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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 958-965
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
A novel cause of mild/moderate hemophilia A: mutations scattered
in the factor VIII C1 domain reduce factor VIII binding to von
Willebrand factor
Marc Jacquemin,
Renaud Lavend'homme,
Abdellah Benhida,
Beatrijs Vanzieleghem,
Roseline d'Oiron,
Jean-Maurice Lavergne,
Hans H. Brackmann,
Rainer Schwaab,
Thierry VandenDriessche,
Marinee K. L. Chuah,
Marc Hoylaerts,
Jean Guy G. Gilles,
Kathelijne Peerlinck,
Jos Vermylen, and
Jean-Marie R. Saint-Remy
From the Center for Molecular and Vascular Biology, University of
Leuven, Leuven, Belgium; Hôpital Bicêtre, le
Kremlin-Bicêtre, AP-HP, France; Institut für Experimentelle
Hämatologie und Transfusionsmedizin, Bonn, Germany; and the
Center for Transgene Technology and Gene Therapy, Flanders
Interuniversity Institute for Biotechnology, University of Leuven,
Leuven, Belgium.
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Abstract |
The mechanisms responsible for the low factor VIII (fVIII) activity
in the plasma of patients with mild/moderate hemophilia A are poorly
understood. In such patients, we have identified a series of fVIII
mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and
Pro2153Gln) clustered in the C1 domain and associated with reduced
binding of fVIII to von Willebrand factor (vWf). For each patient
plasma, the specific activity of mutated fVIII was close to
that of normal fVIII. Scatchard analysis showed that the affinity for
vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His
fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively,
when compared with normal fVIII. Given the importance of vWf for the
stability of fVIII in plasma, these findings suggested that the
reduction of fVIII binding to vWf resulting from the above-mentioned
mutations could contribute to patients' low fVIII plasma levels. We,
therefore, analyzed the effect of vWf on fVIII production by Chinese
hamster ovary (CHO) cells transfected with expression vectors for
recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and
Arg2150His fVIII. These 3 mutations impaired the vWf-dependent
accumulation of functional fVIII in culture medium. Analysis of fVIII
production by transiently transfected CHO cells indicated that, in
addition to the impaired stabilization by vWf, the secretion of
functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and
6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII.
These findings indicate that C1-domain mutations resulting in reduced
fVIII binding to vWf are an important cause of mild/moderate hemophilia A.
(Blood. 2000;96:958-965)
© 2000 by The American Society of Hematology.
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Introduction |
Hemophilia A is a bleeding disorder caused by a
deficiency or dysfunction of coagulation factor VIII (fVIII). Patients
affected by the severe form of the disease suffer from spontaneous
bleedings, whereas in mild/moderate hemophilia A (fVIII activity
0.01-0.4 IU/mL) bleeding occurs after minor trauma or surgery. Although the alterations of the fVIII molecule leading to severe hemophilia A
are well characterized, only scarce information is available about
the molecular mechanisms responsible for mild/moderate hemophilia A.1
The mature fVIII molecule is a heterodimer of 2332 amino acids, which
circulates as a heavy and a light chain noncovalently bound by a
divalent metal bridge.2 fVIII contains 3 types of domains,
the A domains, homologous to ceruloplasmin, a unique B domain, and 2 C
domains, homologous to the phospholipid binding protein discoidin. The
heavy chain is made up by the A1 and A2 domains and by variable lengths
of the B domain. The light chain consists of the A3, C1, and C2
domains.2
In plasma, fVIII circulates bound to von Willebrand factor (vWf). The
latter interaction is crucial for fVIII stability as indicated by the
low fVIII levels observed in patients with severe von Willebrand
disease (vWD)3 and in patients with vWD type 2 Normandy,
with altered binding of vWf to fVIII.4
In vitro experiments have established that vWf is required for stable
association of fVIII heavy and light chains, efficient fVIII
production,5,6 and protection from enzymatic degradation, notably by activated protein C and factor Xa.7,8 vWf also prevents fVIII binding onto phospholipids.9-11 Two fVIII
regions were shown to be involved in fVIII binding to vWf: the
amino-terminal part of the A3 domain and the C2
domain.12-18
Recent observations19,20 have suggested that residues
located outside the A3 and C2 domains could also contribute to the binding of fVIII to vWf. Thus, mutation Arg2150His in the C1 domain alters the binding of an inhibitor antibody that recognizes fVIII only
in the presence of vWf. Analysis of Arg2150His fVIII binding to vWf
indicated that this substitution reduced the binding of fVIII to vWf,
which prevented the binding of the antibodies recognizing fVIII bound
to vWf.20 In addition, a human monoclonal antibody to the
fVIII C1 domain was shown to inhibit fVIII binding to
vWf.21 Residues located in the C1 domain could, therefore,
interfere, directly or indirectly, with fVIII binding to vWf. To
address this question, we examined the interaction between vWf and
fVIII variants carrying a point mutation in the fVIII C1 domain,
resulting in mild/moderate hemophilia A.
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Patients, materials, and methods |
Reagents and buffers
Full-length human recombinant (r)fVIII (specific activity of 4000 IU/mg) was generously provided by Hyland (Glendale, CA). Ortho-phenylenediamine, phenylmethylsulfonyl fluoride (PMSF), and
avidin-peroxidase were purchased from Sigma Chemical Co (St. Louis,
MO). Coatest Factor VIII was bought from Chromogenix AB (Mölndal,
Sweden) and Factor VIII Chromogenic Assay from Dade (Düdingen, Switzerland).
Patients
Plasma was obtained from normal donors, from patients with mild
hemophilia A, and from a patient with vWD type 2 with compound heterozygosity for a substitution Arg91Gln on one allele and very low
levels of messenger RNA (mRNA) from the second vWf
allele.22 A full clinical description of patient LE has
been given elsewhere.19 Institutionally approved
informed consent was obtained from all patients and normal subjects.
Protein preparation
vWf was purified by gel filtration chromatography in the presence of
0.5 mol/L CaCl2 from commercially available Factor VIII vWf
SD (Belgian Red Cross) as previously described.21 rfVIII produced by stably transfected Chinese hamster ovary (CHO) cell lines
incubated in conditioned medium devoid of vWf and fetal calf serum
(FCS) was partially purified by ion exchange chromatography. Conditioned medium was applied on a Mono Q Fast Flow column (Pharmacia) and washed with 20 mmol/L HEPES, 150 mmol/L NaCl, CaCl2 5 mmol/L, pH 7.2 (HEPES buffer). The proteins were eluted with HEPES
buffer, supplemented with 1 mol/L NaCl and 0.1% Tween-80, stabilized
by addition of 0.1% bovine serum albumin (BSA), concentrated using solid polyethylene glycol, dialyzed against HEPES buffer, and stored at
 80°C.
Measurement of fVIII binding to vWf in plasma
fVIII binding to vWf in plasma was evaluated by using a
centrifugation assay.20 Thus, the vWf-specific moab75H4B12
immunoglobulin M (IgM) antibody was coupled to CNBr-activated
Sepharose-4B beads (Pharmacia Biotech), according to the
manufacturer's instruction. Plasma samples were diluted 10-fold in
Tris 50 mmol/L, NaCl 0.15 mol/L, BSA 1%, Tween-80 0.1%, pH 7.4 (TBS-BSA); 50 µL of each diluted plasma were mixed with 50 µL of
the anti-vWf Sepharose (50% suspension), or of a control Sepharose,
for an incubation of 90 minutes at 4°C. After centrifugation, the
amount of fVIII present in the supernatant of the anti-vWf and control
Sepharose was evaluated in a chromogenic assay (Coatest Factor VIII),
according to the manufacturer's recommendations except that, when
required, plasma samples were diluted in TBS-BSA. Initial experiments
had established that TBS-BSA was optimal to prevent nonspecific binding of plasma fVIII to Sepharose and could be used to dilute plasma in the
Coatest factor VIII. fVIII bound to vWf was calculated by subtracting
the amount of fVIII in the supernatant of the anti-vWf Sepharose from
that present in the supernatant of the control Sepharose.
To evaluate the binding of rfVIII to vWf in plasma, rfVIII was added at
a final concentration of 0.1 IU/mL to the plasma of a patient with
severe hemophilia A. After 30 minutes of incubation at 20°C, the
fraction of fVIII bound to vWf was measured as above.
Scatchard analysis of rfVIII binding to vWf
Sepharose beads coated with vWf were prepared by mixing 200 µL of
the anti-vWf moab75H4B12 Sepharose (50% suspension) with 200 µL vWf
(1.8, 5.5, 16.6, or 50 µg/mL) or control buffer for 60 minutes at
20°C. After centrifugation, the amount of vWf present in the
supernatant of the anti-vWf moab75H4B12 Sepharose was evaluated by
enzyme-linked immunosorbent assay (ELISA). The fraction of vWf bound to
Sepharose was calculated by subtracting the amount of vWf recovered in
the supernatant of anti-vWf Sepharose from the total vWf added to the
assay. After 5 washes in TBS-BSA, vWf Sepharose was resuspended in 800 µL TBS-BSA.
rfVIII (50 µL) at 0.05 to 5.5 nmol/L was incubated for 90 minutes at
20°C with 50 µL vWf Sepharose or 50 µL control Sepharose. After
centrifugation for 10 seconds at 10 000g, total and free fVIII
were measured by a chromogenic assay in the supernatants of control and
vWf Sepharose, respectively. Bound fVIII was calculated by subtracting
free fVIII from total fVIII. Initial experiments with normal rfVIII
established that fVIII binding was evaluable with the use of a fVIII
chromogenic assay provided that bound fVIII was greater than 5% and
less than 95% of total fVIII.
Immunoassays
Plasma fVIII antigen levels were measured by ELISA with the use of
the Immunozym FVIII:Ag test (Immuno AG, Vienna, Austria) following the
manufacturer's recommendations.
rfVIII levels were measured by ELISA, according to published
methods,19 using moab F4H12 or F15B12, recognizing the A1
or A2 domain, respectively, as capture antibodies. Bound fVIII was detected by the addition of a mixture of moab 13, F8D6, F29A1, and
F14A12.19 vWf levels were measured by ELISA, according to published methods.21
Plasmid mutagenesis
Mutagenesis was performed within the mammalian expression vector
pGCSamF8EN23 (provided by R.A. Morgan) coding for the B domain-less fVIII complementary DNA (cDNA). Mutant plasmids were generated through oligonucleotide site-directed mutagenesis, utilizing the polymerase chain reaction.24 Codon n°2098 was
mutated from ATC to AGC, predicting an amino acid change from I to S. Codon n°2119 was mutated from TCC to TAC, predicting an amino acid
change from S to Y. Codon n°2150 was mutated from CGT to CAT,
predicting an R to H amino acid change. All mutated cDNAs were
controlled by sequencing in both directions, using an ABI (Genetic
Analyser 3.10 PerkinElmer).
CHO cell transfection
To establish cell lines expressing wild-type or mutant fVIII, CHO
cells were transfected with pGCSamF8EN or the mutated vector by using
FUGENE 6 (Boehringer Mannheim, Brussels, Belgium), according to the
manufacturer's instructions. The cell lines producing the highest
fVIII amounts were expanded and subcloned twice.
FVIII production by transiently transfected CHO cells
Transient transfections were performed in a similar manner. CHO
cells (8 × 104 cells/well) were seeded in 6-well
plates (Life Technologies), using MEMAlpha Medium (MEM ; Life
Technologies Ltd, Paisley, UK) supplemented with 10% FCS (MEM-FCS).
After 24 hours of incubation, a transfection mixture of 0.5 µg DNA in
10 µL of Tris-EDTA, 100 µL OPTIMEM, and 2 µL FUGENE 6 was applied
to the cells. After 48 hours, the cells were washed twice with MEM,
and the culture medium was replaced by MEM supplemented with
Nutridoma-CS (Boehringer Mannheim, Germany) and 3 mmol/L sodium
butyrate (MEM-Nutridoma). After 16 hours, the conditioned medium was
harvested, centrifuged to remove cell debris, and assayed for fVIII
activity. This conditioned medium was used because preliminary
experiments had shown that higher fVIII levels could thus be obtained
in the absence of serum and of vWf. Seventy-two hours after
transfection, cells incubated in MEM-Nutridoma were washed in
methionine-free MEM and incubated for 30 minutes in methionine-free
medium containing 0.5 mCi/mL of [35S]methionine
(Amersham) and aprotinin (0.5%; Sigma).5 Intracellular levels of fVIII translation products were evaluated by
immunoprecipitation, followed by SDS-PAGE and
autoradiography.25
After 30 minutes of incubation, the cells were harvested and lysed in
150 mmol/L NaCl, 50 mmol/L Tris, pH 7.4, 0.05% (w/v) SDS, 1% (v/v)
NP-40, 1 mmol/L PMSF, and 1 mg/mL soybean trypsin inhibitor
(Tris-NP-40). Equal volumes of cell extracts were incubated with 500 µL human antibody at 2 µg/mL in Tris-NP-40 supplemented with 0.25%
gelatin and 5% BSA (Tris-NP-40-Gel-BSA). The tubes were gently rocked
for 2 hours at 4°C. A 50% solution of Protein A Sepharose (20 µL) was then added to the antigen/antibody mixture and incubated for
1 hour at 4°C on a rocking platform. Sepharose beads were
centrifuged and washed twice with Tris-NP-40-Gel-BSA and once in 10 mmol/L Tris, pH 7.5, 1% v/v NP40. Bound antigen/antibody complexes
were eluted from the beads by boiling for 4 minutes in 30 µL of SDS
gel loading buffer. An aliquot of 25 µL was analyzed by 8% (w/v)
PAGE and visualized by autoradiography.
The relative concentrations of the fVIII primary translation products
were evaluated by comparison with serial dilutions of immunoprecipitates from a reference [35S]-labeled cell extract.
mRNA quantification
Total RNA was isolated from 106 transiently transfected
CHO cells by using the TRIzol Reagent (Life Technologies, Gaithersburg, MD). After DNase treatment of the RNA samples, a first strand cDNA
synthesis was carried out with the Superscript Preamplification system,
according to the instructions of the manufacturer (Life Technologies).
Quantitative real-time PCR was carried out with the use of
gene-specific double fluorescently labeled probes in a 7700 Sequence Detector (PE Applied Biosystems, Norwalk, CT). 6-Carboxyfluorescein, FAM, and JOE, were used as the 3' fluorescent reporter for fVIII and hypoxanthine guanine phosphoribosyl transferase (HPRT),
respectively, whereas tetramethylrhodamine (TAMRA) was used as the
5' quencher. The following primer and probe sequences were used:
HPRT forward primer, 5'-TTATCAGACTGAAGAGCTACTGTAATGATC-3';
HPRT reverse primer, 5'-TTACCAGTGTCAATTATATCTTCAACAATC-3';
HPRT probe,
5'-TAMRA-TGAGAGATCATCTCCACCAATAACTTTTATGTCCC-JOE-3'; fVIII
forward primer, 5'-AACCGAAGCTGGTACCTCACA-3'; fVIII reverse primer, 5'-GGATCCTCAAGCTGCACTCC-3'; and fVIII probe,
5'-TAMRA-AGAATATACAACGCTTTCTCCCCAATCC-FAM-3'. All probes
were designed to span exon junctions in the fully processed message to
prevent reporting of amplification of any possible contaminating
genomic DNA. The cDNA samples underwent a denaturating step of 10 minutes at 95°C and 40 cycles of 15 seconds at 95°C and 1 minute at 60°C, yielding an amplicon of 126 and 74 base pairs (bp)
for HPRT and fVIII, respectively.
fVIII immunoprecipitation and immunoblot analysis
Normal and mutant fVIII (1 IU) were immunoprecipitated with the
human monoclonal antibody BO2C11,26 according to published methods. Immunoprecipitates were analyzed by Western blotting with the
use of moab12 and F14A12.
fVIII production in presence and absence of vWf
To evaluate the stabilization of fVIII by vWf, cell cultures were
performed, according to published methods.5 Stably
transfected CHO cells were seeded at 5 × 104 per
well in 48-well plates and incubated overnight at 37°C in MEM
supplemented with 10% FCS. The cells were then washed 3 times and the
cell culture medium was replaced by Opti-MEM (Life Technologies) with 3 mmol/L sodium butyrate, supplemented or not with various amounts of
vWf. After 16 hours at 37°C, the cell culture supernatants were
harvested and assayed for fVIII activity.
Statistical analysis
The significance of the differences was determined by using the
Student t test for unpaired values.
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Results |
Binding of plasma fVIII to vWf
Although mutations in the fVIII C1 domain are commonly found in
patients with mild/moderate hemophilia,1 no role(s) has yet
been ascribed to the C1 domain in fVIII synthesis, secretion, stability, or cofactor function. To evaluate whether mutations in the
C1 domain resulted in a reduction of fVIII binding to vWf, plasmas of
patients with mild/moderate hemophilia A with such mutations were
analyzed. Tenfold diluted plasmas were incubated with Sepharose coupled
to an anti-vWf antibody. At equilibrium, the Sepharose was centrifuged,
and unbound vWf and fVIII were measured. In all experiments, more than
95% vWf was captured on the Sepharose. For 7 normal plasmas and a pool
of normal plasmas, the fraction of bound fVIII was comprised between
71.1% and 80.9% (X = 73.1, SD = 3.7) (Figure
1A). To rule out an effect of the fVIII:c
plasma level in the assay system, the plasma of a normal donor was
diluted 10- or 50-fold in the plasma of a patient with severe
hemophilia A. The fraction of fVIII bound to vWf was 76% ± 3% and
81% ± 2%, respectively (ie, within the normal range of the
assay). In a further control experiment, the plasma of a patient with
vWD type 2 Normandy showed strongly reduced fVIII binding in the assay
system (35% ± 11%).
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| Fig 1.
Binding of plasma fVIII to vWf.
Sepharose beads coated with anti-vWf moab75H4B12 were incubated with
10-fold diluted plasma. The beads were centrifuged, and fVIII present
in supernatants was measured in a chromogenic assay. Control
experiments were carried out with uncoated Sepharose beads. Results are
expressed as the percentage of fVIII bound to vWf (Bound
fVIII(%) = [fVIII in the supernatant of control Sepharose fVIII
in the supernatant of anti-VWF Sepharose] × 100/fVIII in the
supernatant of control Sepharose). (A) Normal donor plasma fVIII; (B)
plasma of patients with mild/moderate hemophilia A with mutations in
the fVIII A1 or A2 domain; (C) plasma of a patient with mild/moderate
hemophilia A with mutation in the fVIII A3 domain; (D) plasmas with
fVIII mutations in the C1 domain. Each value represents the mean of at
least 2 experiments and SDs are as indicated. Values of vWf-bound fVIII
significantly lower than that of normal fVIII are indicated:
*P < .025; **P < .01.
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fVIII with mutations Ile312Val, Gly479Arg, Leu625Val, and Asn2019Ser in
the A1, A2, and A3 domain, respectively, bound to vWf to the same
extent as normal fVIII (Figure 1 B and C). As shown in Figure 1D, of 7 variants with mutations in the C1 domain resulting in mild/moderate
hemophilia A, only 2 showed a normal binding to vWf. The substitutions
Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln in the
C1 domain were characterized by a smaller fraction of bound fVIII
(range 13%-67%).
For the 5 mutations associated with reduced fVIII/vWf binding, the
levels of fVIII:Ag and fVIII:c were closely related, indicating that
the specific activity of the mutated fVIII molecules was close to that
of normal fVIII (Table 1). Hence, the
mechanisms responsible for reduced fVIII levels in the plasma of these
patients were likely to involve either reduced synthesis and/or
secretion or reduced stability in plasma. These questions were
addressed with the use of rfVIII variants produced by CHO cells
transfected with cDNA expression vectors for B domain-deleted fVIII.
Binding of Ile2098Ser, Ser2119Tyr, Arg2150His rfVIII
mutants to vWf
Three representative fVIII mutants were selected for these
experiments: Ser2119Tyr (ie, the mutant with the lowest binding to
vWf), Ile2098Ser, and Arg2150His. An expression vector coding for a B
domain-deleted fVIII was selected, because it allowed for a
higher fVIII production than a vector encoding the complete fVIII
gene.23
The affinity of rfVIII variants for vWf was compared by Scatchard
analysis. Initial experiments with normal rfVIII established that fVIII
levels evaluable with the use of a fVIII chromogenic assay were
obtained with concentrations of vWf ranging from 0.2 to 6 nmol/L. By
contrast, with the mutant fVIII preparations, valid data were obtained
only when using a concentration of vWf of 6 nmol/L.
The apparent dissociation constant (KD) of the
binding of normal rfVIII to vWf was 1.0 ± 0.3 nmol/L (Table
2), as determined from binding experiments
with the use of different concentrations of vWf insolubilized on
Sepharose (Figure 2A). This
determination is in agreement with published
values (KD = 0.49 ± 0.12
nmol/L).27 The calculated number of fVIII binding sites per
vWf monomer was 0.4. Such a low ratio could be attributed to either
degraded molecules in the vWf preparation or to limited accessibility
of fVIII molecules for insolubilized vWf. The latter hypothesis is more
likely, because similar results were obtained by using different vWf
preparations (plasma-derived or recombinant vWf preparations, data not
shown).
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| Fig 2.
Scatchard analysis of normal and mutant rfVIII binding to
vWf.
(A) Normal rfVIII (0.05 to 5.5 nmol/L) was incubated for 90 minutes at
room temperature with vWf insolubilized on Sepharose ( , 0.2 nmol/L;
 , 2.4 nmol/L;  , 5.9 nmol/L) or with control Sepharose. After
centrifugation, free (F) and total (T) fVIII were measured by a
chromogenic assay in the supernatant of control and vWf Sepharose,
respectively. Bound (B) fVIII was calculated by subtracting free from
total fVIII. The concentration of fVIII bound to vWf (B) is plotted
against the ratio of bound/free (B/F) fVIII. Plain lines represent
linear regression analysis of experimental data points. One experiment
representative of 3 separate evaluations is shown. (B) Mutant rfVIIIs
( , Ile2098Ser;  , Ser2119Tyr;  , Arg2150His) were incubated at
various concentrations (0.05-5.3 nmol/L) with vWf-bound Sepharose (5.9 nmol/L). The concentration of fVIII bound to vWf (B) is plotted against
the ratio of bound/free (B/F) fVIII. Plain lines represent linear
regression analysis of experimental data points. Dotted lines represent
the linear regression analysis of experimental data points and of an
arbitrary point, corresponding to the maximal number of fVIII binding
sites on vWf. This number was calculated as the intersection with the x
axis of the line corresponding to the linear regression analysis of
experimental data points of normal and Arg2150His rfVIII binding to
vWf. One experiment representative of 3 separate evaluations is
shown.
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Analysis of the binding of Arg2150His to insolubilized vWf (6 nmol/L)
indicated that the dissociation KD was 3.3-fold higher than
that of normal fVIII. The binding stoichiometry at saturation was
similar to that determined for normal fVIII (0.4 fVIII molecule per vWf
monomer). Under the same experimental conditions, the binding
of Ile2098Ser or Ser2119Tyr fVIII to vWf was too low to provide enough
data points to calculate the number of binding sites at saturation and
the KD of the reaction. For these 2 mutants, the KD
was, therefore, determined by taking into account an arbitrary point corresponding to the number of fVIII binding sites present on
insolubilized vWf. This number was derived from the Scatchard analyses
of the binding of normal and Arg2150His fVIII to vWf (Figure 2A-B). By
using this method, the calculated KD of Ser2119Tyr and
Ile2098Ser fVIII binding to vWf was about 80-fold and 8-fold higher
than that of normal fVIII, respectively (Table 2), in agreement with
the reduced fVIII/vWf interaction observed in plasma containing these
mutants (Figure 1).
To evaluate the vWf-binding capacity of rfVIII molecules in
plasma, a fixed concentration of fVIII:c (0.1 IU/mL) was added to the
plasma of a patient with severe hemophilia A. After 30 minutes of
incubation at 20°C, the plasmas were diluted 10-fold and the
fraction of fVIII bound to vWf was determined after incubation with a
Sepharose coated with a murine anti-vWf monoclonal antibody. As shown
in Table 2, the binding to vWf of normal rfVIII was in accordance with
the binding of normal plasma fVIII when assayed under similar
conditions (Figure 1).
The fractions of mutated rfVIII molecules bound to vWf were
significantly reduced by comparison to normal rfVIII (Table 2) and were
within the same range of magnitude as the fractions of mutant fVIII
molecules associated to vWf in patients' plasma (Figure 1). Thus, for
recombinant and plasma fVIII, the lowest association with vWf was
observed with Ser2119Tyr mutant, in close agreement with the low
affinity for vWf determined for this mutant by Scatchard analysis
(Table 2).
Subunit composition and specific activity of mutant
rfVIII molecules
The reduced binding to vWf could still be due to an alteration of
the mutant rfVIII molecules. Therefore, the physical state of the fVIII
molecules was analyzed by Western blot, and the specific activity of
fVIII mutants was determined with the use of a fVIII functional assay
and ELISA.
The subunit composition of purified rfVIII was analyzed by Western
blotting analysis. To eliminate BSA present in the fVIII preparations,
and which altered the migration of the fVIII chains on SDS-PAGE, fVIII
molecules were immunoprecipitated before analysis. To carry out the
precipitation, the human monoclonal antibody BO2C11 was selected for
its high affinity for fVIII26 and because of its ability to
completely inhibit the functional activity of the 3 fVIII mutants,
which was taken as a further indication that the mutations had not
altered the epitope recognized by the antibody.
The amounts of fVIII light chain were similar in preparations of normal
and mutant molecules when identical amounts of fVIII were used as
adjusted by functional activity (Figure 3).
This finding excluded that isolated light chains able to bind to vWf that could have interfered within the binding experiments reported above. The amounts of heavy chain were also similar for the 4 rfVIII,
indicating that the proportion of light chains associated to heavy
chains were similar in normal and mutant proteins. This finding
strongly suggested that there was no difference between normal and mutant fVIII in the cleavage of the single-chain form of
fVIII. All fVIII preparations also contained an additional fragment
with a molecular weight of 50 kd. The amounts of the latter were
similar in all preparations of normal and mutated fVIII (Figure 3).
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| Fig 3.
Western blot analysis of recombinant normal and purified
fVIII.
Partially purified normal and mutant rfVIII molecules were
immunoprecipitated by using the human monoclonal antibody BO2C11 bound
on protein A Sepharose and analyzed by SDS-PAGE followed by Western
blotting. Mouse anti-fVIII moab 12 and F14A12 were used to detect fVIII
fragments.
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To evaluate the specific activity of the rfVIII molecules, fVIII levels
measured in chromogenic assays were compared with fVIII antigen levels
determined in ELISA. To avoid a bias because of mutation-induced
alteration of epitopes or because of degradation fragments, fVIII
antigen levels were evaluated in 3 different ELISAs, using capture
antibodies recognizing either the light chain, the A1 domain, or the A2
domain, respectively. Concordant results were obtained by the 3 assays,
and the specific activities (fVIII:c/fVIII:Ag) of the 3 recombinant
molecules were close to that of normal rfVIII (Table 2) and of their
plasma counterparts (Table 1). Taken together, these data excluded that
alterations of the rfVIII molecules could have introduced a bias in the
evaluation of the vWf binding capacity of mutant rfVIII molecules.
In vitro production of fVIII Ile2098Ser, Ser2119Tyr, and
Arg2150His fVIII
A reduction of fVIII secretion is an important physiopathological
mechanism responsible for hemophilia A. Two other
laboratories28,29 demonstrated that mutations responsible
for mild/moderate hemophilia A and located at residue Arg2307 in the
fVIII C2 domain strongly reduced the fVIII secretion rate. We,
therefore, determined whether, in addition to reduction of fVIII
binding to vWf the mutations Ile2098Ser, Ser2119Tyr, and Arg2150His
could also result in a reduced fVIII synthesis and/or secretion.
The experiments were performed by using CHO cells transiently
transfected with expression vectors for normal or mutated B domain-deleted fVIII. In all experiments, CHO cells were washed 48 hours posttransfection and incubated in conditioned medium devoid of vWf.
The fVIII mRNA steady state levels were evaluated by real-time
quantitative PCR in CHO cell extracts 64 hours after transfection. As
shown in Table 3, the fVIII mRNA levels
were similar in cells transfected with cDNA expression vectors for
Ser2119Tyr, Ile2098, Arg2150His, and normal fVIII.
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Table 3.
mRNA steady-state levels, translation, and secretion of
normal and mutated fVIII by transiently transfected CHO cells
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The efficiency of fVIII translation was evaluated in duplicate cell
cultures, by short labeling with [35S]methionine and
immunoprecipitation of total cell extracts with a pool of anti-fVIII
antibodies. Intracellular mutants were detected as a single-chain form,
migrating at approximately 170 kd, as for normal fVIII (Figure
4). The intensity of the bands
corresponding to the 3 mutants and normal fVIII varied slightly (Figure
4). However, there was no consistent effect of these mutations on the
rate of fVIII protein translation when compared with the corresponding fVIII mRNA steady-state levels (Table 3).
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| Fig 4.
Normal and mutant fVIII translation efficiencies.
CHO cells transiently transfected with normal and mutant fVIII cDNA
expression vectors were labeled with [35S]methionine for
30 minutes. Cell extracts were analyzed by immunoprecipitation with a
pool of monoclonal antibodies to fVIII light and heavy chains.
Molecular weight markers are shown on the left. One experiment
representative of 3 separate evaluations is shown.
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The rate of functional fVIII secretion by transiently transfected CHO
cells was assessed by measuring fVIII:c activity accumulated in
conditioned medium devoid of vWf during the 16 hours preceding [35S]methionine labeling or RNA extraction. Relative to
the fVIII primary translation products, the rate of accumulation of
Ser2119Tyr was equal to that of normal fVIII (Table 3),
whereas those of Ile2098Ser and Arg2150His were reduced about twofold
and sixfold, respectively. In control experiments, normal or mutant
rfVIII were incubated at 37°C for various periods of time in the
presence of CHO cells, and the kinetics of disappearance of fVIII from the conditioned culture medium were evaluated by using a fVIII chromogenic assay. The half-times of disappearance of mutant and normal
fVIII were similar (data not shown), excluding that differences in the
stability and/or rate of clearance between normal and mutant rfVIII
could significantly affect accumulation of functional fVIII in the
absence of vWf. Accordingly, the rate of accumulation of fVIII in
transfected CHO cell conditioned medium likely reflects the rate of
fVIII secretion.
fVIII accumulation in the presence of vWf
As fVIII/vWf association is crucial for in vivo fVIII stability, it
was likely that the alteration of fVIII binding to vWf observed for the
3 mutants characterized above reduced the stability of fVIII in plasma.
To determine in vitro whether alteration of fVIII/vWf interaction
resulted in a reduction of fVIII production and/or stability, we
assessed the accumulation of fVIII in the conditioned medium of stably
transfected CHO cell lines producing Ser2119Tyr, Ile2098Ser, and
Arg2150His fVIII in the presence of various concentrations of vWf.
As shown in Figure 5, the accumulation of
normal fVIII was tripled in the presence of 1 µg/mL vWf. The maximal
(ninefold) increase of fVIII concentration was achieved with 25 µg/mL
vWf. By contrast, the concentration of the 3 mutants was enhanced to a
lesser extent by vWf. For Ser2119Tyr, Ile2098Ser, and Arg2150His fVIII
a 3-fold fVIII increase was achieved with vWf concentrations 16-fold,
8-fold, and 3-fold higher, respectively, than for normal fVIII (Figure
5). Thus, for the 3 mutants, the deficient accumulation in the presence
of vWf closely reflected the reduced fVIII binding to vWf, as evaluated
in plasma (Figure 1) or in a purified system (Figure 2B; Table 2).
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| Fig 5.
vWf-dependent rfVIII accumulation in conditioned medium
of transfected CHO cells.
CHO cells transfected with expression vectors for normal (),
Ile2098Ser (), Ser2119Tyr (), and Arg2150His () fVIII were
incubated for 16 hours in the presence of various concentrations of
vWf, as indicated. fVIII activity was determined in a chromogenic
assay. Results were expressed as the ratio between fVIII:c levels in
the presence of vWf to that in the absence of vWf. One representative
experiment of 3 is shown.
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Discussion |
Although many point mutations located in the C1 domain result in
mild/moderate hemophilia A, the function of the C1 domain is still
completely unknown. The observation that mutations located in C1 alter
the binding of fVIII to vWf is remarkable given that this region of the
fVIII molecule is not considered to participate in fVIII binding to vWf.
By antibody inhibition assays and recombinant fragment binding assays,
2 regions of the fVIII molecule have been shown to contribute to
binding to vWf: a portion of the A3 domain and the C2
domain.12-18 The affinity of the separated A3 and C2
domains was respectively 60 and 200 nmol/L, whereas the affinity of the complete light chain was 2 nmol/L.18
Although the role of C2 in fVIII binding to vWf is well established,
mutations of the C2 domain leading to alteration of fVIII binding to
vWf have not been identified so far. Early evaluation of Arg2307Gln
fVIII suggested that the mutation altered fVIII binding to
vWf,30 but analysis of the rate of fVIII synthesis showed
that the low fVIII levels were due to the retention of mutated fVIII in
the endoplasmic reticulum, which prevented efficient secretion.28,29
In the A3 domain, the only mutations identified so far that result in
reduced fVIII binding to vWf are substitutions of tyrosine residue
1680, which prevent a sulphation critical for fVIII binding to
vWf.31,32 A possible reason for the difficulty to detect such mutation(s) may be that, in contrast to the C1 domain, the A3 and
C2 domains also mediate interactions of fVIII with either coagulation
factors or phospholipids. Hence, mutations in the latter domains may
frequently be associated to alteration of fVIII cofactor activity in
addition to potential disturbance of fVIII binding to vWf.
After the initial description of a patient with mild hemophilia A with
an Arg2150His substitution in the C1 domain and reduced fVIII binding
to vWf,20 a systematic screening was undertaken on plasmas
of patients with C1 mutations to determine whether this represented a
significant mechanism that could account for the mild hemophilia A
phenotype. Analysis of fVIII/vWf interactions in such plasmas
identified 4 other mutations in addition to Arg2150His (ie, Ile2098Ser,
Ser2119Tyr, Asn2129Ser, and Pro2153Gln), which resulted in a
qualitatively impaired fVIII binding to vWf.
Albeit performed with plasma, the method used for the screening does
not allow us to determine the affinity of fVIII binding to vWf or the
fraction of fVIII bound to vWf in plasma in vivo. Indeed, the
evaluation of the fraction of vWf-bound fVIII requires the initial
capture of vWf on Sepharose coated with anti-vWf monoclonal antibody.
To allow for a complete capture of vWf molecules, plasma had to be
diluted 10-fold, which displaces the equilibrium toward dissociation of
fVIII from vWf. Binding of vWf to Sepharose may also alter the
accessibility of some vWf monomers, thereby further displacing the
equilibrium toward dissociation of the fVIII/vWf complex. Matrix
effect, such as ionic strength, calcium ion concentrations, and
detergent, may also theoretically modify fVIII/vWf interaction in
comparison to native undiluted plasma.
Other patient-related parameters, such as the concentration of vWf, are
likely to significantly influence the evaluation of fVIII binding to
vWf in plasma. This is, in fact, illustrated by the observation that,
for 3 patients carrying the Arg2150His substitution, the fraction of
fVIII bound to vWf varied from 40% to 67%. We, therefore, analyzed in
a purified system the affinity of selected rfVIII mutants for vWf.
Scatchard analysis of rfVIII binding to vWf confirmed that the 3 mutants Ile2098Ser, Ser2119Tyr, and Arg2150His had a reduced affinity
for vWf. The 80-fold reduced affinity of Ser2119Tyr rfVIII for vWf is
in agreement with the severely impaired fVIII/vWf interactions observed
in the plasma of a patient carrying this mutation, suggesting that,
despite all the caveats theoretically associated with the evaluation of fVIII/vWf interactions in plasma, this assay provides results representative of relative affinities of mutated fVIII molecules. Conversely, the modest 3.3-fold reduction of Arg2150His rfVIII affinity
for vWf indicates that the analysis of fVIII/vWf complex in plasma is
very sensitive for the detection of fVIII variants with
reduced binding to vWf.
It is of interest to relate the mutations resulting in reduced fVIII
binding to vWf to the recently proposed a 3-dimensional model of the
fVIII C1 domain.33 Four of the 5 mutations occur in
residues that are conserved in the discoidin family and are, therefore,
more likely to play a structural role in the C1 domain than to be
involved directly in intermolecular interactions with vWf. Ser2119 is
located at the interface between the A3 and C1 domains, and the
substitution to Y disrupts the domain interface and the global
structure of fVIII. Asn2129 is found in a surface loop, whereas Arg2150
and Pro2153 are located in the central  -sandwich core of the C1
domain. Mutations of these residues are, therefore, expected to
destabilize the C1 domain and potentially result in misfolded protein.
Mutation of Arg2150 to H is predicted to be particularly
disruptive,33 which is a possible explanation for its
reduced rate of secretion. The fifth mutation affects Ile 2098, a
residue that is not conserved in the discoidin family, although residue
hydrophobicity at this location is maintained in the C1 and C2 domains
of factor V and in the C2 domain of fVIII. Ile2098 is located in a loop
buried in the hydrophobic core of the C1 domain, and substitution
Ile2098 to S is, therefore, also predicted to result in destabilization
and/or impaired folding of the fVIII molecule.34
The mechanism by which C1 influences the affinity of fVIII for vWf is
not yet elucidated. It is possible that the reduction of fVIII binding
is due to an impairment of contact between C1 and vWf residues. This
hypothesis would be compatible with recent observations, showing that a
human monoclonal antibody recognizing the C1 domain, LE2E9, is able to
completely inhibit fVIII binding to vWf.21 Fab fragments of
LE2E9 are also able to interfere with fVIII binding to vWf.
Conversely, mutations in the C1 domain could have an indirect effect,
by altering other regions of the fVIII molecule mediating the contact
with vWf, notably within the A3 or C2 domains. The prediction of the
structural effects of mutation Ser2119Tyr is compatible with such a
phenomenon because of its peculiar location at the A3-C1
interface.33
Given the importance of vWf for fVIII stability in plasma, the reduced
binding of mutated fVIII to vWf could be responsible for the reduced
fVIII levels found in the plasma of the patients. Alternatively, the
synthesis/secretion rate of fVIII could be altered. Analysis of fVIII
production in the absence of vWf by transiently transfected CHO cells
indicated that the accumulation of functional Ile2098Ser and Arg2150His
fVIII molecules was reduced about 2-fold and 6-fold, respectively, by
comparison to normal and Ser2119Tyr fVIII. Because, in the absence of
vWf, mutant and normal fVIII had a similar stability in the presence of
native CHO cells, the reduced rates of accumulation of
Ile2098Ser and Arg2150His fVIII in transfected CHO cells
conditioned medium likely reflect a reduced rate in the secretion of
functional fVIII molecules.
When fVIII production by CHO cells was evaluated in the presence of
vWf, the vWf-dependent increase in accumulation of fVIII in the culture
medium was reduced for the mutants compared with normal fVIII. These
results support the concept that the reduced binding to vWf observed in
plasma contributes to the low fVIII levels of the patients. Noteworthy,
in this in vitro culture system, the addition of vWf resulted in only a
9-fold increase of the concentration of normal fVIII. This increase is
in contrast to the situation of patients with severe vWD, in whom the
fVIII levels are reduced more than 20-fold.3 This reduction
is likely due to the fact that clearance mechanisms or inactivation
phenomena at play in vivo are absent or less effective in the in vitro
cell culture system. Accordingly, it can be expected that the reduction of fVIII stabilization observed in vitro with Arg2150His and Ile2098Ser fVIII is even more significant in vivo.
These findings demonstrate that mutations in the fVIII C1 domain
leading to a reduced fVIII binding to vWf without alteration of fVIII
procoagulant activity are an important cause of mild/moderate hemophilia A.
| |
Footnotes |
Submitted September 9, 1999; accepted March 23, 2000.
Supported in part by research grant G.0292.98 from the Flemish Research
Foundation and by grant Schw 752/1-1 from the Deutsche Forschungsgemeinschaft.
J.V. is holder of the Dr Jean Choay Chair for Hemostasis Research.
Reprints: Marc Jacquemin, Center for Molecular and Vascular
Biology, University of Leuven, Campus Gasthuisberg, O&N, Herestraat 49, B-3000 Leuven, Belgium; e-mail: marc.jacquemin{at}med.kuleuven.ac.be.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction