An Arg760Cys mutation in the consensus sequence of the von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant

We describe a von Willebrand disease (VWD) variant characterized by the persistence of von Willebrand factor (VWF) propeptide as a result of a C>T transition at nucleotide 2527 in exon 17 of the VWF gene. This mutation, which was present in the proband and his father, predicts the substitution of Cys for Arg at position 760 of pre-pro-VWF, 4 residues before the propeptide cleavage site belonging to a consensus sequence for substrate recognition by the processing enzyme paired dibasic amino acid-cleaving enzyme (PACE)/furin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) documented the presence of both processed and unprocessed VWF in the patient's plasma, with unprocessed VWF relatively less represented. The patient's hemostatic phenotype was characterized by a mild decrease in plasma factor VIII (FVIII) and VWF, a decrease in plasma VWF multimers, and a mild reduction in the FVIII binding capacity of VWF. The FVIII binding defect was more pronounced in the proband than in the father because he also inherited the type 2N Arg91Gln mutation from his mother. The persistence of VWF propeptide did not impair VWF synthesis because platelet VWF content was normal, nor did it compromise VWF storage in endothelial cells, because of the normal post-1-deamino-8-D-arginine vasopressin (DDAVP) increase in plasma VWF. Coexpression of wild-type and Arg760Cys VWF into a Furin-producing BHK cell line resulted in decreased VWF secretion and a defect in the FVIII binding capacity of VWF, together with the persistence of VWF propeptide. These findings confirm that a normal consensus sequence for VWF propeptide cleavage and efficient cleavage are required in vivo for normal FVIII binding capacity of VWF.     

Identification of new type 2B von Willebrand disease mutations: Arg543Gln, Arg545Pro and Arg578Leu

We report the identification in five patients (three families) affected with type 2B von Willebrand disease (VWD) of three heterozygous nucleotide substitutions at the codon for arginine 543, 545 and 578 of the mature von Willebrand factor (VWF) subunit resulting in a glutamine, proline and leucine substitution, respectively. These mutations are located in the A1 loop where prevalent type 2B mutations (Arg543Trp, Arg545Cys and Arg578Gln) have been already identified at the same positions. By in vitro mutagenesis of full-length cDNA of VWF and transient expression in Cos-7 cells, we have shown that the six corresponding mutated recombinant VWFs (Gln543, Trp543, Cys545, Pro545, Leu578 and Gln578 rVWF) exhibited quantitatively normal expression and normal multimeric pattern but increased ristocetin- and botrocetin-induced binding to platelets as compared with that for wild-type rVWF. The two mutations at position 545 induced the greatest reactivity for GPIb of corresponding rVWFs as compared to the two mutations at positions 543 and 578.     

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.     

Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor

We compared the properties of plasma von Willebrand factor (vWF) from normal individuals and from two patients with type IIA (Glu875Lys) and type IIB (duplication of Met 540) von Willebrand disease (vWD) with the corresponding fully multimerized recombinant proteins. We included cryosupernatant from normal human plasma and type IIA plasma (Cys509Arg). Functions of vWF were analyzed by binding assays to platelets in the presence of ristocetin or botrocetin. Parameters of binding (number of binding sites per vWF subunit, and dissociation constant Kd) were quantitatively estimated from the binding isotherms of 125I-botrocetin or glycocalicin to vWF, independently of the size of the multimers. We found that ristocetin- or botrocetin-induced binding to platelets was correlated in all cases with the size of vWF multimers. In the absence of inducer, only type IIB rvWF Met-Met540 spontaneously bound to platelets. No significant difference of binding of purified botrocetin to vWF was found between normal and patients' plasma, or between wild-type rvWF (rvWF-WT) and rvWF-Lys875. In contrast, affinity of botrocetin for type IIB rvWF Met-Met540 was decreased. Botrocetin-induced binding of glycocalicin to vWF from all plasma and cryosupernatant was similar. Compared with rvWF-WT, binding of glycocalicin to rvWF-Lys875 was normal. In contrast, the affinity for type IIB rvWF Met-Met540 was 10-fold greater. Thus, our data suggest that, in the patients tested, the abnormal IIA phenotype results from the lack of large-sized multimers and is independent of the point mutations. In contrast, the type IIB mutation is directly involved by providing a conformation to the vWF subunits that allows the high molecular weight multimers to spontaneously interact with platelet glycoprotein Ib.     

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.     

The defective interaction between von Willebrand factor and factor VIII in a patient with type 1 von Willebrand disease is caused by substitution of Arg19 and His54 in mature von Willebrand factor

In this report we describe the further investigation of the von Willebrand factor (vWF)/FVIII interaction in a type 1 von Willebrand disease patient characterized by discrepant VIII:C levels as determined by one-stage and two-stage VIII:C assays. A solid-phase binding assay shows that this patient's plasma vWF is moderately defective in capturing recombinant FVIII. Sequence analysis of the FVIII-binding domain encoded by the vWF mRNA of the affected individual identified mutations in both vWF alleles. In allele A, the mutations C2344T and T2451A result in the substitution of Trp for Arg19 (R19W) and of G1n for His54 (H54Q) in mature vWF, respectively. This allele also contains a reported polymorphism (A2365G, Thr26Ala). Allele B, which is underexpressed at the RNA level, contains a one-nucleotide deletion in the FVIII-binding domain (delta G2515) that results in the premature termination of translation. Analysis of the binding of FVIII by full- length vWF transiently expressed in COS-7 cells confirms that the combined R19W and H54Q substitutions are the cause of the defective vWF/FVIII interaction in this patient. The FVIII-binding defect of vWF containing either mutation alone is approximately half that of the double mutant, which suggests that the effect of these mutations is additive. The mutant proteins are recognized equally well by vWF monoclonal antibodies MBC105.4, 32B12, and 31H3, which block the binding of FVIII by vWF, indicating that amino acids Arg19, Thr26, and His54 are not critical residues in the epitopes of these antibodies.     

Assay of the von Willebrand factor (VWF) propeptide to identify patients with type 1 von Willebrand disease with decreased VWF survival

Type 1 von Willebrand disease (VWD) is characterized by a partial quantitative deficiency of von Willebrand factor (VWF). Few VWF gene mutations have been identified that cause dominant type 1 VWD. The decreased survival of VWF in plasma has recently been identified as a novel mechanism for type 1 VWD. We report 4 families with moderately severe type 1 VWD characterized by low plasma VWF:Ag and FVIII:C levels, proportionately low VWF:RCo, and dominant inheritance. A decreased survival of VWF in affected individuals was identified with VWF half-lives of 1 to 3 hours, whereas the half-life of VWF propeptide (VWFpp) was normal. DNA sequencing revealed a single (heterozygous) VWF mutation in affected individuals, S2179F in 2 families, and W1144G in 2 families, neither of which has been previously reported. We show that the ratio of steady-state plasma VWFpp to VWF:Ag can be used to identify patients with a shortened VWF half-life. An increased ratio distinguished affected from unaffected individuals in all families. A significantly increased VWFpp/VWF:Ag ratio together with reduced VWF:Ag may indicate the presence of a true genetic defect and decreased VWF survival phenotype. This phenotype may require an altered clinical therapeutic approach, and we propose to refer to this phenotype as type-1C VWD.     

Compound heterozygosity for two novel mutations (1203insG/Y1456X) in the von Willebrand factor gene causing type 3 von Willebrand disease

A 23-year-old Chinese woman with severe von Willebrand factor (VWF) deficiency and her parents were investigated by PCR/direct sequencing of the VWF gene. The patient was found to be compound heterozygous for two novel null mutations. The first was a microinsertion in exon 8 (1203insG) that introduced a frameshift at codon 298 leading to premature translational termination at codon 302. The second was a C to A transversion in exon 28 which resulted in the replacement of tyrosine 562 by a stop codon (Y1456X). The failure to amplify VWF cDNA from the patient by semi-nested PCR is consistent with the induction of nonsense-mediated mRNA decay.     

A monoclonal antibody (B724) to von Willebrand factor recognizing an epitope within the Al disulphide loop (Cys509-Cys695) discriminates between type 2A and type 2B von Willebrand disease

Monoclonal antibody (MoAb) B724 to von Willebrand factor (vWF) completely inhibits its interaction with heparin, sulphatides and botrocetin and consequently botrocetin-induced binding of vWF to platelets. MoAb B724 has no effect on the binding of vWF to collagen or to ristocetin-treated platelets nor on vWF-dependent platelet aggregation induced with ristocetin and asialo-vWF-mediated platelet aggregation. MoAb B724 preferentially recognizes a conformation of native vWF, in solution, or immobilized through a coated antibody. It exhibits a markedly lower affinity for vWF immobilized onto collagen or plastic surfaces. Using proteolytic fragments of vWF, B724 epitope was localized within the 512–673 sequence of the Al disulphide loop of vWF, MoAb B724 was used as second antibody in a two-site ELISA to test a series of patients with type 1, 2 A, 2B and 2N vWD or haemophilia A and recombinant wild type or mutated vWFs. Results were compared with those obtained by control ELISAs performed using polyclonal antibodies. Using MoAb B724, strikingly lower levels of vWFAg were observed in plasma from most patients with type 2B vWD, and in seven out of the eight rvWF mutated close to or within the Al disulphide loop. Therefore MoAb B724, which interferes with this loop involved in the function of vWF, appears to be a useful tool for rapid screening of conformational changes in this region.     

Characterization of von Willebrand factor gene defects in two unrelated patients with type IIC von Willebrand disease

Genetic studies were performed in two unrelated patients with the IIC phenotype of von Willebrand disease (vWD) characterized by the increased concentration of the protomeric form of von Willebrand factor (vWF). In patient B, the sequencing of both exons 15 and 16 of the vWF gene showed two sequence alterations: a 3-bp insertion in exon 15 resulting in the insertion of a Glycine at position 625 (625insGly) and a 2-bp deletion in exon 16 leading to a premature translational stop at codon 711 (711 ter), at the heterozygote state. Patient A was found homozygous for a single point mutation also localized in exon 15 and responsible for the substitution Cys623Trp. These candidate mutations were not found in a panel of 96 normal chromosomes, suggesting a causal relationship with IIC vWD phenotypic expression. The composite heterozygote or homozygote state of both patients supports the recessive mode of inheritance already described for this phenotype. Furthermore, the localization of these gene defects in the D2 domain of vWF propeptide, known to play an important role in vWF multimerization, provides another argument in favor of their causative effect regarding the peculiar multimeric pattern of vWF in these patients.     

Analysis of Arg834Gln and Val902Glu type 2A von Willebrand disease mutations: studies with recombinant von Willebrand factor and correlation with patient characteristics

Type 2A von Willebrand disease (vWD), the most common qualitative form of vWD, is characterized by a relative decrease in circulating intermediate and high molecular weight (HMW) multimers. We studied the biosynthesis of recombinant von Willebrand factor (vWF) containing each of two type 2A vWD mutations previously reported by us, Arg834Gln and Val902Glu. The structure of recombinant Arg834Gln vWF within transfected COS-7 cells and the secretion of HMW multimers were similar to wild type vWF. The normal transport and secretion of Arg834Gln vWF, categorizes it as a group II type 2A mutation. In contrast, the Val90- 2Glu mutation resulted in intracellular proteolysis of vWF with the generation of a 176-kD fragment and retention of vWF between the endoplasmic reticulum and the Golgi complex. Moreover, the 176-kD fragment was also increased in plasma from patients with the Val902Glu mutation. Significantly impaired secretion and intracellular proteolysis of Val902Glu vWF categorizes a new sub-group of type 2A mutations. The intracellular proteolysis of vWF Val902Glu explains the lack of response to 1-deamino 8-D-arginine vasopressin (DDAVP) in patients who carry the mutation.     

von Willebrand factor and von Willebrand disease [published erratum appears in Blood 1988 Mar;71(3):830]

Progress has occurred in the past several years in the understanding of the structure and function of von Willebrand factor (vWF). This multimeric glycoprotein exhibits a dual role, that of mediating platelet adhesion and aggregation onto thrombogenic surfaces, and that of functioning as carrier in plasma for the factor VIII procoagulant protein. New insights into the nature of the several functional domains of vWF have led to the identification of the regions of the molecule that interact with factor VIII, heparin, the glycoprotein lb of platelets, and collagen. Alterations of vWF are the cause of von Willebrand disease (vWD), a congenital bleeding disorder. In the majority of patients, the plasma levels of vWF are decreased, but there is no demonstrable structural or functional alteration of the protein. In other patients, however, the structure of vWF is abnormal. This review summarizes the current knowledge on vWF and vWD.     

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.     

An apparently silent nucleotide substitution (c.7056C>T) in the von Willebrand factor gene is responsible for type 1 von Willebrand disease

Background

Nucleotide variations not changing protein sequences are considered silent mutations; accumulating data suggest that they can, however, be important in human diseases.

Design and Methods

We report an altered splicing process induced by a silent substitution (c.7056C>T) in the von Willebrand factor gene in a case of type 1 von Willebrand disease originally classified as lacking von Willebrand factor mutations.

Results

The c.7056C>T synonymous substitution introduces a new donor splice site within exon 41, leading to messenger RNA lacking nucleotides 7055-7081 (c.7055_7081del). The encoded von Willebrand factor protein is predicted to lack amino acids 2352-2360 in the B2 domain. The patient’s von Willebrand disease phenotype was characterized by reduced plasma and platelet von Willebrand factor, which was normal in function and multimer structure. In vitro expression studies demonstrated that co-transfection of equimolar c.7055_7081del and wild-type von Willebrand factor (mimicking the patient’s heterozygous state) induced a 50% lower von Willebrand factor secretion than the wild type, while almost no von Willebrand factor secretion was seen with the mutated von Willebrand factor alone. The secreted von Willebrand factor was structurally and functionally normal, suggesting that the c.7056C>T substitution behaves like a loss-of-function allele.

Conclusions

This is the first report of a synonymous von Willebrand factor substitution being responsible for von Willebrand disease. Our findings suggest the need to reconsider the role of von Willebrand factor polymorphisms in von Willebrand disease.     

An Arg545----Cys545 substitution mutation of the von Willebrand factor in type IIB von Willebrand's disease.

Type IIB is a special variant of von Willebrand's disease, characterized by an abnormal von Willebrand factor which shows an increased interaction with platelets. This interaction sometimes causes platelet aggregation and thrombocytopenia in vivo. It involves the glycoprotein-Ib (GPIb) receptor on platelets and corresponding GPIb-binding sites in the von Willebrand factor. We here demonstrate a C----T mutation at codon 1308 of the von Willebrand factor gene in 2 related patients with IIB von Willebrand's disease. The transition gives rise to a substitution of arginine by cysteine at position 545 of the mature von Willebrand factor subunit. This position is close to the GPIb- as well as the collagen- and heparin-binding domains of the von Willebrand factor. The mutation may change the conformation of the molecule in this region and activate the GPIb-binding domain, which is normally not exposed in the von Willebrand factor of circulating blood.     

Type 2M:Milwaukee-1 von Willebrand disease: an in-frame deletion in the Cys509-Cys695 loop of the von Willebrand factor A1 domain causes deficient binding of von Willebrand factor to platelets

This report examines the genetic basis of a variant form of moderately severe von Willebrand disease (vWD) characterized by low plasma von Willebrand factor antigen (vWF:Ag) levels and normal multimerization, typical of type 1 vWD, but disproportionately-low agonist-mediated platelet-binding activity. We identified an in-frame deletion in vWF exon 28 in three generations of affected family members, who are heterozygous for this mutation. The deletion of nucleotides 4,173-4,205 results in the loss of amino acids Arg629-Gln639 in the Cys509-Cys695 loop of the A1 domain in mature vWF. The secreted mutant vWF showed a normal multimeric profile but did not bind to platelets in the presence of optimal concentrations of either ristocetin or botrocetin. The mutant vWF also failed to interact with heparin, and with vWF monoclonal antibody AvW3, which blocks the binding of vWF to GPlb. In addition, mutant vWF showed reduced secretion from transfected cells concomitant with increased intracellular levels. These results confirm that the deletion is the genetic defect responsible for the reduced interaction of vWF with platelets. We have designated this new variant type 2M:Milwaukee-1 vWD. Our analysis suggests that the potential frequency of this phenotype in individuals diagnosed with type 1 vWD is about 0.5%.     

Comparative analysis of type 2b von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets

von Willebrand factor (vWF) is a multimeric glycoprotein that forms an adhesive link following vascular injury between the vessel wall and its primary ligand on the platelet surface, glycoprotein Ib (GpIb). Type 2b von Willebrand disease (vWD) is a qualitative form of vWD resulting from enhanced binding of vWF to platelets. Molecular characterization of the vWF gene in patients with type 2b vWD has resulted in identification of a panel of mutations associated with this disorder, all clustered within the GpIb binding domain in exon 28 of the vWF gene. We have expressed six of the most common type 2b vWD mutations in recombinant vWF and show that each mutation produces a similar increase in vWF binding to platelets in the absence or presence of ristocetin. Furthermore, expression of more than one type 2b vWD mutation in the same molecule (cis) or in different molecules within the same multimer (trans) failed to produce an increase in vWF platelet binding compared with any of the individually expressed mutations. Taken together, these data support the hypothesis that the vWF GpIb binding domain can adopt either a discrete "on" or "off" conformation, with most type 2b vWD mutations resulting in vWF locked in the on conformation. This model may have relevance to other adhesive proteins containing type A domains.