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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4663-4670
A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly
Decreases Its Ability to Bind Factor VIII and Affects Its
Multimerization
By
S. Jorieux,
C. Gaucher,
J. Goudemand, and
C. Mazurier
From the Laboratoire de Recherche sur l'Hémostase, Laboratoire
Français du Fractionnement et des Biotechnologies, Lille, France;
and the Laboratoire d'Hématologie, Centre Hospitalier
Régional Universitaire de Lille, Lille, France.
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ABSTRACT |
In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF)
is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for
factor VIII (FVIII). In this report, we describe the case of a vWD
patient who has an abnormal vWF multimers distribution associated with
a markedly decreased vWF ability to bind FVIII. Sequencing analysis of
patient's vWF gene showed, at heterozygous state, a G A
transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a CT transition, changing the
codon for Arg 896 into a stop codon. His sister who has a subnormal vWF
level, but a normal FVIII/vWF interaction, was found to be heterozygous
for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the
candidate (Asn116) missense mutation was expressed in COS-7 cells. The
expression level of Asn116rvWF was significantly decreased compared
with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly
impaired as shown by the decrease in high molecular weight forms. The
FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the
defective FVIII/vWF interaction and the impaired multimeric pattern
observed in the patient's vWF. The monoclonal antibody 31H3 against
D' domain of vWF (epitope aa 66-76) that partially inhibits the FVIII
binding and recognizes only nonreduced vWF, showed a decreased ability
to bind Asn116rvWF when used as capture-antibody in enzyme-linked
immunosorbent assay (ELISA). This result suggests that a potential
conformation change in the D' domain is induced by the Asp116Asn
substitution, which is localized in the D3 domain.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
VON WILLEBRAND FACTOR (vWF) is a large
multimeric glycoprotein, synthesized in endothelial cells and
megakaryocytes, which plays two main hemostatic roles: it mediates
platelet adhesion and aggregation to the damaged vessel wall under
conditions of high shear rate, and it is the carrier of procoagulant
factor VIII (FVIII), an essential cofactor in the generation of
activated factor X (FXa) (for review, see Meyer and
Girma1). The vWF gene, located on chromosome 12, contains
52 exons and is transcribed into an 8.7-kb mRNA, which encodes a 2,813 amino acids (aa) precursor, named pre-pro-vWF, consisting of a 22-aa
signal peptide, a 741-aa propeptide, and a 2,050-aa mature vWF
subunit.2,3 The analysis of the aa sequence for vWF
precursor showed that over 90% of the sequence is contained within
repeats of four types of homologous domains designated A
to D and arranged as follows:
D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2.4 In plasma, vWF is
present as a series of multimers composed of a 250-kD
subunit that ranges in size from 500 kD (protomer) to over 15,000 kD.
The multimeric structure of vWF is crucial in platelet adhesion and
aggregation, the larger multimers being the most
functional.5 The different functions of vWF reside on
well-characterized domains of the subunit. The FVIII binding site has
been localized on a 31-kD tryptic fragment that contains the first 272 aa residues of the mature vWF subunit.6,7 This fragment is
encoded by the exons 18 to 23 and is composed of the D' (aa 6 to 102)
and a part of the D3 (aa 103 to 479) homologous domains. The
interaction between FVIII and vWF is necessary for normal survival of
FVIII in blood circulation8,9 and stabilization of
recombinant FVIII in cell culture media.10 Furthermore, vWF protects FVIII from inactivation by activated protein
C11,12 and prevents FVIII activation and inactivation by
FXa.13,14 vWF also acts as a cofactor for
thrombin-catalyzed cleavage of the FVIII light chain.15
Von Willebrand disease (vWD), the most common cause for inherited
bleeding disorder, with an estimated prevalence of up to 1% of the
general population, is heterogeneous and results from quantitative
and/or qualitative defects of vWF.16 Type 1 vWD is
characterized by a dominant inheritance and a more or less pronounced
quantitative defect of vWF, while type 3 vWD is recessive and
associated with extremely low levels or undetectable vWF. Among type 2 vWD, defined by a qualitative vWF abnormality, the type 2N refers to
patients with a markedly decreased binding to FVIII, but a normal
multimeric pattern.17 This variant form of vWD is
characterized by a disproportionately low level of FVIII as compared
with normal or subnormal vWF level and is recessively inherited.18 Until now, type 2N vWD patients have been
found homozygous or compound heterozygous for seven missense mutations (Thr28Met, Arg91Gln, Arg53Trp, Arg19Trp, His54Gln, Glu24Lys, and Gly22Glu) localized in the D' domain.19 For six of these
mutations, the expression of mutated recombinant vWF (rvWF) has
confirmed that the corresponding single aa substitution results in
decreased FVIII binding capacity.20-25 Furthermore, a
subgroup of type 2N vWD patients was characterized by coinheritance of
one allele containing a type 2N mutation with a vWD type 1 or type 3 mutation on the other allele.21,22,24-28
We report here the case of a vWD patient who has an abnormal vWF
multimer distribution and a marked decrease in vWF ability to bind
FVIII. The nucleotide (nt) sequencing of polymerase chain reaction
(PCR)-amplified fragments of exons 18 to 28 from the patient's vWF
gene showed, on one allele, an nt transition in exon 20, which changes
the aspartic acid residue 116 of mature subunit into asparagine
(Asp116Asn). Furthermore, an nt substitution was also detected at
heterozygous state, in exon 28, changing the codon for arginine residue
896 into a stop codon. The expression in mammalian cells of the
full-length vWF cDNA showed that the Asp116Asn substitution is
responsible for both FVIII binding defect and abnormal multimerization
of vWF. The use of monoclonal antibodies (MoAbs) directed to the
N-terminal part of mature vWF provided indirect evidence of a potential
conformation change in the D' domain induced by this Asp116Asn
substitution localized in the D3 domain.
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MATERIALS AND METHODS |
Case report.
The patient, a man born in 1924, was referred to one of us in August
1984 before osteoarthritis surgery, because of a prolonged activated
partial thromboplastin time (APTT) (49 seconds v
39 seconds for normal plasma) and borderline bleeding time (10 minutes). In the past, the patient experienced bleeding symptoms after
tooth extraction and puncture of femoral artery, which was followed by
the development of an important hematoma. After he was diagnosed as a
type 2 vWD patient, other surgeries (total hip replacement, tooth
extraction, pacemaker implantation) were performed under treatment with
vWF concentrates (LFB, Lille, France) without any bleeding problem. The
biological investigations performed on four occasions are reported in
Table 1. Plasma FVIII activity (FVIII:C) has ranged from 0.16 to 0.42 U/mL (normal, 0.5 to 1.5 U/mL). The vWF antigen (vWF:Ag) and ristocetin
cofactor (vWF:RCo) activity levels have ranged from 0.22 to 0.55 U/mL
(normal, 0.5 to 1.5) and from 0.10 to 0.25 U/mL (normal, 0.5 to 1.5),
respectively. In addition, the patient suffers from
noninsulin-dependent diabetes, hypertension, unstable angina, and
obesity.
His sister, born in 1932, also had bleeding complications after a
hysterectomy and described frequent ecchymosis. A recent blood sample
showed that she has a normal level of FVIII:C (0.54 U/mL) and slightly
reduced levels of vWF:Ag (0.36 U/mL) and vWF:RCo (0.48 U/mL).
DNA sequence analysis.
Genomic DNA was extracted from peripheral blood leukocytes and the
exons 18 to 27 of vWF gene were amplified by PCR using, as primers,
oligonucleotides localized in adjacent introns of which the sequences
were derived from the gene sequence data already published.2 Exon 28 was amplified as previously described
in detail.29 The use of 5 -biotinylated PCR primers
and the purification of single-stranded DNA on streptavidin-coated
magnetic beads (Dynal, Oslo, Norway) according to the manufacturer's
instructions, allowed subsequent direct solid-phase sequencing of each
amplified exon using the T7 sequencing kit (Pharmacia, Uppsala,
Sweden).
Site-directed mutagenesis.
The construction of expression vector pSVvWF, containing the
full-length cDNA for human vWF has been previously
described.29 The vWF cDNA nucleotides (nt) are numbered
beginning at the A of the initiating methionine codon.30
Plasmid pSV116N was derived from pSVvWF by introducing a GA
transition at nt 2635, using the Transformer site-directed mutagenesis
kit (Clontech, Palo Alto, CA). The oligonucleotides used for
mutagenesis were as follows: 5-CCTCACCTTCAACGGGCTCAAA-3 (vWF nt 2625-2646, substitution is underlined) and
5-AGTTTCCCAGCTTCTTATTTTGATG-3 (vWF nt 5101-5125). The
first primer was used to introduce the desired mutation into pSVvWF
used as template and the second primer to destroy the unique previously
created NheI restriction site of the plasmid for the purpose of
selection. Clones containing the desired mutation were identified by
allele-specific PCR, and the nt substitution was confirmed by nt
sequence analysis.
Transient expression of vWF.
COS-7 cells were transfected with plasmids pSVvWF and pSV116N by the
diethyl aminoethyl (DEAE)-dextran method, as previously described.29 Forty hours after transfection, cells were
cultured for 72 hours in serum-free Dulbecco's modified Eagle's
medium. The concentration of vWF:Ag in conditioned media was measured by enzyme-linked immunosorbent assay (ELISA) using rabbit anti-vWF polyclonal antibodies.31
FVIII binding assay of vWF.
FVIII binding to vWF was assayed as described previously.25
Briefly, increasing amounts of vWF were immobilized by binding to
anti-vWF polyclonal antibody-coated microplates. Recombinant FVIII
(Cutter, Biological Miles Inc, Berkeley, CA) was incubated with the
captured vWF, and the bound FVIII was then measured by adding the
reagents for a chromogenic assay of FVIII-dependent Factor X activation
(Diagnostica Stago, Asnières, France). The amounts of vWF
captured by immobilized antibodies were then quantified by
ELISA.31
Comparative recognition of vWF by various MoAbs.
MoAbs 32B12 and 31H3 have been selected among a panel of MoAbs directed
towards the N-terminal part of the vWF mature subunit. MoAb 32B12 is a
potent inhibitor of the FVIII/vWF interaction and recognizes reduced
vWF. MoAb 31H3, which is a moderate inhibitor of the FVIII binding to
vWF, is sensitive to the reduction of vWF. The epitopes of MoAbs 32B12
and 31H3 have been precisely mapped to aa residues 51 to 60 and 66 to
76, respectively.32 Moreover, both MoAbs recognize all
multimeric forms, whatever the degree of multimerization of vWF
(unpublished observation). The ability of these MoAbs to capture plasma
or recombinant vWF (rvWF) was tested as described
previously,25 except that MoAbs were used at 5 µg/mL for
coating. The results are expressed as percent of the absorbance, the
values obtained with normal plasma and wild-type (WT) rvWF being
considered 100%.
Plasmin digestion of vWF.
Plasmin digestion of vWF was performed as described
previously33 with minor modifications. vWF was
immunoisolated with anti-vWF MoAb 21A11 covalently linked to
Sepharose-4B beads and the digestion was performed for 4 hours at
37°C after adding 0.045 U of bovine plasmin (Sigma, Chemical Co, St
Louis, MO).
Protein characterization.
Ristocetin-induced binding of vWF to formalin-fixed platelets was
assayed as previously described.34 The multimeric structure of plasma, platelet, or recombinant vWF was determined by 0.1% sodium
dodecyl sulfate (SDS)-1.5% agarose gel electrophoresis as previously
described,35 except that vWF was detected by anti-vWF polyclonal antibodies conjugated to alkaline-phosphatase. The percentages of the protomer and multimers were deduced from
computerized scanning of the areas of the peaks obtained with a Helena
pC24 densitometer.35 The subunit composition
of rvWF was analyzed on vWF immunoisolated with rabbit anti-vWF
polyclonal antibodies. The bound proteins were recovered by boiling for
5 minutes in electrophoresis sample buffer (10 mmol/L Tris, pH 8.0, 1 mmol/L EDTA, 2.5% SDS) containing 5% 2-mercaptoethanol and analyzed
by electrophoresis on a 5% to 9% gradient polyacrylamide gel. After electrotransfer onto nitrocellulose as previously
described,36 vWF was visualized with anti-vWF polyclonal
antibodies (Dako, Glostrup, Denmark), followed by peroxidase-conjugated
antirabbit IgG, which was subsequently revealed by the enhanced
chemiluminescence method (Amersham, Buckinghamshire, UK).
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RESULTS |
Abnormal features of the patient's vWF.
The patient's plasma was characterized by several biological
abnormalities. The vWF:Ag level was decreased or borderline and always
higher than the vWF:RCo level (Table 1).
Plasma vWF binding to ristocetin activated platelets was decreased as
compared with a pool of normal plasmas (data not shown). In addition,
electrophoretic pattern obtained in 2.5% agarose gel displayed a
moderate decrease in high molecular weight (HMW) multimers and a loss
of satellite bands (data not shown). At first, these data brought us to
classify this patient as a type IIE vWD. At the time of the last
examination, the patient's FVIII:C level (0.16 U/mL) was decreased out
of proportion of vWF:Ag level (0.48 U/mL) leading to a suspicion of
type 2N vWD. In agreement with this assumption, the patient's vWF
showed a markedly decreased ability to bind FVIII
(Fig 1A). Furthermore, the patient's
plasma and platelet vWF analyzed by 1.5% agarose gel electrophoresis,
exhibited a loss of the largest multimeric forms as compared with a
pool of normal plasmas and a normal platelet lysate, respectively (Fig
1B). To accurately define the multimeric defect observed in the
patient's plasma vWF, the percentage of protomer and HMW forms were
calculated from the densitometric scanning of multimeric patterns
(Table 2). As compared with normal individual plasma, the patient's plasma vWF was consistently
characterized by a significant decrease in HMW  5-mer and 10-mer
by 25.8% and 40%, respectively, associated with a relative increase
in protomer.
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| Fig 1.
Features of the patient's vWF. (A) FVIII binding ability
of plasma vWF. Serial dilutions of plasma samples were incubated into a
microtiter plate coated with anti-vWF polyclonal antibodies. A constant
amount of recombinant FVIII (0.1 U/mL) was then added and the activity
of FVIII bound to immobilized vWF was determined using a chromogenic
assay. The amounts of vWF captured by immobilized antibodies were
measured by ELISA using a peroxidase-conjugated MoAb as
described.25 Plasma samples : ( ), pool of normal
plasmas; ( ), patient's plasma. Each dilution sample was analyzed in
duplicate and the results were averaged. (B) Multimeric pattern of
normal and patient's vWF. Plasma and platelet lysate samples were
electrophoresed in SDS-1.5% agarose gel and vWF was visualized with
alkaline-phosphatase conjugated anti-vWF polyclonal antibodies. Lane 1, pool of normal plasmas; lane 2, patient's plasma; lane 3, patient's
platelet lysate; and lane 4, normal platelet lysate.
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Interestingly, his sister who has proportionately low vWF:Ag and
vWF:RCo levels, was found to have normal binding of vWF to FVIII, as
well as normal multimeric distribution of plasma vWF.
Identification of candidate mutations in the vWF gene.
To identify the abnormalities in vWF gene responsible for the FVIII
binding defect and the abnormal multimeric pattern, PCR-amplified fragments of exons 18 to 28 were sequenced (exons 29 through 52 of the
patient's vWF gene were not sequenced). A single nt change was
identified in exon 20, consisting of a GA transition at
position 2635, which modifies the encoded aa residue from aspartic acid to asparagine at position 116 of the mature vWF subunit
(Fig 2A). Because both nucleotides G and A
were identified on the sequencing gel, the patient is heterozygous for
the Asp116Asn candidate mutation. In contrast, this mutation was not
detected in the patient's sister.
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| Fig 2.
Identification of two mutations in patient's vWF gene.
Part of the nucleotide sequence gels of amplified vWF exon 20 (A) and
exon 28 (B) for both normal control (c) and patient (p) are shown. The
asterisks localize the point mutations: (A) the indicated
G2635A transition alters the encoded sequence from Asp 116 to
Asn; (B) the indicated C4975T transition alters the encoded
sequence from Arg896 to stop codon.
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As shown on Fig 2B, a CT substitution was detected in exon 28 at position 4975, changing the codon for arginine in position 896 of
mature vWF into a stop codon. The patient is also heterozygous for this
nonsense mutation. The C4975T substitution, which creates a DdeI
restriction site, was detected by digestion of PCR-amplified exon 28 fragment at heterozygous state in the patient's sister (data not
shown).
Binding of FVIII to recombinant vWF.
To determine whether the Asp116Asn substitution alone could account for
the defect in FVIII/vWF interaction, the corresponding mutated rvWF was
expressed. Plasmids pSVvWF and pSV116N were transiently expressed in
COS-7 cells in seven separate experiments and quantitative analysis of
secreted rvWF, using ELISA, were performed on the conditioned media.
The vWF:Ag concentration of WT rvWF present in the culture media was 80 ± 22 mU/mL (mean ± standard deviation [SD], n = 7), while the
concentration of Asn116rvWF was significantly decreased to 37 ± 10 mU/mL. The hybrid rvWF, obtained by cotransfection of a
1:1 ratio of WT and mutated plasmids, was secreted in the conditioned
media at the intermediate concentration of 59.4 ± 18 mU/mL.
The ability of the expressed rvWF to bind FVIII was next examined using
a solid-phase binding assay as described in experimental procedures
(Fig 3). As normal plasma vWF, the WT rvWF
bound FVIII in a dose-dependent manner. The mutated Asn116rvWF showed a
markedly decreased FVIII binding affinity, whereas comparable amounts
of rvWF were captured by the coated anti-vWF polyclonal antibodies. The
hybrid Asp/Asn116rvWF gave an intermediate FVIII binding curve between
that of WT and Asn116 rvWFs.
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| Fig 3.
Binding of FVIII to recombinant vWF. The ability of WT or
mutated rvWF to bind FVIII was determined as described in Fig 1A.
Conditioned media samples: (), WT rvWF; () Asn116rvWF; ( )
hybrid Asp/Asn116rvWF. Each dilution sample was analyzed in duplicate
and the results were averaged. Similar results were consistently
observed when three independent tansfection media were tested in three
separate experiments.
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Structural characterization of mutated vWF.
As the multimeric pattern of the patient's plasma vWF was
significantly altered, electrophoretic study of Asn116rvWF was
performed to check the potential impact of Asp116Asn substitution on
the multimerization. In comparison to WT rvWF, Asn116rvWF showed a marked decrease in HMW 5-mer (27% v 69% for WT rvWF),
which is correlated to a relative increase in the amount of protomer (38% for mutated rvWF, as compared with 9% for WT rvWF)
(Fig 4). These data give evidence that the
multimerization abnormalities previously characterized in the
patient's plasma were reproduced in mutated rvWF, but to a more
pronounced extent. However, the pattern of hybrid Asn116/WT rvWF
appeared normal (Fig 4).
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| Fig 4.
Multimeric analysis of rvWF by SDS-1.5% agarose gel
electrophoresis. Lane 1, WT rvWF; lane 2, Asn116rvWF; lane 3, hybrid
Asp/Asn116rvWF.
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On SDS-polyacrylamide gel electrophoresis under reducing conditions,
immunoisolated WT, mutated, and hybrid rvWFs showed a subunit band at
225 kD similar to that obtained with both normal and patient's plasma
vWF. In all of the rvWF proteins, no pro-vWF subunit was detected that
is consistent with full processing in COS-7 cells. Moreover, minor
bands corresponding to proteolytic fragments of vWF were detected at
the same level in normal and patient's plasmas (data not shown).
Conformation of the N-terminal part of mutated vWF.
To explain the FVIII binding capacity defect of plasma and rvWF
harboring the mutation Asp116Asn, we sought a potential conformation change of the N-terminal part of the mature subunit. Immunoisolated plasma and recombinant vWFs (normal and mutated) were hydrolyzed by
plasmin and analyzed by SDS-polyacrylamide gel electrophoresis under
nonreducing conditions. The electrophoretic mobility of the N-terminal
plasmin fragment, which has an apparent molecular weight of 31 kD, was
similar for normal and patient's plasma vWF. Moreover, the
amino-terminal plasmin fragments obtained with WT rvWF, Asn116rvWF, and
hybrid Asp/Asn116rvWF displayed the same electrophoretic mobility as
compared with plasma fragments (data not shown). We also performed
antigen-capture ELISA with anti-vWF MoAbs that inhibit the binding of
FVIII. MoAb 32B12, which recognizes reduced vWF, bound in a similar way
normal and patient's plasma vWF, as well as WT and mutated rvWFs
(Fig 5). In contrast, the recognition of
the MoAb 31H3, which fails to bind reduced vWF, was decreased for the
patient's plasma, as well as for mutated and hybrid rvWFs to 41.2% ± 2.8%, 30.5% ± 4.7%, and 68.4% ± 11.3%, respectively,
as compared with normal plasma and WT proteins (Fig 5).
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| Fig 5.
Recognition of plasma and recombinant vWF by MoAbs.
Plasma samples and conditioned media containing 0.01 U/mL vWF:Ag were
incubated for 1 hour at 37°C into microtiter plate coated with
() MoAb 32B12 or ( ) MoAb 31H3. After washing, the bound vWF was
detected with peroxidase-conjugated anti-vWF polyclonal antibodies.
Values are expressed as a percent of the absorbance obtained with
normal plasma or WT rvWF. Each value is the mean (± SD) of
three experiments.
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DISCUSSION |
Since the identification and confirmation of the first type 2N vWD
mutation, ie, Thr28Met,20,36 six other missense mutations (Arg19Trp, Gly22Glu, Gly24Lys, Arg53Trp, His54Gln, and Arg91Gln) have
been reported in patients characterized by a markedly decreased capacity of vWF to bind FVIII (database through the Internet at hppt :
//mmg2.im.med.umich.edu/vWF). All of these mutations are localized in
the first 102 aa residues of mature vWF subunit, which correspond to
the D' domain (aa 764-865 of pre-pro-vWF).
We report here the case of a patient who has been first classified as
having type IIE vWD based on a discrepancy between vWF:Ag and vWF:RCo
levels and a slightly disturbed vWF multimers distribution. In the
context of a complete routine biologic analysis, a reduced FVIII level
disproportionate to the vWF level underscored by a marked decrease in
vWF ability to bind FVIII led us to consider a type 2N vWD. Despite the
defective FVIII/vWF interaction, the discrepancy between plasma FVIII
and vWF levels is not constant in our patient. The variations in the
FVIII and vWF may be explained by the different diseases from which our
patient suffers that influence plasma vWF and FVIII levels. The nt
sequence analysis of exons 18 to 28 of vWF gene allowed us to identify
in this patient both a nt transition in exon 20, changing Asp 116 into
Asn and a nt transition in exon 28, changing the encoded Arg896 into a stop codon. The patient was heterozygous for these two mutations. To
show that the Asp116Asn substitution is responsible for the observed
FVIII binding defect, the corresponding rvWF was expressed by COS-7
cells. The Asn116rvWF exhibited a dramatic decrease in the ability to
bind FVIII, while hybrid rvWF, resulting from the cotransfection with
WT and mutated vWF cDNA, showed a moderately decreased binding. These
data suggest that the Asp116Asn substitution, like the other type 2N
vWD mutations already described, is recessive. Although the patient is
heterozygous for the Asp116Asn mutation, the plasma vWF behavior was
more consistent with Asn116rvWF than with hybrid Asp/Asn116rvWF,
suggesting that the second allele not mutated in exon 20 is
underexpressed. The nonsense mutation detected in exon 28 has been
previously identified by Zhang et al,37 at homozygous
state, in a type 3 vWD patient and, at heterozygous state, associated
with mild type 1 vWD phenotype. In agreement with these data, the
patient's sister who was found to be heterozygous for the Arg896ter
mutation has a moderate plasma vWF deficiency (0.36 U/mL) and a normal
FVIII/vWF interaction. Thus, the propositus described here is likely a
compound heterozygous type 3/2N vWD patient with the Asp116Asn mutation
on one allele and the Arg896ter mutation inducing the lack of normal
vWF expression on the second allele. When transiently expressed by
COS-7 cells, the accumulation of Asn116rvWF in conditioned media was
half that of WT rvWF. Charged to alanine scanning mutagenesis has
previously shown that the substitution of Ala for Asp 116 also induces
a secretion defect of mutated vWF protein expressed by 293/Tag cells
(David Ginsburg, personal communication, October 1996). These data
confirm the importance of aa residue at position 116 in the secretion
of rvWF protein. Therefore, in addition to the FVIII binding defect,
the Asp116Asn substitution appeared to induce a secretion impairment, which might also explain the low level of plasma vWF:Ag initially found
in our patient.
Besides the FVIII binding defect, the patient's plasma vWF displayed
platelet-dependent function impairment, characterized by a lower
vWF:RCo than vWF:Ag level and a moderate decrease in vWF binding to
ristocetin-activated platelets, which seems to be related to the
abnormal multimeric pattern.
In an attempt to gain more information on the origin of the patient's
vWF multimeric impairment, structural studies were performed on mutated
rvWF. Analysis by SDS-polyacrylamide gel electrophoresis under reducing
conditions indicated that WT and mutated rvWF proteins had similar
subunit composition and proteolytic patterns and were both secreted as
a processed form corresponding to the mature subunit. The possibility
that rvWF was less stable outside of the cell was rendered unlikely by
immunoblot analysis showing no detectable degradation product in
agreement with the normal patient's plasma vWF pattern. However, the
multimeric structure of Asn116rvWF analyzed by SDS-agarose gel
electrophoresis was strikingly different from that of WT rvWF. The
amount of HMW multimers was significantly decreased, while the relative
amount of protomer was increased. The multimeric profile of hybrid
Asp/Asn116rvWF being similar to the one of WT rvWF, it appears that the
Asp116Asn mutation does not exert a dominant effect on WT vWF in COS-7
cells. Therefore, the Asn116rvWF expressed by COS-7 cells reproduced the multimerization impairment found in the patient's plasma vWF, but
to a larger extent. However the pattern of patient's platelet vWF,
which displayed a lack of HMW multimers, was closer to that of
Asn116rvWF. The difference in the extent of the loss of HMW multimers
between plasma and platelet vWF is difficult to explain.
To define the mechanism by which the Asp116Asn substitution acts on
FVIII binding and multimerization of vWF, we investigated the
hypothesis of a conformation change. For this purpose, MoAbs directed
to the N-terminal part of the mature vWF subunit were used. MoAb 32B12,
a potent inhibitor of the FVIII/vWF interaction, which is not sensitive
to the reduction of vWF, recognized equally well WT and mutated vWF. In
contrast, MoAb 31H3, a moderate inhibitor of the FVIII binding to vWF,
which only recognizes nonreduced vWF, showed a similar decreased
capacity to capture the patient's plasma vWF and Asn116rvWF in
correlation with the absence of expression of the allele not mutated in
exon 20. Moreover, as the epitope of MoAb 31H3 has been previously
mapped with synthetic peptides to aa residues 66 to 76 in D'
domain,32 these data suggest that Asp116, localized in the
D3 domain of the vWF, either belongs to or is required for the native
conformation of FVIII binding site and epitope of MoAb 31H3.
The potential conformation change induced by the Asp116Asn mutation may
explain the loss of FVIII binding capacity of vWF. Indeed, two other
mutations (Gly22Glu, Thr28Met) localized in D' domain, have been
previously shown to induce a conformation change leading to a FVIII
binding defect.25,36 The FVIII binding domain is
constituted of at least three aa sequences (aa 19-28, 53-54, 91-95), as
supported by naturally-occuring mutations and epitope mapping of
anti-vWF MoAbs inhibiting the FVIII/vWF
interaction.32,38,39 The stabilization of this domain by
disulfide bridges is required,6 but we suggest that aa
residues other than cysteine are also necessary to maintain the
functional conformation of this site.
The multimerization of vWF is a complex process, which involves
different parts of the precursor (pro-vWF) subunit. Previous studies
have shown that the propeptide, as well as D' and D3 domains of the
mature subunit, are required for dimer assembly.40 The prosequence composed of the two homologous D1 and D2 domains contains two consensus sequences that are similar to those of the active site of
disulfide isomerases that catalyzes thiol protein disulfide interchange.41 The D3 domain contains sulfhydryl groups,
which are involved both in intra- and intermolecular disulfides
bridges.42,43 In contrast, all of the cysteine residues of
D' domain are involved in intrachain disulfide bonds, and the possible
role of this domain in multimerization is so far unknown. The potential
conformation change induced by the Asp116Asn substitution may affect
the accessibility necessary to promote the interchain disulfide bridges
involved in the assembly of pro-vWF dimers into multimers. Recent
studies by Eikenboom et al44 have shown that the Cys386Arg
mutation, detected in a patient classified as type 1 vWD, induces a
secretion defect linked to a decrease in the concentration of the
largest vWF multimers. Our data confirm the key role of the D3 domain in the multimerization and secretion of vWF, but further studies are
required to elucidate the mechanisms involved.
In conclusion, this report describes the first naturally-occurring
mutation localized in the D3 domain, which affects both the FVIII
binding and the structure, as well as the level of vWF. Thus, this
peculiar mutation causes an unusual phenotype. As type 2N refers to
variants with decreased affinity of vWF for FVIII, but normal vWF
multimeric pattern, and type 2A refers to variants with decreased
platelet-dependent function associated with the absence of HMW vWF
multimers,45 we propose to classify Asp116Asn mutation as
type 2N (A) vWD. The patient described here, who is a compound
heterozygote with type 3 mutation (Arg896ter) on one allele and the
Asp116Asn mutation on the other one, may be classified as type 3/2N (A)
vWD.
| |
ACKNOWLEDGMENT |
We thank Cutter Biological Miles Inc for the generous gift of
recombinant FVIII. We are grateful to S. Belmont and V. Barylo for
their excellent technical assistance and to V. Tancré for typing
the manuscript.
| |
FOOTNOTES |
Submitted November 19, 1997;
accepted August 5, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to S. Jorieux, Unité de Recherche, LFB,
59 rue de Trévise, BP 2006, 59011 Lille cédex, France.
| |
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Abstract
Introduction