From the Departments of Pathology and
Medicine, Vanderbilt University, Nashville, Tennessee 37232 and the
§ Cardiovascular and Urogenital Diseases Center of
Excellence, GlaxoSmithKline,
King of Prussia, Pennsylvania 19406
Received for publication, December 13, 2002
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ABSTRACT |
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During hemostasis, factor IX is activated
to factor IXa Coagulation factor IX
(FIX,1 EC 3.4.21.22) is the
zymogen precursor of a plasma serine protease, factor IXa FIX is a 56-kDa polypeptide comprised of C-terminal trypsin-like
catalytic (heavy chain) domain and an N-terminal noncatalytic (light
chain) region separated by an 11-kDa activation peptide (1, 11). Two
proteolytic cleavages are required to liberate the activation peptide
from the remainder of the molecule to produce FIXa This evidence not withstanding, there are some data to support a role
for the Gla domain in FIX activation by factor XIa. A Gla domain
mutation at amino acid 4 associated with hemophilia B interferes with
factor XIa-mediated activation of FIX (26), as does failure to remove
the FIX propeptide from the N terminus (27). Monoclonal antibodies
directed against FIX-Gla block activation of FIX by factor XIa (28,
29). However, the antibodies interfere with other activities such as
FIX activation by factor VIIa/tissue factor and factor X activation by
FIXa Plasma and Recombinant FIX Proteins--
Plasma-derived FIX,
FIXa Purification of Recombinant Proteins--
Proteins were purified
from conditioned medium by monoclonal antibody affinity chromatography.
Antibodies were linked to 5 ml of Affi-Gel 10 (Bio-Rad) at 3 mg of
IgG/ml of gel. For rFIX, the antibody used was humanized murine
monoclonal IgG SB 249417 (GlaxoSmithKline, King of Prussia, PA), a
calcium-dependent antibody that recognizes the properly
Recombinant Factor XIa-Ala557--
Preparation
of recombinant factor XI has been described previously (31). Briefly,
HEK293 cells were transfected with an expression construct consisting
of the human factor XI cDNA in pJVCMV to generate stable expressing
clones, and recombinant factor XI was purified from conditioned medium
by affinity chromatography using monoclonal IgG 1G5.12 (31). This
process was used to generate a recombinant variant of factor XI in
which the active site serine residue at amino acid position 557 was changed to alanine (factor XI-Ala557) by site-directed
mutagenesis. Factor XI-Ala557 was converted to the
"active" form (factor XIa-Ala557) by
diluting to 300 µg/ml in TBS containing 5 µg/ml human factor XIIa
(Enzyme Research Laboratory) and incubating at 37 °C. Conversion of
the 80-kDa zymogen to the 45-kDa heavy and 35-kDa light chains of the
"activated" species was followed by reducing SDS-PAGE. Factor
XIa-Ala557 was separated from factor XIIa by repurification
over the 1G5.12 column.
Determination of Specific Activities of Zymogen and Active
Recombinant Proteins in Plasma Clotting Assays--
Activated partial
thromboplastin time (aPTT) assays were performed as follows. The
protein to be tested (plasma FIX, rFIX, rFIX-des FIX and FIXa
Analyte (FIX, FIXa
SPR experiments on the effect of the FIX Gla domain-specific antibody
SB 249417 on the FIX-factor XIa interaction were performed similarly,
with the following exceptions. The concentration of analyte (FIX) was
fixed at 1 µM, and various concentrations of SB 249417 (0.01-5.0 µM) were mixed with the analyte and incubated for 5 min at room temperature prior to injecting into the flow cells.
The data were fit to a single site competition model, and the
inhibition constant (Ki) was calculated by
nonlinear regression (GraphPad Prism, version 3.0, GraphPad, San Diego).
Effect of Monoclonal Antibody SB 249417 on Factor IX Activation
by Factor XIa--
Inhibition of FIX activation by factor XIa was
assessed by a colorimetric assay. A reaction mixture containing 200 nM FIX, 1 nM factor XIa, and varying
concentrations of SB 249417 (1 nM to 10 µM)
in reaction buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2.5 mM CaCl2, and 0.05%
CHAPS) was incubated at 37 °C for 30 min. After incubation, FIXa Time Courses of FIX Activation followed by Western Immunoblot and
SDS-PAGE--
Plasma-derived or recombinant proteins were diluted to
100 nM in TBS containing 2.5 mM
CaCl2, and the solution was warmed to 37 °C in a water
bath. Reactions were started by the addition of plasma-derived factor
XIa to a final concentration of 1 nM. At time points
between 0 and 60 min, 30-µl volumes were removed and mixed with 10 µl of 4× nonreducing SDS-sample buffer (500 nM Tris-HCl,
pH 6.8, 40% glycerol, 10% SDS). Samples were size fractionated on
12% polyacrylamide gels followed by transfer to nitrocellulose
membranes. Blots were developed with goat anti-human FIX polyclonal IgG
(Affinity Biologicals) using an ECL chemiluminescence Western blotting
detection kit (Amersham Biosciences). The intensities of bands on
autoradiographs for Western blots of recombinant protein activation by
factor XIa were measured using a Bio-Rad Imaging Densitometer model
GS-670. Measurements for bands representing the zymogen (56 kDa band)
and active protease (45 kDa band) were determined for each lane. For
each lane, the value for the active protease was divided by the sum of
the signals for the zymogen and active protease for each time point to
determine the percent zymogen converted to protease.
Cleavage of FIX by Russell's viper venom protease (RVVP) was assessed
as follows. Plasma-derived or recombinant FIX proteins were diluted to
1.0 µM in TBS containing 2.5 mM
CaCl2, and the solution was warmed to 37 °C in a water
bath. Reactions were started by he addition of RVVP (Enzyme Research
Laboratory) to a final concentration of 15 nM. At time
points between 0 and 120 min, 30-µl volumes were removed and mixed
with 10 µl of 4× reducing SDS-sample buffer (500 nM
Tris-HCl, pH 6.8, 40% glycerol, 10% SDS, 10% 2-mercaptoethanol).
Samples were size fractionated on 12% polyacrylamide gels, and gels
were stained with GelCode Blue stain reagent (Pierce).
Binding of FIX and FIXa
A significant concern when using SPR to study the binding of a
substrate to its enzyme is, of course, that the substrate may be
converted to product by the enzyme on the surface of the flow cell. In
the case under consideration, this could confound interpretation of
results for binding of FIX to factor XIa. To address this issue, we
prepared a recombinant version of factor XIa, factor
XIa-Ala557, in which the active site serine of the
catalytic domain was replaced with alanine. Wild type factor XIa
expressed in HEK293 cells has been shown to have activity similar to
that of plasma-derived factor XIa in plasma and purified protein based
assays (31, 36). As expected, factor XIa-Ala557 lacks
activity in plasma clotting assays and does not cleave the factor XIa
chromogenic substrate S-2366 (data not shown). The binding of FIX and
FIXa Recombinant FIX Proteins and Activity in Plasma Clotting
Assays--
To determine the importance of the FIX Gla domain in
binding to, and activation by, factor XIa, recombinant versions of FIX with altered Gla domains were prepared (Fig.
2A). Recombinant proteins were
expressed in the human fibroblast cell line HEK293 because this line
has been shown to
The aPTT assay requires FIX to be activated by factor XIa and the
FIXa Recombinant FIX Protein Binding to Factor XIa Studied with
SPR--
Recombinant proteins were tested by SPR for their ability to
bind to factor XIa (Fig. 2C). rFIX binds to plasma factor
XIa with a Kd of 107 nM, a value
similar to those obtained for plasma-derived FIX and FIXa
The humanized murine monoclonal antibody SB 249417 recognizes the FIX
Gla domain in a calcium-dependent manner
(32)2 and is sensitive to
conformational changes to the domain which accompany incomplete
FIX Activation by Factor XIa and RVVP--
Initially, we examined
the capacity of SB 249417 to inhibit FIX activation by factor XIa using
a chromogenic substrate assay. Consistent with results from SPR studies
(Fig. 3A), SB 249417 is a potent inhibitor of FIX activation
by factor XIa, with a Ki of 33 nM
(Fig. 3B). The chromogenic substrate assay used to follow
this reaction is relatively insensitive to FIXa
The venom of Dabois russelli (Russell's viper) contains a
protease, RVVP, which cleaves FIX between Arg180 and
Val181 at the C terminus of the activation peptide to
produce an active FIX intermediate called FIXa Activation of FIX, a key step in the formation and maintenance of
a fibrin clot, is controlled by at least two distinct mechanisms. Initiation of fibrin formation involves binding of factor VIIa in
plasma to the membrane protein tissue factor at a site of blood vessel
injury (2, 3). Factor VIIa/TF activates factor X and FIX in reactions
that require calcium and a phospholipid surface (13, 40, 41). Activated
factor X (factor Xa) then converts prothrombin to thrombin, which
initiates fibrin formation. It is postulated that factor VIIa/TF is
inhibited relatively early in the coagulation process by the TF pathway
inhibitor (2, 42). FIXa Factor XI has an unusual structure for a coagulation protease. The
protein is a disulfide bond linked dimer of two identical 80-kDa
polypeptides (25, 45) and lacks the Gla domain that is a characteristic
feature of other coagulation proteases (24). The noncatalytic
N-terminal "heavy chain" portion of the factor XI polypeptide
comprised of four 90-91-amino acid repeats called apple domains
(A1-A4 from the N terminus), which mediate binding to proteins,
platelets, and glycosaminoglycans (25, 31, 36, 46, 47). Using
recombinant factor XI in which apple domains were replaced with
corresponding domains from the functionally distinct protein plasma
prekallikrein, it was determined that factor XI A3 likely contains a
binding site for FIX (31, 35). Studies based on peptide mimicry suggest
that the C-terminal half of A2 is also involved in FIX binding (47).
Zymogen factor XI, unlike factor XIa, does not cleave small chromogenic
substrates. This indicates that conversion of zymogen to active
protease involves a conformational change that gives small molecules,
as well as portions of FIX, access to the catalytic active site. The
SPR studies demonstrate that conversion to factor XIa is also required for binding of FIX and FIXa The structural elements on FIX required for binding to factor XIa have
not been clearly defined. The noncatalytic portion of the molecule
contains (from N terminus to C terminus) the Gla domain, a short
aromatic stack region, two EGF-like domains, and the activation peptide
(3). Some Gla domain mutations causing hemophilia B are associated with
poor factor XIa mediated activation, as is the failure to remove the
propeptide from the N terminus of the molecule (26, 27). Similarly,
single amino acid substitutions in the EGF-1 (factor IX New London)
(51) and EGF-2 (52) domains associated with cross-reactive
material-positive hemophilia B are activated poorly by factor XIa.
However, all of these mutations interfere with other activities of FIX
in addition to activation by factor XIa. A rFIX protein containing the
factor VII EGF1 domain is activated normally by factor XIa (53, 54),
whereas a study employing alanine scanning mutagenesis on portions of
the EGF2 domain identified only a single amino acid (position 89) that appeared to be necessary for normal activation by factor XIa (55).
Using SPR, plasma coagulation, antibody inhibition, and purified
protein activation assays we have demonstrated that the FIX Gla domain
either contains all or a portion of the factor XIa binding site or is
required for the proper conformation of the binding site. The results
with chimera rFIX/VII-Gla are particularly intriguing. In its active
form, this molecule appears to activate factor X reasonably well in a
plasma clotting assay and is activated by the snake venom protease RVVP
in a manner similar to wild type FIX. This indicates that rFIX/VII-Gla
is structurally and catalytically similar to FIX and strongly suggests
that the failure of the protein to demonstrate activity in a standard
aPTT assay is because of a specific defect in factor XIa-mediated
activation. SPR confirmed that this protein has a profound defect in
binding to factor XIa. Activation of factor X by FIXa The notion that a Gla domain may be involved in a binding interaction
not involving phospholipid has precedent. Amino acids 3-11 in the FIX
Gla domain are critical for high affinity binding of the protein to
aortic endothelial cells (57). It appears that this interaction is
caused by binding of FIX to collagen IV and not phospholipid in the
cell membrane (58). Using scanning force microscopy, Wolberg and
colleagues (59) demonstrated that FIX binds specifically to collagen IV
in the later molecule collagenous domain. Regan and co-workers (60)
have shown that the protein C Gla domain is involved in a
protein-protein interaction with the protein C receptor on endothelial
cells. A recombinant prothrombin molecule containing the protein C Gla
domain bound specifically to cells expressing the protein C receptor.
Along similar lines, Lockett and Mast (61) presented data suggesting
that an interaction between the C terminus of TF pathway inhibitor and
the factor Xa Gla domain is required for proper TF pathway inhibitor
mediated-inhibition of factor Xa. Thus, Gla domain involvement in
protein-protein interactions may be common.
by factor VIIa and factor XIa. The glutamic acid-rich
-carboxyglutamic acid (Gla) domain of factor IX is involved in
phospholipid binding and is required for activation by factor VIIa. In
contrast, activation by factor XIa is not
phospholipid-dependent, raising questions about the
importance of the Gla for this reaction. We examined binding of factors
IX and IXa
to factor XIa by surface plasmon resonance. Plasma
factors IX and IXa
bind to factor XIa with Kd values of 120 ± 11 nM and
110 ± 8 nM, respectively. Recombinant factor IX bound
to factor XIa with a Kd of 107 nM,
whereas factor IX with a factor VII Gla domain (rFIX/VII-Gla) and
factor IX expressed in the presence of warfarin (rFIX-des
) did not
bind. An anti-factor IX Gla monoclonal antibody was a potent inhibitor
of factor IX binding to factor XIa (Ki 34 nM) and activation by factor XIa (Ki 33 nM). In activated partial
thromboplastin time clotting assays, the specific activities of plasma
and recombinant factor IX were comparable (200 and 150 units/mg),
whereas rFIX/VII-Gla activity was low (<2 units/mg). In contrast,
recombinant factor IXa
and activated rFIX/VIIa-Gla had similar
activities (80 and 60% of plasma factor IXa
), indicating that both
proteases activate factor X and that the poor activity of zymogen
rFIX/VII-Gla was caused by a specific defect in activation by factor
XIa. The data demonstrate that factor XIa binds with comparable
affinity to factors IX and IXa
and that the interactions are
dependent on the factor IX Gla domain.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(FIXa
),
which is required for formation and maintenance of a fibrin clot at a
site of blood vessel injury (1-3). The importance of this protein to
normal hemostasis is demonstrated by the severe bleeding abnormality (hemophilia B) associated with its deficiency state (4, 5). FIX is a
member of a family of proteases, including the hemostasis-related proteins prothrombin, factor VII, factor X, and protein C (the "vitamin-K dependent proteases"), which require specific
post-translational modifications for normal activity (3, 6). At the N
terminus of the mature forms of these proteins is a region rich in
glutamic acid called the
-carboxyglutamic acid or "Gla" domain.
Glutamic acid residues in the Gla domain are modified by the addition
of a carboxyl group to the
-carbons in a reaction catalyzed by the vitamin K-dependent enzyme
-glutamyl carboxylase (6, 7). Proper
-carboxylation is required for Gla domain binding to calcium and phospholipid, two properties that are indispensable for proper protease activity during coagulation (8). In vivo, Gla
domain-dependent protease-substrate interactions take place
on the phospholipid membranes of damaged cells and activated platelets.
Binding to phospholipid accelerates the conversion of substrate to
product by decreasing the Km for the reactions
several orders of magnitude (8-10). The use of coumarin compounds as
therapeutic anticoagulants is based on their ability to interfere with
vitamin K metabolism, causing incomplete
-carboxylation of the Gla
domains of prothrombin and factors VII, IX, and X (8). In addition to
phospholipid, protease-substrate interactions involving vitamin
K-dependent proteins require a protein cofactor, which
further improves catalytic efficiency by increasing the
kcat for the reactions (9).
(11, 12).
Activation may occur by two distinct mechanisms mediated by the plasma
serine proteases factor VIIa (EC 3.4.21.21) and factor XIa (EC
3.4.21.27) (2, 3, 13, 14). Activation of FIX by factor VIIa is a
typical coagulation protease-substrate interaction requiring calcium,
phospholipid, and a protein cofactor (the membrane protein tissue
factor) (13, 15). In this reaction the Gla domains of both FIX and
factor VIIa form critical interactions with the phospholipid surface
(16-20). The importance of the FIX Gla domain to FIX activation by
factor XIa is less certain because the reaction appears to involve a
mechanism distinctly different from typical vitamin
K-dependent protease-substrate interactions. Although
calcium is required (1, 21, 22), phospholipid has little influence on
the process (1, 11, 23). Indeed, factor XIa lacks a Gla domain,
suggesting that it may interact poorly with phospholipid (3, 24, 25).
Furthermore, a protein cofactor has not been identified for FIX
activation by factor XIa, at least when the reaction occurs in liquid
plasma. These observations raise the possibility that the FIX Gla
domain is not required for activation by factor XIa.
. Therefore, nonspecific steric interference cannot be ruled out
as the mechanism of action. In this report we describe studies on FIX
binding to factor XIa. The work demonstrates that both FIX and FIXa
bind to factor XIa but not zymogen factor XI and that the FIX Gla
domain plays a critical role in the interactions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and FIXa
were purchased from Enzyme Research Laboratories
(South Bend, IN). The cDNAs for human wild type FIX and chimera
FIX/VII-Gla were gifts from Dr. Darrell Stafford, University of North
Carolina, Chapel Hill (30). FIX/VII-Gla contains the factor VII signal
peptide, propeptide, Gla domain, and aromatic stack region (factor VII
amino acids 1-48) and the FIX epidermal growth factor-like (EGF) 1 and
2 domains, activation peptide, and catalytic domain (factor IX amino
acids 50-415). cDNAs were ligated into the mammalian expression
vector pJVCMV, which contains a cytomegalovirus promoter (31). 50 million HEK293 cells (a human fetal kidney fibroblast line, ATCC CRL
1573) were cotransfected with 40 µg of expression construct and 2 µg of plasmid RSVneo, which contains a gene conferring resistance to
neomycin. Transfection was by electroporation (Electrocell Manipulator
600 BTX, San Diego). Transfected cells were grown in Dulbecco's
modified Eagle's medium with 5% fetal bovine serum containing the
neomycin analog G418 at 500 µg/ml. G418-resistant clones were
transferred to 96-well culture plates, and supernatants were tested for
protein expression by enzyme-linked immunosorbent assay using goat
anti-human FIX antibodies (Affinity Biologicals, Hamilton, ON).
Expressing clones were expanded in 175-cm2 culture flasks,
and the medium was changed to serum-free Cellgro Complete Medium
(Mediatech, Herdon, VA) supplemented with 10 µg/ml vitamin
K1 (phytonadione, Abbot). The medium was exchanged every 48-72 h. Conditioned medium was supplemented with benzamidine to a
final concentration of 5 mM and stored at
20 °C
pending purification. Recombinant proteins are designated by the prefix "r" to distinguish them from plasma-derived proteins (no prefix). To generate rFIX that is incompletely
-carboxylated (rFIX-des
), transfected HEK293 cells expressing rFIX were grown in medium supplemented with 5 µg/ml sodium warfarin (Sigma) instead of vitamin K.
-carboxylated FIX Gla domain (32). One to two liters of conditioned
medium were run across the column, followed by washing with 25 mM Tris-HCl, pH 7.5, 100 mM NaCl (TBS) containing 2.5 mM CaCl2. Elution was with TBS
containing 25 mM EDTA. For rFIX-des
and rFIX/VII-Gla, a
monoclonal murine IgG against the FIX catalytic domain (kindly provided
by Dr. George Broze, Washington University, St. Louis, MO) was used.
Washing was with TBS containing 2.5 mM CaCl2,
and elution was with TBS containing 2.5 mM
CaCl2 and 2.0 M sodium thiocyanate. Protein containing fractions from elutions were pooled and concentrated in an
Amicon concentrator, dialyzed against TBS, and stored at
80 °C.
Protein concentration was determined by dye binding assay (Bio-Rad).
, or rFIX/VII-Gla)
was diluted to 5 µg/ml in TBS containing 0.1% bovine serum albumin
(TBSA). Serial dilutions of the 5 µg/ml stocks were prepared in TBSA
prior to testing in the aPTT assay. 65 µl of FIX-deficient plasma
(Diagnostica Stago, Asnieres-sur-Seine, France), 65 µl of the protein
to be tested, and 65 µl of PTT-A reagent (Diagnostica Stago) were
mixed in a fibrin cup and incubated at 37 °C for 5 min. 65 µl of
25 mM CaCl2 was added, and the time to clot
formation was determined on a fibrometer (DataClot II, Helena
Laboratories, Beaumont, TX). Results were compared with serial
dilutions of pooled normal plasma (George King, Overland Park, KS). By
definition, undiluted normal plasma has a FIX activity of 1 unit/ml or
100%. For modified partial thromboplastin time (modified PTT) assays,
proteases to be tested (plasma FIXa
, rFIXa
, or rFIXa
/VII-Gla)
were diluted to 5 µg/ml in TBSA, and serial dilutions were prepared
as described above. 65 µl of FIX-deficient plasma, 65 µl of rabbit
brain cephalin prepared by the method of Bell and Alton (33), and 65 µl of the protein to be tested were mixed in a fibrin cup warmed to
37 °C. 30 s later, 65 µl of 25 mM
CaCl2 was added, and the time to clot formation was
determined on the fibrometer. Results were compared with those for
plasma FIXa
, which was arbitrarily assigned an activity of
100%.
Binding to Factor XI and Factor XIa Studied by
Surface Plasmon Resonance (SPR)--
SPR studies were performed on a
dual flow cell Biacore X device (Biacore, Inc., Uppsala, Sweden). The
zymogen or activated versions of plasma-derived factor XI or
recombinant factor XI-Ala557 were immobilized on a
carboxymethyl dextran (CM5) surface using amine coupling chemistry. The
surface of the flow cell was activated by injection of a mixture of
N-hydroxysuccinimide and EDAC for 5 min at a rate of 10 µl/min. Factor XI or XIa at 50 µg/ml in sodium acetate buffer, pH
5.5, was manually injected onto the activated surface. Finally, the
remaining active sites on the flow cell were blocked by injecting 1 M ethanolamine for 5 min. A flow cell to assess nonspecific
background binding was prepared using plasma kallikrein instead of
factor XIa. Plasma kallikrein is structurally highly similar to factor
XIa but interacts poorly with FIX (25, 31, 34).
, rFIX, rFIX-des
, or rFIX/VII-Gla) was injected
across the flow cells at varying concentrations (4 nM to 2 µM) in HEPES-buffered saline (10 mM HEPES, pH
7.4, 150 mM NaCl, 0.005% polysorbate 20) containing 2.0 mM CaCl2 at a flow rate of 35 µl/min. A 2-min
association time was determined to be adequate for plasma-derived FIX,
FIXa
, and wild type rFIX and was used for all subsequent
experiments. Removal of bound analyte to regenerate the flow cell was
accomplished by infusing HEPES-buffered saline containing 3 mM EDTA. Flow cells were equilibrated with HEPES-buffered
saline containing 2.0 mM CaCl2 prior to
subsequent runs. A similar procedure was used for determining
nonspecific binding of analyte to kallikrein-coated flow cells. Data
obtained for analyte binding to immobilized factor XI/XIa were
corrected for nonspecific binding by subtracting the kallikrein flow
cell signal from the factor XI/XIa flow cell signal obtained with the same analyte. BIAevaluation software provided by the manufacturer was
used for data analysis. The BIAcore equilibrium analysis method was
used for all interactions. Briefly, the response at equilibrium (Rueq) for each concentration of analyte was
determined as above. A nonlinear regression routine was used to
determine the Kd by fitting the data to the 1:1
interaction steady-state affinity model,
where C represents the concentration of analyte and
Rmax the maximal binding capacity.
(Eq. 1)
generation was determined by adding the reaction mixture to an equal
volume of a detection mixture comprising reaction buffer and 66%
ethylene glycol containing 2 mM FIXa
p-nitroanilide-conjugated peptide substrate S299 (American
Diagnostica, Greenwich, CT). After incubating for 10 min at 37 °C,
the release of p-nitroanilide as a measure of FIXa activity
was determined at a wavelength of 405 nm using a spectrophotometric
microplate reader. In the absence of FIX in the assay, no
p-nitroanilide signal was detected. Data were fit to a
single site competition model, and an inhibition constant
(Ki) was calculated by nonlinear regression
(GraphPad Prism).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to Factor XIa Studied with
SPR--
Initially, binding of plasma-derived FIX in solution to
immobilized plasma-derived factor XIa was studied. FIX concentrations between 4 nM and 2 µM were tested using 2-min
association and 90-s dissociation times. FIX rapidly associates with,
and dissociates from, factor XIa (Fig.
1A) in a process that is
dependent on calcium (data not shown). Dissociation appears to be
nearly complete, indicating that little nonspecific irreversible
binding to components of the flow cell is occurring. A plot of plasma
FIX bound to factor XIa as a function of FIX concentration is shown in
Fig. 1B (open circles). The plot was derived from
data in Fig. 1A corrected for nonspecific binding as
determined by the simultaneous infusion of the identical concentrations
of FIX across the reference flow cell containing immobilized plasma
kallikrein. Certain characteristics of the FIX-factor XIa interaction
(the rapid dissociation rate in particular) preclude a kinetic fit of
the data. We used equilibrium binding analysis to determine the binding
constant, where binding at steady state is plotted against the
concentration of FIX, and Kd is determined in
the traditional manner as the concentration of analyte (FIX) occupying
50% of available binding sites. Using this method, a
Kd for the FIX-factor XIa binding interaction of 120 ± 11 nM was obtained. This result is in
reasonably good agreement with published values of
Km for activation of FIX by factor XIa (160-180
nM) determined by chromogenic substrate assay (31, 35).
Interestingly, FIXa
also bound to plasma factor XIa with a
Kd of 110 ± 8 nM (Fig.
1B, open squares). Apparently liberation of the
activation peptide during FIX activation does not alter the affinity of
the protein for factor XIa. Neither FIX nor FIXa
bound to zymogen
factor XI (Fig. 1B, closed circles and
closed squares), indicating that factor XI undergoes
conformational changes upon conversion to factor XIa which exposes the
FIX binding site.
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Fig. 1.
SPR studies of binding interactions between
FIX or FIXa and factor XI or XIa.
A, FIX binding to factor XIa. Human plasma-derived factor
XIa was immobilized on a carboxymethyl dextran flow cell surface as
described under "Experimental Procedures." Human plasma-derived FIX
at concentrations from 4 nm to 2 µM in HEPES-buffered
saline containing 2.5 mM CaCl2 and 0.005%
polysorbate 20 was injected across the flow cell using 2-min
association and 90-s dissociation times. The traces, from
bottom to top, represent FIX at concentrations of
0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1,000, and 2000 nM. B, SPR studies of human plasma-derived FIX
and FIXa
binding to immobilized plasma-derived factor XI and factor
XIa.
, FIX binding to factor XIa;
, FIXa
binding to factor
XIa;
, FIX binding to factor XI; and
, FIXa
binding to factor
XI. C, SPR studies of human plasma-derived FIX and FIXa
binding to recombinant factor XI/XIa-Ala557.
, FIX
binding to factor XIa-Ala557;
, FIXa
binding to
factor XIa-Ala557;
, FIX binding to factor
XI-Ala557; and
, FIXa
binding to factor
XI-Ala557. Data have been corrected for nonspecific binding
and analyzed as described under "Experimental Procedures." The data
shown in the figure are representative runs for each experiment. All
experiments were performed in triplicate.
to factor XIa-Ala557 was studied by SPR in the same
manner as binding to plasma-derived factor XIa (Fig. 1C,
open circles and squares). The
Kd values for binding of FIX and FIXa
to
factor XIa-Ala557 (152 ± 41 and 129 ± 27 nM, respectively) are comparable with those obtained with
plasma-derived factor XIa. Again, FIX and FIXa
do not bind to
uncleaved "zymogen" factor XI-Ala557 (Fig.
1C, closed circles and squares). The
results indicate that conversion of FIX to FIXa
by immobilized
factor XIa, if it occurs, does not influence the results of the binding
assays appreciably. The studies demonstrate that both zymogen and
activated FIX bind to factor XIa with similar affinity and that a
catalytically functional factor Xla molecule is not required for binding.
-carboxylate properly a high percentage of
expressed recombinant vitamin K-dependent protein (37). In
a standard aPTT assay, plasma-derived FIX demonstrated a specific
activity of 200 units/mg (1 unit equaling the FIX activity in 1 ml of
normal plasma). rFIX expressed in the presence of vitamin K had a
specific activity of 75% (150 units/mg) of that of plasma FIX in the
aPTT assay. FIX expressed in the presence of the vitamin K antagonist
warfarin (rFIX-des
) demonstrated significantly reduced activity
(<1% normal activity or <2 units/mg of protein) when tested under
the same conditions. As shown in Fig. 2B, the presence of
warfarin in the cell culture results in a protein that is not recognized by a monoclonal antibody (SB 249417) that requires calcium
and a properly
-carboxylated FIX Gla domain for protein recognition.
This demonstrates that adding warfarin to the cell culture system
effectively interferes with
-carboxylation of the Gla domain. rFIX
in which the Gla domain has been replaced by the corresponding domain
from factor VII (rFIX/VII-Gla; Fig. 2A) was expressed in the
presence of vitamin K. This chimeric protein also demonstrated poor
activity in the aPTT assay (<1% normal activity or <2 units/mg of
protein).
View larger version (29K):
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Fig. 2.
Recombinant FIX proteins. A,
nonreducing SDS-PAGE (12% gel) of recombinant proteins (~200 ng)
purified by monoclonal antibody affinity chromatography. Staining is
with GelCode Blue. The positions of molecular mass standards are shown
on the left of the figure. It is not clear why rFIX/VII-Gla
appears as two bands; however, this was a consistent finding in several
protein preparations. B, Western blots of rFIX expressed in
HEK293 cells grown in the presence of 10 µg/ml vitamin K
(VK) or 3 µM warfarin (W). The blot
on the left was developed with goat polyclonal anti-human
factor IX IgG and the blot on the right with humanized
murine monoclonal antibody SB 249417, which recognizes the properly
-carboxylated FIX Gla domain. C, binding of rFIX,
rFIX/VII-Gla, or rFIX-des
to plasma factor XIa studied with SPR.
rFIX concentrations between 4 nM and 1 µM
were tested.
, rFIX;
, rFIX-des
; and
, rFIX/VII-Gla.
subsequently generated to activate factor X. Poor activity in
this assay could, therefore, reflect a defect in one or both of these
steps. To define further the abnormalities in rFIX-des
and
rFIX/VII-Gla, we attempted to activate recombinant proteins by
incubating them with a high concentration (100 nM) of
factor XIa before testing them in clotting assays (modified PTT assay).
This step removes the requirement for activation by factor XIa from the
clotting process. rFIX-des
could not be activated to FIXa
by
prolonged incubation with high concentrations of factor XIa.
rFIX/VII-Gla was completely converted to the active form; however, it
was noted that activation was considerably slower than with wild type
rFIX (see below) or plasma FIX. In the modified PTT assay, rFIXa
demonstrated ~80% of the activity of plasma-derived FIXa
,
consistent with results from the conventional aPTT assay. Interestingly, rFIXa
/VII-Gla also demonstrated significant activity in this assay (~60% of the activity of plasma FIXa
). This
suggests that the factor VII Gla domain is a reasonably good substitute for the FIX Gla domain when FIXa
is incorporated into the factor Xase complex in plasma with zymogen factor X, factor VIIIa, calcium, and phospholipid. Furthermore, the finding strongly indicates that the
poor performance of rFIX/VII-Gla in the aPTT assay is caused by a
specific defect in activation by factor XIa rather than a global
abnormality of protein structure which affects multiple FIX functions.
(Fig. 1,
B and C). In contrast, neither rFIX-des
nor
rFIX/VII-Gla demonstrated binding above background (Fig.
2C). These data demonstrate that proper
-carboxylation of
the Gla domain is required for FIX binding to factor XIa and that the factor VII Gla domain is not an adequate substitute in this interaction.
-carboxylation (Fig. 2B). Furthermore, the antibody does
not recognize rFIX/VII-Gla in Western blot assays or enzyme-linked
immunosorbent assays (data not shown), indicating that binding involves
epitopes specific to the FIX Gla domain. The effect of SB 249417 on FIX
binding to factor XIa was examined using SPR. As shown in Fig.
3A, the antibody is a potent
inhibitor of binding, with a Ki of 34 nM (EC50 320 nM).
View larger version (12K):
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Fig. 3.
Monoclonal antibody SB 249417 interferes with
interactions between FIX and factor XIa. A, SPR
studies. The binding of 1 µM FIX to immobilized factor
XIa was measured in the presence of SB 249417 at concentrations between
0.010 and 5 µM. B, antibody SB 249417 interferes with FIX activation by factor XIa. 200 nM FIX
was incubated with 1 nM factor XIa in the presence of 1 nM to 10 µM SB 249417 and 2.5 mM
CaCl2 at 37 °C for 30 min. After incubation, FIXa
generation was determined by chromogenic substrate assay as described
under "Experimental Procedures." The experiment was run in
duplicate. Data points represent means for two runs. Data in
A and B were fit to a single site competition
model, and an inhibition constant (Ki) was
calculated by nonlinear regression.
and, therefore, may
not detect low levels of activation. For this reason, conversion of
rFIX molecules to rFIXa
by a low concentration of factor XIa (1 nM) was examined by western immunoblot (Fig.
4). As can be seen in Fig. 4A,
zymogen wild type rFIX undergoes nearly complete conversion to rFIXa
within 1 h under the conditions of the assay (Fig. 4A).
No activation of rFIX-des
was observed (Fig. 4B). rFIX/VII-Gla appears to be slowly converted to FIXa
, with a small increase in the activated form detectable at late time points (Fig.
4C). This finding is consistent with the earlier observation that prolonged incubation of rFIX/VII-Gla with high concentrations of
factor XI will eventually result in complete conversion to the active
protease. This indicates that the activation cleavage sites on
rFIX/VII-Gla are accessible to factor XIa. Progress curves for
activation of the recombinant proteins were constructed using densitometry measurements from Western blot autoradiographs (Fig. 4D). The initial slopes of the progress curves (first 5 min
for rFIX, 60 min for rFIX-des
and rFIX/VII-Gla) were 4.1 nM/min for activation of rFIX, 0.2 nM/min for
activation of rFIX/VII-Gla, and 0.0 nM/min for rFIX-des
.
Thus, the initial rate of activation of rFIX was ~20-fold greater
than for rFIX/VII-Gla.
View larger version (27K):
[in a new window]
Fig. 4.
rFIX activation by factor XIa. Shown are
Western immunoblots of time courses of recombinant rFIX (A),
rFIX-des (B), or rFIX/VII-Gla (C) activated by
plasma-derived factor XIa. 100 nM purified recombinant
protein was incubated with 1 nM factor XIa in TBS
containing 2.5 mM CaCl2. Samples at various
time points (indicated across the top of the figure in min)
were collected into nonreducing SDS-sample buffer and then size
fractionated on SDS-PAGE (12%) gels, followed by transfer to
nitrocellulose. FIX and FIXa
were detected by a goat polyclonal
anti-human factor IX antibody and chemiluminescence. Standards for FIX
and FIXa
are shown on the right of each panel.
D, progress curves for activation of recombinant proteins by
factor XIa. Data were derived from densitometry measurements of the
Western blots in A-C, as described under "Experimental
Procedures."
, rFIX;
, rFIX-des
; and
, rFIX/VII-Gla. Note
that the progress curve for rFIX includes data points for 0.25, 1.5, and 3 min which are not shown in A.
(38). As with the
activation of FIX by factor XIa, this reaction is enhanced by calcium
but not by phospholipid (39). rFIX proteins were incubated in the
presence of purified RVVP and calcium (Fig.
5). rFIX and rFIX/VII-Gla are converted
to FIXa
in a similar manner, consistent with this reaction not
having a specific requirement for the FIX Gla domain. Furthermore, this
experiment demonstrates that the conformation of rFIX/VII-Gla is
sufficiently like FIX that it interacts properly with RVVP. In
contrast, rFIX-des
is not cleaved by RVVP, indicating that poor
-carboxylation induces significant enough conformational changes in
the protein to interfere with activation. Significant structural
alteration induced by poor
-carboxylation could explain our
inability to activate rFIX-des
with high concentrations of factor
XIa (100 nM), even with prolonged periods of incubation (>12 h).
View larger version (53K):
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Fig. 5.
FIX conversion to FIXa
by RVVP. rFIX (A), rFIX-des
(B), or
rFIX/VII-Gla (C) (1 µM) was incubated with
purified factor X-activating protease from 15 nM RVVP in
TBS containing 2.5 mM CaCl2. At designated time
points (indicated across the top of the figure in min)
samples were removed to reducing SDS-sample buffer, followed by size
fractionation on SDS-PAGE (12% gels). Gels were stained with GelCode
Blue. The positions of molecular mass standards are indicated on the
left of the panels. The positions of standards
for zymogen FIX (fIX), the large fragment of FIXa
representing the light chain and activation peptide (IXa
LC + AP), and the catalytic heavy chain domain of FIXa
(HC) are shown on the right of each
panel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and its cofactor, factor VIIIa, would be
required for generation of factor Xa to sustain coagulation after
inhibition of factor VIIa/TF. In this model, bleeding in hemophilia
(congenital deficiency of factor VIII or FIX) is caused by poorly
sustained factor X activation rather than a failure to initiate fibrin
formation (2, 43). Given the severity of the bleeding disorders
associated with complete deficiencies of FIX and factor VII, it appears
that FIX activation by factor VIIa is important for most hemostatic challenges. In contrast, factor XI deficiency is associated with bleeding that typically requires a more severe hemostatic insult (trauma or surgery) (44). FIX activation via factor XIa, therefore, is
most likely required in certain situations to supplement FIXa
produced through factor VIIa/TF.
to the protease. Binding sites for FIX
are probably masked in the zymogen and become available for binding
upon activation. Catalytic activity is not required for the binding
interaction because factor XIa lacking an active site serine residue
binds normally to FIX and FIXa
. It is interesting that both FIX and
FIXa
bind to factor XIa. This indicates that the activation peptide
is not required for the binding interaction, which is consistent with a
report showing that rFIX lacking an activation peptide is activated by
factor XIa (48). Furthermore, it supports the notion that binding of
FIX and factor XIa involves interactions remote from the activation
cleavage sites. The importance of the dimeric structure of factor XIa
to FIX binding and activation is not entirely clear. In solution, a
monomeric variant of factor XIa activates FIX with kinetic parameters
that are similar to those for activation by wild type factor XIa (31).
However, recent work suggests that the physiologic environment for this reaction may be the surface of activated platelets (46, 49, 50). In
this environment, the dimeric structure of factor XI is necessary for
normal FIX activation, possibly because one polypeptide of the dimer is
required for binding to the platelet, whereas the other interacts with
FIX (46, 49, 50).
is a
phospholipid-dependent reaction involving the Gla domains
of both proteins (8). It would appear that the Gla domain of factor VII
is similar enough to that of FIX to substitute for it in this reaction.
In contrast, factor VII-Gla cannot substitute for FIX-Gla during FIX
activation by factor XIa, a reaction not influenced by phospholipid.
Liebman and co-workers (28) demonstrated that a Fab fragment of a
monoclonal antibody that interacts with the factor IX Gla domain
phospholipid-binding epitope inhibited activation of factor IX by
factor XIa. This group postulated that factor XIa may have a surface
domain with features similar to those of phospholipid vesicles which
may mediate the interaction with factor IX. However, phospholipid,
although not enhancing FIX activation by factor XIa, does not inhibit
the reaction either (23).3
This suggests that the FIX Gla domain may contain one or more epitopes
for a protein-protein interaction with factor XIa and that the epitopes
are distinct from the phospholipid-binding elements on the domain. This
is not to say that FIX binding to phospholipid is not relevant to
activation of the protein by factor XIa. As mentioned above, a
physiologic site for the reaction may well be the surface of activated
platelets. Although factor XIa binds to platelets largely through a
protein-protein interaction involving glycoprotein 1b (56), it is
likely that FIX would bind to the platelet surface, at least in part,
through a phospholipid-protein interaction involving the Gla domain.
Work is under way to identify specific residues in the Gla domain
involved in the interaction between FIX and factor XIa, to determine
whether they are distinct from those required for phospholipid binding.
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ACKNOWLEDGEMENTS |
---|
We thank Mao-Fu Sun for technical expertise and Jean McClure for graphics work and preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant HL58837 from the NHLBI, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Established investigator of the American Heart Association. To whom correspondence should be addressed: Division of Hematology/Oncology, Vanderbilt University, 777 Preston Research Bldg., 2220 Pierce Ave., Nashville, TN 37232-6305. Tel.: 615-936-1505; Fax: 615-936-3853; E-mail: dave.gailani@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M212748200
2 J. Toomey and D. Gailani, unpublished observations.
3 D. Gailani, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FIX, factor IX;
aPTT, activated partial thromboplastin time;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate;
EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
EGF, epidermal
growth factor;
FIX-des, FIX that is incompletely
-carboxylated;
Gla,
-carboxyglutamic acid;
HEK, human embryonic kidney;
r prefix, recombinant;
RVVP, Russell's viper venom protease;
SPR, surface
plasmon resonance;
TBS, Tris-buffered saline;
TF, tissue factor.
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