Diagnosis of inherited von Willebrand disease: comparison of two methodologies and analysis of the discrepancies

Diagnostics of von Willebrand disease (VWD) includes assessment of factor VIII (FVIII) coagulant activity, von Willebrand factor (VWF) antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), and more specific tests as multimeric and genetic analyses are necessary for the correct VWD classification. The ACL AcuStar? analyzer introduces chemiluminescence (CL) technology in detection of VWD with automated VWF:Ag and VWF:RCo assays. Compare VWF:Ag‐ELISA and VWF:RCo by aggregometry conventional assays with new CL VWF:Ag‐IL and VWF:RCo‐IL assays, investigate the ability to make accurate VWD diagnosis and concordance with multimeric and genetic analyses. 146 patients with congenital VWD (51 Type 1; 34 Type2A; 16 Type 2B; 31 Type 2M; 5 Type 2N; 9 Type 3) and 30 healthy normal subjects were included. A comparison was made between CL and conventional methods. Diagnostic evaluation included: VWF:RCo/VWF:Ag ratio, multimeric distribution (sodium dodecyl sulfate [SDS]‐agarose gel) of VWF and genetic analysis in 110 of 146 patients. CL and conventional methods revealed good correlation. Kappa test agreement diagnosis was >0.8. CL diagnostic sensitivity was 100% and specificity 97%. Multimeric and genetic analysis were of help in clarifying 13 discrepancies of diagnosis between methods, of which six discrepancies were explained by lack of conventional methods′ sensibility. CL methodology can detect VWD and discriminate between type 1, 3 and variant forms and offers an automated, faster, sensitive and less cumbersome method when compared to conventional assays, in particular VWF:RCo by aggregometry. In some cases, even with all phenotype and genetic analyses, discrepancies exist in the classification of VWD.     

Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD

von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)-based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα(235Y;239V)) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.     

A two‐centre comparative evaluation of new automated assays for von Willebrand factor ristocetin cofactor activity and antigen

von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two‐centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter‐assay imprecision (AcuStar: CV% range 3.3–6.9; TOP500: CV% range 2.6–6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6–173.9 IU dL^?1; VWF:RCo 43.1–191.5 IU dL^?1) and patients (range: VWF:Ag <0.3–115.1 IU dL^?1; VWF:RCo <0.5–57.2 IU dL^?1) showed a good correlation between the two VWF:Ag and VWF:RCo methods (r_s = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients' samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL^?1 vs. 2.2 IU dL^?1 and 0.5 IU dL^?1 vs. 4.4 IU dL^?1 respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non‐specialized and reference laboratories.     

Diagnostic challenges in patients with bleeding phenotype and von Willebrand exon 28 polymorphism p.D1472H

Exon 28 polymorphism p.D1472H is associated with significantly lower von Willebrand Ristocetin cofactor activity (VWF:RCoF) to von Willebrand antigen (VWF:Ag) ratio compared to normal, but has been reported as not conferring haemorrhagic risk. The impact of this polymorphism while assessing symptomatic patients for von Willebrand disease (VWD) has not been previously analysed. We retrospectively reviewed charts of children with clinically significant bleeding and abnormal VW panel who underwent VW exon 28 analysis. Twenty‐three of 63 patients studied had p.D1472H. Of these 23 patients, 6 with borderline low VWF:RCo were given provisional diagnosis of VWD type 1 by treating physicians, which could be alternatively explained as due to the effect of p.D1472H. None of the patients with low VWF:RCo, decreased VWF:RCo/VWF:Ag ratio and p.D1472H had VWD type 2M mutations identified. This study illustrates the challenge in diagnosing VWD using ristocetin‐based VW assay in symptomatic patients with p.D1472H.     

von Willebrand disease R1374C: Type 2A or 2M? A challenge to the revised classification. High frequency in the northwest of Spain (Galicia)

Patients initially diagnosed with type 1 von Willebrand disease (VWD) have been reclassified as type 2 after a more exhaustive analysis in several studies. Our study's objectives were (1) to reanalyze patients that were previously diagnosed as type 1 to achieve a more accurate diagnosis and (2) to compare the von Willebrand factor (VWF) ristocetin cofactor assay (VWF:RCo) and the VWF collagen binding assay (VWF:CB) in order to evaluate the possibility of replacing the former assay with the latter in the diagnosis of VWD. Twenty-one patients from two large unrelated families and 104 normal controls were studied. VWF:Ag, VWF:RCo, FVIII coagulant activity (FVIII:C), bleeding time (BT), PFA(100), and multimeric analysis of VWF were tested. Genetic analysis by sequencing exon 28 on the VWF gene was also carried out. Patients presented lower levels of VWF:Ag and VWF:RCo, a dissociation between VWF:RCo/VWF:Ag, and the presence of all sizes of multimers in plasma VWF. The results for VWF:CB varied depending on the type of collagen used. The genetic analysis showed that the mutation R1374C is responsible for type 2M VWD. A high frequency of the R1374C mutation is observed in northwestern Spain (Galicia). Some types of 2M VWD are misdiagnosed as type 1 VWD. The VWF:CB (with type I collagen) assay was unable to discriminate defective platelet binding of the R1374C VWF. This confirms that VWF:CB cannot substitute for VWF:RCo, and both should be tested when diagnosing VWD.     

Comparison of a new chemiluminescent immunoassay for von Willebrand factor activity with the ristocetin cofactor‐induced platelet agglutination method

Measuring von Willebrand factor (VWF) activity is essential for the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra‐ and inter‐assay variabilities. A new automated chemiluminescent immunoassay VWF activity has recently become commercially available (HemosIL AcuStar von Willebrand Factor Ristocetin Cofactor Activity). The main objective of this study was to evaluate this new method and to compare it with the VWF:RCo assay as the reference method. We studied 91 samples, 18 healthy volunteers samples and 73 samples from patients (VWF:RCo level <50 IU dL^?1): 29 type 1 VWD, 13 type 2A, 5 type 2B, 5 type 2M, 3 type 2N, 5 type 3, 4 type 3 under treatment, 5 type 3 carriers and 4 samples with other pathologies. The HemosIL AcuStar VWF:RCo assay was 96% sensitive and 100% specific for detecting VWF abnormalities. The good analytical performance, and the sensitivity and specificity of HemosIL AcuStar VWF:RCo to detect VWF deficiency renders it a suitable method for VWD screening.     

Von Willebrand factor collagen binding activity in the diagnosis of von Willebrand disease: an alternative to ristocetin co‐factor activity?

The capability of von Willebrand factor (VWF) to bind platelet glycoprotein Ib (GPIb) and promote platelet plug formation is currently evaluated in vitro using the ristocetin co-factor activity (VWF:RCo) assay. The replacement of this cumbersome and not always reproducible test with the collagen binding activity of VWF (VWF:CBA) has been attempted with controversial results. To evaluate the capacity of VWF:CBA to identify classic and variant von Willebrand disease (VWD) compared with VWF:RCo, we studied 10 type 2A and 12 type 2B VWD patients, together with 30 type 1 VWD patients with reduced platelet VWF content. In both 2A and 2B VWD, VWF:CBA and VWF:RCo were decreased, but that of VWF:CBA was more consistent. The difference was more evident when values were expressed as a ratio, obtained by normalizing VWF:CBA and VWF:RCo with the VWF antigen value; the ratio for VWF:CBA was always below 0.2, while that for VWF:RCo was greater than 0.4, and in no patient was the VWF:CBA value higher than VWF:RCo. In contrast, in type 1 VWD, the decrease in VWF:CBA was similar to that seen in VWF:RCo with the ratios always within the normal range. To better investigate the relationship between VWF:CBA and VWF:RCo, and the representation of large/intermediate VWF multimers, to which both tests are sensitive, 1-deamino-cys-8-D-arginine-vasopressin (DDAVP) was infused in type 2A and 2B VWD patients. The differences between the two tests were even more evident after DDAVP, and in type 2A, even though large multimers were persistently decreased, VWF:RCo was normalized, while VWF:CBA remained defective. These findings clearly indicate that VWF:CBA detects the absence of large and intermediate VWF multimers better than VWF:RCo. Hence, we suggest adding VWF:CBA to the panel of tests employed in the diagnosis of VWD. Moreover, owing to the difficulty in performing VWF:RCo and its low reproducibility, we suggest that, when necessary, VWF:CBA may be substituted for VWF:RCo.     

Changing insights in the diagnosis and classification of autosomal recessive and dominant von Willebrand diseases 1980-2015

The European Clinical Laboratory and Molecular (ECLM) criteria define 10 distinct Willebrand diseases (VWD): recessive type 3, severe 1, 2C and 2N; dominant VWD type 1 secretion/clearance defect, 2A, 2B, 2E, 2M and 2D; and mild type 1 VWD (usually carriers of recessive VWD). Recessive severe 1 and 2C VWD are characterized by secretion and multimerization defects caused by mutations in the D1-D2 domain. Recessive 2N VWD is a mild hemophilia due to D’-FVIII-von Willebrand factor (VWF) binding site mutations. Dominant 2E VWD caused by heterozygous missense mutations in the D3 domain is featured by a secretion-clearance-multimerization VWF defect. Dominant VWD type 2M due to loss of function mutations in the A1 domain is characterized by decreased ristocetin-induced platelet aggregation and VWF:RCo, normal VWF multimers and VWF:CB, a poor response of VWF:RCo and good response of VWF:CB to desmopressin (DDAVP). Dominant VWD type 2A induced by heterozygous mutations in the A2 domain results in hypersensitivity of VWF for proteolysis by ADAMTS13 into VWF degradation products, resulting in loss of large VWF multimers with triplet structure of each individual VWF band. Dominant VWD type 2B due to a gain of function mutation in the A1 domain is featured by spontaneous interaction between platelet glycoprotein Ib (GPIb) and mutated VWF A1 followed by increased proteolysis with loss of large VWF multimers and presence of each VWF band. A new category of dominant VWD type 1 secretion or clearance defect due to mutations in the D3 domain or D4-C1-C5 domains consists of two groups: Those with normal or smeary pattern of VWF multimers.     

Prevalence of von Willebrand disease in women with iron deficiency anaemia and menorrhagia in Taiwan

Summary.  Iron deficiency anaemia (IDA) is a frequently encountered disease, which can be attributed to menorrhagia. Most female patients with von Willebrand disease (VWD) have menorrhagia. The aim of this study was to investigate the prevalence of VWD in women with both IDA and menorrhagia in Taiwan. From January to December 2005 and November 2006 to January 2007, 56 consecutive patients with both IDA and menorrhagia were enrolled in this study. Their median age was 41 years (range 18–53). IDA was diagnosed by anaemia plus either low ferritin or transferrin saturation. Menorrhagia was evaluated by patient's menses history. Both von Willebrand factor antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) were measured for each patient. Bleeding time (BT) and platelet function analyser (PFA)-100 assay were determined as ancillary tests. The VWD diagnosis was established if: (i) both VWF:Ag (<50%) and VWF:RCo (<50%) were low; (ii) either VWF:Ag or VWF:RCo was low plus prolonged BT or prolonged PFA closure times. VWF multimer analysis was performed for subtype confirmation of VWD. Nine of the 56 (16.1%) patients were identified to have VWD. VWD patients with menorrhagia might develop IDA at younger age (34.3 vs. 39.7, P  = 0.09) and had more IDA recurrence (75% vs. 16%, P  = 0.03) than those patients without VWD. Of the eight VWD patients with VWF multimer analyses, all were revealed to have type I VWD. Our study demonstrates that VWD was not uncommon in women with both IDA and menorrhagia in Taiwan.     

Molecular Genetics of Type 2 von Willebrand Disease

Type 2 von Willebrand disease (VWD) is characterized by a wide heterogeneity of functional and structural defects. These abnormalities' cause either defective von Willebrand factor (VWF)-dependent platelet function in subtypes 2A, 2B, and 2M or defective VWF-factor VIII (FVIII) binding in subtype 2N. The diagnoses of types 2A, 2B, and 2M VWD may be guided by the observation of disproportionately low levels of ristocetin cofactor activity or collagen-binding capacity relative to VWF antigen. The abnormal platelet-dependent function is often associated with the absence of high molecular weight (HMW) multimers (type 2A, type 2B), but the HMW multimers may also be present (type 2M, some type 2B), and supranormal multimers may exist ("Vicenza" variant). The observation of a low FVIII-to-VWF:Ag ratio is a hallmark of type 2N VWD. in which the FVIII levels depend on the severity of the FVIII-binding defect. Today, the identification of mutations in particular domains of the pre-pro-VWF is helpful in classifying these variants and providing further insight into the structure-function relationship and the biosynthesis of VWF. Thus, mutations in the D2 domain, involved in the multimerization process, are found in patients with type 2A, formerly named IIC VWD. Mutations located in the D' domain or in the N terminus of the D3 domain define type 2N VWD. Mutations in the D3 domain characterize Vicenza and IIE patients. Mutations in the A1 domain may modify the binding of VWF multimers to platelets, either increasing (type 2B) or decreasing (type 2M, 2A/2M) the affinity of VWF for platelets. In type 2A VWD, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding, impairing VWF secretion or increasing its susceptibility to proteolysis. Finally, a mutation localized in the carboxy-terminus CK domain, which is crucial for the dimerization of the VWF subunit, has been identified in a rare subtype 2A, formerly named IID.     

Use of the collagen-binding assay for von Willebrand factor in the analysis of type 2M von Willebrand disease: a comparison with the ristocetin cofactor assay

This study compares the utility of two functional assays for von Willebrand factor (VWF), the ristocetin cofactor assay (VWF:RCo) and the collagen-binding assay (VWF:CBA). We analysed a group of 32 patients with type 2 von Willebrand disease (VWD) (25 patients with type 2M, six with type 2A and one with type 2B) and 22 normal control subjects. VWF:RCo/VWF antigen (VWF:Ag) ratios and VWF:CBA/VWF:Ag ratios were compared between the patient and control groups. In the six patients with type 2A VWD, both VWF:RCo/VWF:Ag ratios and VWF:CBA/VWF:Ag ratios were discordant (< or = 0.7). In the 25 type 2M VWD patients, the VWF:CBA/VWF:Ag ratios were concordant (> 0.7), but the VWF:RCo/VWF:CBA ratios were discordant (< or = 0.7) (P = 0.001) compared with control subjects. Thus, VWF:RCo/VWF:Ag ratios were discordant in both type 2M and 2A VWD patient groups indicating a functional abnormality. However, VWF:CBA/VWF:Ag ratios were discordant in the type 2A VWD group but not in the type 2M VWD group. Our study showed that VWF:CBA is sensitive to functional variants associated with the loss of high-molecular-weight multimers, i.e. type 2A and 2B in VWD, but the assay was unable to discriminate defective platelet-binding VWD variants with normal multimeric patterns such as type 2M VWD. It was concluded that the VWF:CBA assay should be used in association with rather than as a replacement for the VWF:RCo assay.     

A new mutation,S1285F,within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and botrocetin causing type 2M von Willebrand disease

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or botrocetin-induced binding of VWF to platelets in association with a decreased binding to botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to botrocetin and GPIb.     

Measurement of von Willebrand factor binding to a recombinant fragment of glycoprotein Ibalpha in an enzyme-linked immunosorbent assay-based method: performances in patients with type 2B von Willebrand disease

Type 2B von Willebrand disease (VWD) is characterised by an increased affinity of von Willebrand factor (VWF) for its platelet receptor glycoprotein Ib (GPIb). This feature is usually studied in vitro by a ristocetin-dependent VWF platelet-binding assay, which has some limitations as it requires [e.g. (radio)-labelled anti-VWF antibodies and normal formaldehyde-fixed platelets]. We, here, extended the applicability of an enzyme-linked immunosorbent assay-based method previously described for the measurement of ristocetin co-factor activity that used a recombinant fragment of GPIb (rfGPIb alpha) and horseradish peroxidase-labelled rabbit anti-human VWF antibodies for measuring the captured ristocetin-VWF complexes on the rfGPIb alpha. Thirty-one type 2B VWD patients from 15 families with eight different known mutations were studied. VWF in plasma from 28 of these patients bound better than normal VWF at 0.2 mg/ml ristocetin, with the ratio, optical density (OD) patient/OD normal pool plasma, higher than 1.8. For two of the three other patients with no enhanced response of plasma VWF, the platelet lysate VWF showed an enhanced binding capacity; for the last patient, the results in other members of the family are unequivocal. We conclude that, this new method for measurement of plasma or platelet VWF-binding capacity offers great advantages for correct type 2B VWD diagnosis.     

A comparative in vitro evaluation of six von Willebrand factor concentrates

The efficacy of von Willebrand factor (VWF) concentrates for treatment of von Willebrand disease (VWD) is dependent on their content of VWF and factor VIII (FVIII). STUDY OBJECTIVES: To measure the content and quality of VWF and FVIII in six VWF concentrates: Haemate-P (Aventis Behring), Immunate (Baxter Bioscience), Koate (Bayer Corp.), 8Y (BPL), Innobrand (LFB) and Facteur Willebrand (LFB). METHODS: The VWF antigen content (VWF:Ag), ristocetin cofactor activity (VWF:RCo), collagen-binding activity (VWF:CB), VWF multimers with electrophoresis and densitometry, FVIII activity and total protein content. RESULTS: Specific activity (VWF:RCo/total protein) varied considerably (4.7-129.5 IU mg(-1)). Activity measures, VWF:RCo and VWF:CB, correlated well, but we found no correlation between any of these and VWF:Ag. The content of high-molecular weight multimer (HMWM) was normal or close to normal in Haemate-P, Innobrand and Facteur Willebrand, moderately reduced in Koate and 8Y, and significantly reduced in Immunate. The HMWM content correlated significantly with the VWF:RCo/VWF:Ag ratio. Only Haemate-P, Innobrand and Facteur Willebrand had VWF:RCo/VWF:Ag ratios >0.7. We found large differences in the content of FVIII and in the FVIII/VWF:RCo ratio. Facteur Willebrand had the lowest (0.02) and Immunate the highest (6.00) ratio. CONCLUSION: Treating physicians must be aware of the large differences between different VWF concentrates and the potential clinical implications. Concentrates lacking HMWM are probably less efficient for mucosal bleedings. FVIII is most important for surgical bleedings, but concentrates with high FVIII/VWF-ratio may induce very high FVIII levels with increased risk of thrombosis. A low FVIII content may be preferable except in case of acute surgery.     

Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease

In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU mL^?1 per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels (FVIII:C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16–60 years), and duration of follow‐up ranged from 5 to 26 years (mean 11 years). Patients had 2–10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII:C at time of diagnosis were 0.44 IU mL^?1 0.34 IU mL^?1 and 0.75 IU mL^?1. At last follow‐up, the mean VWF:Ag, VWF:RCo and FVIII:C were significantly increased to 0.71 IU L^?1, 0.56 IU mL^?1 and 0.90 IU mL^?1 (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and FVIII: C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL^?1 (0.21–0.39, CI 95%, P < 0.0001), 0.20 IU mL^?1 per decade (0.13–0.27, CI 95%, P = 0.0001) and 0.20 IU mL^?1 (0.11–0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age‐related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels.     

Comprehensive evaluation of haemostatic function in von Willebrand disease patients using a microchip‐based flow chamber system

The diagnosis of von Willebrand disease (VWD) is difficult due to the wide spectrum of clinical phenotypes associated with this disorder. We have analysed and characterized haemostatic function in VWD patients using a microchip‐based flow chamber system. Microchips coated with either collagen [platelet (PL)‐chip] or collagen/thromboplastin [atherome (AR)‐chip] were used to evaluate platelet thrombus formation at 1000 s^?1 and fibrin‐rich platelet thrombus formation at 240 s^?1 respectively. Blood samples from an asymptomatic patient with VWD type 1 [von Willebrand factor (VWF): RCo 3.2%; bleeding score (BS 2] displayed normal thrombus formation in both PL‐ and AR‐chips, whereas blood from a symptomatic type 1 patient (VWF: RCo 14%, BS 9) had significantly delayed capillary occlusion. Nearly complete suppression of the flow pressure increase was observed in symptomatic patients with VWD type 2A (BS 13) and 2N (BS 27), whereas no flow pressure was found for the type 3 patient (BS 6). Fibrin‐rich platelet thrombus formation was only weakly increased by the in vitro addition of factor VIII (FVIII) to blood samples from the type 3 patient, but was normalized by the addition of VWF/FVIII. The in vivo effects of treatment with desmopressin or VWF/FVIII for the symptomatic patients were analysed using two types of microchips. The PL‐chip was highly sensitive for patients’ VWF‐mediated platelet functions, whereas the AR‐chip allowed assessment of overall haemostatic ability, including sensitivity to both VWF and FVIII. The combined analysis with PL‐ and AR‐chips may be potentially useful for the diagnosis of VWD based on clinical phenotypes, and for monitoring drug effects.     

Development of an ELISA method for testing VWF ristocetin cofactor activity with improved sensitivity and reliability in the diagnosis of von Willebrand disease

Objectives: Current methods in assessing von Willebrand factor (VWF) ristocetin cofactor activity for Von Willebrand’s disease (VWD) diagnosis include platelet agglutination by aggregometer or macroscopic slide examination, which are both time‐consuming with suboptimal interassay and intra‐assay variation. The purpose of this study is to establish a sensitive assay to detect VWF:RCo activity and evaluate its performance in VWD diagnosis. Methods: We have established a sensitive VWF:RCo–ELISA method using a monoclonal antibody, SZ‐151, to immobilize the recombinant fragment of platelet glycoprotein Ib (rfGPIbα). VWF was captured by rfGPIbα in the presence of ristocetin, and then detected by HRP‐conjugated rabbit anti‐human VWF IgG. We tested the VWF:RCo level by this VWF:RCo–ELISA in 25 patients with different types of VWD and 36 healthy donors, and compared this method to a previously reported ELISA using 2D4 coating antibody. Results: The sensitivity of VWF:RCo–ELISA was greatly improved with this assay (0.008 IU/dL compared to 0.031 IU/dL by 2D4 antibody). The interassay and intra‐assay coefficient variation were 8% and 12%, respectively. The mean values (ranges) of VWF:RCo in patients with type 1, type 2A, type 2B, type 2M, and type 3 of VWD and control group are 31.8 (22.3–56.9), 4.8 (0.6–11.8), 8.6 (1.6–19.7), 3.9 (1.0–6.8), 1.0 (0.5–1.6), and 91.5 (47.3–169.2) IU/dL, respectively. The corresponding ratios (ranges) of VWF:RCo / VWF:Ag are 0.83 (0.70–1.16), 0.27 (0.08–0.58), 0.31 (0.15–0.40), 0.18 (0.14–0.21), 0.52 (0.13–1.19), and 0.92 (0.62–1.26). Conclusion: The VWF:RCo–ELISA using monoclonal anti‐rfGPIbα antibody SZ‐151 showed improved sensitivity and reliability in detecting VWF:RCo activity, and its clinical application would facilitate the diagnosis and classification of VWD.     

Autosomal dominant C1149R von Willebrand disease: phenotypic findings and their implications

Background

Mutation C1149R in the von Willebrand factor (VWF) gene has been thought to cause autosomal dominant severe type 1 von Willebrand disease (VWD).

Design and Methods

Eight patients from three unrelated families with this mutation were included in the present study who had distinct VWF abnormalities, not described in earlier studies.

Results

The patients showed notably low levels of VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding (VWF:CB), and a reduced ristocetin-induced platelet aggregation (RIPA). VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios were lower than 0.7. At basal conditions, all the VWF multimers were decreased in plasma, with a clearly lower relative proportion of the high molecular weight VWF multimers (HMWM). In high-resolution agarose gels, a large decrease in the relative proportions of the satellite bands was seen. The patients had a brief good response to desmopressin (DDAVP) administration, but the released VWF half-life was shorter than normal, indicating an accelerated clearance of their VWF. Platelet VWF was abnormal.

Conclusions

We conclude from the results obtained in these patients for plasma phenotypic data that this mutation should be classified as a VWD type 2A (IIE). DDAVP therapy may be somewhat helpful for this mutation, at least for mild to moderate bleeding. These data provide evidence that for VWD classification factors other than basal VWF, such as DDAVP response and platelet VWF, should be considered.     

N1421K mutation in the glycoprotein Ib binding domain impairs ristocetin- and botrocetin-mediated binding of von Willebrand factor to platelets

von Willebrand disease (VWD) is a common inheritable bleeding disorder caused by deficiency of von Willebrand Factor (VWF), which is involved in platelet adhesion and aggregation. We report a family consisting of three patients with VWD characterized by an apparently normal multimeric pattern, moderately decreased plasma factor VIII (FVIII) and VWF levels, and disproportionately low-plasma VWF:RCo levels. The patients were found to be heterozygous for the novel N1421K mutation, caused by a 4263C > G transversion in exon 28 of the VWF gene coding for the A1 domain. Botrocetin- and ristocetin-mediated binding of plasma VWF to GPIb were reduced in the patients. In vitro mutagenesis and expression in COS-7 cells confirmed the impairment of the mutant in botrocetin- and ristocetin-mediated VWF binding to GPIb. VWF collagen binding capacity was unaffected in plasma from the heterozygous individuals as well as in medium from transfected COS-7 cells. Our findings indicate that the N1421K substitution in the VWF affects the GPIb binding site or a recognition element by a conformational change of the A1 domain.