Getting at the variable expressivity of von Willebrand disease

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by abnormalities of von Willebrand factor (VWF). VWF levels vary widely in the general population, and this variation is likely to be a major factor accounting for the incomplete penetrance and variable expressivity of VWD. In addition, variation in VWF level may play an important role in determining the risk of venous thrombosis. A large component of the variation in VWF level in the general population has been shown to be attributable to genetic factors. This review will focus on the current understanding of the genetic causes for variation in VWF level, and will highlight future directions for getting at the variable expressivity of von Willebrand disease.     

Mutations in von Willebrand factor multimerization domains are not a common cause of classical type 1 von Willebrand disease

Type 1 von Willebrand disease (vWD) is an autosomal dominant bleeding disorder of variable penetrance. It is characterised by a mild to moderate bleeding tendency and a quantitative deficiency of von Willebrand factor (vWF) with the full range of vWF multimers. Few mutations have been described which account for the mode of inheritance in dominant vWD type 1. We screened the vWF multimerization domains (regions D1-D3 of the vWF gene) of 12 unrelated patients with dominant vWD type 1 to investigate the hypothesis that multimerization of vWF sub-units may be inhibited or reduced by a "dominant negative" mechanism. Platelet-derived RNA was reverse transcribed and the resulting vWF cDNA amplified by the polymerase chain reaction (PCR) in a series of overlapping fragments. These were subjected to a combination of single-strand conformation polymorphism (SSCP) and heteroduplex analysis. This approach identified mobility shifts on acrylamide gels that represented 12 distinct SSCP and/or heteroduplex patterns in our patient group. DNA sequencing of the region encompassing each mobility shift showed these variants to represent previously described polymorphisms within the vWF coding sequence. Examination in all 12 patients for the previously described G3389T and T3445C mutations proved negative. The molecular pathology of classical type 1 vWD remains enigmatic, mutations having been identified in only a small minority of patients. A common mechanism underlying this disease state has still to be elucidated.     

An investigation of the von Willebrand factor genotype in UK patients diagnosed to have type 1 von Willebrand disease

Forty families diagnosed by UK centres to have type 1 VWD were recruited. Following review, six families were re-diagnosed to have type 2 VWD, one to have a platelet storage pool disorder, and one family was determined to be unaffected. Direct DNA sequencing of the promoter region and all exons and intronic boundaries of the VWF gene identified six mutations likely to be causative of VWD in index cases of nine of the 32 (28%) confirmed type 1 VWD families. These included R1205H (3614G > A) VWD Vicenza, P1648fsX45 (4944delT), D141G (422A > G) and three splice site mutations: 3108 + 5G > A, 7437 + 1G > A and 3379 + 1G > A. The Y1584C (4751A > G) polymorphism was present in eight additional families. No significant VWF gene mutation or polymorphism was identified in 15 of the 32 type 1VWD index cases (47%). Haplotype studies were performed using a panel of VWF polymorphisms to investigate the segregation in families of VWD phenotype with the VWF gene. In 13 of the 32 families it was likely that VWD segregated with the VWF gene. In eight families (25%) VWD clearly did not segregate with the VWF gene. We suggest that mutation screening of the VWF gene has limited general utility in genetic diagnostic and family studies in type 1 VWD. If genetic studies are performed, the incomplete penetrance and variable expressivity of type 1 VWD must be taken into account. Unless linkage of VWD phenotype with the VWF gene can be clearly demonstrated, the results of any genetic family studies should be interpreted with caution.     

Survival of von Willebrand factor released following DDAVP in a type 1 von Willebrand disease cohort: influence of glycosylation, proteolysis and gene mutations

Reduced plasma survival of von Willebrand factor (VWF) may contribute towards the pathogenesis of type 1 von Willebrand disease (VWD). However, little is known about mechanism(s) of VWF clearance and factors that may affect it. The half-life of VWF-related parameters following the administration of DDAVP was measured in 26 patients with type 1 VWD and 10 haemophilia A controls. Binding of lectins Ricinus communis (RCA-I) and Erythina crystagalli (ECA) agglutinins to VWF and VWF susceptibility to ADAMTS-13-mediated proteolysis were investigated. Sequence analysis of targeted regions of the VWF gene was performed to inspect for mutations that have been associated with increased clearance. Post-DDAVP clearance of VWF was increased approximately three-fold in the type 1 VWD cohort overall. However this was not shown to consistently associate with steady-state VWF antigen (VWF:Ag) levels. Furthermore, increased VWF clearance was not consistently associated with increased ratios of VWF propeptide (VWFpp) to VWF:Ag indicating that a normal ratio does not necessarily reflect normal post-DDAVP survival in type 1 VWD patients. RCA-I and ECA binding to VWF were increased in type 1 VWD patients and, although inversely correlated with VWF levels, this was independent of VWF clearance. There was no association between VWF clearance and ADAMTS-13-mediated proteolysis. Three novel candidate mutations with an increased clearance phenotype were identified. The data are consistent with heterogeneity in pathogenic mechanisms in type 1 VWD and are consistent with type 1 VWD representing a complex genetic trait.     

Platelet adhesion to collagen in subtypes of type I von Willebrand's disease is dependent on platelet von Willebrand factor

Von Willebrand's disease type I, characterized by low levels of factor VIII coagulant activity (VIII: C), von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (RiCof) (1), can be subdivided on the basis of platelet von Willebrand factor into subtype platelet normal, platelet discordant, and platelet low (2). We have investigated the contribution of platelet von Willebrand factor in these various subtypes to platelet adhesion using the rectangular perfusion chamber of Sakariassen et al. (3) with fibrillar collagen or a fibroblast matrix as adhesive surfaces. Platelet adhesion to fibrillar collagen was decreased in all subtypes of von Willebrand's disease, but not as low as in severe von Willebrand's disease. A close correlation was observed between platelet adhesion to collagen and plasma vWF:Ag in severe von Willebrand's disease, subtype platelet low, subtype platelet discordant, and normal controls. The platelet adhesion in subtype platelet normal was higher than expected from the plasma vWF:Ag level. Perfusions in which washed platelets were added to a human albumin solution together with red blood cells gave similar adhesion values in subtype platelet normal and normal controls; adhesion was decreased in subtype platelet discordant, and the lowest values were found in subtype platelet low and in severe von Willebrand's disease. These data indicate that platelet von Willebrand factor may contribute to platelet adhesion, when plasma von Willebrand factor is low. Perfusion studies over a fibroblast matrix gave similar low adhesion values for subtype platelet low and platelet normal, indicating that the contribution of platelet von Willebrand factor can only be observed on a strongly activating surface such as fibrillar collagen.     

Abnormality of the N-terminal portion of von Willebrand factor in type IIA and IIC von Willebrand disease

We have established a new analytical method which allows the characterization of von Willebrand factor (vWF) degradation fragments in minute amounts (10 microliters) of plasma, without the need for immunopurification of vWF. Plasma vWF was hydrolysed by S aureus V-8 protease (V-8 protease) and the cleaved fragments separated by SDS-agarose gel electrophoresis followed by staining with 125I-labeled polyclonal or monoclonal antibodies against vWF and autoradiography. Quantification of the amount of each product was estimated by counting the incorporated radioactivity following excision. V-8 protease limitedly hydrolysed vWF in normal as well as type I von Willebrand disease (vWD) plasma and produced two distinct fragments with identical electrophoretic and antigenic characteristics to those produced from purified vWF, i.e. a C-terminal SpII and a series of N-terminal SpIII fragments (SpIIIa, b and c). The method was applied to further characterize the molecular abnormalities of vWF in eighteen patients with type II vWD. In seven individuals with type IIA and five patients with type IIC, SpIII appeared significantly modified as compared to normal. In type IIA, there was a marked decrease or absence of SpIIIa and an increase of SpIIIb and c. In type IIC, SpIIIb was lacking. In three patients with type IIB and in three patients with type IID, there was no significant modification of SpIII. In all cases, SpII was apparently not modified. In conclusion, the molecular abnormality of vWF in type IIA and IIC vWD appears to reside in SpIII, the N-terminal portion of the vWF-subunit (residues 1 to 1,365).     

Major haemorrhage related to surgery in patients with type 1 and possible type 1 von Willebrand disease

Patients with von Willebrand disease (VWD) frequently bleed under a challenge. The aim of our study was to identify predictive markers of perioperative major haemorrhage in type 1 (VWF:RCo = 15-30 IU dl(-1)) and possible type 1 (VWF:RCo = 31-49 IU dl(-1)) VWD patients. We recorded perioperative bleeding complications previous to diagnosis and laboratory parameters in 311 patients with 498 surgical procedures. The patients were grouped according to the absence (A) or presence (B) of perioperative major haemorrhages. Eighty-one patients (26%) and 87 surgical procedures (17.5%) presented major haemorrhages associated with surgeries. There was no difference between the percentage of type 1 and possible type 1 VWD patients who had major haemorrhages (32.6% and 24.8% respectively; p = ns). No difference in the prevalence of O blood group, age, gender, positive family history and laboratory test results (FVIII and VWF) was observed, independent of the haemorrhagic tendency. Bleeding after tooth extraction was the most frequent clinical feature observed in patients with perioperative major haemorrhages. The bleeding score and the number of bleeding sites (> or = 3) were not predictors of major haemorrhage associated with surgery. Caesarean section and adenotonsillectomy showed the highest frequency of major haemorrhages (24.6% and 22.3%, respectively). In conclusion, type 1 and possible type 1 VWD patients showed similar incidence of perioperative major haemorrhages. Laboratory tests and positive family history did not prove to be effective at predicting major haemorrhages in patients that had either type 1 or possible type 1 VWD. The history of bleeding after tooth extraction could define risk factors of major haemorrhage.     

Bleeding tendency and efficacy of anti-haemorrhagic treatments in patients with type 1 von Willebrand disease and increased von Willebrand factor clearance

Accelerated clearance of von Willebrand factor (VWF) has been recently identified as a major pathophysiologic mechanism inducing low VWF in some patients with von Willebrand disease (VWD). The frequency of bleeding and the best treatment of these patients have never been evaluated prospectively in large series of patients. It was the aim of the present study to prospectively evaluate clinical events of 60 heterozygous patients with VWD Vicenza (VWD-VI) carrying R1205H VWF mutation and 23 with C1130F mutation, both characterised by markedly increased VWF clearance. During 71 months of follow-up, 65% of patients with VWD-VI and 61% with C1130F required treatment. The rate of spontaneous bleeding requiring consultation/treatment was 7.5/100 patients-year in patients with C1130F mutation vs. 1.9/100 patients-year in those with R1205H (p=0.004). This difference persisted also by multivariate analysis adjusted for sex, age and blood group (hazard ratio [HR]=3.3 for C1130F, 95% confidence interval [CI] 1.16-9.27) and females were at greater risk of bleeding (HR=3, 95%CI 1.01-9.93) because of menorrhagia. Only 3/15 (20 %) women in fertile age with VWD-VI compared to 8/9 (89 %) with C1130F mutation required consultation/treatment for menorrhagia (iron supplementation, combined oral contraceptives, tranexamic acid). Almost all dental extractions, minor surgeries and deliveries occurring during follow-up were successfully managed with desmopressin. Major surgery required factor VIII/VWF concentrates, but a few cases benefited from desmopressin. In conclusion, similar to patients with type 1 VWD, also in patients with increased VWF clearance desmopressin maintains a major therapeutic role.     

Carrier detection in severe (type III) von Willebrand disease using two intragenic restriction fragment length polymorphisms

DNA from a family with a female member affected with severe (type III) vWD was analysed using three restriction enzymes and a partial vWF cDNA probe. Two restriction fragment length polymorphisms (RFLPs) detected with the enzymes Bgl II and Xba I proved to be informative in this family. A 36.0 Kb allele demonstrated with the enzyme Xba I was rare in the general population but very important in this family for segregation analysis of the alleles and their association with the putative defective chromosome. The propositus was homozygous for the 36.0 Kb Xba I polymorphic band and heterozygous for the Bgl II polymorphism. She was the only member of the family showing this allelic pattern. The linkage of the alleles could be determined because her mother was homozygous for the 9.0 Kb Bgl II polymorphism but heterozygous for the Xba I polymorphism. The segregation of the alleles could be traced to the proband's son and a niece. The genotypic analysis revealed that her niece could be considered as carrying a defective gene for severe vWD.     

Platelet glycoprotein Ibalpha polymorphisms modulate the risk for myocardial infarction

Platelet glycoprotein Iba (GPIba) gene polymorphisms have been reported to affect the risk of developing coronary heart disease. Here, within the GPIba gene, we determine the association between the variable number of tandem repeats (VNTR), the -5C/T Kozak sequence dimorphism, and the human platelet antigen (HPA)-2 polymorphisms with occurrence of myocardial infarction (MI). Patients (n=180) presenting survivors of MI were compared to 180 controls matched by age, gender, and race. Carriers of VNTR-CD genotype had a 2-fold higher risk for MI compared to controls. The prevalence of VNTR-BC was lower among patients than among controls (P=.007). These data are in agreement with recent reports of increased plug formation by human platelets containing VNTRCD but no other VNTR genotypes. Among patients, the number of vessels severely occluded was greater among carriers of the D-allele (P=.019) or VNTR-CD (P=.026) and lower among carriers of the C-allele (P=.003) or VNTR-CC (P=.0009) compared to non-carriers of these alleles. No influence was seen with the Kozak or HPA-2 polymorphisms. Determination of VNTR of the GPIba gene may prove useful for identifying high-risk individuals for MI.     

von Willebrand disease in a pediatric-based population--comparison of type 1 diagnostic criteria and use of the PFA-100 and a von Willebrand factor/collagen-binding assay

Definitive diagnosis of type 1 von Willebrand Disease (VWD) remains a problem. Provisional consensus guidelines for the diagnosis of definite and possible type 1 VWD were prepared by the Scientific Subcommittee on von Willebrand factor (VWF) of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) during the 1996 annual meeting for the specific purpose of further evaluation in retrospective and prospective studies by a Working Party on Diagnostic Criteria (1996 Annual Report of the SSC/ISTH Subcommittee on VWF). In the first phase of this study, we compared 2 definitions of type 1 VWD. each with 3 criteria: significant bleeding history, laboratory investigations, and family history. Using the ISTH consensus guidelines for type 1 VWD definition, significantly fewer patients were diagnosed with definite type 1 disease as compared to our "in house" Hospital for Sick Children (HSC) criteria (4 vs. 31). While we recognize that the provisional ISTH consensus guidelines were not intended for clinical use, we believe that the results of our studies are of interest and will assist in any future refinements to the ISTH guidelines. In the second phase of this study, we investigated the utility of 2 new tests, a laboratory screening test and a functional test, for VWD in our well characterized, pediatric-based population. The Platelet Function Analyzer (PFA-100) provides an in vitro measure of primary hemostasis under conditions of high shear, using disposable cartridges containing collagen and either epinephrine or ADP. All tested subjects with types 2 or 3 VWD had prolonged PFA-100 closure times (CTs) with both cartridge types (n = 17) and prolonged bleeding times (n = 14). In subjects with definite type 1 VWD, 20/24 (83%) had prolonged CTs with the collagen/ADP cartridge (19/24 (79%) with collagen/epinephrine), compared with 7/26 (27%) with prolonged bleeding times. In subjects with definite types 1, 2, or 3 VWD, collagen/ADP CTs were abnormal in 37/41 subjects, giving an overall sensitivity of 90%. With this high sensitivity, the PFA-100 is a better screening test for VWD than the bleeding time. We also tested a VWF collagen-binding assay (VWF:CBA) as a functional test for VWF, in comparison with the more routinely-used ristocetin cofactor assay (VWF:RC0). The VWF:CBA is based on an ELISA technique, which has the potential to be more reproducible than the VWF:RC0. We found that the VWF:CBA detected 43/49 (88%) subjects with definite types 1, 2, or 3 VWD, performing as well as the VWF:RC0, that detected 42/48 (88%). We also showed that, used in conjunction with VWF antigen levels, the VWF:CBA may be useful in classification of VWD subtypes.     

Identification of a new type 2M von Willebrand disease mutation also at position 1324 of von Willebrand factor

Type 2M von Willebrand disease (VWD) refers to variants with decreased platelet-dependent function that is not associated with the loss of high molecular weight (HMW) von Willebrand factor (VWF) multimers. This category includes the so-called "phenotype B" responsible for inexistent ristocetin-induced but normal botrocetin-induced binding of VWF to platelet glycoprotein lb. The missense mutation G1324S was identified in the first patient reported to display "phenotype B". We report here on the identification in four members of a French family of a missense mutation also affecting this glycine residue but changing it into an alanine residue. These individuals are heterozygous for this mutation and two of them display an additional quantitative VWF deficiency resulting from a stop codon at position 2470. After transient transfection in Cos-7 cells, the mutated recombinant protein harbouring the G1324A substitution was shown to exhibit normal multimers and inexistent ristocetin-induced but normal botrocetin-induced binding to GPIb, confirming the classification of this new mutation as a type 2M VWD mutation.     

Three distinct candidate point mutations of the von Willebrand factor gene in four patients with type IIA von Willebrand disease.

Type IIA von Willebrand disease (vWD) is the most common type II vWD and is characterized by the selective loss of large and intermediate sized multimers. One explanation for this disorder has been postulated to be a qualitative defect in von Willebrand factor (vWF) which results in increased susceptibility to proteolysis at the bond between residues Tyr842 and Met843. Four missense mutations that may cause type IIA vWD have recently been identified near the cleavage site. We analyzed the molecular basis for type IIA vWD in six patients. A 512 bp DNA sequence spanning the proteolytic cleavage site was targeted for PCR amplification and sequencing. We exploited a difference in restriction sites between the vWF gene and the pseudogene and have designed allele-specific oligomer used with PCR to distinguish these two genes. Three candidate missense mutations; Ser743 (TCG)----Leu (TTG), Leu799 (CTG)----Pro (CCG), and Arg834 (CGG)----Trp (TGG) were identified in 4 out of 6 patients. The amino acid substitution at Arg834 has been reported previously, but the other substitutions at Ser743 and Leu799 are novel candidate mutations locating 99 and 43 amino acids to the N-terminal side of the cleavage site, respectively. Our results indicate that amino acid substitutions located relatively distant from the cleavage site may also be involved in type IIA vWD.     

Treatment of von Willebrand disease

In von Willebrand disease, there are two main options for the treatment of spontaneous bleeding episodes and for bleeding prophylaxis: desmopressin and transfusional therapy with plasma products. Desmopressin is the treatment of choice for most patients with type 1, who account for approximately 70 to 80 per cent of all cases with the disease. This non-transfusional hemostatic agent raises endogenous factor VIII and von Willebrand factor three- to fivefold and thereby transiently corrects both the intrinsic coagulation and primary hemostasis defects. In patients with the more severe type 3 and in the majority of those with type 2 desmopressin is not effective or is contraindicated, so that it is usually necessary to resort to plasma concentrates containing factor VIII and von Willebrand factor. Concentrates treated with virus inactivation methods should be preferred to cryoprecipitate because they are equally effective and perceived as safer.     

Platelet membrane glycoprotein V: characterization of the thrombin-sensitive glycoprotein from human platelets

Human platelets contain a single membrane glycoprotein which is susceptible to thrombin proteolysis, glycoprotein V. We have purified 1 mg of glycoprotein V from 10(13) platelets using a combination of gel filtration, hydroxylapatite and ion-exchange chromatographies. Glycoprotein V has a blocked amino-terminus. Following proteolysis by human alpha-thrombin, a major fragment, termed glycoprotein Vf1, had the sequence Gly-Pro-Phe-X-Arg-Pro-Ala-Ala-Asp-Glu-Ser-Val-Glu-Ala-Pro-Val-Asn-Gln-Al a-Glu- Ala-Pro-. The purified glycoprotein was not a substrate for human gamma-thrombin. Glycoprotein V contained 17.5% carbohydrate, with the majority of the carbohydrate consisting of neutral hexoses. Deglycosylated glycoprotein V had a molecular weight of 57.5 kDa compared to the glycosylated protein's 82 kDa and the deglycosylated protein was recognized by polyclonal antibodies raised against glycoprotein V. Immunoelectrophoresis of human and rat platelets and megakaryocytes gave a single immunoreactive band, with the rat glycoprotein having a slightly larger molecular mass. Glycoprotein V is most likely an integral membrane protein.