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The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due
to Missense Mutations Within the A2-Domain of Factor VIII
By Kagehiro Amano, Rita Sarkar, Susan Pemberton, Geoffrey Kemball-Cook,
Haig H. Kazazian, Jr, and Randal J. Kaufman
Factor VIII (FVIII) is the protein defective in the bleeding
disorder hemophilia A. Approximately 5% of hemophilia A
patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive.
The majority of genetic alterations that result in CRMpositive hemophilia A are missense mutations within the
A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we
constructed six mutations within the FVIII cDNA that were
previously found in five CRM-positive hemophilia A patients
(R527W, S558F, I566T, V634A, and V634M) and one CRMreduced hemophilia A patient (DeltaF652/3). The specific
activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated
with published data for the patients plasma-derived FVIII,
confirming the basis of the genetic defect. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells
showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to
wild-type FVIII. The majority of the DeltaF652/3 mutant was
defective in secretion and was degraded within the cell. All
mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had
a reduced mobility because of use of an introduced potential
N-linked glycosylation site that was confirmed by
N-glycanase digestion. To evaluate interaction of FVIII with
factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565,
previously shown to be a factor IXa interaction site. The
concentration of peptide required for 50% inhibition of FVIII
activity (IC50) was reduced for the I566T (800 mmol/L) and
the S558F (960 mmol/L) mutants compared with wild-type
FVIII (G2,000 mmol/L). N-glycanase digestion increased I566T
mutant FVIII activity and increased its IC50 for the peptide
(1,400 mmol/L). In comparison to S558F, a more conservative
mutant (S558A) had a sixfold increased specific activity that
also correlated with an increased IC50 for the peptide. These
results provided support that the defects in the I566T and
S558F FVIII molecules are caused by steric hindrance for
interaction with factor IXa.
r 1998 by The American Society of Hematology.
H
ranging in size from 90 to 220 kD and a carboxy-terminal light
chain fragment of 80 kD. Thrombin activates FVIII by cleaving
the 90 kD heavy chain (A1-A2) into 50-kD (A1) and 43-kD
(A2) fragments, and the 80-kD light chain to a 73-kD (A3-C1C2) fragment.7 The active form of FVIII is a metal-ion linked
heterotrimer of A1, A2, and A3-C1-C2.8,9
Approximately 5% of hemophilia A patients have normal
levels of dysfunctional FVIII protein and are termed crossreacting material (CRM)-positive. CRM-positive patients have
considerable amounts of FVIII protein in their plasma (at least
30% of the normal amount), but the protein is nonfunctional; ie,
the FVIII activity is much less than the FVIII plasma protein
level. In contrast, FVIII antigen is not detected in CRMnegative patients. Patients with CRM-reduced hemophilia A
have reduced plasma FVIII antigen levels regardless of activity
levels. Some CRM-reduced patients also display lower activity
values compared with the plasma antigen levels.10,11 Approximately 40% of the CRM-positive and CRM-reduced hemophilia A patients contain missense mutations within the A2domain.12 The A2-domain consists of approximately 330 amino
acids or approximately 15% of the entire amino acid sequence
of FVIII, indicating that a selective clustering of missense
mutations occur within this region that result in hemophilia A.
Several reports have supported that the A2-domain subunit is
required for FVIII procoagulant activity. The thrombinactivated heterotrimer exhibited a pH-dependent dissociation of
the A2 subunit from the complex that correlated with a loss in
procoagulant activity.13 The difference in stability between
porcine and human FVIIIa correlates with increased affinity of
the porcine A2-domain compared with the human A2-domain
for the A1/A3-C1-C2 heterodimer.8,14 Furthermore, in-frame
deletion of the A2-domain in recombinant FVIII yielded a
secreted protein that did not exhibit procoagulant activity.
However, either cotransfection of this A2-domain deletion
EMOPHILIA A is caused by a quantitative or qualitative
deficiency of plasma factor VIII (FVIII), a cofactor for
factor IXa in the proteolytic activation of factor X to factor
Xa.1,2 FVIII is synthesized as a single chain polypeptide of
2,351 amino acids from which a 19–amino acid signal peptide is
cleaved, and has a domain organization of A1-A2-B-A3-C1C2.3,4 The A-domains are homologous to the A-domains of
ceruloplasmin,5 a copper binding plasma protein, suggesting a
possible role in metal-ion binding. The C-domains are homologous to phospholipid-binding proteins, suggesting a role in
phospholipid interaction.6 The B-domain displays no significant
homology to any known protein. FVIII is proteolytically
processed on secretion from the cell to form heterodimers
composed of a series of amino-terminal heavy chain fragments
From The Howard Hughes Medical Institute and the Department of
Biological Chemistry and University of Michigan Medical Center, Ann
Arbor, MI; the Department of Genetics, University of Pennsylvania
School of Medicine, Philadelphia, PA; the Haemostasis Research
Group, Medical Research Council Clinical Sciences Centre, London,
UK; and the Department of Clinical Pathology, Tokyo Medical College,
Tokyo, Japan.
Submitted March 17, 1997; accepted September 15, 1997.
Supported by National Institutes of Health Grants No. HL53777 and
HL52173 (R.J.K.) and the Tokyo Medical College (K.A.).
Address reprint requests to Randal J. Kaufman, PhD, Department of
Biological Chemistry and the Howard Hughes Medical Institute,
University of Michigan Medical Center, 4554 Medical Science Research Building II, 1150 W. Medical Center Drive, Ann Arbor, MI
48109-0650.
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9102-0034$3.00/0
538
Blood, Vol 91, No 2 (January 15), 1998: pp 538-548
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MISSENSE MUTATIONS IN FACTOR VIII A2-DOMAIN
mutant with an A2-domain expression vector or addition of
purified A2-domain fragment itself restored procoagulant activity for the A2-domain deletion molecule.15 Although the
A2-domain is essential for procoagulant activity, it is not
understood how the A2 subunit contributes to tenase complex
activity. Recent work using synthetic peptides showed that the
amino acid region 558 to 565 within the FVIII A2 subunit
represents a factor IXa interaction site.16,17
Results from site-directed mutagenesis studies of human
recombinant FVIII have contributed to our understanding of its
structure-function relationships. For example, missense mutations introduced at the thrombin cleavage sites showed that
cleavages at R372 and R1689 are required for full functional
activity18 consistent with the results from analysis of naturally
occurring mutations that result in hemophilia A.19-24 To date
there is only one report describing the characterization of a
missense mutation within the A2-domain that resulted in a
defective protein. In that situation, plasma FVIII from a patient
that contained the missense mutation of isoleucine to threonine
at residue 566, creating a novel N-linked glycosylation site at
asparagine residue 564, was defective in procoagulant activity.25 In this report, we have studied the mechanistic basis for
missense mutations within A2-domain affecting molecular
interactions required for FVIII function. Site-directed mutagenesis was used to create six different mutations within the
A2-domain that were previously shown to correlate with
defective circulating FVIII protein in patients with severe to
mild hemophilia A. Functional characterization of these mutant
proteins, expressed in transiently transfected COS-1 cells,
showed that I566T and S558F FVIII missense mutations have
reduced specific activity that can be attributed to reduced
interaction with factor IXa.
MATERIALS AND METHODS
Materials. FVIII-deficient plasma and normal pooled human plasma
were obtained from George King Biomedical Inc (Overland Park, KS).
Monoclonal antibody to the heavy chain of FVIII (F8) coupled to
CL4B-sepharose was a gift from Debra Pittman (Genetics Institute Inc,
Cambridge, MA). ESH-4 and ESH-8 antibodies were purchased from
American Diagnostica Inc (Greenwich, CT). Activated partial thromboplastin (Automated APTT reagent) was purchased from General
Diagnostics Organon Teknika Corporation (Durham, NC). COAMATIC
chromogenic FVIII activity assay kit was purchased from Pharmacia
Hepar (Franklin, OH). Soybean trypsin inhibitor, phenylmethylsulfonylfluoride (PMSF) and aprotinin were purchased from Boehringer,
Mannheim GmbH (Mannheim, Germany). Human a-thrombin and
O-Phenylenediamine Dihydrochloride (OPD) and N-a-Leu-Leunorleucinal (ALLN) were purchased from Sigma Chemical Co (St
Louis, MO). [35S]-methionine (.1,000 Ci/mmol) was purchased from
Amersham Life Science Inc (Arlington Heights, IL). Dulbecco’s
Modified Eagle’s Medium (DMEM), methionine-free DMEM, Biotin
N-Hydroxy Succinimide Ester, and Streptavidin-Horseradish Peroxidase Conjugate were obtained from GIBCO BRL (Gaithersburg, MD).
Fetal bovine serum was purchased from PAA Laboratories Inc (Newport Beach, CA). Recombinant N-glycanase was purchased from
Genzyme DIAGNOSTICS (Cambridge, MA). Patient’s plasma containing FVIII variant I566T (designated as ARC-22 or JH-11725 by the
Holland Laboratory, American Red Cross Blood Services, or the Center
for Medical Genetics, Johns Hopkins University School of Medicine,
respectively) was generously supplied by Carol Kasper (Orthopedic
Hospital, Los Angeles, CA) for research purposes.
539
Plasmid construction. Mutagenesis was performed within the mammalian expression vector pMT226 containing the wild-type (WT) full
length FVIII cDNA. Mutant plasmids were generated through oligonucleotide site-directed mutagenesis using the gapped-heteroduplex
procedure or the polymerase chain reaction as described previously.27-29
Codon 527 was mutated from CGG to TGG predicting an amino acid
change from arginine to tryptophan, and the resultant mutant plasmid
was designated R527W. Codon 558 was mutated from TCT to either
TTT or GCA predicting an amino acid change from serine to either
phenylalanine or alanine, respectively, and the resultant mutant plasmids were designated S558F and S558A. Codon 566 was mutated from
ATA to ACA predicting an amino acid change from isoleucine to
threonine, and the resultant mutant plasmid was designated I566T.
Codon 634 was mutated from GTG to either GCG or ATG predicting an
amino acid change from valine to either alanine or methionine,
respectively, and the resultant mutant plasmids were designated V634A
and V634M. Either codon 652 or 653, which are both TTC coding
phenylalanine, was deleted and the resultant 3-bp deletion mutant
plasmid was designated DeltaF652/3. The mutations were confirmed by
DNA sequence analysis using the dideoxy nucleotide sequencing
method.30
DNA transfection and analysis. Plasmid DNA was transfected into
COS-1 cells by the diethyl aminoethyl-dextran procedure as described.27 After 40 hours the cells were fed fresh medium containing
10% heat-inactivated fetal bovine serum and samples of conditioned
medium (CM) were harvested at 60 hours posttransfection for FVIII
assay. Subsequently, protein synthesis and secretion were analyzed by
metabolically labeling cells for 20 minutes with [35S]-methionine,
followed by a chase for 4 hours in medium containing a 100-fold excess
of unlabeled methionine and 0.02% aprotinin.27 Cell extracts (CE) were
prepared by lysis in Nonidet P-40 lysis buffer.27 For analysis of the
effect of cysteine protease inhibition, increasing amounts of ALLN were
included in the chase medium. CE and CM containing labeled proteins
were harvested as described previously31 and immunoprecipitated with
F8 antibody coupled to CL-4B sepharose. Immunoprecipitated proteins
were washed with phosphate buffered saline (PBS) containing Triton
X-100 and resuspended in 50 mmol/L Tris-HCl pH 7.5, 150 mmol/L
NaCl, 2.5 mmol/L CaCl2, and 5% glycerol (buffer A). Immunoprecipitated proteins from conditioned medium were resuspended in buffer A
and treated with or without 2 U/mL of thrombin at 37°C for 30 minutes.
Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under reducing conditions and visualized
by fluorography after treatment with En3Hance (Dupont, Boston, MA).
FVIII assay. FVIII activities were measured in a one stage clotting
assay using FVIII-deficient plasma as substrate or by the COAMATIC
chromogenic assay according to the manufacturer. One unit of FVIII
activity is that amount measured in 1 mL of normal human pooled
plasma. FVIII antigen was quantitated by a sandwich enzyme-linked
immunosorbent assay (ELISA)32 using two monoclonal antibodies to
the FVIII light chain. Purified recombinant FVIII protein was used as a
standard.
Protein purification. Partially purified protein was obtained from
200 mL of conditioned medium from COS-1 cells transfected with
FVIII WT, S558F, S558A, I566T, and V634A by immunoaffinity
chromatography as described previously.31 The bound FVIII was eluted
in buffer containing 60% ethylene glycol and concentrated by dialysis
against a 10% polyethylene glycol (MW 15K-20K) containing buffer33
and stored at 270°C.
N-glycanase digestion of WT and I566T mutant FVIII. Immunoprecipitated FVIII WT and I566T mutant from radiolabeled CM was
resuspended with buffer A and divided into three aliquots for incubation
in the absence or presence of thrombin or thrombin and subsequently
N-Glycanase. Human thrombin was added to a final concentration of 5
U/mL. Recombinant N-Glycanase was added to a final concentration of
10 U/mL in the presence of 4% Nonidet P-40 and 20 mmol/L PMSF.
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540
AMANO ET AL
Table 1. Comparison of Phenotype of Hemophilia A Patients and Expression of Their Respective Recombinant Proteins
Mutation
Exon
Classification
R527W
S558F
I566T
V634A
V634M
DeltaF652/3
S558A
11
11
12
13
13
13
CRM-pos
CRM-pos
CRM-pos
CRM-pos
CRM-pos
CRM-red
Nucleotide
Change
CGG-TGG
TCT-TTT
ATA-ACA
GTG-GCG
GTG-ATG
deletion of TTC
TCT-GCA
Patients’ Plasma
(% of normal)
COS CM
(% of WT)
Phenotype
Activity
Antigen
Activity
Antigen
Reference
mild
mild
severe-moderate
mild
severe
severe
9.5-38
21
,1-4
5
,1
1
43-245
175
154-200
138
175
12
37 6 5
661
463
462
363
162
45 6 5
51 6 12
57 6 9
72 6 19
71 6 11
104 6 14
42 6 3
79 6 13
12, 35
12
25, 35
12
12
12
The activity and antigen in COS CM represent mean 6 SD of the three independent transfections.
The resulting polypeptides were separated by SDS-PAGE and visualized by autoradiography as described previously.
FVIII activity of samples from patient’s plasma and conditioned
medium were measured after N-Glycanase digestion. Patient’s plasma
was incubated with recombinant N-Glycanase (final concentration 10
U/mL) at 37°C. FVIII activity after increasing periods of time was
measured by the one stage clotting assay. Normal pooled plasma was
used under the same conditions as a control for this assay. For the
evaluation of recombinant FVIII protein, CM was mixed with an equal
volume of FVIII-deficient plasma to approximate physiological conditions. This sample was treated with recombinant N-Glycanase and
FVIII activity was measured as described previously. WT FVIII
conditioned medium was also mixed with FVIII-deficient plasma as a
control in this assay.
Peptide inhibition of intrinsic factor Xase activity. The synthetic
peptide SVDQRGNQ, which corresponds to FVIII residues 558 to 565,
and a scrambled version of this peptide, NGSQDQRV, were synthesized
by the University of Michigan Protein and Carbohydrate Structure
Facility (Ann Arbor, MI). Both peptides were greater than 90% pure as
determined by high-liquid-performance-chromatography analysis and
their identity was confirmed by mass spectrometry. The effect of
synthetic peptides on intrinsic factor Xase activity was evaluated by the
Factor Xa generation assay using COAMATIC chromogenic factor VIII
activity assay kit. Partially purified FVIII samples were mixed with
various concentrations of synthetic peptide, then factor reagent containing factor IXa, factor X, thrombin, CaCl2, and phospholipid was added.
After 2 minutes incubation at 37°C, S-2765 chromogenic substrate was
reacted with thrombin inhibitor I-2581, which prevents hydrolysis of
S-2765 by thrombin. Factor Xa activity was determined as initial rate
values by measuring optical density at 405 nm. Exponential best fit
curves were determined from the independent experiments presented.
Molecular modeling. The effects of missense mutations within a
structural model for the A domains of FVIII34 were characterized by
altering residue side chains using the Biopolymer module of InsightII
(Molecular Simulations Inc, Cambridge, UK) and the variant molecules
were reminimized using Discover (Molecular Simulations Inc).
RESULTS
CRM-Positive mutants are synthesized and secreted similarly
to WT FVIII. The profile of the mutations analyzed in this
study is summarized in Table 1.12,25,35 We constructed six
mutations within the A2-domain that correlate with mutations
observed in five CRM-positive and one CRM-reduced hemophilia A. FVIII WT and the A2-domain mutants were compared
by transient DNA transfection of the cDNA expression vectors
into COS-1 monkey cells. At 60 hours after transfection, the
rates of synthesis were analyzed by immunoprecipitation of CE
from [35S]-methionine pulse-labeled cells. Intracellular FVIII
WT was detected in its single chain form and migrated at
approximately 280 kD (Fig 1, lane 1). The primary translation
Fig 1. Synthesis of FVIII WT
and mutants in COS-1 cells. WT
and mutant expression plasmids
were transfected into COS-1
monkey cells. At 60 hours posttransfection, cells were pulselabeled with [35S]-methionine for
20 minutes and cell extracts (CE)
were harvested. Duplicate plates
were chased for 4 hours in medium containing excess unlabeled methionine and then CE
were harvested. Equal volumes
of CE were immunoprecipitated
with anti-FVIII antibody and
equal aliquots were analyzed by
SDS-PAGE. Mock indicates cells
that did not receive plasmid
DNA. The migration of FVIII in
the CE is indicated at the right as
FVIII. Molecular weight size
markers are shown on the left.
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MISSENSE MUTATIONS IN FACTOR VIII A2-DOMAIN
products for each of the A2-domain mutants were detected at
280 kD at similar levels to WT (Fig 1, lanes 2 to 7). Thus, there
was no significant effect of these mutations on the rate of FVIII
protein translation. Analysis of the CE after a 4-hour chase
indicated that FVIII WT and all mutants disappeared from the
CE at approximately similar rates (Fig 1, lanes 10 to 16).
The secretion into the CM was analyzed by immunoprecipitation of CM from [35S]-methionine pulse-labeled transfected
cells chased for 4 hours in medium containing excess unlabeled
methionine. On SDS-PAGE analysis, FVIII WT was detected as
a 300-kD single chain, a 200-kD heavy chain, and an 80-kD
light chain (Fig 2, lane 1). The amount of secreted polypeptides
from DeltaF652/3 mutant transfected cells was greatly reduced
in the CM (Fig 2, lane 11), whereas the five CRM-positive
mutants were secreted at levels similar to FVIII WT (Fig 2,
lanes 3, 5, 7, 9, and 13).
The thrombin cleavage fragments for all mutants, except
I566T, were indistinguishable from WT FVIII. Immunoprecipitated FVIII molecules were treated with thrombin before
analysis by SDS-PAGE. Thrombin cleavage of all mutants,
except I566T, generated the light chain migrating at 73 kD and
the heavy chain derived fragments corresponding to the 50-kD
A1-domain and 43-kD A2-domain (Fig 2, lanes 4, 8, 10, 12, and
14) that were indistinguishable from the FVIII WT (Fig 2, lane
2). However, the A2-domain fragment from the I566T mutant
showed reduced mobility at 46 kD compared with FVIII WT
(Fig 2, lane 6).
Specific activity for each mutant correlated with published
data for the patients’ plasma-derived FVIII. CM from cells
transfected with FVIII WT or the A2-domain mutants were
harvested at 60 hours posttransfection for FVIII assay. The
activity and antigen in the CM of FVIII WT showed 195
mU/mL and 37 ng/mL (average of 3 different transfections),
respectively. The specific activity for the WT FVIII was 5,270
Fig 2. Secretion and thrombin cleavage of WT and mutant
FVIII. The secretion of each mutant was analyzed by immunoprecipitation of conditioned medium
from [35S]-methionine pulse-labeled transfected cells chased for
4 hours in medium containing
excess unlabeled methionine. Immunoprecipitated FVIII molecules
were analyzed by SDS-PAGE before (2) and after (1) thrombin
(IIa) digestion. Mock indicates
cells that did not receive plasmid
DNA. Molecular weight size
markers are shown on the left.
Single chain (Single), heavy chain
(Heavy), and light chain (Light)
are indicated for undigested
samples. A3-C1-C2, A1, and A2
fragments are indicated for digested samples. The symbol *
represents the A2 fragment with
reduced mobility.
541
U/mg, consistent with the value obtained for plasma-derived
FVIII. The activities determined by the COAMATIC assay and
antigen levels (represented as percent of FVIII WT) in the CM
from cells transfected with the A2-domain mutants are compared with the patients’ phenotype (represented as percent of
normal) in Table 1. Compared with FVIII WT, the activity
values obtained by the COAMATIC assay were similar to those
obtained by clotting assay (data not shown). The S558F mutant
CM showed a lower amount of activity than the patient’s
plasma-derived FVIII and the V634M mutant CM showed a
slightly higher amount of activity than the patient. The other
A2-domain mutants exhibited activities similar to the patients’
values. Compared with antigen levels of FVIII WT in the CM or
FVIII in normal plasma, the levels of FVIII antigen in the CM
from most of the FVIII mutant transfected cells were lower than
those obtained in the patients’ plasma. Quantitation of the
specific activity showed that all A2-domain mutants, except for
R527W, had values less than 10% of FVIII WT (Fig 3).
Mutant S558A has increased specific activity over S558F.
As mentioned previously, the amino acid region 558 to 565
within the A2 subunit is proposed to be a factor IXa interaction
site.16 Because of the significant difference in the amino acid
structure between serine and phenylalanine, the S558F mutant
may have a reduced interaction with factor IXa. To test this
possibility, a more conservative mutation of serine to alanine
(S558A) was made. Analysis of pulse-chase [35S]-methionine
labeled CE and CM before and after thrombin cleavage by
SDS-PAGE showed that the S558A mutant was synthesized,
secreted, and cleaved by thrombin similar to FVIII WT and
S558F (Fig 1, lanes 8 and 17; Fig 2, lanes 15 and 16). However,
its specific activity was increased sixfold over the S558F mutant
(Table 1 and Fig 3). The results suggest that eliminating
the large side-chain of phenylalanine increased procoagulant
activity.
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542
Fig 3. The specific activity for WT and mutant FVIII. CM of WT and
mutant FVIII were harvested at 60 hours posttransfection for FVIII
assay. The activity and antigen in the CM were measured by
COAMATIC chromogenic assay and ELISA using an anti-FVIII light
chain antibody, respectively. Specific activity is expressed as percent
of WT. Bars are expressed as a mean 6 standard deviation (SD) of
three independent transfection experiments.
I566T creates a new N-linked glycosylation site that is used.
Substitution of isoleucine at residue 566 for threonine creates a
potential new N-linked glycosylation site at asparagine 564.
Analysis of plasma-derived FVIII from a patient harboring the
I566T mutation suggested that this glycosylation site is used
resulting in reduced FVIII activity.25 The thrombin-derived
A2-domain fragment of the recombinant-derived I566T mutant
had a reduced mobility (Fig 2, lane 6 and Fig 4A, lanes 2 and 5)
consistent with addition of oligosaccharides to asparagine 564.
To show that the reduced mobility results from additional
glycosylation, immunoprecipitated WT and I566T mutant FVIII
from radiolabeled CM were treated with thrombin and
N-glycanase before SDS-PAGE analysis. After N-glycanase
digestion the A2 fragment from I566T comigrated with the WT
A2 fragment (Fig 4A, lanes 3 and 6). This result is consistent
with the previous data characterizing FVIII in patient’s plasma.25
To test whether N-glycanase digestion would increase FVIII
activity for the I566T mutant, we analyzed the FVIII clotting
activity after increasing periods of incubation with N-glycanase.
N-glycanase digestion increased the activity for the I566T
recombinant protein in CM to 30%, and this increase was
similar to that observed for the I566T patient’s plasma-derived
FVIII (Fig 4B).
AMANO ET AL
I566T and S558F FVIII are more sensitive to inhibition by a
synthetic peptide corresponding to a factor IXa interaction site.
The results above show that FVIII activity increased after
removal of either the large side-chain of phenylalanine for the
S558F mutant or of the carbohydrate at N564 for the I566T
mutant. We propose that these moieties may sterically prevent
FVIII interaction with factor IXa. Previously, a synthetic
peptide corresponding to FVIII residues 558 to 565 prevented
Xa generation by factor IXa, presumably because of preventing
factor IXa interaction.16 To measure the factor IXa interaction
with the mutants, we tested the ability of the synthetic peptide
558 to 565 to inhibit FVIII activity.
Equal amounts (4 nmol/L) of partially purified FVIII samples
were mixed with increasing concentrations of synthetic peptide
and assayed for FVIII activity. An exponential best fit curve was
determined from three independent experiments to extrapolate
the concentration of peptide required for 50% inhibition of
activity (IC50) for WT FVIII and for V634A mutant FVIII to be
3,340 (680) µmol/L and 2,240 (6330) µmol/L, respectively
(Fig 5A). In contrast, the IC50 for the I566T and S558F mutant
FVIII molecules were measured to be 800 (6130) µmol/L and
960 (660) µmol/L, respectively. These results support that the
I566T and S558F mutants are more sensitive than WT to
inhibition by the peptide. The inhibition curve for S558A was
shifted right and the IC50 for S558A increased to 1,690 (6300)
µmol/L (Fig 5A). N-glycanase digestion did not change the
inhibition profile of WT FVIII, but increased the IC50 for I566T
to 1,400 (6170) µmol/L (Fig 5B). Thus I566T and S558F were
inhibited at lower concentrations of peptide than WT FVIII.
To confirm that the effect of this peptide was specific for
interaction between factor IXa and FVIII, a scrambled synthetic
peptide having the same composition was used in the same
assay. The scrambled peptide did not inhibit either WT or any
mutant FVIII (Fig 5C). Equal antigenic amounts of each FVIII
protein were used in the peptide inhibition assays. Therefore,
the initial activity for the WT and each mutant was different
because of differences in specific activity. Therefore, we tested
if the amount of initial activity affected the peptide inhibition.
The effects on increasing peptide 558 to 565 on four different
concentrations of WT FVIII were measured. The inhibition
profiles for all samples were indistinguishable (Fig 5D), suggesting that the results did not depend on initial activity of sample.
Intracellular accumulation of DeltaF652/3 mutant in the
presence of inhibitors of intracellular degradation. Although
the DeltaF652/3 mutant was synthesized and chased from the
CE similar to WT FVIII (Fig 1), it displayed significantly
reduced appearance in the conditioned medium (Fig 2). Thus,
this protein was either degraded within the cell or was secreted
and rapidly degraded in the conditioned medium. The cysteineprotease inhibitor, ALLN, inhibits intracellular degradation and
previous studies showed increased intracellular accumulation of
a mutant FVIII (R2307Q) that was inefficiently secreted.29
These results support that this mutant did not accumulate in the
CM because of a block in secretion with subsequent degradation within the secretory pathway and not as a consequence of
instability in the CM. The effect of ALLN on the secretion of
DeltaF652/3 mutant was studied by analyzing [35S]-methionine
labeled FVIII protein. Addition of ALLN to the CM resulted in a
greater accumulation of the DeltaF652/3 in the CE (Fig 6, lanes
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MISSENSE MUTATIONS IN FACTOR VIII A2-DOMAIN
543
Fig 4. Effect of N-glycanase
for I566T mutant FVIII. (A) Immunoprecipitated WT and I566T mutant from radiolabeled CM were
treated with nothing (IIa2,
N-Gly2) or thrombin (IIa1, NGly2) or N-glycanase after thrombin (IIa1, N-Gly1) before SDSPAGE as described in the
Materials and Methods. Molecular weight size markers are
shown on the left. A3-C1-C2, A1,
A2, and mutant A2 fragments
are indicated at the right. (B)
FVIII in conditioned medium as
well as in patient’s plasma were
incubated with 10 U/mL of Nglycanase at 37°C. FVIII activity
after increasing times was determined by one stage clotting assay. Data are plotted as the percent activity of the mutant
compared with the WT-recombinant (X) or plasma-derived (W)
FVIII, respectively. Data represent the average of two independent experiments.
6-8) compared with WT (Fig 6, lanes 2-4). However, ALLN
treatment did not increase the secretion of either WT or
DeltaF652/3 into the CM (Fig 6, lanes 13-18). These results
suggest that defective secretion with subsequent intracellular
degradation is responsible for loss of the mutant FVIII from the
CE, leading to the CRM-reduced phenotype.
DISCUSSION
The study of missense mutations in the FVIII gene that result
in hemophilia A has elucidated the functional requirements for
FVIII activity. Previous studies showed that the A2 subunit is
essential for FVIIIa activity.8,9,13-15 In this study, we have
characterized six different mutants within the A2-domain using
site-directed mutagenesis. The goal of these studies was to
confirm the genetic basis for the phenotype of hemophilia A and
to elucidate the mechanism by which these missense mutations
within the A2-domain result in hemophilia A.
We compared the phenotype of recombinant mutant FVIII
proteins with their respective mutants analyzed in patients’
plasma. SDS-PAGE analysis showed that there was no significant effect of these mutations on the rate of FVIII synthesis and
all the CRM-positive mutants were secreted into the CM similar
to WT. However, the activity in the CM for all the CRMpositive mutants was reduced and correlated with the reduced
activity reported in patients’ plasma. Quantitation of the antigen
levels in the CM by ELISA showed that most mutants also
displayed lower antigen levels in the CM compared with WT
and these were lower than the values obtained from respective
patients’ plasma. We speculate that the synthesis and/or secretion of the FVIII mutants in vivo might be stimulated to
compensate for the low plasma antigen levels. Although there
was variation in the antigen levels, the specific activity of all
mutants, except for the R527W mutant, were very low as
predicted by the hemophilia A phenotype. Interestingly, the
antigen level for the R527W was low as measured by ELISA,
although SDS-PAGE analysis showed similar amounts of FVIII
secretion compared with WT. It is likely that the ELISA
quantitation for this mutant was reduced as a consequence of
poor reactivity with the antibodies used in the ELISA. In this
context, it is essential that quantitation of antigen in patients’
plasma also consider the particular antibodies used in the assay.
The reduced quantity of FVIII antigen measured by ELISA for
the R527W mutant resulted in an apparently higher specific
activity. If we assume the antigen level was similar to WT as
indicated by the characterization of the secretion of the R527W
mutant, then the specific activity for the R527W mutant was
also significantly reduced. Therefore, the analysis of all the
recombinant proteins supports that each mutation is responsible
for the respective patients’ phenotype, confirming the basis of
the genetic defect.
How do the mutations within the A2-domain of FVIII reduce
procoagulant activity? SDS-PAGE analysis showed that all
mutants were susceptible to thrombin cleavage, eliminating
thrombin resistance as a cause for the defect in activity.
However, after thrombin cleavage, all mutants studied displayed no significant increase in activity (data not shown),
indicating a functional defect in factor VIIIa. Previous studies
have identified two regions within FVIII that are likely responsible for factor IXa interaction. First, peptide inhibition studies
suggest that residues 558 to 565 comprise a factor IXa
interaction site.16 In addition, residues 1,778 to 1,840 within the
FVIII light chain were proposed to be involved in factor IXa
binding because an antibody directed against this region
inhibited the binding of factor IXa.36 This second factor IXa
binding site has been more localized to the residues 1,811 to
1,818 by using synthetic peptides.37 Thus, present data support
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544
AMANO ET AL
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
MISSENSE MUTATIONS IN FACTOR VIII A2-DOMAIN
545
Fig 6. Inhibition of intracellular degradation causes intracellular accumulation of DeltaF652/3. Parallel plates of transfected COS-1 monkey
cells were labeled at 60 hours posttransfection with [35S]methionine for 30 minutes, chased for 4 hours in the absence (lanes 2, 6, 10, 13, 16, and
19) or presence of increasing amounts of ALLN (lanes 3, 4, 7, 8, 11, 12, 14, 15, 17, 18, 20, and 21). CE and conditioned medium (CM) were harvested
and equal proportionate volumes of CE and CM were immunoprecipitated with anti-FVIII specific antibody for analysis by SDS-PAGE. Mock
indicates cells that did not receive plasmid DNA. Molecular weight size markers are shown on the left of each CE and CM.
two interaction sites for factor IXa. It has been suggested that
the binding site on the FVIII light chain is responsible for
complex assembly via the first EGF-like domain of factor IXa,38
whereas the factor IXa interaction with the A2-domain might
induce changes within the factor IXa active site.17 In this study
we have shown that mutants I566T and S558F are defective in
factor IXa interaction by an assay that measured the functional
consequence of the FVIII-factor IXa interaction. We confirmed
that I566T creates a new N-linked glycosylation site that is used
and this prevents appearance of FVIII activity. In addition,
removal of the large side-chain of phenylalanine for the S558F
mutant, by mutagenesis of S558 to alanine, improved the FVIII
activity. The activities of the I566T and S558F mutants were
inhibited at lower concentrations of peptide 558 to 565 than WT
FVIII or V634A. In our experiments, inhibition of WT FVIII
required approximately 2 mmol/L peptide, compared with the
value of 0.1 mmol/L previously reported.16 The greater amount
of peptide required to inhibit WT FVIII in our assays may be
because of the higher concentration of factor IXa (4 nmol/L)
present in our assays compared with those of Fay et al16 (1
nmol/L). Either removal of the bulky phenylalanine side chain
at residue 558 in mutant S558F by mutation of S558A or
removal of N-linked oligosaccharides in the I566T mutant by
N-glycanase increased both the specific activity of the FVIII
and the IC50 of the peptide. These results support the hypothesis that the defects in the I566T and S558F FVIII molecules are
;
Fig 5. Inhibition of intrinsic factor Xase activity by synthetic peptide 558 to 565. (A) Equal amounts (4 nmol/L) of partially purified FVIII
samples were mixed with increasing concentrations of synthetic peptide and assayed for FVIII activity as described in the Materials and
Methods. The data represent the average of three independent experiments. FVIII activity for each molecule is expressed as a percent of that
obtained in the absence of the peptide. The symbols represent WT FVIII (W), S558F (Q), S558A (S), I566T (M) and V634A (U). (B) WT and I566T
mutant FVIII were treated with N-glycanase for 3 hours at 37°C, then samples were tested in the peptide inhibition assay. The results represent
the average of three (filled symbols) and two (open symbols) independent experiments. The symbols represent WT FVIII (W), WT FVIII with
N-glycanase (X), I566T (M) and I566T with N-glycanase (N). (C) Equal amounts (4 nmol/L) of partially purified FVIII samples were mixed with
increasing concentrations of scrambled synthetic peptide and assayed for FVIII activity as described in the Materials and Methods. Two
independent experiments are shown for WT FVIII (W), S558F (Q), I566T (M) and V634A (U). (D) Four different concentrations of WT FVIII (4
nmol/L [W], 0.8 nmol/L [M], 0.4 nmol/L [Q], 0.08 nmol/L [U]) were tested in the peptide inhibition assay. The results are the average of two
independent experiments.
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
546
caused by a reduced specific activity resulting from steric
hindrance for interaction with factor IXa.
A structural model of the FVIII A domains based on the
3-angstrom structure of ceruloplasmin showed that both sequences 558 to 565 and 1,811 to 1,818 are surface exposed and
oriented toward the same direction.34 Structural modeling of
I566T indicates that side-chain of the new N-glycosylation site
(N564) is surface exposed and lies within the proposed factor
IXa binding site (Fig 7A). Modeling of S558F shows that
residue 558 is surface exposed and the substitution of serine to
phenylalanine introduces a bulky hydrophobic side chain,
AMANO ET AL
altering the conformation of the loop in that region and leading
to steric strain around the proposed factor IXa binding site (Fig
7B). Thus, addition of a bulky side-chain or N-linked oligosaccharide would be expected to prevent, or severely reduce, the
interaction with factor IXa.
In contrast to the missense mutations S558F and I566T, the
mechanism of the molecular defect of the CRM-positive
mutants R527W, V634A, and V634M remains unknown. Because these mutants were efficiently secreted as heterodimers,
they are likely not significantly defective in protein folding or
chain assembly. V634A was inhibited by peptide 558 to 565
Fig 7. Structural model of
FVIII mutations I566T and S558F.
(A) Homology model of the triplicated A domains of FVIII is shown
as viewed perpendicular to threefold axis with ‘‘top’’ of molecule
to top of Fig Red, blue, and green
represent A1, A2, and A3 subunit, respectively. Binding loop
of factor IXa is shown as magenta CPK spheres. N564 (new
N-glycosylation site) and T566
are colored by atom (carbon,
green; hydrogen, white; oxygen,
red; nitrogen, blue). (B) Factor
IXa binding loop 558 to 565
shown in CPK spheres: residue
558 colored by atom, 559 to 565
in gray. Blue ribbons represent
the alpha-carbon trace of neighboring residues in the A2 domain. Left, WT S558; Right, variant F558 (reminimized).
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
MISSENSE MUTATIONS IN FACTOR VIII A2-DOMAIN
similar to WT FVIII, suggesting no defect in its factor IXa
interaction. Additional studies are required to elucidate the
mechanism responsible for the absence of procoagulant activity
for these mutants.
In contrast to the CRM-positive mutants studied, the secretion of the one CRM-reduced mutant DeltaF652/3 was reduced.
According to the structural model, residues 652 and 653 are
close to the A3-domain and at the interface of the A2 and A3
subunit.39 Therefore, deletion of this residue might prevent
formation of the tightly packed structure of FVIII, resulting in
aberrant folding and a block to transport through the secretory
pathway. Previously, R2307Q mutant FVIII protein showed
reduced secretion with increased intracellular degradation,
although the low level of secreted protein displayed WT specific
activity.29 DeltaF652/3 also displayed defective secretion; however, the low level of secreted mutant protein had, in addition, a
low specific activity. Inhibition of intracellular degradation
resulted in intracellular accumulation of the DeltaF652/3 mutant; however, it was not secreted, presumably as a result of
failure to bypass the ‘‘quality control’’ machinery of the
secretory pathway. Thus, even if it were possible to rescue
secretion of this mutant, it would not likely improve the severity
of hemophilia A caused by the DeltaF652/3 mutation because of
its reduced specific activity.
ACKNOWLEDGMENT
We thank Peter Lenting for helpful comments regarding the peptide
inhibition experiments, Debra Pittman for assistance in the early phase
of this study, and Steven Pipe for valuable discussion throughout the
course of these studies.
REFERENCES
1. Hoyer LW: The factor VIII complex structure and function. Blood
58:1, 1981
2. Davie EW, Fujikawa K, Kisiel W: The coagulation cascade:
Initiation, maintenance, and regulation. Biochemistry 30:10363, 1991
3. Vehar GA, Keyt B, Eaton D, Rodriguez H, O’Brien DP, Rotblat F,
Oppermann H, Keck R, Wood WI, Harkins RN, Tuddenham EGD,
Lawn RM, Capon DJ: Structure of human factor VIII. Nature 312:337,
1984
4. Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL,
Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Orr EC, Amphlett
GW, Foster WB, Coe ML, Knutson GJ, Fass DN, Hewick RM:
Molecular cloning of a cDNA encoding human antihemophilic factor.
Nature 312:342, 1984
5. Ortel TL, Takahashi N, Putnam FW: Structural model of human
ceruloplasmin based on internal triplication, hydrophilic/hydrophobic
character, and secondary structure of domains. Proc Natl Acad Sci USA
81:4761, 1984
6. Stubbs JD, Lekutis C, Singer KL, Bui A, Yuzuki D, Srinivasan U,
Parry G: cDNA cloning of a mouse mammary epithelial cell surface
protein reveals the existence of epidermal growth factor-like domains
linked to factor VIII-like sequences. Proc Natl Acad Sci USA 87:8417,
1990
7. Eaton D, Rodriguez H, Vehar GA: Proteolytic processing of
human factor VIII. Correlation of specific cleavages by thrombin, factor
Xa, and activated protein C with activation and inactivation of factor
VIII coagulant activity. Biochemistry 25:505, 1986
8. Lollar P, Parker ET: Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog. J
Biol Chem 266:12481, 1991
547
9. Fay PJ, Haidaris PJ, Smudzin TM: Human factor VIIIa subunit
structure. J Biol Chem 266:8957, 1991
10. Hoyer LW, Breckenridge RT: Immunologic studies of antihemophilic factor (AHF, Factor VIII): Cross-reacting material in a genetic
variant of hemophilia A. Blood 32:962, 1968
11. Lazarchick J, Hoyer LW: Immunoradiometric measurement of
the factor VIII procoagulant antigen. J Clin Invest 62:1048, 1978
12. McGinniss MJ, Kazazian HH Jr, Hoyer LW, Bi L, Inaba H,
Antonarakis SE: Spectrum of mutations in CRM-positive and CRMreduced hemophilia A. Genomics 15:392, 1993
13. Lollar P, Parker CG: pH-dependent denaturation of thrombinactivated porcine factor VIII. J Biol Chem 265:1688, 1990
14. Lollar P, Parker ET, Fay PJ: Coagulant properties of hybrid
human/porcine factor VIII molecules. J Biol Chem 267:23652, 1992
15. Pittman DD, Millenson M, Marquette K, Bauer K, Kaufman RJ:
A2 domain of human recombinant-derived factor VIII is required for
procoagulant activity but not for thrombin cleavage. Blood 79:389,
1992
16. Fay PJ, Beattie T, Huggins CF, Regan LM: Factor VIIIa A2
subunit residues 558 to 565 represent a factor IXa interactive site. J Biol
Chem 269:20522, 1994
17. O’Brien LM, Medved LV, Fay PJ: Localization of factor IXa and
factor VIII interactive site. J Biol Chem 270:27087, 1995
18. Pittman DD, Kaufman RJ: Proteolytic requirements for activation and inactivation of antihemophilic factor (Factor VIII). Proc Natl
Acad Sci USA 85:2429, 1988
19. Gitschier J, Kogan S, Levinson B, Tuddenham EGD: Mutations
of factor VIII cleavage sites in hemophilia A. Blood 72:1022, 1988
20. Arai M, Inaba H, Higuchi M, Antonarakis SE, Kazazian HH Jr,
Fujimaki M, Hoyer LW: Direct characterization of factor VIII in
plasma: Detection of a mutation altering a thrombin cleavage site
(arginine-372 Æ histidine). Proc Natl Acad Sci USA 86:4277, 1989
21. Arai M, Higuchi M, Antonarakis SE, Kazazian HH Jr, Phillips JA
III, Janco RL, Hoyer LW: Characterization of a thrombin cleavage site
mutation (Arg 1689 to Cys) in the factor VIII gene of two unrelated
patients with cross-reacting material-positive hemophilia A. Blood
75:384, 1990
22. Shima M, Ware J, Yoshioka A, Fukui H, Fulcher CA: An arginine
to cysteine amino acid substitution at a critical thrombin cleavage site in
a dysfunctional factor VIII molecule. Blood 74:1612, 1989
23. Higuchi M, Wong C, Kochan L, Olek K, Aronis S, Kasper CK,
Kazazian HH Jr, Antonarakis SE: Characterization of mutations in the
factor VIII gene by direct sequencing of amplified genomic DNA.
Genomics 6:65, 1990
24. Pattinson JK, McVey JH, Boon M, Ajani A, Tuddenham EGD:
CRM1 haemophilia A due to a missense mutation (372 Æ Cys) at the
internal heavy chain thrombin cleavage site. Br J Haematol 75:73, 1990
25. Aly AM, Higuchi M, Kasper CK, Kazazian HH Jr, Antonarakis
SE, Hoyer LW: Hemophilia A due to mutations that create new
N-glycosylation sites. Proc Natl Acad Sci USA 89:4933, 1992
26. Kaufman RJ: Vectors used for expression in mammalian cells, in
Goeddel DV (eds): Methods in Enzymology (vol 185). San Diego, CA,
Academic Press, 1990, p 487
27. Pittman DD, Kaufman RJ: Site-directed mutagenesis and expression of coagulation factors VIII and V in mammalian cells, in Lorand L,
Mann KG (eds): Methods in Enzymology (vol 222). San Diego, CA,
Academic Press, 1993, p 236
28. Toole JJ, Pittman DD, Orr EC, Murtha P, Wasley LC, Kaufman
RJ: A large region (,95kDA) of human factor VIII is dispensable for in
vitro activity. Proc Natl Acad Sci USA 83:5939, 1986
29. Pipe SW, Kaufman RJ: Factor VIII C2 domain missense
mutations exhibit defective trafficking of biologically functional proteins. J Biol Chem 271:25671, 1996
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
548
30. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain
terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977
31. Michnick DA, Pittman DD, Wise RJ, Kaufman RJ: Identification
of individual tyrosine sulfation sites within factor VIII required for
optimal activity and efficient thrombin cleavage. J Biol Chem 269:
20095, 1994
32. Zatloukal K, Cotten M, Berger M, Schmidt W, Wagener E,
Birnstiel ML: In vivo production of human factor VIII in mice after
intrasplenic implantation of primary fibroblasts transfected by receptormediated, adenovirus-augmented gene delivery. Proc Natl Acad Sci
USA 91:5148, 1994
33. Fay PJ, Beattie T, Regan LM, O’Brien LM, Kaufman RJ: Model
for the factor VIIIa-dependent decay of the intrinsic factor Xase: Role of
subunit dissociation and factor Xa-catalyzed proteolysis. J Biol Chem
271:6027, 1996
34. Pemberton S, Lindley P, Zaitsev V, Card G, Tuddenham EGD,
Kemball-Cook G: A molecular model for the triplicated A domains of
human factor VIII based on the crystal structure of human caeruloplasmin. Blood 89:2413, 1997
AMANO ET AL
35. Kemball-Cook G, Tuddenham EGD: The factor VIII mutation
database on the World Wide Web: The haemophilia A mutation search
test and resource site. HAMSTeRS update (Version 3.0). Nucleic Acids
Res 25:128, 1997 (URL: http://europium.mrc.rpms.ac.uk)
36. Lenting PJ, Donath MJSH, van Mourik JA, Mertens K: Identification of a binding site for blood coagulation factor IXa on the light
chain of human factor VIII. J Biol Chem 269:7150, 1996
37. Lenting PJ, van de Loo JWHP, Donath MJSH, van Mourik JA,
Mertens K: The sequence Glu 1811 -Lys 1818 of human blood coagulation
factor VIII comprises a binding site for activated factor IX. J Biol Chem
271:1935, 1996
38. Lenting PJ, Christophe OD, ter Maat H, Rees DJG, Mertens K:
Calcium binding to the first epidermal growth factor-like domain of
human blood coagulation factor IX promotes enzyme activity and factor
VIII light chain. J Biol Chem 271:25332, 1996
39. Pan Y, DeFay T, Gitschier J, Cohen FE: Proposed structure of the
A domains of factor VIII by homology modeling. Nat Structure Biol
2:740, 1995
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
1998 91: 538-548
The Molecular Basis for Cross-Reacting Material−Positive Hemophilia A
Due to Missense Mutations Within the A2-Domain of Factor VIII
Kagehiro Amano, Rita Sarkar, Susan Pemberton, Geoffrey Kemball-Cook, Haig H. Kazazian, Jr and
Randal J. Kaufman
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