Importance of Genotyping in von Willebrand Disease to Elucidate Pathogenic Mechanisms and Variability in Phenotype.

Genotyping is not routinely performed at diagnosis of von Willebrand disease (VWD). Therefore, the association between genetic variants and pathogenic mechanism or the clinical and laboratory phenotype is unknown in most patients, especially in type 1 VWD. To investigate whether genotyping adds to a better understanding of the pathogenic mechanisms and variability in phenotype, we analyzed the VWF gene in 390 well-defined VWD patients, included in the WiN study. A VWF gene variant was found in 155 patients (61.5%) with type 1, 122 patients (98.4%) with type 2, and 14 patients (100%) with type 3 VWD. Forty-eight variants were novel. For each VWF gene variant, the pathogenic mechanisms associated with reduced VWF levels was investigated using the FVIII:C/VWF:Ag and VWFpp/VWF:Ag ratios. In type 1 VWD, reduced synthesis or secretion of VWF was most frequently found in patients with nonsense variants, frameshift variants, and deletions, whereas rapid clearance of VWF was mainly found in patients with missense variants. Furthermore, type 1 VWD patients with and without a VWF gene variant were clearly distinct in their clinical features such as age of diagnosis, laboratory phenotype, and bleeding phenotype. In type 2 VWD, 81% of variants were associated with an increased clearance of VWF. To conclude, we identified the pathogenic mechanisms associated with various VWF gene variants in type 1, 2, and 3 VWD patients. Additionally, major differences in the phenotype of type 1 VWD patients with and without a variant were observed, which may be of importance for clinical management.

Desmopressin response depends on the presence and type of genetic variants in patients with type 1 and type 2 von Willebrand disease.

Patients with type 1 and type 2 von Willebrand disease (VWD) can be treated with desmopressin. Although a previous study has shown that the location of the causative VWF gene variant is associated with desmopressin response in type 1 VWD, the association between variants in the VWF gene and desmopressin response is not yet fully understood. Our primary aim was to compare desmopressin response in type 1 VWD patients with and without a VWF gene variant. Secondly, we investigated whether desmopressin response depends on specific VWF gene variants in type 1 and type 2 VWD. We included 250 patients from the Willebrand in the Netherlands study: 72 type 1 without a VWF gene variant, 108 type 1 with a variant, 45 type 2A, 16 type 2M, and 9 type 2N patients. VWF gene was analyzed with ion semiconductor sequencing and Multiplex Ligation-dependent Probe Amplification. Complete response to desmopressin was observed in all type 1 VWD patients without a variant, 64.3% of type 1 patients with a variant, and 31.3% of type 2 patients (P < .001). Despite a large interindividual variability in desmopressin response, patients with the same variant had comparable desmopressin responses. For instance, in 6 type 1 patients with exon 4 to 5 deletion, mean VWF activity at 1 hour after desmopressin was 0.81 IU/mL, with a coefficient of variation of 22.9%. In conclusion, all type 1 VWD patients without a VWF gene variant respond to desmopressin. In type 1 and type 2 VWD patients with a VWF variant, desmopressin response highly depends on the VWF gene variants.

Von Willebrand Factor Multimer Densitometric Analysis: Validation of the Clinical Accuracy and Clinical Implications in Von Willebrand Disease.

Von Willebrand factor (VWF) multimer analysis is important in the classification of von Willebrand disease (VWD). Current visual VWF multimer analysis is time consuming and inaccurate in detecting subtle changes in multimer patterns. Although VWF multimer densitometric analysis may be useful, the accuracy needs further investigation before it can be widely applied. In this study we aimed to validate VWF multimer densitometric analysis in a large cohort of VWD patients and to identify patient characteristics associated with densitometric outcomes. Patients were included from the Willebrand in the Netherlands (WiN) study, in which a bleeding score (BS) was obtained, and blood was drawn. For multimer analysis, citrated blood was separated on an agarose gel and visualized by Western blotting. IMAGEJ was used to generate densitometric images and medium-large VWF multimer index was calculated. We included 560 VWD patients: 328 type 1, 211 type 2, and 21 type 3 patients. Medium-large VWF multimer index performed excellent in distinguishing visually classified normal VWF multimers from reduced high-molecular-weight (HMW) multimers (area under the curve [AUC]: 0.96 [0.94-0.98], P < 0.001), normal multimers from absence of HMW multimers (AUC 1.00 [1.00-1.00], P < 0.001), and type 2A and 2B from type 2M and 2N (AUC: 0.96 [0.94-0.99], P < 0.001). Additionally, higher medium-large VWF multimer index was associated with lower BS in type 1 VWD: ß = -7.6 (-13.0 to -2.1), P = 0.007, adjusted for confounders. Densitometric analysis of VWF multimers had an excellent accuracy compared with visual multimer analysis and may contribute to a better understanding of the clinical features such as the bleeding phenotype of VWD patients.

ADAMTS-13 and bleeding phenotype in von Willebrand disease.

BACKGROUND: The bleeding phenotype of von Willebrand disease (VWD) varies highly between patients and can only partly be explained by von Willebrand factor (VWF) parameters. By cleaving large VWF multimers into smaller, less active multimers, ADAMTS-13 is an important regulator of VWF activity. However, it is unknown what the role of ADAMTS-13 is in individuals with VWD. OBJECTIVES: We therefore studied how ADAMTS-13 activity is associated with the laboratory and bleeding phenotype in individuals with VWD. METHODS: We measured ADAMTS-13 activity using the fluorescence resonance energy transfer substrate VWF 73 assay in 638 individuals with VWD in the nationwide cross-sectional Willebrand in the Netherlands study and in 36 healthy controls. The bleeding phenotype was assessed using the Tosetto bleeding score. RESULTS: ADAMTS-13 activity was similar in individuals with VWD (109% ± 20.6%) and controls (110% ± 19.7%). ADAMTS-13 activity was higher in individuals with VWD with type 3 than those with type 1 (mean difference, 11.8%; 95% confidence interval [CI], 2.9%-20.8%) or type 2 (mean difference, 16.1%; 95% CI, 7.1%-25.1%). ADAMTS-13 activity was not associated with the Tosetto bleeding score (0.1 Tosetto bleeding score increase per 10% ADAMTS-13 increase, 95% CI, -0.2 to 0.3). Furthermore, ADAMTS-13 activity did not differ between individuals with and without a bleeding event during the year preceding blood sampling (mean difference, 1.4%; 95% CI, -2.1% to 4.9%). CONCLUSION: ADAMTS-13 activity was highest in individuals with type 3 VWD, but it had only minor associations with VWF parameters. ADAMTS-13 activity does not influence the bleeding phenotype in individuals with VWD.

Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequent VWF single nucleotide polymorphisms to inhibit mutant VWF. The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes.

von Willebrand factor and factor VIII levels after desmopressin are associated with bleeding phenotype in type 1 VWD.

The bleeding phenotype of patients with type 1 von Willebrand disease (VWD) is very heterogeneous. We hypothesized that this heterogeneity may partly be explained by variability in response of von Willebrand factor (VWF) and factor VIII (FVIII) levels to stress during hemostatic challenges. We therefore investigated whether VWF and FVIII levels after administration of desmopressin, which mimic in vivo hemostatic response during hemostatic challenges, explain the heterogeneity in bleeding phenotype of patients with type 1 VWD. We performed a retrospective cohort study in 122 patients with type 1 VWD. All patients received a test dose of desmopressin shortly after diagnosis. Patients' mean age was 47 ± 14 years, and the mean Tosetto bleeding score was 10 ± 7. Higher FVIII activity during the complete time course after desmopressin administration (1, 3, and 5-6 hours), and higher VWF and FVIII levels combined at 3 hours after desmopressin administration, were associated with a lower bleeding score: ß = -0.9 (-1.7; -0.1) and ß = -1.2 (-1.9; -0.5), respectively, adjusted for age, sex, body mass index (BMI), and comorbidities. Patients with FVIII activity in the highest quartile 3 hours after desmopressin administration had a much lower bleeding score compared with patients in the other 3 quartiles (ß = -5.1 [-8.2; -2.0]) and also had a lower chance of an abnormal bleeding score (odds ratio = 0.2 [0.1-0.5]), both adjusted for age, sex, BMI, and comorbidities. In conclusion, VWF and FVIII levels after desmopressin administration, which mimic hemostatic response to hemostatic challenges, are associated with the bleeding phenotype of patients with type 1 VWD. This may partly explain the variability in bleeding phenotype of these patients.

Von Willebrand factor and ADAMTS13 activity in relation to risk of dementia: a population-based study.

Low ADAMTS13 activity is associated with an increased risk of cardiovascular disease, which is generally attributed to its proteolytic effects on Von Willebrand factor (VWF). Cardiovascular health is an important determinant of cognitive decline, but the association of either VWF or ADAMTS13 with risk of dementia is unknown. Between 1997-2002, we measured VWF antigen and ADAMTS13 activity in 6055 participants of the population-based Rotterdam Study (mean age 69.3 years, 57.2% women). At baseline, 85 participants had dementia, and during 15 years of follow-up 821 developed dementia. Higher VWF was associated with prevalence and risk of dementia, unaffected by concurrent ADAMTS13 activity, but estimates strongly attenuated over time and were no longer statistically significant at 4 years of follow-up (relative risks [95% CI] per standard deviation increase- cross-sectional: 1.37 [1.06-1.77], and longitudinal: 1.05 [0.97-1.14]). In contrast, low ADAMTS13 was associated with increased risk of dementia throughout follow-up (hazard ratio per SD decrease- 1.16 [1.06-1.28]), which alike for ischaemic stroke, was modified by the presence of diabetes (P-interaction = 0.003). In conclusion, higher VWF and low ADAMTS13 activity are associated with increased risk of dementia, but differences in time-course and lack of synergistic effects may indicate in part independent underlying mechanisms.

von Willebrand Factor, ADAMTS13 Activity, and Decline in Kidney Function: A Population-Based Cohort Study.

BACKGROUND: Altered levels of von Willebrand factor (vWF) and ADAMTS13 can promote thrombosis and disturb blood flow in kidney microcirculations. We investigated the association of serum vWF:ADAMTS13 ratio in relation to decline in kidney function. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 2,479 individuals (mean age, 65.1±5.9 [SD] years; 43% men) from the population-based Rotterdam Study. PREDICTORS: vWF, ADAMTS13, and vWF:ADAMTS13 ratio. OUTCOMES & MEASUREMENTS: Annual decline in estimated glomerular filtration rate (eGFR), halving of eGFR, and new-onset eGFR<60mL/min/1.73m2 were assessed. RESULTS: During a median follow-up of 11 (range, 7.81-13.57) years, 500 cases of new-onset eGFR<60mL/min/1.73m2 occurred. The population had a mean eGFR decline of 0.96±0.92mL/min/1.73m2 per year. Higher vWF:ADAMTS13 ratio was associated with steeper annual decline in eGFR (difference, -0.06 [95% CI, -0.09 to -0.02] mL/min/1.73m2 per year) and higher risk for new-onset eGFR<60mL/min/1.73m2 (OR, 1.13; 95% CI, 1.01-1.27). Likewise, higher vWF:ADAMTS13 ratio was associated with higher risk for halving of eGFR (OR, 1.40; 95% CI, 1.02-1.93). After adjustment for cardiovascular risk factors and blood group, effect estimates remained the same. LIMITATIONS: No data available for albuminuria. Participants were classified based on a single measurement of vWF and ADAMTS13. CONCLUSIONS: In this population-based study, we showed that higher vWF:ADAMTS13 ratio is associated with decline in kidney function, suggesting a role of elevated prothrombotic factors in the development and progression of kidney disease.

Bleeding spectrum in children with moderate or severe von Willebrand disease: Relevance of pediatric-specific bleeding.

The bleeding phenotype of children with von Willebrand disease (VWD) needs to be characterized in detail to facilitate diagnosis during childhood and aid in the planning and assessment of treatment strategies. The objective was to evaluate the occurrence, type, and severity of bleeding in a large cohort of children with moderate and severe VWD. We included 113 children (aged 0-16 years) with Type 1 (n = 60), 2 (n = 44), and 3 (n = 9) VWD with von Willebrand factor (VWF) antigen and/or VWF ristocetin cofactor levels ≤ 30 U/dL from a nation-wide cross-sectional study ("Willebrand in the Netherlands" study). Bleeding severity and frequency were determined using the International Society on Thrombosis and Hemostasis-Bleeding Assessment Tool (ISTH-BAT) with supplementary pediatric-specific bleeding symptoms (umbilical stump bleeding, cephalohematoma, cheek hematoma, conjunctival bleeding, postcircumcision and postvenipuncture bleeding). We found that all 26 postmenarche girls experienced menorrhagia. Other common bleedings were cutaneous (81%), oropharyngeal (64%), prolonged bleeding from minor wounds (58%), and epistaxis (56%). Pediatric-specific bleeding symptoms were present in 44% of patients. ISTH-BAT bleeding score was higher in index cases than in affected family members (median, 12.0 vs. 6.5, P < 0.001), higher in Type 3 VWD than in Type 2 or 1 (17.0 vs. 10.5 or 6.5, P < 0.001) and higher in children with severe (<10 U/dL) than moderate VWD (10-30 U/dL) (11.0 vs. 7.0, P < 0.001). Frequency of any bleeding, epistaxis, and oral cavity was higher in types 2 and 3 than in Type 1 VWD and was associated with VWF levels. We conclude that pediatric-specific bleeding symptoms occurred in a large proportion of children with moderate or severe VWD and should be included when evaluating children for VWD.

Genetic variants in the ADAMTS13 and SUPT3H genes are associated with ADAMTS13 activity.

A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) cleaves von Willebrand factor, reducing its prothrombotic activity. The genetic determinants of ADAMTS13 activity remain unclear. We performed a genome-wide association study of ADAMTS13 activity in the Rotterdam Study, a population-based cohort study. We used imputed genotypes of common variants in a discovery sample of 3443 individuals and replication sample of 2025 individuals. We examined rare exonic variant associations in ADAMTS13 in 1609 individuals using an exome array. rs41314453 in ADAMTS13 was associated with ADAMTS13 activity in both our discovery (ß, -20.2%; P = 1.3 × 10(-33)) and replication sample (P = 3.3 × 10(-34)), and explained 3.6% to 6.5% of the variance. In the combined analysis of our discovery and replication samples, there were 2 further independent associations at the ADAMTS13 locus: rs3118667 (ß, 3.0; P = 9.6 × 10(-21)) and rs139911703 (ß, -11.6; P = 3.6 × 10(-8)). In addition, rs10456544 in SUPT3H was associated with a 4.2 increase in ADAMTS13 activity (P = 1.13.6 × 10(-8)). Finally, we found 3 independent associations with rare coding variants in ADAMTS13: rs148312697 (ß, -32.2%; P = 3.7 × 10(-6)), rs142572218 (ß, -46.0%; P = 3.9 × 10(-5)), and rs36222275 (ß, -13.9%; P = 2.9 × 10(-3)). In conclusion, we identified rs41314453 as the main genetic determinant of ADAMTS13 activity, and we present preliminary findings for further associations at the ADAMTS13 and SUPT3H loci.