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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 156-163
An Arg/Ser Substitution in the Second Epidermal Growth Factor-Like
Module of Factor IX Introduces an O-Linked Carbohydrate and
Markedly Impairs Activation by Factor XIa and Factor VIIa/Tissue
Factor and Catalytic Efficiency of Factor IXa
By
Mark S. Hertzberg,
Sandra L. Facey, and
Philip J. Hogg
From the Department of Haematology, Westmead Hospital and University
of Sydney, Sydney, New South Wales; and the Centre for Thrombosis and
Vascular Research, School of Pathology, University of New South Wales,
Sydney, Australia.
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ABSTRACT |
Factor IXR94S is a naturally occurring hemophilia B defect, which
results from an Arg 94 to Ser mutation in the second epidermal growth
factor (EGF)-like module of factor IX. Recombinant factor IXR94S was
activated by factor XIa/calcium with an  50-fold reduced rate and by
factor VIIa/tissue factor/phospholipid/calcium with an 20-fold
reduced rate compared with wild-type factor IX. The apparent molecular
mass of the light chain of factor IXaR94S was 6 kD
higher than that of plasma or wild-type factor IX, which was not
corrected by N-glycosidase F digestion. This result indicated the
presence of additional O-linked carbohydrate in the mutant light chain,
probably at new Ser 94. The initial rate of activation of factor X by
factor IXaR94S in the presence of polylysine was 7% ± 1% of the
initial rate of activation of factor X by plasma factor IXa, and the
kc/Km for activation of factor X by factor IXaR94S/factor VIIIa/phospholipid/calcium was 4% ± 1% of the
kc/Km for activation of factor X by plasma
factor IXa/factor VIIIa/phospholipid/calcium. The reduced efficiency of
activation of factor X by factor IXaR94S in the tenase enzyme complex
was due to a 58-fold ± 12-fold decrease in kcat with
little effect on Km. In conclusion, the R94S mutation had
introduced an O-linked carbohydrate, which markedly impaired both
activation by factor XIa and turnover of factor X in the tenase enzyme complex.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
FACTOR IX is a vitamin K-dependent
coagulation protein whose deficiency results in hemophilia
B.1 Factor IX circulates in plasma as an inactive single
chain glycoprotein of 415 amino acids and participates in both the
intrinsic and extrinsic coagulation pathways. After initiation of
coagulation, factor IX can be activated to a two-chain serine
proteinase factor IXa by either factor XIa or factor VIIa/tissue
factor.2 Activation of factor IX by either pathway occurs
by cleavage of the Arg145-Ala146 and Arg180-Val181 peptide bonds to
yield a light and heavy chain linked by two disulfide bonds.
Subsequently, the enzyme factor IXa forms a macromolecular complex with
its cofactor factor VIIIa, to activate factor X to factor Xa in the
presence of calcium and a negatively charged phospholipid
surface.3,4
Factor IX is synthesized in the liver as a precursor molecule of 461 amino acids containing a 28-residue signal peptide and an 18-residue
propeptide, both of which are proteolytically cleaved before
secretion.5,6 The mature molecule consists of an N-terminal  -carboxyglutamic acid-rich domain (Gla domain) followed by a short
hydrophobic amino acid stack domain, two epidermal growth factor
(EGF)-like modules, an activation peptide region, and a serine
proteinase module.5,6 The first EGF module
participates in factor VIIIa and factor X binding and is required for
activation of factor IX by factor VIIa/tissue factor.7-13
The second EGF module and the serine proteinase module have also been
proposed to participate in factor VIIIa and factor X
binding,11,14,15 although studies using proteolytic
fragments of factor IX have argued against a direct binding function
for the second EGF module.16,17
Approximately 35% of individuals with hemophilia B possess
immunologically normal levels of factor IX antigen, but reduced levels
of factor IX coagulant activity.18 These variants are designated cross-reacting material positive (CRM+). The
analysis of naturally occurring CRM+ mutants provides
valuable information concerning structure-function relationships of
factor IX. While a number of CRM+ mutants have been
characterized to date, few have been reported to occur in the second
EGF module.19 One of these, Factor IX Fukuoka, contains an
Asp92 to His substitution and was shown to result in defective binding
to factor X.20 This mutation occurred at a residue, which
is highly conserved among all vitamin K-dependent coagulation proteins.
We now report functional characterization of the first identified
naturally occurring mutation involving a factor IX-specific residue
within the second EGF module, in which Arg94 is replaced by Ser (factor
IXR94S), resulting in a moderate form of hemophilia B. We show that the
R94S mutation markedly impaired both activation by factor XIa and
factor VIIa/tissue factor and turnover of factor X in the tenase enzyme
complex and present evidence that the mutation introduced a site for
O-linked glycosylation in the second EGF module.
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MATERIALS AND METHODS |
Materials.
Plasma-derived human coagulation factors VIIa, IX, X, XIa, thrombin, as
well as coagulation protein from Russell's viper venom (RVV-X) and
human tissue factor were purchased from Enzyme Research Laboratories
(South Bend, IN). Dialyzed fetal calf serum and L-polylysine were
obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). The
chromogenic substrate, MeO-CO-D-CHG-Gly-Arg-p-nitroanilide (CHG-GR-pNA,
Spectrozyme FXa), was obtained from American Diagnostica (Greenwich,
CT), human factor IX-deficient plasma and activated partial
thromboplastin time (APTT) reagent from Organon Teknika (Durham, NC),
and Lipofectin from GIBCO-BRL (Gaithersburg, MD). Rabbit polyclonal
anti-factor IX antibodies and peroxidase-conjugated goat polyclonal
anti-factor IX antibodies were purchased from Dako (Carpinteria, CA).
Kind gifts used in this study were: Factor VIII from Dr William Drohan
(American Red Cross, Rockville, MD); Sepharose-coupled
calcium-dependent monoclonal antibody (A-7) directed against the light
chain of factor IX from Dr Kenneth Smith (University of New Mexico
School of Medicine, Albuquerque); factor IX/factor
X-binding protein from the venom of Trimesesurus flavoviridis
(T flavoviridis) (IX/X-bp) and rabbit anti-IX/X-bp polyclonal antibody from Dr T. Morita (Meiji College of Pharmacy, Tanashi-Shi, Tokyo, Japan); calcium-dependent monoclonal
antibody (JK-IX) directed against the light chain of factor IX from Dr Teriuko Sugo (Jichi Medical School, Tochigi, Japan); monoclonal antibody C10D directed against the heavy chain of factor IX from Dr
Arthur Thompson (University of Washington, Seattle). pED mammalian expression vector was from Genetics Institute (Cambridge, MA). Phospholipid vesicles (85:15, PC:PS) were prepared as described previously.14
Construction of mutant cDNA.
The cDNA for human factor IX in pUC12, mutated to provide convenient
cloning sites at the boundaries of the aromatic amino acid stack and
first EGF module, the first EGF and second EGF modules, and the second
EGF and serine proteinase modules was prepared
previously.14 The mutated factor IX cDNA, inserted into the
PstI site of pUC12, has an SalI site at the boundary between the aromatic amino acid stack and first EGF modules, and a
KpnI site between the second EGF and serine proteinase modules. Using polymerase chain reaction (PCR) mutagenesis by
overlap extension,21 Arg 94 (AGA) was mutated to a Ser
(AGT), such that the final PCR-derived product could be cloned into the
SalI and KpnI sites of the factor IX cDNA. The mutant
was characterized by DNA sequencing, and the cDNA encoding factor
IXR94S was subcloned into the PstI site of the mammalian
expression vector, pED.22
Cell culture and expression.
Dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cells
(CHO-DUKX-B11)23 were grown in a modified Eagle's medium supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, 100 U/mL
of penicillin and streptomycin, and 5 µg/mL vitamin K. A total of 20 µg of plasmid DNA containing the cDNA encoding wild-type factor IX or
mutant factor IXR94S was transfected into the cells using Lipofectin.
Three days after transfection, cells were selected for DHFR expression
using medium deficient in ribonucleosides. Surviving foci were isolated
using cloning rings and allowed to grow to confluence in 24-well
plates. The presence of factor IX protein secreted into tissue culture
medium was screened by a sandwich enzyme-linked immunosorbent assay
(ELISA) procedure. Briefly, plates were coated with rabbit polyclonal
antifactor IX antibodies, blocked with 5% bovine serum albumin (BSA),
and incubated with medium derived from isolated transfected cells. The
presence of human factor IX was confirmed using peroxidase-conjugated goat polyclonal anti-human factor IX antibody. Those clones expressing the highest concentration of wild-type factor IX or factor IXR94S were
isolated and used for large-scale tissue culture.
Purification of recombinant wild-type factor IX and factor IXR94S.
Supernatant from CHO cells expressing the recombinant wild-type factor
IX or factor IXR94S was collected, made 2 mmol/L in benzamidine, and
concentrated eightfold to 10-fold using an Amicon 0.22-µm spiral
ultrafiltration cartridge (Amicon, Inc, Beverly, MA). The concentrated
supernatant was made 10 mmol/L in CaCl2 and applied to a
column containing Sepharose-coupled calcium-dependent A7
monoclonal antibody, which has been used previously to purify -carboxylated factor IX.24 The column was washed with
0.02 mol/L Tris/HCl, pH 7.4, 0.15 mol/L NaCl (TBS) containing 10 mmol/L CaCl2 and then with 0.02 mol/L Tris/HCl, pH 7.4, 1 mol/L
NaCl containing 10 mmol/L CaCl2. After equilibration of the
column in TBS containing 10 mmol/L CaCl2, protein was
eluted with TBS containing 20 mmol/L EDTA. Fractions containing eluted
protein, as determined by absorbance at 280 nm, were pooled, dialyzed
against TBS, and concentrated using an Amicon Centriprep 30 microconcentrator. Protein concentration was measured by
BCA assay (Pierce, Rockford, IL) using purified plasma factor IX as a
standard. Concentrated proteins were stored at  70°C.
Clotting assays.
Wild-type factor IX and factor IXR94S were characterized functionally
for activity in plasma-based clotting assays. A one-stage APTT was
performed using human factor IX-deficient plasma, rabbit brain
phospholipid, and micronized silica.25 Clotting activity of
the recombinant proteins was calculated from a standard curve generated
using purified plasma human factor IX and activity expressed as a
percentage of plasma factor IX coagulant activity.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blotting.
Samples were separated on 12.5% or 5% to 15% SDS-PAGE under reducing
conditions.26 Samples were reduced by boiling with 20 mmol/L dithiothreitol for 5 minutes and the cysteines blocked using 40 mmol/L iodoacetamide. The proteins were either stained with Coomassie
Brilliant Blue or transferred to Immobilon-P membranes (Millipore,
Bedford, MA) and developed and visualized using chemiluminescence according to the manufacturer's instructions (DuPont, Boston, MA).
Primary murine monoclonal antibodies were used at 5 µg/mL and
secondary rabbit antimouse IgG horseradish peroxidase-conjugated antibodies (Dako) were used at 1:2,000 dilution.
Binding of factor IX to IX/X-bp.
IX/X-bp is an anticoagulant protein isolated from the venom of T
flavoviridis, which binds the Gla domain of vitamin K-dependent proteins in a calcium-dependent manner.27 The binding assay was performed as described by Nishimura et al.20
Binding of factor IX to phospholipid vesicles.
Binding of factor IX to phospholipid vesicles was measured by
separating bound from free factor IX in an air-fuge.28
Factor IX (10 µg/mL) and 75:25 DOPC:DOPS vesicles (15 µmol/L) was added to polyallomer centrifuge tubes containing 0.05 mol/L HEPES, 0.125 mol/L NaCl, 10 mmol/L CaCl2 or 10 mmol/L
EDTA, 5 mg/mL BSA, pH 7.4 buffer. The reactions were incubated for 20 minutes and the phospholipid vesicles sedimented in an air-fuge
(Beckman, Sydney, Australia) for 20 minutes. The
centrifugation sedimented  95% of the phospholipid vesicles. The
supernatant was aspirated and the free factor IX was determined by
converting factor IX to IXa with factor XIa. Factor IX and XIa were
incubated at a weight ratio of 10:1 in binding buffer for 3 hours at
37°C. Nonspecific binding of factor IX to control tubes not
containing phospholipid vesicles represented  10% of the binding to
phospholipid vesicles. The moles of factor IX bound to phospholipid
vesicles was calculated from the total factor IX and the ratio of bound
versus total factor IX.
Activation of factor IX by factor XIa.
Purified human factor XIa (5 or 18 nmol/L) was incubated with plasma
factor IX or factor IXR94S (2.7 µmol/L) in TBS containing 10 mmol/L
CaCl2 at 37°C. At discrete time intervals, aliquots were quenched with 20 mmol/L EDTA and resolved on SDS-PAGE under reducing conditions. Proteins were visualized by staining with Coomassie Brilliant Blue or transferred to Immobilon-P membranes and
Western blotted using monoclonal antibodies directed against either the
light chain (JKIX) or heavy chain (C10D) of factor IX.
Activation of factor IX by factor VIIa/tissue factor.
Purified human factor VIIa (2 nmol/L), tissue factor (4 nmol/L), and
75:25 DOPC:DOPS vesicles (1 mmol/L) was incubated with plasma factor IX
or factor IXR94S (0.5 µmol/L) in TBS containing 5 mmol/L
CaCl2 at 37°C. The factor VIIa/tissue
factor/DOPC:DOPS/Ca2+ enzyme complex was established by
incubation for 10 minutes before addition of factor IX to start the
reaction. At discrete time intervals, aliquots were quenched with 20 mmol/L EDTA and resolved on SDS-PAGE under reducing conditions.
Proteins were transferred to Immobilon-P membranes and blotted with
monoclonal antibodies directed against the heavy chain (C10D) of factor IX.
Hydrolysis of CHG-GR-pNA by factor IXa.
Plasma factor IXa or factor IXaR94S (0.05 to 1 µmol/L) was incubated
with CHG-GR-pNA (2.5 mmol/L) in TBS containing 0.1% BSA. The increase
in absorbance at 405 nm was continuously monitored using a Thermomax
Kinetic Microplate Reader (Molecular Devices, Palo Alto, CA). The
initial rates were calculated at substrate consumption of less than
10%.
Activation of factor X by factor IXa or factor IXaR94S in the
presence of polylysine.
The catalytic efficiency of activation of factor X by plasma factor IXa
or factor IXaR94S was measured in the presence of polylysine. Factor
IXa (50 nmol/L) was incubated with 60 nmol/L polylysine in 0.1 mol/L
triethanolamine, 0.1 mol/L NaCl, 0.1% polyethylene glycol (PEG) 6000, pH 9.0 buffer29,30 at 37°C. Factor X was added to a
final concentration of 0.6 µmol/L to start the reaction. At discrete
time intervals, samples were removed and diluted 1 in 25 into a
solution of 300 µmol/L CHG-GR-pNA in the triethanolamine buffer. This
solution was incubated for 5 minutes and the reaction was stopped by
adding an equal volume of 50% acetic acid. The absorbance at 405 nm
was a function of the amount of factor Xa generated and increased
linearly with time. A standard curve of known factor Xa concentrations
was used to determine factor Xa activity generated, which was then
plotted as a function of time.
Activation of factor X by plasma factor IXa or factor IXaR94S in the
tenase enzyme complex.
The ability of plasma factor IXa or factor IXaR94S to assemble with
factor VIIIa on a phospholipid surface and to catalyze factor X
activation was studied using the purified proteins. Plasma factor IXa
(0.17 nmol/L) or factor IXaR94S (1.5 nmol/L) was incubated with human
factor X (0 to 100 nmol/L), human factor VIIIa (14 U/mL), phospholipid
vesicles (35 µmol/L) and Ca2+ (5 mmol/L) in TBS
containing 0.1% BSA at 37°C. At discrete time intervals, 25 µL-aliquots were taken and added to microtiter wells containing 25 µL of TBS containing 0.1% BSA and 0.5 mol/L EDTA. Factor VIII had
been preactivated with 2 nmol/L thrombin for 2 minutes and the thrombin
neutralized by addition of 50 nmol/L hirudin.31 The initial
velocities of factor Xa generation were measured using CHG-GR-pNA as
described above. The apparent kinetic parameters for activation of
factor X by plasma factor IXa or factor IXaR94S in the tenase enzyme
complex were determined by fitting of the data to the Michaelis-Menten
equation using nonlinear regression analysis (Scientist, Salt Lake
City, UT).
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RESULTS |
Expression and purification of recombinant wild-type factor IX and
factor IXR94S.
Recombinant wild-type factor IX and mutant factor IXR94S were stably
expressed in CHO cells and purified by affinity chromatography using a
calcium-dependent monoclonal antibody (A7) directed against the Gla
domain of the protein. Purified recombinant proteins were resolved on
SDS-PAGE and visualized by staining with Coomassie Brilliant Blue
(Fig 1). Both proteins migrated as a single
band under reducing conditions, although factor IXR94S migrated
marginally slower than recombinant wild-type factor IX. This was
probably due to the presence of additional carbohydrate in the mutant
protein (see below).
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| Fig 1.
SDS-PAGE analysis of purified recombinant wild-type
factor IX and factor IXR94S. Recombinant proteins (3 µg) were
resolved on 12.5% SDS-PAGE under reducing conditions and stained with
Coomassie Blue.
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Coagulant activity of plasma and wild-type factor IX and factor
IXR94S.
In a plasma-based APTT assay, the coagulant activity of wild-type
recombinant factor IX was 96% ± 8% that of purified plasma factor
IX. In contrast, factor IXR94S exhibited coagulant activity, which was
6% ± 1% that of plasma factor IX.
Comparison of the Gla-domains of plasma and wild-type factor IX and
factor IXR94S.
The anticoagulant protein IX/X-bp has been previously shown to be a
probe for monitoring the conformational state of the Gla domain of
factor IX.27 Factor IXR94S bound IX/X-bp with the same
affinity as plasma or wild-type factor IX
(Fig 2A). Also, factor IXpl, IXwt, and
IXR94S bound to 75:25 DOPC:DOPS vesicles to the same extent (Fig 2B).
No specific binding to phospholipid vesicles was observed in buffer
containing 10 mmol/L EDTA (not shown). These results implied that the
Gla-domains of the recombinant factor IX proteins were intact and
functional.
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| Fig 2.
Comparison of the Gla-domains of plasma factor IX,
wild-type factor IX, and factor IXR94S. (A) Increasing
moles of factor IXpl ( ), factor IXwt ( ), or factor
IXR94S ( ) were coated onto microtiter wells and then incubated with
15 µg/mL of IX/X-bp in the presence of 5 mmol/L Ca2+ in
TBS. The binding of IX/X-bp to factor IX was measured by ELISA. The
three factor IX proteins bound IX/X-bp equally well. (B) Binding of
factor IX to 75:25 DOPC:DOPS vesicles. The three factor IX proteins
bound phospholipid vesicles equally well. The bars and errors represent
the mean and standard deviation (SD) of three experiments.
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Activation of plasma and wild-type factor IX and factor IXR94S by
factor XIa.
Factor IXpl, IXwt, and IXR94S were activated by factor XIa at a molar
enzyme:substrate ratio of 1:500 (Fig 3).
Samples of the reactions were resolved on reducing SDS-PAGE and stained
with Coomassie Brilliant Blue. Plasma and wild-type factor IX were activated at equivalent rates producing identical heavy chain and light
chain cleavage products. Factor IXR94S, however, was activated at a
substantially reduced rate and the light chain was not apparent using
Coomassie Brilliant Blue staining (not shown).
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| Fig 3.
SDS-PAGE analysis of factor XIa activation of plasma
factor IX and wild-type factor IX. Factor IXpl (A) or IXwt (B) (2.7 µmol/L) was incubated with factor XIa (5 nmol/L) in TBS containing 10 mmol/L Ca2+ at 37°C. At discrete time intervals,
aliquots (3 µg) of the reactions were resolved on 12.5% SDS-PAGE
under reducing conditions and stained with Coomassie Blue.
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To investigate the fate of the light chain of IXR94S, factor IXpl and
IXR94S were activated by factor XIa at a molar enzyme:substrate ratio
of 1:150 and the activation products examined by Western blot using
monoclonal antibodies recognizing either the heavy (Fig 4) or light
(Fig 5) chains of factor IXa.
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| Fig 4.
Western blot analysis of factor XIa activation of plasma
factor IX and factor IXR94S using a factor IXa heavy chain antibody.
Factor IXpl (A) or IXR94S (B) (2.7 µmol/L) was activated by factor
XIa (18 nmol/L) in TBS containing 10 mmol/L Ca2+ at
37°C. The activation products were sampled at the indicated time
intervals, resolved on reducing 12.5% SDS-PAGE, and transferred to
Immobilon-P polyvinylidene fluoride (PVDF) membrane. The membrane was
blotted with the C10D monoclonal antibody, which recognizes the heavy
chain (HC) of factor IXa, and developed using chemiluminescence. The
positions of intact factor IX, IX (HC + AP) and IXaHC are
indicated. The positions of the molecular weight markers are indicated
at left.
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| Fig 5.
Western blot analysis of factor XIa activation of plasma
factor IX and factor IXR94S using a factor IXa light chain antibody.
Factor IXpl (A) or IXR94S (B) (2.7 µmol/L) was activated by factor
XIa (18 nmol/L) in TBS containing 10 mmol/L Ca2+ at
37°C. The activation products were sampled at the indicated time
intervals, resolved on reducing 12.5% SDS-PAGE, and transferred to
PVDF membrane. The membrane was blotted with the JKIX monoclonal
antibody, which recognizes the light chain (LC) of factor IXa, and
developed using chemiluminescence. The positions of intact factor IX
and IXaLC are indicated. The dotted arrow in (B) indicates the usual
migration position of the factor IXa light chain. The positions of the
molecular weight markers are indicated at left.
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Factor IXpl was activated completely within 1 to 2 minutes (Fig 4A),
while factor IXR94S was activated at a much slower rate (Fig 4B).
Densitometric analysis of the 31-kD heavy chain bands indicated that
factor IXR94S was activated by factor XIa at approximately 2% of the
rate of activation of factor IXpl or IXwt. An intermediate heavy chain
product with an approximate molecular mass of 44 kD was observed with
factor XIa activation of factor IXR94S (Fig 4B). This product, termed
factor IX , consists of the heavy chain plus activation peptide (HC + AP) and its presence indicates that the light chain (LC) is cleaved
from the AP at the Arg145-Ala146 peptide bond. The activation peptide
is cleaved from the heavy chain at the Arg180-Val181 peptide bond.
The light chain of factor IXaR94S migrated at an apparent molecular
mass of 30 kD (Fig 5B), which is significantly higher than the light
chain of plasma (or wild-type) factor IX, which migrated at an apparent
molecular mass of 24 kD (Fig 5A). Moreover, the light chain of factor
IXaR94S migrated at a molecular mass which is virtually identical to
that of the heavy chain. This would account for the apparent absence of
the light chain of factor IXR94S on Coomassie-stained SDS-PAGE. The
intact mutant protein also migrated at a slightly higher molecular mass
than the wild-type protein (Fig 1). The increase in the observed
molecular mass of the intact mutant protein and its activated light
chain was probably due to the presence of additional carbohydrate in
the light chain of factor IXR94S. Digestion of factor IXaR94S with
N-glycosidase F did not reduce the molecular weight of the mutant light
chain to that of plasma factor IXa light chain (not shown), indicating that the carbohydrate was not N-linked. This finding implied that the
R94S mutation had introduced a site for O-glycosylation.
Activation of plasma and wild-type factor IX and factor VIIa/tissue
factor.
Factor IXpl, IXwt, and IXR94S were activated by factor VIIa/tissue
factor/DOPC:DOPS/Ca2+ enzyme complex at a molar
enzyme:substrate ratio of 1:250. Samples of the reactions were resolved
on reducing SDS-PAGE and Western blotted using the factor IXa
heavy chain (C10D) antibody. Plasma and wild-type factor IX were
activated at equivalent rates (not shown), which were similar to that
described for activation of plasma factor IX by Lawson and
Mann.32 Factor IXR94S, however, was activated at a
substantially reduced rate (Fig 6).
Densitometric analysis of the 31-kD heavy chain bands indicated that
factor IXR94S was activated by factor VIIa/tissue
factor/DOPC:DOPS/Ca2+ at approximately 5% of the rate of
activation of factor IXpl or IXwt.
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| Fig 6.
Western blot analysis of factor VIIa/tissue
factor/DOPC:DOPS/Ca2+ enzyme complex activation of plasma
factor IX and factor IXR94S using a factor IXa heavy chain antibody.
Factor IXpl (A) or IXR94S (B) was activated by factor VIIa/tissue
factor/DOPC:DOPS/Ca2+ enzyme complex in TBS at 37°C.
The reaction component concentrations were: 0.5 µmol/L factor IXpl or
IXR94S, 2 nmol/L factor VIIa, 4 nmol/L tissue factor, 1 mmol/L
DOPC:DOPS, and 5 mmol/L Ca2+. The activation products
were sampled at the indicated time intervals, resolved on reducing 5%
to 15% SDS-PAGE, and transferred to PVDF membrane. The membrane was
blotted with the C10D monoclonal antibody, which recognizes the heavy
chain (HC) of factor IXa, and developed using chemiluminescence. The
positions of intact factor IX and IXaHC are indicated. The positions of
the molecular weight markers are indicated at left.
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Amidolytic activity of plasma factor IXa and factor IXaR94S.
Factor IXa cleaves the chromogenic substrate CHG-GR-pNA, albeit with
low catalytic efficiency. Plasma factor IXa and factor IXaR94S
hydrolyzed CHG-GR-pNA with initial rates of 1.51 ± 0.06 min 1 and 1.68 ± 0.02 min1,
respectively (Fig 7).
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| Fig 7.
Comparison of the amidolytic activity of plasma factor
IXa and factor IXaR94S. Varying concentrations of plasma factor IXa
() or factor IXaR94S () were incubated with 2.5 mmol/L CHG-GR-pNA
in TBS containing 0.1% BSA, and the increase in absorbance at 405 nm
with time was measured. The initial velocity of p-nitroaniline
formation is expressed as a function of enzyme concentration. The solid
lines represent the linear regression fit to the data. The observed
rate of hydrolysis of CHG-GR-pNA by plasma factor IXa and factor
IXaR94S was 1.51 ± 0.06 min1 and 1.68 ± 0.02 min1, respectively.
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Activation of factor X by plasma factor IXa or factor IXaR94S in the
presence of polylysine.
Polylysine can partially substitute for phospholipid and factor VIIIa
in the tenase enzyme complex. The efficiency of factor X activation by
factor IXa is enhanced at least 300-fold by polylysine.29 The initial rate of activation of factor X by factor IXaR94S in the
presence of polylysine was 0.19 ± 0.01 nmol/L/min1, compared with the initial rate of
activation of factor X by plasma-derived factor IXa of 2.54 ± 0.07 nmol/L/min1 (Fig 8).
Therefore, the efficiency of activation of factor X by factor IXaR94S
in the presence of polylysine was 7% ± 1% of the efficiency of
activation by plasma factor IXa.
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| Fig 8.
Comparison of the factor X-activating activity of plasma
factor IXa and factor IXaR94S in the presence of polylysine. Human
factor X (600 nmol/L) was activated by 50 nmol/L plasma factor IXa
() or factor IXaR94S () in the presence of polylysine (60 nmol/L)
in 0.1 mol/L triethanolamine, 0.1 mol/L NaCl, 0.1% PEG 6000, pH 9.0 buffer at 37°C. At discrete time intervals, aliquots of the
reactions were assayed for factor Xa concentration using CHG-GR-pNA.
The results are expressed as concentration of factor Xa formed as a
function of time. The solid lines represent the linear regression fit
to the data. The initial rate of factor X activation by plasma factor
IXa and factor IXaR94S was 2.54 ± 0.07 nmol/L/min1 and
0.19 ± 0.01 nmol/L/min1, respectively.
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Activation of factor X by plasma factor IXa or factor IXaR94S in the
tenase enzyme complex.
The catalytic efficiency of factor IXaR94S in the tenase enzyme complex
was compared with plasma factor IXa. The factor VIIIa concentration
dependence for activation of factor X by either plasma factor IXa or
factor IXaR94S in the tenase enzyme complex was first determined. A
factor VIIIa concentration of 2 U/mL gave half-maximal activity for
activation by plasma factor IXa, while essentially maximal activity was
achieved with 6 U/mL (not shown). A factor VIIIa concentration of
either 6 U/mL or 14 U/mL made no change to the rate of activation of
factor X by factor IXaR94S, implying that the concentration of factor
VIIIa required for half-maximal activity was <6 U/mL. Therefore, to
ensure that the rate of the tenase reaction was not limited by factor
VIIIa, a factor VIIIa concentration of 14 U/mL was chosen for
estimation of the kinetic parameters for factor X activation. The
Km and kcat for activation of factor X by
either plasma factor IXa or factor IXaR94S was estimated from the data
shown in Fig 9.
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| Fig 9.
Comparison of the catalytic activity of plasma factor IXa
and factor IXaR94S in the tenase enzyme complex. Plasma factor IXa
(0.17 nmol/L, , ___) or factor IXaR94S (1.5 nmol/L, ,
 -) was incubated with factor X (0 to 100 nmol/L) in the presence of
phospholipid vesicles (35 µmol/L), factor VIIIa (14 U/mL) and
Ca2+ (5 mmol/L) in TBS containing 0.1% BSA at 37°C.
At discrete time intervals, aliquots of the reactions were quenched
with 50 mmol/L EDTA and factor Xa quantitated using CHG-GR-pNA. The
data has been normalized with respect to enzyme concentration by
expressing the ordinate axis as initial velocity, vi,
divided by the factor IXa or factor IXaR94S concentration. The lines
represent the best fit of the data to the Michaelis-Menten equation
using nonlinear regression analysis. The kinetic parameters are
summarized in Table 1.
|
|
The kinetic parameters for factor X activation are listed in
Table 1. The parameters for plasma factor
IXa were similar to those determined by van Diejen et al.33
It is apparent that the Km for factor X is effectively the
same for either plasma factor IXa or factor IXaR94S. However, the
kcat for cleavage of factor X by factor IXaR94S is 58-fold ± 12-fold slower than that for plasma factor IXa.
View this table:
[in this window]
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|
Table 1.
Kinetic Parameters for Activation of Factor X by Plasma
Factor IXa and Factor IXaR94S in the Tenase Enzyme Complex
|
|
| |
DISCUSSION |
The purpose of this study was to evaluate the structural and kinetic
properties of a variant factor IX molecule in which there is a single
amino acid substitution at a factor IX-specific residue (Arg94 to Ser)
in the second EGF module. This mutation results in a moderately severe
Hemophilia B and is characterized by a factor IX clotting activity of
1% to 2% and a factor IX antigen level of 84%.34 This
factor IX mutation was significant for two reasons. First, this is only
the second characterized case of a naturally occurring mutation in the
second EGF module in which the antigenic level is normal, but
functional clotting activity is significantly impaired. Second, it is
the first mutation described at a factor IX-specific residue in the
second EGF module and so has the potential to report on factor
IX-specific interactions such as with factor VIII and factor X.
Factor IXR94S was activated by factor XIa/calcium at a rate, which was
2% that of plasma factor IX, and by factor VIIa/tissue factor/phospholipid/calcium at a rate which was 5% that of plasma factor IX. This is the first amino acid substitution identified in
factor IX in which there is a significant reduction in the rate of
cleavage by factor XIa. Factor IX New London, which is caused by a
Pro50 to Glu substitution in the first EGF module, was originally
thought to result in a reduced rate of activation by factor
XIa,35 but has since been shown to exhibit normal activation rates.13 Activation of factor IXR94S was
complete, albeit slow. This is in contrast to the virtual absence of
factor XIa cleavage of the Arg145-Ala-146 peptide bond in factor IX
Chapel Hill in which Arg145 is replaced by His.29 Despite
this mutation, the clotting activity of factor IX Chapel Hill is 20%
that of factor IXa and the clinical bleeding tendency is very
mild.29
One other mutation in the second EGF module of factor IX has been
characterized to date. Factor IX Fukuoka, which has a Asn92 to His
mutation in the second EGF module, is characterized by 3% clotting
activity and 64% factor IX antigen levels. In contrast to the R94S
mutation, the N92H mutation results in a normal rate of activation by
factor XIa, but a reduced rate of turnover of factor X in the tenase
enzyme complex.
The apparent molecular mass of the light chain of factor IXaR94S was
6 kD higher than the light chain of plasma or wild-type factor IXa.
The apparent molecular mass of the intact factor IXR94S was also higher
than wild-type factor IX. This finding implied that the R94S mutation
had resulted in incorporation of additional carbohydrate into the light
chain. There are four sites of attachment of O-linked oligosaccharides
in plasma factor IX involving Ser53 and Ser61 within the first EGF
module, which are uniformly glycosylated,36 and Thr159 and
Thr169 in the activation peptide (residues 145-180), which are
partially glycosylated.37 The O-linked carbohydrates in the
first EGF module may play a role in receptor-ligand
interaction.38,39 The new carbohydrate in the light chain
of factor IXR94S is probably O-linked to the new Ser. The R94S mutation
creates a Gly-Ser-Cys motif, which is identical to that at Ser61 in the
first EGF domain. Ser61 is O-fucosidically linked to the
tetrasaccharide, NeuAc(2  6)Gal(1
4)GlcNAc(1 3)Fuc1 O-Ser.36,38 Moreover, O-glyconase, which cleaves Gal1
3GalNAc-Ser/Thr linkages, does not cleave the
O-fucosidically linked tetrasaccharide at Ser61,36,38 and
O-glyconase digestion did not correct the difference in the molecular
weights between the mutant and wild-type light factor IX chains (not
shown). In addition, digestion of factor IXaR94S with N-glycosidase F,
which hydrolyzes all types of N-glycan chains from mammalian
glycoproteins, also did not correct the difference in the molecular
weights between the mutant and wild-type light chains (not shown).
These results support a fucosidically linked saccharide at Ser94 in the
R94S mutant. Jentoft40 has shown that a major function of
O-glycosylation is to induce a specific conformation. Therefore, it is
likely that the presence of an O-linked carbohydrate at Ser94 in the
second EGF module will perturb the tertiary structure of this region.
The amidolytic activity of factor IXaR94S was the same as plasma factor
IXa. This finding suggested that the environment of the active site of
the serine proteinase module was not perturbed by the R94S mutation,
although perturbation at exosites was not excluded by this result. In
contrast, the initial rate of activation of factor X by factor IXaR94S
in the presence of polylysine was 7% of the initial rate of activation
of factor X by plasma factor IXa. It is unlikely that this result was
due to an impaired Gla domain, as the plasma and recombinant proteins
bound a calcium-dependent monoclonal antibody, the Gla-dependent
IX/X-binding protein, and phospholipid vesicles to the same extent.
The kc/Km for activation of factor X by factor
IXaR94S/factor VIIIa/phospholipid/calcium was 4% of the
kc/Km for activation of factor X by plasma
factor IXa/factor VIIIa/phospholipid/calcium. The reduced catalytic
efficiency of factor IXaR94S in the tenase enzyme complex was due to a
58-fold decrease in kcat with little effect on
Km. These results do not exclude the possibility that factor IXaR94S may have bound with slightly reduced affinity or nonproductively to factor VIIIa in the tenase complex, which could have
accounted for some of the reduced catalytic efficiency of factor X
activation by tenase. However, the magnitude of reduction in the
initial rate of activation of factor X by factor IXaR94S compared with
plasma factor IX in the absence of factor VIIIa (7% of the initial
rate for plasma factor IXa) was effectively the same as the magnitude
of reduction in kc/Km for activation of factor
X in the presence of factor VIIIa and phospholipid (4% of the
kc/Km for plasma factor IXa). Taken together,
these results implied that the reduced rate of turnover of factor X was
not due to impaired binding of factor IXaR94S to factor VIIIa in the tenase complex.
The findings reported herein suggest that the second EGF module of
factor IXa is important for catalytically productive binding of factor
X. In other words, the mutation in second EGF domain of factor IXa may
have perturbed the nature, but not the overall affinity of factor X
binding such that it was poorly activated by the factor IXa serine
proteinase domain. Further evidence for this proposal comes from the
work of Nishimura et al.20 These investigators
characterized factor IX Fukuoka, which has a Asn92 to His mutation in
the second EGF module. This mutation caused a moderate form of
hemophilia, which was characterized by 3% clotting activity and 64%
factor IX antigen levels. The N92H mutation results in a reduced rate
of activation of factor X in the presence of polylysine (8% of the
initial rate for plasma factor IXa) and a reduced
kc/Km for activation of factor X in the tenase
complex (2.3% of the kc/Km for plasma factor
IXa). The reduced catalytic efficiency of factor IXa Fukuoka in the
tenase enzyme complex is due to a 1,000-fold decrease in
kcat and a 24-fold decrease in Km. Therefore,
the major effect of the N92H mutation is a reduced rate of turnover of
factor X in the tenase enzyme complex. These findings are in general
agreement with the results for the R94S mutation described herein. It
is also possible that the R94S mutation perturbed the catalytic
activity of the serine proteinase domain of the mutant factor IXa,
although this seems unlikely.
Christophe et al41 have suggested that the two EGF modules
in factor IX associate electrostatically via Glu78 in the first module
and Arg94 in the second module and that this interaction is important
for binding of the factor VIII light chain. However, it is not known
whether the second EGF module facilitates binding of factor VIII to the
first module or whether factor VIII binding spans both EGF modules.
In conclusion, the R94S mutation appeared to have resulted in the
incorporation of an additional O-linked carbohydrate in the light
chain, probably at the new Ser in the second EGF module, which markedly
impaired both activations by both factor XIa and factor VIIa/tissue
factor and turnover of factor X in the tenase enzyme complex.
| |
FOOTNOTES |
Submitted May 26, 1998; accepted March 1, 1999.
Supported by grants from the National Health and Medical Research
Council of Australia.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mark S. Hertzberg, MB, BS, PhD, Department
of Haematology, Level 2, ICPMR, Westmead Hospital, Westmead, NSW 2145, Australia; e-mail: markh{at}westmed.wh.su.edu.au.
| |
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ABSTRACT
INTRODUCTION