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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3139-3145
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Département Recherche et Développement,
Laboratoire Français du Fractionnement et des Biotechnologies,
Lille; Centre hospitalier universitaire (CHU), Nantes; Centre
hospitalier régional universitaire (CHRU), Hôpital Huriez,
Lille; and INSERM U143, Hôpital Bicêtre, Le
Kremlin-Bicêtre, France.
We report 2 new mutations identified in 3 patients and characterized
by the markedly decreased affinity of von Willebrand factor (vWF) for
factor VIII (FVIII). Patients 2 and 3, who have a typical type 2N
phenotype, were found to be compound heterozygous for Arg91Gln and
Cys25Tyr or Cys95Phe, respectively. Patient 1, who is the first cousin
of patient 2, had an FVIII binding defect of vWF, low levels of vWF,
and multimeric impairment. She was found to be compound heterozygous
for the mutations Cys25Tyr and a stop codon (D93ter) in exon 4. Transient expression of recombinant vWF (rvWF) containing either
Cys25Tyr or Cys95Phe mutations resulted in mutated rvWF with markedly
reduced FVIII binding ability, multimeric structure impairment, and a
significant decrease in the vWF expression level. Moreover, the use of
anti-vWF monoclonal antibodies that inhibit the FVIII binding showed
that these 2 mutations likely induce a conformational change in the
D' domain. These results show that the native
conformation of the D' domain of vWF is not only required for
FVIII binding but also for normal multimerization and optimal secretion.
(Blood. 2000;95:3139-3145)
von Willebrand factor (vWF) is a large, multimeric
glycoprotein synthesized by megakaryocytes and endothelial cells in a
precursor form, named pre-pro-vWF, consisting of a 22-amino acid (aa)
signal peptide, a 741-aa propeptide, and a 2050-aa mature vWF
subunit.1 After several processing steps, including
dimerization, glycosylation, sulfation, multimerization, and
proteolytic cleavage of propeptide,2 vWF is secreted and
circulates in plasma as a series of high molecular weight (HMW)
multimers composed of disulfide-linked mature subunits that range from
500 to more than 15 000 kd.3 vWF plays 2 main hemostatic
roles: it mediates platelet adhesion to the subendothelium and platelet
aggregation at the site of damaged vessel walls, and it serves as
carrier protein for factor VIII (FVIII).4 Defects of vWF
result in von Willebrand disease (vWD), an autosomal bleeding disorder
classified into 3 main types.5 Type 1 refers to a partial
quantitative deficiency of vWF and is often dominant, whereas type 3, inherited in a recessive manner, is characterized by extremely low
levels of or undetectable vWF. Type 2 includes all qualitative
deficiencies of vWF and is subdivided into 4 subtypes. Among type 2 vWD, type 2N is inherited in a recessive manner and is initially
characterized by a normal multimeric pattern and normal
platelet-dependent function but a markedly decreased affinity of vWF
for FVIII, leading to a secondary deficiency in plasma FVIII
levels.5,6
Formation of a noncovalent complex with vWF is required for the normal
survival of FVIII in the blood circulation.7,8 The
association between FVIII and vWF shows characteristics of both
electrostatic and hydrophobic interactions.9 Binding
domains within each protein have been identified. The light chain of
FVIII contains 2 distinct regions that associate with vWF. One is an acidic cluster in the amino terminus of A3 domain (aa
1670-1684),10-13 and the other is localized in the C2
domain (aa 2173-2332).14,15 On vWF, the FVIII binding site
resides within the tryptic fragment consisting of the first 272 aa of
the mature subunit.16,17 This part of vWF, named SpIII-T4,
contains 24 cysteine residues, all of which are involved in
intrasubunit disulfide bonds required for the functional conformation
of this domain.16 Furthermore, the epitopes of anti-vWF
monoclonal antibodies (mAbs), which block FVIII binding to vWF, have
been mapped to aa residues in position 2-53,18
51-60,19 and 78-96.20 These data,
suggesting that the FVIII binding site consists of discontinuous
sequences, were reinforced by the identification of
molecular gene defects found in patients with type 2N vWD. To date,
several missense mutations reported in the database (Ginsburg and
Sadler21; Internet Web page
http://mmg2.im.med.umich.edu/vWF) have been localized on aa sequences
previously defined as epitopes of mAbs, which inhibit FVIII/vWF
interaction. Affected persons with normal vWF levels were generally
found to be either homozygous or compound heterozygous for type 2N vWD
mutations, in agreement with the recessive inheritance pattern of this
variant disease. Another group of patients is characterized by
moderately decreased vWF levels, whereas FVIII levels are
disproportionately low. Such patients are compound heterozygous for
type 2N and type 3 vWD mutations localized on separate
alleles.6
In this paper, 2 new candidate mutations affecting cysteine residues 25 and 95 in the mature vWF subunit were identified in the vWF gene of 3 patients classified as type 2N vWD. These mutations were characterized
by expression in COS 7 cells of the corresponding mutated rvWF
(Tyr25rvWF and Phe95rvWF). In addition to the FVIII/vWF interaction
defect, both mutations gave rise to a secretion defect and to an
abnormal multimeric pattern of rvWF. The use of mAbs directed to the
N-terminal part of vWF aroused suspicion of conformational change,
which was emphasized by the detection of free sulfhydryl groups.
Reagents
Routine coagulation tests
Molecular genetic studies Genomic DNA was extracted from peripheral blood leukocytes, and exons 18 to 23 of vWF gene were amplified by polymerase chain reaction (PCR) as described previously.23 After treatment by shrimp alkaline phosphatase and exonuclease I (according to the manufacturer's instructions) to remove any remaining dNTPs and primers, the PCR products were directly sequenced using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham).
Restriction enzyme analysis The nucleotide substitution in exon 20 at codon 854 of the vWF cDNA destroys an MspI site. PCR-amplified exon 20 was digested with MspI, according to the manufacturer's recommendation, then electrophoresed in agarose gel. The fragment resulting from the loss of an MspI restriction site was cut from the gel and sequenced as described above.Plasmid constructions The previously described plasmid pSVvWF, containing the full-length cDNA of human vWF, has been used as a template for the construction of the mutant plasmids. Plasmids pSV25Tyr, pSV95Phe, and pSV91Gln were derived from pSVvWF by introducing a G A transition at
nucleotide (nt) 2363, a GT transversion at nt 2573, and a GA transition at nt 2561, respectively, using the Transformer site-directed mutagenesis kit (Clontech). The oligonucleotide primers
used for mutagenesis are listed in Table 1.
The primers 25A, 95A, and 91B were used to introduce the desired
mutation into pSVvWF, and the primer NheI-O was used to destroy the
unique, previously created NheI restriction site of the plasmid for the purpose of selection. Clones containing the desired mutation were digested by HindIII and NotI, and the resultant
fragment was cloned into pSVvWF treated with the same restriction
enzymes. The oligonucleotide sequence between HindIII (nt 2235)
and NotI (nt 2880) restriction sites of all constructs was
verified by sequencing as described above.
Expression of recombinant vWF COS-7 cells were transiently transfected with plasmids pSVvWF (wild-type) and pSV25Tyr, pSV91Gln, and pSV95Phe (mutants) using the diethylaminoethyl-dextran method and culture conditions previously described.24 Forty hours after transfection, cells were incubated in serum-free Dulbecco's modified Eagle's medium for 72 hours. Then cell-conditioned media were collected into a final concentration of 5 mmol/L EDTA and 2 mmol/L PMSF. Expression levels in conditioned media were measured by enzyme linked immunosorbent assay (ELISA).25Monoclonal antibodies All mAbs were purified from ascites fluid by chromatography on protein A Sepharose CL-4B according to the manufacturer's instructions. Directed to the A2 domain of FVIII heavy chain (aa 372-740), mAb 52H7 was biotinylated and used to detect bound FVIII in the FVIII binding assay. Biotinylation of mAb 52H7 was performed using BAC-NHS as follows: purified IgG at a concentration of 2 mg/mL in Tris-buffered saline (Tris 25 mmol/L, NaCl 150 mmol/L, pH 7.35) was gently mixed with 100-fold molar excess of freshly prepared biotinylation reagent. The reaction was allowed to proceed for 2.5 hours at 22°C and was followed by gel filtration on a NAP5 column equilibrated in Tris-buffered saline. Fractions containing the biotinylated IgG were pooled and stored at 4°C.FVIII binding assay The binding of FVIII to vWF was assayed as previously described26 with minor modifications. Briefly, increasing amounts of vWF were immobilized by binding to anti-vWF mAb 9311A2-coated microplates. Recombinant FVIII (Baxter Healthcare, Glendale, CA) was incubated with the immobilized vWF, and the bound rFVIII was then measured by incubating for 1 hour at 37°C successively 100 µL biotinylated mAb 52H7 and 100 µL streptavidin peroxidase conjugate (1:1000 dilution). Peroxidase activity was determined using O-phenylenediamine substrate. The absorbance at 492 nm was then read using a Vmax microplate reader (Molecular Devices, Menlo Park, CA). After washing, the relative amount of immobilized vWF in each well was also measured by ELISA using peroxidase-conjugated mAb 333A9 (aa 911-1365) as a detector and a pool of normal plasma as a standard.Comparative recognition of vWF by mAbs The ability of mAbs to capture wild-type (WT) or mutant rvWF was examined by ELISA as previously described.26 Absorbance at 492 nm was converted to mU/mL vWF using the standard curve generated for each capture mAb using dilutions of a pool of normal plasma.Detection of free cysteine residues EZ-link biotin-HPDP (Pierce) is a biotinylation reagent that reacts specifically with thiol groups. Biotinylation of rvWF was carried out according to the manufacturer's instructions with minor modifications. Conditioned medium (0.5 mL) was applied to a NAP5 column equilibrated in phosphate-buffered saline (PBS)-EDTA (20 mmol/L phosphate buffer, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA), and the amount of recovered proteins was quantified using the BCA protein assay reagent. On a part of the recovered proteins, 3 mol/L guanidine HCl was added and incubated for 30 minutes before biotin-HPDP was added. Biotin-HPDP was added at 0.1 mmol/mg protein, and biotinylation was performed by incubation for 90 minutes at room temperature. The excess of reagent was removed by gel filtration onto a NAP5 column. Serial dilutions of eluate analyzed in duplicate were incubated for 1 hour at 37°C in microtiter wells coated with anti-vWF polyclonal antibodies. Wells were then washed in PBS-T, and the amount of immobilized rvWF was detected by anti-vWF peroxidase-conjugated polyclonal antibodies, whereas biotin was detected by streptavidin-peroxidase (1:1000 dilution). After additional washing with PBS-T, O-phenylenediamine substrate was added to the wells, and the reaction was stopped by the addition of 1 mol/L H2SO4.
Case report and phenotypic analysis Three patients from 2 unrelated families were referred to us for mild to moderate bleeding disorders. Routine biologic data are summarized in Table 2. Patient 1, a woman born in 1972 who has a moderate mucosal bleeding, shows a prolonged bleeding time and decreased levels of plasma vWF associated with disproportionately lower FVIII levels. The patient's mother and sister, who are asymptomatic, have normal FVIII levels (98 and 66 U/dL, respectively) but a borderline (50 U/dL) and slightly decreased (42 U/dL) plasma vWF level, respectively. Patient 2, a woman born in 1977 and the first cousin of patient 1, had mild bleeding after tonsillectomy and adenoidectomy. She has normal bleeding time and normal vWF levels but moderately decreased FVIII levels. Patient 3, a woman born in 1929, has had a lifelong history of bleeding. She had epistaxis, excessive bleeding after tooth extraction, and was treated in 1980 with intermediate-purity FVIII concentrate for hysterectomy. In 1990, she was found seropositive for hepatitis C markers. She was treated with vWF concentrate (facteur Willebrand-LFB) for liver biopsy in 1996 and for mastectomy in 1999. The current biologic data of this patient indicate normal bleeding time and normal plasma vWF levels but significantly decreased FVIII levels. However, 20 years ago, the newly diagnosed FVIII deficiency was associated with borderline plasma vWF levels (54 U/dL vWF:Ag; 50 U/dL vWF:RCo).
Identification of mutations
Expression of recombinant vWF To determine the effect of the missense mutations on vWF structure and function, site-directed mutagenesis was used to introduce G2363A, G2573T, and G2561A nt substitutions individually into the full-length vWF expression vector. After 5 separate transient expression experiments in COS-7 cells of WT and mutant plasmids, the secretion amounts of rvWF in conditioned media were quantified. As shown in Figure 2, WT and Gln91rvWF were secreted efficiently, achieving a concentration of 10.6 ± 0.4 U/dL and 10.7 ± 1.6 U/dL (mean ± SD), respectively. In contrast, the secretion level of Tyr25rvWF was significantly reduced to 4.7 ± 0.2 U/dL, whereas hybrid Tyr25/Gln91 and Tyr25/WT rvWF, obtained by cotransfection of a 1:1 ratio of corresponding plasmids, were secreted to 5.9 ± 0.4 and 6.6 ± 0.06 U/dL, respectively. The Cys95Phe mutation induced a poor secretion level of rvWF, reaching a concentration of only 2.0 ± 0.3 U/dL in conditioned media. Secretion levels of hybrid Phe95/Gln91 and Phe95/WT rvWF were both decreased to approximately 5.4 U/dL.
Effect of mutations on FVIII binding capacity of rvWF The ability of the mutated rvWF to bind FVIII was measured using the solid-phase binding assay described in "Materials and methods." Mutated Tyr25rvWF failed to bind FVIII, whereas the slope value for the regression line obtained with Gln91rvWF was reduced to 10% compared with WT rvWF (Figure 3A). The hybrid Tyr25/Gln91 rvWF gave an FVIII binding regression line intermediate between that of Tyr25 and Gln91rvWF. As expected, the FVIII binding capacity of hybrid Tyr25/WT rvWF was decreased to 50% of WT, in agreement with the recessive feature of type 2N mutations.
Multimeric pattern of rvWF For both WT and Gln91rvWF, multimers varied in size from protomer to HMW multimers (15 mer or more) (Figure 4A, lanes 1 and 3). The Tyr25rvWF gave an abnormal multimeric profile characterized on one hand by a slight decrease in multimers by 5 mer or more and on the other hand by a smeared feature of HMW multimers (Figure 4A, lane 2). However, the pattern of hybrid Tyr25/Gln91 and Tyr25/WT rvWF appeared normal (Figure 4A, lanes 4 and 5).
Detection of free cysteine residues
Recognition of rvWF by mAbs
In this report, we describe 2 new missense mutations within the
N-terminal part of the mature vWF subunit that were identified in 3 patients and were characterized by a discrepancy between FVIII and vWF
levels, consistent with a markedly decreased capacity of vWF to bind
FVIII. A Tyr substitution for Cys at position 25 of the mature vWF
subunit was detected, at heterozygous state, in the vWF gene of the
related patients 1 and 2. Patient 1 appeared to be a compound
heterozygote for 2 recessive mutations: 1 quantitative (D93ter) type 3 mutation inherited from her mother and 1 qualitative (Cys25Tyr) type 2N
mutation inherited from her father. In patient 2, the first cousin of
patient 1, the second allele was found to contain the Arg91Gln type 2N
mutation.28 This frequent mutation was also found in
patient 3 in association with a Phe95 for Cys substitution originating
from the other allele. The phenotype of patients 2 and 3, who are
compound heterozygotes with 2 type 2N mutations, is typical of type 2N
vWD We thank Baxter Healthcare Corporation for the generous gift of
recombinant FVIII. We also thank V. Barylo, S. Belmont, and D. Hoguet
for their excellent technical assistance, and V. Tancré for
typing the manuscript.
Submitted July 14, 1999; accepted January 18, 2000.
Reprints: S. Jorieux, Département Recherche et
Développement, Laboratoire Français du Fractionnement et
des Biotechnologies, 59, rue de Trévise, BP 2006, 59,011 Lille
cédex, France.
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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Top
Abstract
Introduction
-33P] dATP and 2 µmol cold dCTP, dGTP,
and dTTP. The resultant labeled multiplexes were heat denaturated in
the presence of 50% formamide, run on a 0.5 × MDE
nondenaturing sequencing gel for 16 hours at 700 V, and visualized by
overnight autoradiography.
) WT; (
) Tyr25;
(
) Gln91; (
) hybrid Tyr25/Gln91; (
) hybrid Tyr25/WT. (B)
Mutant and hybrid Phe95rvWF: (
, 
ie, vWF was normal except for a markedly decreased affinity for
FVIII.
