-26). Concerning FX, there is evidence that the light chain of FXa contributes in the interaction of the enzyme with its cofactor (27, 28). However, it is unknown whether the Gla and
the first EGF domains interact directly with FVa in the prothrombin
activation complex or whether they position the catalytic domain at a
correct distance above the phospholipid membrane. Several
investigations have suggested by studying the effect of mutations in TF
that TF-FVIIa complex binds to the Gla domain of FX (29, 30), and there
is evidence that the first EGF-like domain of FX is required for the
activation of the substrate by the TF-FVIIa complex (31). From all
these analyses it has been suggested that the same residues from FX and
FIX within the Gla and EGF1 domains are involved in these interactions.
In the present study, the contribution of the Gla and first EGF domains
in FX-specific reconnaissance by the TF-FVIIa complex and in the
prothrombin activation complex was addressed using chimeric recombinant
FX containing either the Gla domain or the first EGF domain of FIX.
There are two main reasons for the choice of FIX for exchanging FX
homologous domains, the structural similarities between the two
molecules and their respective cofactors, as well as the high
similitude of the blood coagulation reactions in which they
participate. The purified recombinant proteins were then compared with
normal FX with regard to their activation by TF-FVIIa complex and to
their properties as activated serine proteases in the TF-FVIIa-TFPI-FXa
and FXa-FVa complexes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
H-D-Phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline
dihydrochloride
(H-D-Phe-Pip-Arg-p-NA·2HCl),
product name S-2238, and N-a-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline-dihydrochloride (N-a-Z-D-Arg-Gly-Arg-p-NA), product
name S-2765, were from Chromogenix (Mölndal, Sweden). Bovine
serum albumin (BSA), sodium pyruvate, vitamin K1, Ponceau
S, Dulbecco's modified Eagle's medium, soybean trypsin inhibitor,
L-
-phosphatidyl-L-serine (from bovine
brain), and L-
-phosphatidylcholine (from egg yolk) were
obtained from Sigma. Fetal calf serum was purchased from ABCYS (Paris,
France). Fungizone (amphotericin B/deoxycholate),
penicillin/streptomycin, and geneticin G-418 sulfate were obtained from
Invitrogen. Microtiter plates for enzyme-linked immunosorbent assay
(ELISA) (MaxiSorpTM surface), culture flasks, and cell
factories were from Nunc (Roskilde, Denmark). Polystyrene microtiter
plates were from Greiner Merck Eurolab (Strasbourg, France).
ImmobilonTM polyvinylidene difluoride membranes for Western
blotting applications were obtained from Millipore
(Saint-Quentin-Yvelines, France). Madin-Darby canine kidney cells were
purchased from ATCC (Manassas, VA). Benzamidine was from Acros
Organics. Biotinyl-
-aminocaproyl-D-glutamic acid
glycylarginine chloromethyl ketone was purchased from Hematologic Technologies, Inc. (Essex Junction, VT). CNBr-activated Sepharose 4 Fast Flow, HiTrapTM Q column, benzamidine-Sepharose, and
Q-Sepharose Fast Flow were obtained from Amersham Biosciences AB.
Factor X immunodepleted plasma, Owren-Koller buffer, and rabbit brain
thromboplastin-C reagent were purchased from Diagnostica Stago
(Asnières, France).
Proteins--
Mouse monoclonal anti-FX antibody KB-FX008 was
prepared in our laboratory as described previously (32, 33). KB-FX008
was found as being directed against FX protease domain by Western blot.
Polyclonal antibodies against FX conjugated or not with horseradish
peroxidase were obtained from Dako (Dakopatts, Glostrup, Denmark).
Purified human plasma-derived FX (pd-FX), FX-activating enzyme from
Russell's viper venom (RVV-X), bovine antithrombin III, human
prothrombin, human thrombin, and recombinant human TF were obtained
from Kordia (Leiden, The Netherlands). TFPI was obtained from American
Diagnostica (Andresy, France). Human FVa was purchased from Hematologic
Technologies, Inc. (Essex Junction, VT). High purity recombinant human
FVIIa was from Novo Nordisk A/S (NovoSeven®, Bagsvaerd, Denmark).
Protein Concentrations--
ELISA using anti-FX polyclonal
antibodies assayed pd-FX protein. FX was expressed in units, where 1 unit represents the amount in 1 ml of normal human plasma. Recombinant
FX proteins and pd-FX were assayed by ELISA employing coated mouse
monoclonal antibody KB-FX008. Bound FX proteins were detected using
peroxidase-conjugated polyclonal FX antibodies. Proteins were
quantified by the method of Bradford (34), using BSA as a standard.
Molar concentrations of FXa, and
-thrombin were determined by active
site titration (35).
Recombinant FX Derivatives--
Plasmids encoding wt-FX,
FX/FIX(Gla), and FX/FIX(EGF1) have been described previously (36) as
well as the domain borders of the chimeras. The FIX and FX domains are
expressed by the chimera names, i.e. FX/FIX(Gla) contains
the Gla domain and the hydrophobic stack of FIX and FX/FIX(EGF1)
contains the EGF1 domain of FIX. All DNA constructions were expressed
by Madin-Darby canine kidney cells maintained in cell factories with
Dulbecco's modified Eagle's medium supplemented with 2,5% fetal calf
serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml
vitamin K1, and 1 µg/ml amphotericin B/0.8 µg/ml
deoxycholate. FX containing medium was harvested every 48 h.
Benzamidine was added to a final concentration of 10 mM, and medium was centrifuged (6,000 × g), passed over
cellulose acetate membranes (0.22 µm) to eliminate cell debris, and
stored at
80 °C. Conditioned medium was thawed at 37 °C. EDTA
was added to a concentration of 5 mM. The medium was
diluted to bring the final NaCl concentration to 60 mM. The
mixture was then stirred at room temperature for 30 min with
QAE-Sephadex A-50 beads to achieve a final concentration of 0.25%
(w/v). Beads were washed before eluting with 50 mM Tris (pH
7.4), 500 mM NaCl, and 10 mM benzamidine.
Recombinant FX (ELISA) contained in eluted fraction was then
concentrated by precipitation with two successive steps of 40 and 70%
saturated ammonium sulfate. Proteins obtained after the second
precipitation step were diluted in 50 mM Tris (pH 7.4) and
100 mM NaCl and immediately dialyzed against 50 mM Tris (pH 7.4), and 100 mM NaCl, containing
10 mM benzamidine, and 5 mM EDTA. Insoluble
proteins were discarded by centrifugation (6,000 × g
for 30 min). Recombinant FX was purified from the soluble proteins by
immunoaffinity chromatography using monoclonal anti-human FX antibody
KB-FX008. After adsorption, the antibody column was washed with 50 mM Tris (pH 7.4), 100 mM NaCl, containing 10 mM benzamidine followed by elution of bound recombinant FX
with 0.1 M glycine (pH 2.5), and 10 mM
benzamidine. The protein solution was immediately neutralized with 2 M Tris (pH 7.4). Recombinant FX containing fractions were
combined and dialyzed against 50 mM Tris (pH 7.4), 100 mM NaCl, containing 10 mM benzamidine. If present, residual contaminants were removed by Q-Sepharose Fast Flow
chromatography in 50 mM Tris (pH 7.4), 100 mM
NaCl, containing 10 mM benzamidine, using a linear NaCl
gradient (0.05-1 M) for elution. The eluate was dialyzed
extensively against 50 mM Tris (pH 7.4), 100 mM
NaCl and stored at
80 °C. Before any analysis, a final pass over a
benzamidine-Sepharose column equilibrated with 50 mM Tris
(pH 7.4) and 100 mM NaCl was used to eliminate trace
contaminants of FXa that may have been generated during production or
purification of the recombinant protein.
Amino Acid Sequence Analysis--
Purified recombinant FX
derivatives were reduced and loaded onto a 15% SDS-polyacrylamide gel.
The resolved proteins were transferred to an Immobilon membrane and
stained with Ponceau S. The light chains were excised and sequenced
using an Applied Biosystem Procise model 494 sequencer in the
sequencing facility of the Institut de Biologie et Chimie des
Protéines (Lyon, France).
Activation of Recombinant FX Derivatives by
RVV-X--
Recombinant FX derivatives and wt-FX were activated by
RVV-X (37). RVV-X (1 mg) was coupled to 1 ml of CNBr-activated
Sepharose 4B according to manufacturer's instructions. Recombinant FX
derivatives (1 µM) were incubated with coupled RVV-X (30 nM) in 50 mM Tris (pH 7.4), 100 mM
NaCl containing 10 mM CaCl2. After 2 h,
the reaction was stopped by addition of 15 mM EDTA.
Activated recombinant FX derivatives were dialyzed against 50 mM Tris (pH 7.4) and then with 50 mM Tris (pH
7.4), 100 mM NaCl and loaded on a benzamidine-Sepharose column equilibrated in the same buffer. After washing, bound activated FX was eluted with 50 mM Tris (pH 7.4), 100 mM
NaCl containing 5 mM benzamidine. Fractions containing FXa
were pooled and precipitated by the addition of solid ammonium sulfate
to 80% saturation. The precipitated proteins were diluted in 50 mM Tris (pH 7.4) and 100 mM NaCl, and the
protein solution was immediately dialyzed against the same buffer
containing 50% glycerol (v/v) and stored at
20 °C until use. The
active site concentrations of activated recombinant FX derivatives were
determined by titration with known concentration of antithrombin in the
presence of heparin, and an active site-specific assay using
biotinyl-
-aminocaproyl-D-glutamic acid glycylarginine
chloromethyl ketone as described previously (28, 38, 39).
Concentrations of activated recombinant FX derivatives and activated
wt-FX were found to correlate between the two methods.
Plasma-based Coagulant Activities--
The activity of
non-activated derivatives of FX and wt-FX were functionally
characterized in a prothrombin time assay as described previously (40)
with slight modifications. Recombinant FX derivatives or wt-FX were
preincubated 5 min at 37 °C in a fibrometer with FX immunodepleted
human plasma. Clotting was initiated by the addition of rabbit brain
thromboplastin-C reagent and calculated from a standard curve generated
with the clotting times versus the dilutions of pooled
normal plasma. Recombinant FX derivatives and wt-FX clotting activities
were expressed as a percentage of normal activity.
Relipidation of TF Apoprotein--
Recombinant human TF (2.5 µg) in 100 µl of 50 mM Tris (pH 7.4), 100 mM NaCl containing 10 mM CHAPS was mixed with
an equal volume of phospholipid vesicles preparation (PC/PS, 3:1, 2 mM). Phospholipid vesicles (PC/PS, 3:1) of nominal 200 nm
diameter were synthesized by the method of membrane extrusion (41) in 10 mM Hepes (pH 7.5), 100 mM NaCl, 5 mM CaCl2. Phospholipid concentrations were
determined by phosphate analysis. After a 1-h incubation at 37 °C,
the relipidated TF was dialyzed at 4 °C against 50 mM Tris (pH 7.4), 100 mM NaCl. The preparation was kept under
N2 at 4 °C and used within 2 weeks. The functional
concentration of the TF preparation in these vesicles was considered to
be 300 nM, half of its total concentration (42). Throughout
this paper, unless specified, TF refers to this reconstituted preparation.
Amidolytic Activity--
The steady-state kinetics of hydrolysis
of S-2765 by pd-FXa, wt-FXa, and recombinant FXa derivatives were
assayed in 50 mM Tris (pH 7.4), 100 mM NaCl,
containing 2 mg/ml BSA, and 5 mM CaCl2. Kinetic
parameters of substrate hydrolysis were determined employing an enzyme
concentration of 2 nM and various substrate concentrations ranging from 0 to 5 mM. The release of
para-nitroanilide was monitored at 405 nm at 37 °C in a
kinetic microplate reader (Bio-Tek Instruments, Winooski, VT). The
apparent Km and kcat values
for substrate hydrolysis were calculated from the Michaelis-Menten
equation, and the catalytic efficiencies were expressed as the ratio of kcat/Km.
Thrombin Formation--
The rate at which activated recombinant
FX derivatives, wt-FXa, or pd-FXa can activate prothrombin to thrombin
in the presence of phospholipids as a function of FVa concentrations
was compared as described previously with slight modifications (23).
Briefly, phospholipid vesicles (PC/PS, 3:1, 30 µM), and
20 pM FXa were incubated for 5 min with various
concentrations of FVa (0-1 nM). The reaction was started
by the addition of 1 µM prothrombin. The assay was
performed in 50 mM Tris (pH 7.4), 100 mM NaCl,
containing 5 mM CaCl2, and 0.2% (w/v) BSA at
37 °C. Aliquots were taken at specified times, and the reaction was
stopped in EDTA (10 mM final). Thrombin formation in each
was then determined by measuring the amidolytic activity of the samples
toward the synthetic substrate S-2238 (250 µM), in the
presence of 15 µg/ml soybean trypsin inhibitor to inhibit amidase
activity of FXa. During the assay, less than 5% of prothrombin was
converted to thrombin, and thrombin formation was linear. Conversion of
substrate was monitored at 405 nm. Concentrations of thrombin generated
in the activation reactions were determined from a standard curve
prepared from the cleavage rate of S-2238 by known concentrations of
thrombin under the same conditions.
The comparison of the initial rates of prothrombin activation by FXa
derivatives in the presence of subsaturating FVa concentration was
performed as follow. In the absence of phospholipids, thrombin formation was initiated by the addition of 10 µM
prothrombin to a reaction mixture containing 0.5 nM FXa and
75 nM FVa in 50 mM Tris (pH 7.4), 100 mM NaCl, containing 5 mM CaCl2, and
0.2% (w/v) BSA at 37 °C. In the presence of phospholipids, thrombin
formation was initiated by the addition of 1 µM
prothrombin to a reaction mixture containing 20 pM FXa, 250 pM FVa, and 30 µM phospholipids in 50 mM Tris (pH 7.4), 100 mM NaCl, containing 5 mM CaCl2, and 0.2% (w/v) BSA at 37 °C. In
all experiments, aliquots were removed from the reaction mixtures at
specified times and diluted into 10 mM EDTA buffer to stop
the reaction. Thrombin concentration was calculated as described above.
Activation of Recombinant FX Derivatives by FVIIa and Human
TF--
Activation of recombinant FX derivatives by FVIIa were
measured and compared directly to wt-FX activation. The assay was
performed in 50 mM Tris (pH 7.4), 100 mM NaCl,
containing 0.2% (w/v) BSA, and 5 mM CaCl2 at
37 °C. Initial rates of activation by FVIIa and relipidated human TF
were determined as described previously with little modification (43).
Briefly, 60 pM FVIIa was added to 60 pM
relipidated TF in the presence of 30 µM phospholipid vesicles (PC/PS, 3:1). The mixture was incubated at 37 °C for 20 min
in order to establish a FVIIa-TF complex, and the reaction was started
by the addition of various concentrations of FX (0-3 µM). After diverse incubation times, aliquots were taken,
diluted in stop solution containing EDTA (10 mM final), and
assayed for FXa formation employing the synthetic substrate S-2765 (250 µM). Conversion of substrate was monitored at 405 nm. The
concentration of FXa generated in the activation reaction was
determined from a standard curve prepared from the cleavage rate of
S-2765 by known concentrations of active site titrated FXa under the
exact same conditions.
Comparison of the initial rates of FXa derivatives activation by
TF-FVIIa was performed as follow. In the absence of phospholipids, the
reaction was initiated by the addition of 250 nM FX to a
reaction mixture containing 50 nM FVIIa and 250 nM TF solubilized at 37 °C with 5 mM CHAPS
in 50 mM Tris (pH 7.4), 100 mM NaCl, containing 5 mM CaCl2, and 0.2% (w/v) BSA. The mixture
was incubated at 37 °C for 5 min prior to the addition of the
substrate to establish a FVIIa-TF complex. In the presence of
phospholipids, the reaction was initiated at 37 °C by addition of 1 µM FX to a mixture containing 8 nM FVIIa and
0.5 nM TF in the presence of 1 mM phospholipids in 50 mM Tris (pH 7.4), 100 mM NaCl, containing
5 mM CaCl2, and 0.2% (w/v) BSA. As in the
absence of phospholipids, the mixture was incubated at 37 °C for 5 min prior to the addition of the substrate to establish a FVIIa-TF
complex. In all experiments, aliquots were removed from the reaction
mixture at specified times and diluted into 10 mM EDTA
buffer to stop the reaction. FXa concentration was calculated as
described above.
Inhibition of FX Activation by TFPI-FXa--
Inhibition of pd-FX
activation by TFPI-recombinant FXa derivatives was measured as
described previously (44, 45) with minor modifications. Briefly, 25 pM of relipidated TF was incubated with 8 nM
FVIIa for 5 min. FXa generation was started by the addition of 400 nM pd-FX followed by immediate addition of preformed
TFPI-recombinant FXa derivatives (25, 50, and 100 pM).
After various incubation times, aliquots were removed, diluted in a
stop solution containing EDTA (10 mM final), and assayed
for FXa formation using the synthetic substrate S-2765 (500 µM). FXa was then quantified as described above.
The association and dissociation rate constants for the inhibition of TFPI-recombinant FXa derivatives of TF-FVIIa were estimated by fitting experimental data on FX activation by TF-FVIIa to
Equation 1,
|
(Eq. 1)
|
where [FXa]max is the maximal concentration of
FXa; [FXa]t is FXa concentration at a given time point
t, and kobs is the observed first
order rate constant. The apparent second order association rate
constant for TFPI-recombinant FXa derivatives in the inhibition of the
TF-FVIIa complex and the dissociation rate constant were determined as
the slope and the intercept of the y axis, respectively, of
the plot kobs versus TFPI-recombinant FXa derivatives concentration (44).
 |
RESULTS |
Recombinant Proteins--
As reported previously, the employed
expression system produces fully processed recombinant zymogen of the
coagulation system (46) with normal
Ca2+-dependent properties (47). Indeed,
NH2-terminal sequence analysis revealed the expected
sequence of the mature purified recombinant proteins, ANSFLEEMKK for
wt-FX and FX/FIX(EGF1) and YNSGKLEEFV for FX/FIX(Gla), indicating that
the signal sequence and the propeptide have been accurately and
efficiently removed before secretion. In addition, sequence analysis
disclosed that the average yield for the two glutamic acid residues at
the NH2 terminus was less than 5% of the average yield of
the two subsequent residues. Because
-carboxylation reduces the
yield of Glu residues, these data demonstrate that the glutamic
residues were appropriately modified. All recombinant FX proteins were
activated by RVV-X under conditions similar to those of pd-FX. All FX
derivatives could be completely converted into active form, and the
final activated preparations were more than 90% active as determined
by active site titration.
Plasma-based Assay of Function--
The clotting
activity of wt-FX, FX/FIX(Gla), and FX/FIX(EGF1) was estimated in a
prothrombin time assay. Recombinant FX derivatives were incubated with
FX immunodepleted human plasma and clotting initiated by addition of
rabbit brain thromboplastin. Clotting activity of wt-FX was 75 ± 10% of the activity of pooled normal plasma. In contrast, FX/FIX(Gla)
and FX/FIX(EGF1) clotting activities were less than 1 and 6 ± 2%
of the activity of pooled normal plasma, respectively. Because this
global measurement of the dysfunction of the chimera could be due to
abnormal activation and/or catalytic activity, these enzymatic steps
were studied separately using purified components.
Amidolytic Activity--
To explore whether substitutions of the
Gla and EGF1 domains of FX by the corresponding domains of FIX affect
the amidolytic activity of FXa, hydrolysis of various concentrations of
synthetic substrate S-2765 was monitored as described under
"Experimental Procedures." As shown in Table
I, all chimeras display similar rates of
substrate hydrolysis compared with that of wt-FXa or pd-FXa. This
indicates that Gla or EGF1 substitution does not adversely affect the
reactivity of the catalytic triad in the activated FX chimeras.
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Table I
N- -Z-D-Arg-Gly-Arg-pNA (product name S-2765) hydrolysis
by FX chimeras, wt-FXa, and pd-FXa
Hydrolysis of S-2765 by chimeras and normal FXa are measured as
described under "Experimental Procedures."
kcat/Km values are determined at
an enzyme concentration of 2 nM. Mean values ± S.D.
for three experiments are presented.
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|
Prothrombin Activation--
As the chimeric FX variants displayed
a normal reactivity of the catalytic triad, it was of interest to
investigate the influence of the Gla and EGF1 domain substitutions in
prothrombin activation by the FXa-FVa complex. Therefore, FXa chimeras
were compared with wt- and pd-FXa in their ability to activate
prothrombin. The rate of thrombin generation was studied as a function
of increasing concentrations of FVa and in the presence of an excess of
phospholipids. For all FXa tested, thrombin formation was dependent on
FVa concentrations and was saturable (Fig.
1). The apparent Kd
values were 420 ± 38, 405 ± 26, 230 ± 11, and
225 ± 11 pM for pd-FXa, wt-FXa, FX/FIX(Gla), and
FX/FIX(EGF1), respectively, whereas the apparent kcat values were between 15.5 and 19.9 s
1. These experiments show that activated chimeras have
full catalytic activity toward prothrombin in the prothrombinase
complex. In addition, substitution of the Gla or EGF1 domains by the
corresponding domains of FIX even has a positive effect on the apparent
affinity of FVa. To evaluate whether the slight increased affinity of
FVa for the chimeras was related to the presence of phospholipids, the
initial rates of thrombin formation by the chimeras was compared with
that of wt-FXa in the presence of subsaturating concentrations of FVa
with phospholipids (Fig. 2A)
or without phospholipids (Fig. 2B). The same initial rates
of prothrombin activation were obtained for the chimeras and wt-FXa in
the absence of phospholipids (Fig. 2B). A similar rate of
activation was obtained with pd-FXa (data not shown). Thus, the
increased affinity of FVa observed in the experiment depicted in Fig. 1
was due to the presence of phospholipids. In conclusion, substitution
of the Gla or EGF1 domains by the corresponding domains of FIX has no
effect on the apparent affinity of FVa for FXa, and even a slight
increase of affinity was observed in the presence of phospholipids.

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Fig. 1.
Comparison between activated FX chimeras and
activated FX in the rates of thrombin formation as a function of FVa
concentration. Thrombin formation from prothrombin (1 µM) activation by 20 pM pd-FXa (closed
circles), wt-FXa (open circles), FX/FIX(Gla)
(open squares), or FX/FIX(EGF1) (open triangles)
was evaluated at 37 °C in the presence of phospholipid vesicles (30 µM), 5 mM CaCl2, and different
concentrations of FVa. Thrombin formation was quantified as described
under "Experimental Procedures." Data represent the mean values of
three experiments.
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Fig. 2.
Activation of prothrombin by FVa-FXa
complexes in the presence or absence of phospholipids.
A, in the presence of 30 µM phospholipid
vesicles and 250 pM FVa, thrombin formation from
prothrombin (1 µM) activation by 20 pM wt-FXa
(open circles), FX/FIX(Gla) (open squares), or
FX/FIX(EGF1) (open triangles) was measured at 37 °C in
presence of 5 mM CaCl2. B, in the
absence of phospholipids, thrombin formation from prothrombin (10 µM) activation by 0.5 nM wt-FXa (open
circles), FX/FIX(Gla) (open squares), or FX/FIX(EGF1)
(open triangles) was measured at 37 °C in presence of 75 nM FVa and 5 mM CaCl2. Thrombin
formed at each time point was quantified as described under
"Experimental Procedures." Data represent the mean values of three
experiments.
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|
FX Activation by TF-FVIIa Complex--
FX is one natural substrate
of the TF-FVIIa complex, which is considered as the initial enzyme
complex in the cascade following vascular damage. Because several
studies have implicated the Gla domain (28, 48) and the first EGF
domain (31) of FX in the interaction with TF-FVIIa, FX chimeras were
compared with wt-FX for their ability to serve as substrates for the
complex in the presence of phospholipid vesicles and calcium ions. The
apparent kcat of the TF-FVIIa complex toward all
chimeras was slightly decreased compared with that of wt-FX (Table
II). Conversely, the apparent affinity of
FX/FIX(Gla) and FX/FIX(EGF1) for the enzymatic complex was greatly
decreased (Table II). These data indicate that substitution of the Gla
or the EGF1 domains by the corresponding domains of FIX impairs the
binding capacity of FX to the TF-FVIIa. Because Gla domains of FIX and
FX bind to phospholipids which provide a surface for the assembly of
the FVIIa-TF-substrate complex, it is possible that the substitutions
of the Gla and EGF1 domains alter the alignment between the substrate
and TF-FVIIa complex. This possibility was tested by using
detergent-solubilized TF in an assay comparing the initial rates of
chimeras and wt-FX activation by TF-FVIIa. Similarly to the
phospholipids-containing system (Fig.
3A), activation rates of the
chimeras were impaired in the presence of solubilized TF (Fig.
3B). The initial rate was 3.5 ± 1.1% for FX/FIX(Gla)
and 10.8 ± 3.2% for FX/FIX(EGF1) when compared with the rate
obtained for wt-FX (Fig. 3). Wt-FX was activated at a similar rate of
pd-FX (data not shown).
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Table II
Kinetic parameters of FX proteins activation by the TF-FVIIa complex in
the presence of phospholipids
Varying concentrations of recombinant FX proteins (0-3
µM) are mixed at 37 °C with 60 pM FVIIa
and 60 pM relipidated TF, preincubated for 20 min to
establish a TF-FVIIa complex, in presence of 5 mM
CaCl2. FXa formed was determined as described under
"Experimental Procedures." Mean values ± S.D. for three
experiments are presented.
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Fig. 3.
Activation of recombinant FX by the FT-FVIIa
complex in the presence or absence of phospholipids. A,
activation of 1 µM wt-FX (open circles),
FX/FIX(Gla) (open squares), or FX/FIX(EGF1) (open
triangles) was proceeded at 37 °C by 8 nM FVIIa and
500 pM relipidated TF in presence of 5 mM
CaCl2. B, in the absence of phospholipid
vesicles, activation of 250 nM wt-FX (open
circles), FX/FIX(Gla) (open squares), or FX/FIX(EGF1)
(open triangles) was assayed at 37 °C with 50 nM FVIIa and 250 nM detergent-solubilized
full-length TF in the presence of 5 mM CaCl2.
FXa formation was determined as described under "Experimental
Procedures." Data represent the mean values of three
experiments.
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Inhibition of the TF-FVIIa Complex by TFPI-FXa--
Because the
chimeric variants FX/FIX(Gla) and FX/FIX(EGF1) displayed a reduced
affinity for TF-FVIIa, kinetics of TF-FVIIa inhibition by variable
concentrations of preformed TFPI-FXa chimeras was investigated.
Conditions of a limited amount of relipidated TF (25 pM)
and excess of FVIIa (8 nM) were optimal for TF-FVIIa complex formation. TF-FVIIa complex was incubated with substrate pd-FX
(400 nM) and preformed TFPI-FXa variants (25-100
pM). The results of inhibition of
TF-FVIIa-dependent pd-FXa by increasing concentrations of
TFPI-FXa complexes are presented in Fig.
4. The progress curves show a similar
increasing inhibition of TF-FVIIa by increasing concentrations of
TFPI-pd-FX (Fig. 4A) and of TFPI-wt-FX (Fig. 4B).
Conversely, a significantly reduced inhibition was observed in the
presence of the same concentrations of TFPI-FX/FIX(Gla) (Fig.
4C) or TFPI-FX/FIX(EGF1) (Fig. 4D). From the
linear fit of kobs versus TFPI-FXa
concentrations, apparent second order rate association constants and
dissociation rate constants were determined and reported in Table
III. No significant differences were
observed between all the dissociation rate constants. Conversely, the
rate association constants of the TFPI-FX/FIX(Gla) and FX/FIX(EGF1) complexes with TF-FVIIa were about 5-10-fold reduced compared with
that of the TFPI-wt-FXa. Therefore, substitution of the Gla or the EGF1
domains by the corresponding domains of FIX reduced the capacity of
FXa, in complex with TFPI, to inhibit TF-FVIIa activity toward FX by
reducing the capacity of the inhibitor complex to bind to the enzymatic
complex.

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Fig. 4.
Kinetics of inhibition of the TF-FVIIa
complex by variable concentrations of TFPI-FXa complexes. Varying
concentrations of preformed TFPI-FXa complexes are incubated at
37 °C with 25 pM TF-FVIIa complex (made with 25 pM relipidated TF and 8 nM FVIIa) in the
presence of 400 nM pd-FX. The final concentrations of
TFPI-FXa complexes are 25 (open squares), 50 (closed
circles), and 100 pM (open triangles).
Activated pd-FX formed at each time point is determined, and data were
fitted as described under "Experimental Procedures." A typical
experiment for TFPI-pd-FXa (A), TFPI-wt-FXa (B),
TFPI- FXa/FIX(Gla) (C), and TFPI-FXa/FIX(EGF1)
(D) is shown.
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Table III
Association and dissociation rates for TFPI-FXa complexes interaction
with the TF-FVIIa complex
Association rate (k+1) and dissociation rate
(k 1) constants are calculated from the kinetic
(time course) data of one representative experiment depicted in Fig. 4
of the TF-FVIIa complex inhibition by TFPI-FXa complexes as described
under "Experimental Procedures."
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DISCUSSION |
In this study, the role of the Gla and the first EGF-like domains
of FX has been explored. To this end, FX chimeras containing the Gla or
the EGF1 domains of FIX substituting the corresponding domains of FX
were produced. Their properties, regarding different functions of FX,
were compared with those of normal FX.
In the prothrombinase complex FVa displays an apparent affinity for
FX/FIX-Gla and FX/FIX-EGF1 chimeras ~2-fold higher than for normal
FXa (Fig. 1). One possibility is that Gla and EGF1 domains of FXa
interact with FVa. Therefore, the apparent increased affinity of FVa
for the FX chimeras is due to the presence of appropriate binding sites
for the cofactor on the Gla and EGF1 domains of FIX. So far, no
interactions have been described between FVa and FIXa. A second
possibility is that FVa does not interact with the Gla and EGF1 domains
of FXa. Hence, in FX the Gla and EGF1 domains would act as spacers
between phospholipids and the rest of the molecule to match the
cofactor binding sites. Therefore, the presence of the Gla or EGF1
domains of FIX modifies the inclination toward the phospholipid surface
of the EGF2 and protease domains of the chimeras compared with that of
normal FXa. This mechanism is supported by experimental data showing
that in the absence of phospholipids the initial rate of thrombin
formation by the chimeras associated to FVa is identical to those
observed with normal FXa (Fig. 2B). A similar spacer role
has been described previously for FIX in studies using FIX mutated in
the EGF1 domain or in which the corresponding domain of protein C
substituted the EGF1 domain. The mutated FIX molecule displays a
reduced interaction with FVIIIa in the presence of phospholipids but
not in its absence (21). Observation that the FX chimeras displayed a
normal catalytic activity in the presence of FVa (Fig. 2B)
demonstrates that the FVa-binding sites on the chimeras are identical
to those of normal FXa. Thus, because no interactions have been
described between FVa and FIXa, these data imply that the Gla and the
EGF1 domains of FXa do not contain FVa-binding sites and are only
involved in the interaction with the cofactor in the presence of
phospholipids to conform the enzyme toward FVa.
The FX chimeras containing the Gla or the EGF1 domains of FIX are
different from normal FX as substrate for the TF-FVIIa complex in the
presence of phospholipids (Table II). The results indicate that
substitution of one of the two amino-terminal regions of FX by the
corresponding region of FIX affects the apparent affinity of the
substrate for the enzymatic complex. Therefore, these two regions are
involved in the formation of the TF-FVIIa-FX complex, and it can be
suggested that within this ternary complex both domains of FX are
directly or indirectly implicated in the binding to TF-FVIIa. The Gla
domain involvement in the interaction with TF-FVIIa exosites is
supported by several studies. For instance, by studying the
effect of mutations in the carboxyl-terminal domain of TF, an
interaction of this membrane-proximal region with the Gla domain of the
substrates FIX and FX has been proposed (29, 30, 49). Recently, it has
been revealed (31) that FIX and FX interact with TF through, in part,
their EGF1 domains. It is noteworthy that the decreased affinity of the
FX/FIX(Gla) and FX/FIX(EGF1) compared with normal FX for the TF-FVIIa
complex in the presence of phospholipids is associated with a normal
kcat (Table II). These data can be integrated to
the kinetic model for FX activation by TF-FVIIa proposed by
Krishnaswamy and co-workers (50). In their model, substrate recognition
by the TF-FVIIa complex is achieved through two sequential steps.
Initial interactions between TF-FVIIa exosites and complementary sites
on the substrate remote from structures surrounding the scissile bond
are followed by an intramolecular binding step that allows the cleavage
of adjacent structures to dock with the active site of the enzyme prior
to bond cleavage. In the present study, it is observed that in the
absence of phospholipids, the initial rates of activation of the
chimeras were markedly decreased compared with the activation of wt-FX
(Fig. 3). As with the previous data in presence of phospholipids, the
defective activation in their absence is due to defective interaction
of the chimeras with exosites of the TF-FVIIa complex. Taken together,
these results suggest a direct role of the Gla and EGF1 domains in the
interaction with the TF-FVIIa. Thus, despite sequence and structural
homologies of the Gla and the EGF1 domains of FIX and FX and the
probable involvement of the same residues to interact with TF-FVIIa,
the severe defective interactions of the chimeras with TF-FVIIa, in the
presence or absence of phospholipids (Figs. 2 and 3), demonstrate that
these domains cannot be exchanged without altering the substrate
affinity for the enzymatic complex. Two possibilities arise from these
observations. First, the defective interaction of the chimeras with the
enzymatic complex could be due to the involvement of different residues
between FIX and FX domains with TF-FVIIa. However, a docking approach
that proposed a model for the ternary complex TF-FVIIa-FIX has revealed
that residues of FIX interacting with TF-FVIIa are rather conserved in
FX (51). A second possibility is that the substitutions of Gla or EGF1
domains introduce an alteration of the favorable orientation of the
substrate toward the enzymatic complex. The same structural modification is likely the cause of a slight increase of the apparent affinity of FVa for the chimeras in the presence of phospholipids (Fig.
1).
Kinetics of TF-FVIIa inhibition by variable concentrations of preformed
TFPI-FXa show that the substitution of the Gla or the EGF1 domains
markedly reduces the association constants of the FT-FVIIa-TFPI-FXa
quaternary complexes (Table III). This suggests that the defective
interaction of TFPI associated with the activated chimeras with
TF-FVIIa is probably due to an alteration of the favorable orientation
of the inhibitor complex toward the enzymatic complex. These data
indicate that similar interactions contribute to the assembly of FX and
FXa, after complex formation with TFPI, to the TF-FVIIa complex. This
notion is in agreement with a previous study (52) showing that TF
residues, which are important for the activation of FX by the TF-FVIIa
complex, are required for the accelerated inhibition of the TF-FVIIa
complex by TFPI mediated by FXa. These TF residues have been proposed
to interact with the Gla domain of the substrates FIX and FX (29, 30,
49). It has been shown previously (53) that FXa devoid of the Gla domain is not able to support the TFPI-mediated inhibition of TF-FVIIa,
suggesting the importance of FXa Gla domain in the formation of the
TF-FVIIa-TFPI-FXa complex. However, proteolytic fragments of
FX-containing multiple domains are not always reliable for the
identification of binding sites because it has been observed that EGF
domains must be covalently attached to the Gla domain of FX to maintain
their properties (53). Therefore, the approach using FX/FIX chimeras
carrying FIX regions is more appropriate to identify the role of the
Gla and EGF1 domains.
In conclusion, this study demonstrates that the Gla and first EGF-like
domains of FX are not directly involved in the interaction of FXa with
FVa. Experimental data indicate that the FX Gla domain interacts with
the FT-FVIIa complex. Moreover, this domain, together with the first
EGF-like domain, is directly involved in the association of FXa after
complex formation with TFPI to the TF-FVIIa complex. This study also
reveals that in a blood coagulation protein a loss of function can be
counterbalanced by the gain of another one. Thus, a mutation within a
FX molecule, which is responsible for a reduced rate of activation by
the TF-FVIIa complex, could be compensated for by its beneficial effect
on the activated molecule in the presence of its cofactor. There are
other models in nature confirming this paradox. Therefore, it can be
suggested that an individual with no bleeding disorders could possess
such mutations, which would only be detected during coagulation investigation.