Expression of two type 2N von Willebrand disease mutations identified in exon 18 of von Willebrand factor gene.

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factor VIII (FVIII). The FVIII binding site has been localized within the first 272 amino acid residues of mature VWF, encoded by exons 18-23. Two substitutions in exon 18 of VWF gene, inducing candidate mutations Y795C and C804F were identified in the heterozygous state in two French patients who also displayed the frequent R854Q mutation in exon 20. Expression studies in Cos-7 cells showed that these abnormalities, which implicate cysteine residues, induced secretion, multimerization and FVIII binding defects of corresponding recombinant VWF. Results from transfection experiments with R854Q, performed to reproduce the hybrid VWF present in patient plasma, were in agreement with those obtained for patient's plasma VWF. These findings confirm the importance of the VWF D' domain in FVIII binding. In addition, this work shows that exon 18 should preferentially be sequenced in type 2N VWD patients when the frequent R854Q mutation in exon 20 has been excluded or detected in the heterozygous state.

Conformational changes in the D' domain of von Willebrand factor induced by CYS 25 and CYS 95 mutations lead to factor VIII binding defect and multimeric impairment.

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.

Unusual N-glycosylation of a recombinant human erythropoietin expressed in a human lymphoblastoid cell line does not alter its biological properties.

Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycosylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser-126) and involved in the regulation of the level of red blood cells. Today, only one recombinant human Epo (rHuEpo), produced in CHO cell line, is extensively used in therapy to cure severe anemia. The structure of the glycan chains of this rHuEpo slightly differ of those of the urinary human Epo (uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a human lymphoblastoid cell line, named RPMI 1788. In order to fully characterize the Epo-RPMI, structural characterizations of the protein skeleton as well as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of some N-glycan chains, as mainly determined by ESI-MS, revealed some unusual characteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a sialyl Le(x)motif, and 13% contain more than three LacNAc repeats (up to five per molecule). Despite these unusual structural characteristics, the data concerning the in vitro and in vivo biological activities were not impaired when compared to Epo-CHO and uHuEpo.

Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function.

The functional defect caused by substitution of Arg527 (--> Trp) and Arg531 (--> Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII-R527W, the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr511-Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val517-Arg527 are located near the FIXa binding site Ser558-Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558-Gln565 and Val517-Arg527.

Some factor VIII (FVIII) inhibitors recognise a FVIII epitope(s) that is present only on FVIII-vWF complexes.

A mild haemophilia A patient (LE) with an Arg2150His mutation in the C1 domain of the factor VIII (FVIII) light chain was shown to have anti-FVIII antibodies inhibiting wild type but not self FVIII. Polyclonal anti-FVIII antibodies of this patient were purified by affinity adsorption using recombinant FVIII (rFVIII) and/or plasma-derived FVIII-von Willebrand factor (vWF) complexes. A distinct population of antibodies was obtained that bound to FVIII-vWF complexes but not to rFVIII, indicating that an epitope was created by the association of FVIII to vWF. Such antibodies belonged to the IgG2 isotype, but the FVIII epitopes to which they bind could not be mapped with precision due to vWF dependency. Depletion experiments showed that anti-FVIII antibodies recognising FVIII-vWF complex also distinguished wildtype from mutated self FVIII, indicating that the Arg2150His mutation alters the B cell epitope formed by the association of FVIII to vWF. To determine whether the Arg2150His substitution also alters the formation of the FVIII-vWF complex, the interaction between mutated or normal FVIII with vWF was evaluated in plasma. The dissociation rate of mutated FVIII from vWF was found to be significantly increased. The presence of an Arg2150His mutation therefore results in the disappearance of a FVIII B cell epitope generated by the association of FVIII with vWF. Patients carrying such an Arg2150His mutation and receiving infusion of wild-type FVIII may therefore be at risk of developing inhibitors to allogeneic FVIII only.

A novel mutation in the D3 domain of von Willebrand factor markedly decreases its ability to bind factor VIII and affects its multimerization.

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 C-->T 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.

Gene defects in 150 unrelated French cases with type 2 von Willebrand disease: from the patient to the gene. INSERM Network on Molecular Abnormalities in von Willebrand Disease.

Type 2 vWD is defined by qualitative defects of vWF and is subdivided into four subtypes: 2N, 2B, 2A and 2M. The characterization of 150 unrelated French cases with type 2 vWD emphasizes the heterogeneity of this group. In 51 cases of type 2N vWD, new mutations were found not only in the D' domain (Cys25Tyr and Cys95Phe) but also in the D3 domain (Asp116Asn and Cys297Arg). In 42 cases of type 2B vWD, no new mutation was detected. In 45 cases with type 2A phenotype, three new candidate mutations were found in the A2 domain: Gln793Arg, Val841Phe and Leu876Pro. In addition, four new candidate mutations were detected in the A1 domain: Cys509Gly, Arg545His, Arg552Cys and Cys695Tyr. Finally, five new candidate mutations were identified in 12 patients with 2M (or unclassified) phenotype: Leu513Pro, Gly561A1a, Glu596Lys, Arg611Leu and IIe662Phe. For all candidate mutations, expression studies are in progress. This study of a large number of French variants of vWD brings further insight into the relationship between phenotype and genotype.

Evaluation of a new automated ELISA test for von Willebrand factor using two monoclonal antibodies.

A new automated quantitative enzyme linked immunosorbent assay (ELISA) for the detection of human von Willebrand factor (vWF), VIDAS vWF, has been developed for use on the VIDAS analyser (bioMérieux). The two-step capture/tag test relies on two monoclonal antibodies (mAbs), the second one being labelled with alkaline phosphatase. The lower limit of detection of the assay is < 1 IU/dl, and the upper limit of detection is 120 IU/dl. There is no hook effect for concentrations up to 1100 IU/dl. Intra- and inter-assay precision ranges from 3% and 5%. Assays are performed preferentially using citrated plasma and in these conditions the 95% confidence intervals for normal values are 52-154 IU/dl and 60-200 IU/dl for O blood group and non-O blood group subjects, respectively. Using the lower limits of the normal range as the cut-off level, all patients tested with type 1 (n = 29) or 3 (n = 2) von Willebrand disease (vWD) would be accurately classified with the new ELISA. Comparing the VIDAS test with a conventional ELISA gave a good correlation and comparable results with type 1 and 3 vWD patients (n = 31; r = 0.99; y = 0.99x + 0.24), type 2A and 2B vWD patients (n = 20; r = 0.99; y = 1.05x-1.65) and normal subjects (n = 204; r = 0.94; y = 1.06x-2.6).

A patient with type 2N von Willebrand disease is heterozygous for a new mutation: Gly22Glu. Demonstration of a defective expression of the second allele by the use of monoclonal antibodies.

We report the case of a Chinese patient who has subnormal von Willebrand factor (vWF) level and normal vWF multimeric pattern, but a lack of vWF capacity to bind factor VIII (FVIII). Exons 18 to 20 of the patient's vWF gene were analyzed by DGGE and a G2354 --> A substitution which changes the encoded amino acid sequence from Gly22 to Glu was identified. The patient is heterozygous for this substitution, creating a unique Sac I restriction site. Recombinant vWF (rvWF) containing the candidate mutation was transiently expressed in COS-7 cells. It was processed and secreted normally but failed to bind FVIII. FVIII binding ability of hybrid rvWF, obtained by cotransfection of normal and mutated expression vectors and corresponding to a heterozygous genotype, was moderately decreased. To explain this functional discrepancy between patient's plasma vWF and hybrid rvWF, we used anti-vWF monoclonal antibodies (MoAbs) as capture in an enzyme-linked immunosorbent assay test. MoAb 32B12 recognized both wild-type and mutated rvWFs whereas MoAb 418 did not recognize mutated rvWF. Because MoAb 418 also failed to capture the plasma vWF from propositus, it means that his second nonmutated allele is not expressed or expressed at a very low level.

Biological effect of desmopressin in eight patients with type 2N ('Normandy') von Willebrand disease. Collaborative Group.

It is generally thought that the plasma increase in factor VIII (FVIII) after desmopressin (dDAVP) infusion is related to the plasma increase in von Willebrand factor (vWF), which is the plasma carrier for FVIII. The aim of this study was to evaluate the FVIII and vWF responses in patients with type 2N vWD, characterized by the mild FVIII deficiency related to markedly decreased affinity of vWF for FVIII. At different times after one intravenous dose of dDAVP (0.3 or 0.4 microgram/kg) we measured the FVIII coagulant activity, FVIII antigen, vWF antigen and ristocetin cofactor activity, in eight patients with either Arg91Gln or Arg53Trp amino acid substitution in mature vWF. In all the patients, whatever their mutation, the dDAVP infusion resulted in a 2.3 +/- 0.7-fold increase of vWF and a variable rise (9.5 +/- 7.7 times) of FVIII, whereas the vWF capacity to bind FVIII was not improved. The FVIII response was more transient than vWF response, and FVIII half disappearance time was evaluated to be approximately 3 h. The data indicate that the stabilizing effect of vWF on FVIII is not responsible for the FVIII increase induced by dDAVP. The clinical implication of this study is that, in the 2N vWD patients, dDAVP may be a useful prophylactic or curative treatment when the test dose has been shown to be effective to reach a haemostatic FVIII level.

Fine epitope mapping of monoclonal antibodies to the NH2-terminal part of von Willebrand factor (vWF) by using recombinant and synthetic peptides: interest for the localization of the factor VIII binding domain.

Two different approaches were used in order to define the epitope of three monoclonal antibodies (MoAbs) against the NH2-terminal part of the mature subunit of von Willebrand factor (vWF) which contains its factor VIII (FVIII) binding site. First, a vWF cDNA fragment library using the bacteriophage lambda gt11 expression vector was screened with radiolabelled MoAbs. The epitope of each MoAb was defined, following sequence analysis, by the overlapping DNA sequence of immunoreactive clones. MoAb 32B12, a potent inhibitor of FVIII/vWF interaction, binds within the Glu35-Ile81 sequence of vWF subunit. MoAb 14A12, a non-inhibitory antibody, recognizes a sequence within Thr141-Val220. MoAb 31H3, a partial inhibitory antibody, gives no positive clone. In the second method, a panel of 24 synthetic pentadecapeptides corresponding to the first NH2-terminal 105 amino acid residues was used to block the binding of inhibitor MoAbs to immobilized vWF in an ELISA system. The localization of MoAb 32B12 epitope was confirmed and restricted to the Met51-Ala60 sequence. The MoAb 31H3 binding to vWF is inhibited by two synthetic peptides with the overlapping sequence Cys66-Gly76. All these data confirm that the FVIII binding site of vWF is not limited to the binding area (Thr78-Thr96) of the previously described MoAbs inhibiting FVIII/vWF interaction but is composed of several key sequences.

Recombinant von Willebrand factor Arg578-->Gln. A type IIB von Willebrand disease mutation affects binding to glycoprotein Ib but not to collagen or heparin.

von Willebrand factor (vWF) is a multimeric plasma glycoprotein that mediates platelet adhesion to the subendothelium via binding to platelet glycoprotein Ib (GPIb) and to components of the vessel wall. Recently, missense mutations that cause type IIB von Willebrand disease (vWD) were described, clustered within a disulfide loop in the A1 domain of vWF that has binding sites for GPIb, collagen, and heparin. In type IIB vWD, plasma vWF exhibits increased affinity for platelet GPIb, but decreased binding to collagen and heparin. The effect was studied of a type IIB vWD mutation, Arg578-->Gln, on the interaction of vWF with GPIb, collagen, and heparin. Recombinant wild type rvWF and mutant rvWF(R578Q) were expressed in COS-7 cells. Ristocetin-induced binding of rvWF(R578Q) to GPIb was markedly increased compared with rvWF, confirming that the Arg578-->Gln mutation causes the characteristic gain-of-function abnormality of type IIB vWD; botrocetin-induced binding was only slightly increased. Binding to collagen type III and heparin-agarose was compared for rvWF(R578Q) and plasma vWF from patients with four different type IIB mutations: Arg543-->Trp, Arg545-->Cys, Val553-->Met, Arg578-->Gln. For all of the plasma samples, binding to collagen and to heparin was reduced compared with normal plasma. In contrast, binding of rvWF(R578Q) to collagen and heparin was normal compared with wild type rvWF. Therefore, the Arg578-->Gln mutation increases the affinity of vWF for GPIb but does not directly impair vWF interaction with collagen or heparin. Arg578 may therefore be necessary to prevent normal vWF from interacting with GPIb. In type IIB vWD, the defective binding of plasma vWF to collagen and heparin may be secondary to post-synthetic modifications that occur in vivo, such as the loss of high molecular weight vWF multimers.

The mutation Arg (53)----Trp causes von Willebrand disease Normandy by abolishing binding to factor VIII. Studies with recombinant von Willebrand factor.

von Willebrand factor (vWF) and factor VIII (FVIII) circulate in plasma as a noncovalently linked protein complex. The FVIII/vWF interaction is required for the stabilization of procoagulant FVIII activity. Recently, we reported a new variant of von Willebrand disease (vWD) tentatively named "Normandy," characterized by plasma vWF that appears to be structurally and functionally normal except that it does not bind FVIII. Three patients from one family were found to be homozygous for a C----T transition at codon 816 converting Arg 53 to Trp in the mature vWF subunit. To firmly establish a causal relationship between this missense mutation and vWD Normandy phenotype, we have characterized the corresponding recombinant mutant vWF(R53W). Expressed in COS-7 cells or CHO cell lines, normal vWF and vWF(R53W) were processed and formed multimers with equal efficiency. However, vWF(R53W) exhibited the same defect in FVIII binding as did plasma vWF from patients with vWD Normandy, confirming that this mutation is responsible for the vWD Normandy phenotype. These results illustrate the importance of Arg 53 of the mature vWF subunit for the binding of FVIII to vWF, and identify an amino acid residue within a disulfide loop not previously known to be involved in this interaction.

Processing and characterization of recombinant von Willebrand factor expressed in different cell types using a vaccinia virus vector.

The cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgII is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of two point mutations in the von Willebrand factor gene of three families with the 'Normandy' variant of von Willebrand disease.

Plasma von Willebrand factor (vWf) is a multi-domain multimerized glycoprotein which has a dual role in haemostasis: it promotes platelet adhesion to subendothelium and is the carrier of blood coagulation factor VIII (FVIII). We previously characterized a functional defect of vWf, limited to its ability to bind FVIII, in two families whose affected members have the same phenotype that mimics mild haemophilia A and was tentatively named von Willebrand's disease (vWD) 'Normandy'. A homozygous point mutation C----T converting Thr 28 to Met in mature vWf subunit was identified in one of these patients who was born of third-cousin parents. In the present studies we report two unrelated new cases of vWD 'Normandy' and characterize, using the analysis of the vWf gene intron 40 region containing a variable number of tandem repeats, the recessive inheritance of the disease in two affected families without known consanguinity. Exons 18-24 of the vWf gene encoding for the first 311 amino acids of mature vWf subunit were amplified by the polymerase chain reaction method and sequenced. Two new missense mutations, both corresponding to a C----T transition and predicting respectively an Arg 53----Trp and an Arg 91----Gln substitution, were characterized. The three patients from family 1 were homozygous for the first-mentioned mutation while the patient from family 3 was homozygous for the second. The patient from family 2 was found a compound heterozygote for the two mutations. None of the two point mutations reported, both destroying a MspI restriction site, could be detected in DNA from 50 normal controls screened by restriction endonuclease analysis. Our data show that different mutations may be found in patients with the 'Normandy' phenotype. The mutations characterized so far are all localized on the N-terminal region of mature vWf subunit, within or near the major FVIII binding domain, and some of them occur within the epitope of monoclonal antibodies inhibiting the vWf/FVIII interaction. These observations suggest a causal relationship between these mutations and the vWD 'Normandy' phenotype.

Expression of von Willebrand factor "Normandy": an autosomal mutation that mimics hemophilia A.

von Willebrand disease Normandy (vWD Normandy) is a recently described phenotype in which a mutant von Willebrand factor (vWF) appears structurally and functionally normal except that it does not bind to blood coagulation factor VIII. This interaction is required for normal survival of factor VIII in the circulation; consequently, vWD Normandy can present as apparent hemophilia A but with autosomal recessive rather than X chromosome-linked inheritance. A vWF missense mutation, Thr28----Met, was identified in the propositus in or near the factor VIII binding site. The corresponding mutant recombinant vWF(T28M) formed normal multimers and had normal ristocetin cofactor activity. However, vWF(T28M) exhibited the same defect in factor VIII binding as natural vWF Normandy, confirming that this mutation causes the vWD Normandy phenotype. The distinction between hemophilia A and vWD Normandy is clinically important and should be considered in families affected by apparent mild hemophilia A that fail to show strict X chromosome-linked inheritance and, particularly, in potential female carriers with low factor VIII levels attributed to extreme lyonization.

The "Normandy" variant of von Willebrand disease: characterization of a point mutation in the von Willebrand factor gene.

We previously reported a functional defect of von Willebrand factor (vWF) in a new variant of von Willebrand disease (vWD) tentatively named vWD "Normandy." The present work has attempted to characterize the molecular abnormality of this vWF that fails to bind factor VIII (FVIII). The immunopurified vWF from normal and patient's plasma were digested by trypsin and the resulting peptides were compared. The electrophoresis of "vWF Normandy" showed a shift in the band corresponding to a polypeptide from amino acid 1 to 272. Consequently, we performed the molecular analysis of the portion of the vWF gene of this patient encoding this amino acid sequence. Exons 18-24 were amplified by the use of polymerase chain reaction and their nucleotide sequences corresponding to 1.8 kb were determined. Our analysis showed a point mutation C to T at codon 791, resulting in the substitution of Methionine for Threonine at position 28 of the mature vWF subunit. Because this nucleotide substitution destroyed a Mae II restriction site, this mutation was conveniently sought in various individual DNAs. The patterns obtained were consistent with the homozygous and heterozygous state of this mutation in the patient and in her son, respectively, and with its absence in 28 normal individuals. We conclude that Threonine at position 28 in plasma vWF may be crucial for the conformation and FVIII-binding capacity of its cystine-rich N-terminal domain.

In vitro evaluation of a very-high-purity, solvent/detergent-treated, von Willebrand factor concentrate.

A very highly purified von Willebrand factor (vWF) concentrate was analyzed in order to evaluate its suitability for the treatment of von Willebrand's disease (vWD). The functional activity of vWF assessed by its ristocetin cofactor activity (vWF:RCo) correlated with the level of vWF antigen (vWF:Ag), with the vWF:RCo/vWF:Ag ratios ranging from 0.56 to 1.02, and the specific activity being always greater than 50 IU vWF:RCo/mg protein. Electrophoretic analysis showed a normal pattern of high, intermediate and low-molecular-weight multimers of vWF. The biological activity of vWF was also evaluated by studying its ability to bind to collagen and to platelet receptors in the presence of either ristocetin or thrombin. Furthermore, these functional activities of vWF were confirmed by the capacity of this concentrate to induce platelet adhesion to collagen in a perfusion system. Moreover, the vWF present in this preparation was able to bind factor VIII. All these in vitro data suggest that this preparation is likely to be effective in the treatment of vWD patients.

Evidence for a von Willebrand factor defect in factor VIII binding in three members of a family previously misdiagnosed mild haemophilia A and haemophilia A carriers: consequences for therapy and genetic counselling.

A plasma von Willebrand factor (vWf) defect limited to its failure to bind factor VIII (FVIII) was previously characterized in a woman with FVIII deficiency and normal primary haemostasis. By using in vitro tests we found a similar pattern in three siblings of another family previously thought to be affected with mild haemophilia A. Furthermore, a decrease in vWf ability to bind FVIII was found in the parents and the brother of the three patients. This decrease was consistent with heterozygous expression of a recessive vWf gene abnormality. FVIII deficiency was corrected by infusion with a vWf concentrate almost devoid of FVIII coagulant activity. FVIII recovery and half-life thus obtained showed that this treatment was more effective than a FVIII infusion performed by way of comparison. These results indicate that this vWf defect may account for FVIII deficiency in patients without the usual laboratory and clinical features of von Willebrand's disease. Changes in therapy and genetic counselling following the new diagnosis in this family emphasize the need to search for such a vWf defect in patients in whom FVIII deficiency is not obviously X-linked.