Application of genetic testing for the diagnosis of von Willebrand disease.

von Willebrand disease (VWD) is the most frequent inherited bleeding disorder, with an estimated symptomatic prevalence of 1 per 1000 in the general population. VWD is characterized by defects in the quantity, quality, or multimeric structure of von Willebrand factor (VWF), a glycoprotein being hemostatically essential in circulation. VWD is classified into 3 principal types: low VWF/type 1 with partial quantitative deficiency of VWF, type 3 with virtual absence of VWF, and type 2 with functional abnormalities of VWF, being classified as 2A, 2B, 2M, and 2N. A new VWD type has been officially recognized by the ISTH SSC on von Willebrand factor which has also been discussed by the joint ASH/ISTH/NHF/WFH 2021 guidelines (ie, type 1C), indicating patients with quantitative deficiency due to an enhanced VWF clearance. With the advent of next-generation sequencing technologies, the process of genetic diagnosis has substantially changed and improved accuracy. Therefore, nowadays, patients with type 3 and severe type 1 VWD can benefit from genetic testing as much as type 2 VWD. Specifically, genetic testing can be used to confirm or differentiate a VWD diagnosis, as well as to provide genetic counseling. The focus of this manuscript is to discuss the current knowledge on VWD molecular pathophysiology and the application of genetic testing for VWD diagnosis.

Type 2M/2A von Willebrand disease: a shared phenotype between type 2M and 2A.

ABSTRACT: Four variants have been continuously subjected to debate and received different von Willebrand disease (VWD) classifications: p.R1315L, p.R1315C, p.R1374H, and p.R1374C. We chose to comprehensively investigate these variants with full set of VWD tests, protein-modeling predictions and applying structural biology. Patients with p.R1315L, p.R1315C, p.R1374H, and p.R1374C were included. A group with type 2A and 2M was included to better understand similarities and differences. Patients were investigated for phenotypic assays and underlying disease mechanisms. We applied deep protein modeling predictions and structural biology to elucidate the causative effects of variants. Forty-three patients with these variants and 70 with 2A (n = 35) or 2M (n = 35) were studied. Patients with p.R1315L, p.R1374H, or p.R1374C showed a common phenotype between 2M and 2A using von Willebrand factor (VWF):GPIbR/VWF:Ag and VWF:CB/VWF:Ag ratios and VWF multimeric profile, whereas p.R1315C represented a type 2M phenotype. There was an overall reduced VWF synthesis or secretion in 2M and cases with p.R1315L, p.R1374H, and p.R1374C, but not in 2A. Reduced VWF survival was observed in most 2A (77%), 2M (80%), and all 40 cases with p.R1315L, p.R1374H, and p.R1374C. These were the only variants that fall at the interface between the A1-A2 domains. p.R1315L/C mutants induce more compactness and internal mobility, whereas p.R1374H/C display a more extended overall geometry. We propose a new classification of type 2M/2A for p.R1315L, p.R1374H, and p.R1374C because they share a common phenotype with 2M and 2A. Our structural analysis shows the unique location of these variants on the A1-A2 domains and their distinctive effect on VWF.

Clinical and Laboratory Presentation and Underlying Mechanism in Patients with Low VWF.

BACKGROUND: Low von Willebrand factor (VWF) refers to subjects with plasma levels of 30 to 50 IU/dL. The mechanism of low VWF is poorly understood. We chose to determine the clinical presentation, laboratory phenotype, and underlying mechanisms of low VWF. MATERIAL AND METHODS: We included 250 patients characterized with low VWF. The International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT) was used to assess clinical symptoms. To determine the underlying mechanisms of low VWF, we used as markers the VWF propeptide (VWFpp) assay and FVIII:C/VWF:Ag ratio for VWF synthesis and the VWFpp/VWF:Ag ratio for VWF clearance. Results were compared with those of 120 healthy controls. Cases with abnormal screening tests were further evaluated for coagulation factor levels and platelet disorders. RESULTS: The median age of the cohort was 35 years (range 3-85), 21% were children (n = 53), 34% were adult males (n = 85), and 45% (n = 112) were adult females. According to the ISTH-BAT, abnormal bleeding was found in 35% of children, 47% of males, and 49% of females. No association was found between VWF activity levels and ISTH-BAT. Patients showed an overall decreased VWF synthesis/secretion and an enhanced VWF clearance was identified in 33% of them. In 89 patients (36%), there were other hemostasis-related defects, but there was no difference in the ISTH-BAT between the two groups. CONCLUSION: Our findings indicate that reduced VWF synthesis/secretion and enhanced VWF clearance are major mechanisms of low VWF levels. Patients with low VWF have significant bleeding manifestations. While other hemostasis defects occurred together with low VWF, this combination did not exacerbate clinical symptoms.

Population-based prevalence and mutational landscape of von Willebrand disease using large-scale genetic databases.

Von Willebrand disease (VWD) is a common bleeding disorder caused by mutations in the von Willebrand factor gene (VWF). The true global prevalence of VWD has not been accurately established. We estimated the worldwide and within-population prevalence of inherited VWD by analyzing exome and genome data of 141,456 individuals gathered by the genome Aggregation Database (gnomAD). We also extended our data deepening by mining the main databases containing VWF variants i.e., the Leiden Open Variation Database (LOVD) and the Human Gene Mutation Database (HGMD) with the goal to explore the global mutational spectrum of VWD. A total of 4,313 VWF variants were identified in the gnomAD population, of which 505 were predicted to be pathogenic or already reported to be associated with VWD. Among the 282,912 alleles analyzed, 31,785 were affected by the aforementioned variants. The global prevalence of dominant VWD in 1000 individuals was established to be 74 for type 1, 3 for 2A, 3 for 2B and 6 for 2M. The global prevalences for recessive VWD forms (type 2N and type 3) were 0.31 and 0.7 in 1000 individuals, respectively. This comprehensive analysis provided a global mutational landscape of VWF by means of 927 already reported variants in the HGMD and LOVD datasets and 287 novel pathogenic variants identified in the gnomAD. Our results reveal that there is a considerably higher than expected prevalence of putative disease alleles and variants associated with VWD and suggest that a large number of VWD patients are undiagnosed.

Erratum to 'A comparative study in type 2 von Willebrand disease patients using four different platelet-dependent von Willebrand factor assays.' [Research and Practice in Thrombosis and Haemostasis Volume 7, Issue 3, March 2023, 100139].

[This corrects the article DOI: 10.1016/j.rpth.2023.100139.].

A comparative study in patients with type 2 von Willebrand disease using 4 different platelet-dependent von Willebrand factor assays.

Background: Several assays are now available to evaluate platelet-dependent von Willebrand factor (VWF) activity. Objective: To report the results obtained using 4 different assays in patients with von Willebrand disease (VWD) carrying variants mainly in the A1 domain, which is critical for VWF binding to glycoprotein Ib (GPIb) and ristocetin. Methods: We evaluated 4 different assays, 2 gain-of-function mutant GPIb binding (VWF:GPIbM) and 2 ristocetin cofactor (VWF:RCo) assays, in 76 patients with type 2 VWD. Patients and healthy controls were tested using VWF:GPIbM enzyme-linked immunosorbent assay (ELISA), VWF:GPIbM automated, VWF:RCo aggregometric, and VWF:RCo automated assays. Results: There was a good correlation (Pearson's r>0.82) and agreement (Bland-Altman plots assessment) between the 4 assays, although several outliers existed among the type 2B without high-molecular-weight multimers (HMWM). The VWF activity/VWF:antigen ratios, calculated for each assay, were used to establish the percentage of a correct diagnosis of type 2 (ratio<0.60) in these patients: VWF:RCo aggregometric, 2A(100%), 2M(78%), 2M/2A(100%), 2B(68%); VWF:RCo automated, 2A(88%), 2M(89%), 2M/2A(100%), 2B(63%); VWF:GPIbM ELISA, 2A(96%), 2M(67%), 2M/2A(67%), 2B(0%); VWF:GPIbM automated, 2A(73%), 2M(44%), 2M/2A(75%), 2B(84%). In type 2B patients with HMWM, all assays gave a ratio ≥0.60. Conclusion: The VWF:GPIbM-automated assay is the most effective to diagnose as type 2 the 2B variants, whereas the VWF:RCo assays are the most effective in detecting 2M and 2M/2A variants. The VWF:GPIbM ELISA greatly overestimates the activity of the type 2B patients lacking HMWM. In this study, the use of a VWF activity/VWF:antigen ratio cut-off of 0.70 halved the number of misdiagnosed patients.

von Willebrand factor neutralizing and non-neutralizing alloantibodies in 213 subjects with type 3 von Willebrand disease enrolled in 3WINTERS-IPS.

BACKGROUND: Type 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions. OBJECTIVE: The objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS. METHODS: An indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay. RESULTS: In total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14). CONCLUSIONS: Anti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used.

Genetic determinants of enhanced von Willebrand factor clearance from plasma.

BACKGROUND: Enhanced von Willebrand factor (VWF) clearance from plasma is associated with von Willebrand disease (VWD). However, the genetic background of this disease mechanism is not well defined. OBJECTIVE: To determine VWF variants that are associated with reduced VWF survival. METHODS: Two hundred fifty-four patients with VWD (type 1 = 50 and type 2 = 204) were investigated, and the results were compared with 120 healthy controls. The patients were comprehensively characterized for phenotypic and genetic features. The ratio of VWF propeptide (VWFpp)/VWF antigen (VWFpp ratio) was used to establish in each patient the VWF clearance state. RESULTS: Out of 92 variants associated with type 1 (7 were novel) and type 2 VWD, 19 had a VWFpp ratio ranging from 1.7 to 2.2, 24 had a VWFpp ratio between 2.3 and 2.9, and 24 variants had a ratio of ≥3. The VWFpp median ratio in healthy controls was 0.98 (0.55-1.6) so that a cut-off value of >1.6 was considered an indicator of accelerated VWF clearance from plasma. An enhanced VWF clearance was observed in 34% of type 1 cases, 100% of type 1 Vicenza cases, 81% of 2A cases, 77% of 2B cases, 88% of 2M cases, and 36% of 2N cases. CONCLUSIONS: An accelerated VWF clearance was found in most patients with type 2A, 2B, and 2M VWD, with a lower proportion of type 1 and 2N. Sixty-seven different variants alone or in combination with other variants were associated with an increased VWFpp ratio. The variants with the highest VWFpp ratio were mostly located in the D3-A1 VWF domains.

Phenotypic and genetic characterizations of the Milan cohort of von Willebrand disease type 2.

von Willebrand disease (VWD) type 2 is caused by qualitative abnormalities of von Willebrand factor (VWF). This study aimed to determine the genotypic and phenotypic characterizations of a large VWD type 2 cohort from Milan. We included 321 patients (54% female) within 148 unrelated families from 1995 to 2021. Patients were fully characterized using laboratory phenotypic tests, and the genotypic diagnosis was confirmed by target genetic analysis using Sanger sequencing. Patients were diagnosed with type 2A (n = 98; 48 families), 2B (n = 85; 38 families), 2M (n = 112; 50 families), or 2N (n = 26; 12 families). Eighty-two unique VWF variants, including 8 novel variants, were found. The potential pathogenic effect of novel variants was assessed by in silico analysis. Most patients were heterozygous for a single variant (n = 259; 81%), whereas 37 cases (11%) had 2 variants (4 homozygous, 9 in trans, and 24 in cis). Twenty-five patients (8%) had ≥3 variants, mainly as a result of gene conversions. Among the 82 distinct variants identified, 5 different types, including missense (n = 64), gene conversion (n = 10), synonymous (n = 1), deletion (n = 4), and splice (n = 3), were observed. The results from this large cohort showed that VWD type 2 is invariably due to variants that do not prevent the synthesis of the protein, and a vast majority of patients (88%) had missense variants. Given the complexity of type 2 diagnosis and the necessity of performing several phenotypic tests, genetic analysis for patients suspected of having type 2 is beneficial to establish the correct diagnosis.

The dominant p.Thr274Pro mutation in the von Willebrand factor propeptide causes the von Willebrand disease type 1 phenotype in two unrelated patients.

BACKGROUND: von Willebrand factor propeptide (VWFpp) plays an important role in VWF multimerization and storage. VWFpp mutations have been previously associated with types 1, 3 and 2A/IIC von Willebrand disease (VWD). AIMS: To characterize the novel p.Thr274Pro variant identified in two unrelated type 1 VWD patients. METHODS: Phenotype tests were performed to evaluate patients' plasma and platelets following the current ISTH-SSC guidelines. Molecular analysis was performed using next-generation sequencing. The pcDNA3.1-VWF-WT and mutant pcDNA3.1-VWF-Thr274Pro expression vectors were transiently transfected into HEK293 cells to evaluate recombinant (r)VWF constitutive and regulated secretion. For the latter, the transfected cells were stimulated with phorbol-12-myristate-13-acetate. Immunofluorescence staining was performed to assess the localization of WT-rVWF and Thr274Pro-rVWF in endoplasmic reticulum, lysosomes, cis-/trans-Golgi and pseudo-Weibel Palade bodies. RESULTS: Biochemical characterization of patients' plasma samples indicated a type 1 VWD diagnosis. Both patients were heterozygous for the p.Thr274Pro variant. Hybrid Thr274Pro/WT-rVWF showed a secretion reduction of 36±4% according to patients' plasma VWF:Ag levels, whereas Thr274Pro-rVWF secretion was strongly impaired (21±2%). The amount of rVWF in cell lysates was nearly normal for both Thr274P (62±17%) and Thr274Pro/WT-rVWF (72±23%). The regulated secretion was impaired for Thr274Pro/WT-rVWF, whereas Thr274Pro-rVWF was not released at all. Immunofluorescence staining revealed no particular differences between WT and Thr274Pro-rVWF, although Thr274Pro-rVWF showed less pseudo-Weibel Palade bodies with a rounder shape than WT-rVWF. CONCLUSIONS: The novel p.Thr274Pro mutation has a dominant effect and it is responsible of patients' type 1 VWD phenotype through a combined mechanism of reduced synthesis, impaired secretion and multimerization.

Von Willebrand factor propeptide and pathophysiological mechanisms in European and Iranian patients with type 3 von Willebrand disease enrolled in the 3WINTERS-IPS study.

BACKGROUND: Type 3 von Willebrand disease (VWD) is a severe bleeding disorder caused by the virtually complete absence of von Willebrand factor (VWF). Pathophysiological mechanisms of VWD like defective synthesis, secretion, and clearance of VWF have previously been evaluated using ratios of VWF propeptide (VWFpp) over VWF antigen (VWF:Ag) and factor (F)VIII coagulant activity (FVIII:C) over VWF:Ag. OBJECTIVE: To investigate whether the VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios may also be applied to understand the pathophysiological mechanism underlying type 3 VWD and whether VWFpp is associated with bleeding severity. METHODS: European and Iranian type 3 patients were enrolled in the 3WINTERS-IPS study. Plasma samples and buffy coats were collected and a bleeding assessment tool was administered at enrolment. VWF:Ag, VWFpp, FVIII:C, and genetic analyses were performed centrally, to confirm patients' diagnoses. VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were compared among different variant classes using the Mann-Whitney test. Median differences with 95% confidence intervals (CI) were estimated using the Hodges-Lehmann method. VWFpp association with bleeding symptoms was assessed using Spearman's rank correlation. RESULTS: Homozygosity/compound heterozygosity for missense variants showed higher VWFpp level and VWFpp/VWF:Ag ratio than homozygosity/compound heterozygosity for null variants ([VWFpp median difference, 1.4 IU/dl; 95% CI, 0.2-2.7; P = .016]; [VWFpp/VWF:Ag median difference, 1.4; 95% CI, 0-4.2; P = .054]). FVIII: C/VWF:Ag ratio was similarly increased in both. VWFpp level did not correlate with the bleeding symptoms (r = .024; P = .778). CONCLUSIONS: An increased VWFpp/VWF:Ag ratio is indicative of missense variants, whereas FVIII:C/VWF:Ag ratio does not discriminate missense from null alleles. The VWFpp level was not associated with the severity of bleeding phenotype.

Genotypes of European and Iranian patients with type 3 von Willebrand disease enrolled in 3WINTERS-IPS.

Type 3 von Willebrand disease (VWD3) is a rare and severe bleeding disorder characterized by often undetectable von Willebrand factor (VWF) plasma levels, a recessive inheritance pattern, and heterogeneous genotype. The objective of this study was to identify the VWF defects in 265 European and Iranian patients with VWD3 enrolled in 3WINTERS-IPS (Type 3 Von Willebrand International Registries Inhibitor Prospective Study). All analyses were performed in centralized laboratories. The VWF genotype was studied in 231 patients with available DNA (121 [115 families] from Europe [EU], and 110 [91 families] from Iran [IR]). Among 206 unrelated patients, 134 were homozygous (EU/IR = 57/77) and 50 were compound heterozygous (EU/IR = 43/7) for VWF variants. In 22 patients, no or only one variant was found. A total of 154 different VWF variants (EU/IR = 101/58 [5 shared]) were identified among the 379 affected alleles (EU/IR = 210/169), of which 48 (EU/IR = 18/30) were novel. The variants p.Arg1659*, p.Arg1853*, p.Arg2535*, p.Cys275Ser, and delEx1_Ex5 were found in both European and Iranian VWD3 patients. Sixty variants were identified only in a single allele (EU/IR = 50/10), whereas 18 were recurrent (≥3 patients) within 144 affected alleles. Nine large deletions and one large insertion were found. Although most variants predicted null alleles, 21% of patients carried at least 1 missense variant. VWD3 genotype was more heterogeneous in the European population than in the Iranian population, with nearly twice as many different variants. A higher number of novel variants were found in the Iranian VWD3 patients.

Pro-coagulant imbalance in patients with community acquired pneumonia assessed on admission and one month after hospital discharge.

OBJECTIVES: Patients hospitalized because of community-acquired-pneumonia (CAP) are at risk of cardiovascular diseases. Although plasma procoagulant imbalance play a role, mechanisms are not completely understood. We aimed to investigate whether there is a measurable state of procoagulant imbalance following inflammation determined by CAP. METHODS: We analyzed blood from 51 CAP patients at admission and 51 healthy subjects (HS) for (i) pro and anticoagulants, (ii) thrombin generation (TG) with or without thrombomodulin (TM), which is the physiologic activator of the protein C anticoagulant pathway and(iii) by assessing the ratio between von Willebrand-factor (VWF) and its protease ADAMTS13. Thirty patients were re-analyzed one month after discharge when CAP was resolved. RESULTS: Median levels of TG parameters, including the endogenous thrombin potential (ETP), the ETP-TM-ratio (with/without TM), peak-thrombin and velocity index were higher in patients at baseline than HS. In particular, the median (IQR) ETP-TM-ratio in patients vs. HS was 0.88 (0.83-0.91) vs. 0.63 (0.48-0.71), p<0.001. Factor (F)VIII, a potent procoagulant involved in TG was higher in patients at baseline than HS [195 U/dL (100-388) vs. 127(108-145)], p<0.001]. The ratio of VWF/ADAMTS13 was higher at baseline than HS. Cumulatively, the findings indicate a state of pro-coagulant imbalance, which (although reduced), remained high [i.e., ETP-TM-ratio, 0.80 (0.74-0.84); FVIII, 152 U/dL (122-190)] one month after discharge when the infection was resolved. CONCLUSIONS: Patients with CAP possess a state of pro-coagulant imbalance, which remains substantially high, even when the infection is resolved. The findings suggest CAP patients as candidates for antithrombotic prophylaxis even after the resolution of infection. Clinical trials are warranted to assess the benefit/risk ratio of prophylaxis extension.

Increasing levels of von Willebrand factor and factor VIII with age in patients affected by von Willebrand disease.

Essentials VWF and FVIII increase with age in patients affected by VWD. VWF and FVIII increase in type 1 and in low levels of VWF patients. VWF and FVIII do not increase in type 1 Vicenza. FVIII increases in type 2 VWD patients. ABSTRACT: Background Increasing levels of von Willebrand factor (VWF) and factor VIII (FVIII:C) was associated with age in type 1 von Willebrand disease (VWD). Objectives To evaluate VWF and FVIII:C increase with age in a large group of patients with VWD and low levels of VWF, in whom levels were repeatedly measured. Methods Clinical charts from all patients evaluated at the A. Bianchi Bonomi Center between 1970 and 2018 were reviewed and data on VWF and FVIII:C collected. Patients affected by type 3, severe type 1 and 2N VWD were excluded. The repeated measurements were evaluated by linear mixed-effects models. A linear association between age and VWF/FVIII:C was shown after the age of 40 years in the linear mixed models and analyzed by calculating the regression slope coefficient (ß). Results A total of 617 patients were included in the study (314 type 2, 112 type 1, 181 low VWF levels), with a median age at first measurement of 28 years (interquartile range 14/42) and a mean follow-up of 16 years (standard deviation 11). VWF and FVIII:C increased with age in the whole group. The increase became linear after the age of 40 years (3.68 and 7.44 IU/dL per decade for VWF:activity and FVIII:C). In type 2, FVIII:C increased with age, whereas an increase of both VWF:activity and FVIII:C were shown in patients with type 1 VWD and low levels of VWF. Conclusions A differential increase of VWF and FVIII:C with age was shown among in different ages and types of VWD.

The ADAMTS13-von Willebrand factor axis in COVID-19 patients.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is characterized by an increased risk of thromboembolic events, with evidence of microthrombosis in the lungs of deceased patients. OBJECTIVES: To investigate the mechanism of microthrombosis in COVID-19 progression. PATIENTS/METHODS: We assessed von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin-cofactor (VWF:RCo), VWF multimers, VWF propeptide (VWFpp), and ADAMTS13 activity in a cross-sectional study of 50 patients stratified according to their admission to three different intensity of care units: low (requiring high-flow nasal cannula oxygenation, n = 14), intermediate (requiring continuous positive airway pressure devices, n = 17), and high (requiring mechanical ventilation, n = 19). RESULTS: Median VWF:Ag, VWF:RCo, and VWFpp levels were markedly elevated in COVID-19 patients and increased with intensity of care, with VWF:Ag being 268, 386, and 476 IU/dL; VWF:RCo 216, 334, and 388 IU/dL; and VWFpp 156, 172, and 192 IU/dL in patients at low, intermediate, and high intensity of care, respectively. Conversely, the high-to-low molecular-weight VWF multimers ratios progressively decreased with increasing intensity of care, as well as median ADAMTS13 activity levels, which ranged from 82 IU/dL for patients at low intensity of care to 62 and 55 IU/dL for those at intermediate and high intensity of care. CONCLUSIONS: We found a significant alteration of the VWF-ADAMTS13 axis in COVID-19 patients, with an elevated VWF:Ag to ADAMTS13 activity ratio that was strongly associated with disease severity. Such an imbalance enhances the hypercoagulable state of COVID-19 patients and their risk of microthrombosis.

Comparison of von Willebrand factor platelet-binding activity assays: ELISA overreads type 2B with loss of HMW multimers.

BACKGROUND: A number of new assays with different measuring principles are available to measure von Willebrand factor (VWF) glycoprotein Ib (GPIb)-binding activity, but little is known about how these assays might behave differently for subtypes of von Willebrand disease (VWD). OBJECTIVES: The Comparison of Assays to Measure VWF Activity (COMPASS-VWF) study was designed to compare all available VWF GPIb-binding activity assays for VWF. We specifically searched for particular assay behavior differences. PATIENTS/METHODS: To sort out random differences from systematic assay behavior deviations, all assays were performed in different laboratories on the same samples in a blinded fashion. Samples from 53 normal controls and 42 well-characterized VWD patients were reanalyzed in this study to dissect assay-specific discrepancies. RESULTS: No assay behavior differences were found for 53 normal controls. For VWD patients, we found the following systematic assay behavior patterns: (a) All ELISA assays for VWF:GPIbR as well as VWF:GPIbM are insensitive to detect the low VWF activity of VWD type 2B patients with loss of high molecular weight multimers; (b) VWF:Ab assay reports higher activity for the p.V1665E mutation than all other assays; and (c) all ristocetin-based assays (including VWF:RCo using fixed platelets) but the AcuStar assay report discrepantly low VWF activity for the p.P1467S polymorphism. No systematic assay-specific difference was observed for either the particle agglutination VWF:GPIbM assay or the AcuStar assay using magnetic beads. CONCLUSIONS: Different assay principles may lead to discrepant results for certain VWD types or mutations. Therefore, a more extensive study for a large number of patients is needed to better characterize the incidence and relevance of such assay-specific differences.

Bleeding symptoms in patients diagnosed as type 3 von Willebrand disease: Results from 3WINTERS-IPS, an international and collaborative cross-sectional study.

BACKGROUND: Type 3 von Willebrand's disease (VWD) patients present markedly reduced levels of von Willebrand factor and factor VIII. Because of its rarity, the bleeding phenotype of type 3 VWD is poorly described, as compared to type 1 VWD. AIMS: To evaluate the frequency and the severity of bleeding symptoms across age and sex groups in type 3 patients and to compare these with those observed in type 1 VWD patients to investigate any possible clustering of bleeding symptoms within type 3 patients. METHODS: We compared the bleeding phenotype and computed the bleeding score (BS) using the MCMDM-1VWD bleeding questionnaire in patients enrolled in the 3WINTERS-IPS and MCMDM-1VWD studies. RESULTS: In 223 unrelated type 3 VWD patients, both the BS and the number of clinically relevant bleeding symptoms were increased in type 3 as compared to type 1 VWD patients (15 versus 6 and 5 versus 3). Intracranial bleeding, oral cavity, hemarthroses, and deep hematomas were at least five-fold over-represented in type 3 VWD. A more severe bleeding phenotype was evident in patients having von Willebrand factor antigen levels < 20 IU/dL at diagnosis in the two merged cohorts. In type 3 patients, there was an apparent clustering of hemarthrosis with gastrointestinal bleeding and epistaxis, whereas bleeding after surgery or tooth extraction clusters with oral bleeding and menorrhagia. CONCLUSIONS: In the largest cohort of type 3 VWD patients, we were able to describe a distinct clinical phenotype that is associated with the presence of a more severe hemostatic defect.