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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4663-4670
By
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.
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
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.
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.
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 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 G 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).
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
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 G
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.
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
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).
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 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.
Submitted November 19, 1997;
accepted August 5, 1998.
Address reprint requests to S. Jorieux, Unité de Recherche, LFB,
59 rue de Trévise, BP 2006, 59011 Lille cédex, France.
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|
Top
Abstract
Introduction
A
transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C
-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).
5-mer and 
), 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.
)
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.
) 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.