Homozygous type 2N R854W von Willebrand factor is poorly secreted and causes a severe von Willebrand disease phenotype

Summary. Background: von Willebrand disease (VWD) type Normandy (VWD 2N) is caused by mutations at the factor (F)VIII‐binding site of von Willebrand factor (VWF), located in the D′and D3 domains on the N‐terminus of mature VWF. The R854Q mutation is the most frequent cause of this phenotype. Objectives: We report the characterization of a homozygous VWD 2N mutation, R854W, detected in a patient with a severe VWD phenotype. Methods: The plasma VWF phenotype was studied, transient expression of recombinant mutant full‐length VWF in 293 EBNA cells was performed, and the results were compared with those obtained with wild‐type (WT) VWF. Furthermore, expression was also examined in HEK293 cells, which form Weibel–Palade body‐like granules when transfected with WT VWF. Results: The multimer analysis of plasma VWF showed the lack of the typical triplet structure, with the presence of the central band only, and a relative decrease in the high molecular mass multimers. Homozygous expression of recombinant R854W VWF resulted in normal amounts of cellular VWF, but with a severe reduction in secretion into the medium. Severe reductions in FVIII binding to R854W VWF, glycoprotein Ib binding activity and collagen binding of secreted W854 VWF was observed, and reproduced the phenotypic parameters of plasma VWF. In HEK293 cells, homozygous R854W VWF failed to form Weibel–Palade body‐like granules. Conclusions: Our results demonstrate that a homozygous R854W mutation in the D′ domain of VWF induces impaired secretion and activity of the protein, thereby explaining the severe phenotype of the patient.     

Hemorrhagic symptoms and bleeding risk in obligatory carriers of type 3 von Willebrand disease: an international, multicenter study

OBJECTIVES: We undertook an international, multicenter study to describe the clinical picture and to estimate the bleeding risk in a group of obligatory carriers of type 3 von Willebrand disease (VWD). PATIENTS AND METHODS: Obligatory carriers (OC) of type 3 VWD were identified by the presence of offspring with type 3 VWD or by being an offspring of a type 3 patient. Normal controls were age- and sex-matched with the obligatory carriers. A physician-administered standardized questionnaire was used to evaluate hemorrhagic symptoms at presentation. A score system ranging from 0 (no symptom) to 3 (hospitalization, replacement therapy, blood transfusion) was used to quantitate bleeding manifestations. Odds ratios were computed for each symptom. RESULTS: Ten centers participated to the study, enrolling a total of 35 type 3 VWD families, with 70 OC. A total of 215 normal controls and 42 OC for type 1 VWD were also included. About 40% of type 3 OC had at least one bleeding symptom compared to 23% of normal controls and 81.8% of type 1 OC (P < 0.0001 by chi-squared test), showing that type 3 OC clearly represent a distinct population from type 1 OC. The clinical situations associated with an increase of bleeding risk in type 3 OC were epistaxis [odds ratio 3.6; 90% confidence intervals (CI) 1.84-21.5], cutaneous bleeding (odds ratio 5.5; 90% CI 2.5-14.1) and postsurgical bleeding (odds ratio 16.3; 90% CI 4.5-59). The severity of bleeding score correlated with the degree of factor (F) VIII reduction in plasma. CONCLUSIONS: OC for type 3 VWD represent a distinctive population from type 1 OC. These patients, however, present with more frequent bleeding symptoms in comparison to normal controls, especially in case of significantly low FVIII. Desmopressin and/or tranexamic acid might be useful to prevent or treat bleeding in these cases.     

Quantitation of bleeding symptoms in children with von Willebrand disease: use of a standardized pediatric bleeding questionnaire

Summary. Background: Excessive bruising and mucocutaneous bleeding are frequent presenting symptoms in childhood. A detailed bleeding history can distinguish children who may have an inherited bleeding disorder from those who are normal. There is a lack of standardization of such history taking in pediatric practise. Objectives: To assess the performance of a Pediatric Bleeding Questionnaire (PBQ), an adaptation of a standardized adult bleeding questionnaire and score that includes pediatric‐specific bleeding symptoms, in a cohort of children with von Willebrand disease (VWD). Patients/Methods: Bleeding scores were determined by interview, for children with a previous diagnosis of VWD and a control group of unaffected siblings. Results: Bleeding scores were obtained for 100 children with VWD, median age 10.9 years (range, 0.8–17.8 years), and 21 unaffected siblings. Median bleeding score in children with VWD was 7.0 (range, 0–29) and in the control group was 0 (range, ?1–2). Bleeding score varied within and between each VWD type: definite type 1, n = 40, median, 9.0 (range, 2–18); possible type 1, n = 38, median, 2.0 (0–15); type 2, n = 6, median, 14.0 (3–17); and type 3, n = 16, median, 12.0 (4–29). Bleeding scores in affected children correlated with age (Spearman’s correlation coefficient, 0.35; P = 0.0004). The most frequent clinically significant bleeding symptoms were surgical bleeding, bleeding after tooth extraction and menorrhagia. Post‐circumcision bleeding, cephalohematoma, macroscopic hematuria and umbilical stump bleeding were clinically significant in 32% (of circumcised males), 4%, 4% and 3% of children, respectively. Conclusions: The PBQ provides a standardized quantitation of bleeding severity in children with VWD.     

Reduced von Willebrand factor survival in von Willebrand disease: pathophysiologic and clinical relevance

Summary.  Von Willebrand disease (VWD) is characterized by a wide heterogeneity of clinical and laboratory phenotypes. The complexity of the phenotype is further increased by a highly variable removal rate of von Willebrand factor (VWF) released by desmopressin, which is independent of post-infusion peak level. After the initial demonstration that a reduced VWF survival is present in patients with R1205H mutation (VWD Vicenza), several other mutations, mostly occurring in the VWF D3 domain, have been shown to be associated with accelerated removal of released VWF. Normal subjects with O blood group show reduced survival after desmopressin, underlining the role of different VWF glycosylation present in ABO blood group. Recent evidence suggests that liver and spleen macrophages are responsible for VWF clearance through uptake and endocellular degradation, but it is still not known why some VWF mutants are more prone to increased clearance.     

The discriminant power of bleeding history for the diagnosis of type 1 von Willebrand disease: an international, multicenter study

OBJECTIVE: The aim of this study was the validation of the criteria defining a significant mucocutaneous-bleeding history in type 1 von Willebrand disease (VWD). SUBJECTS AND METHODS: To avoid selection bias, 42 obligatory carriers (OC) of type 1 VWD were identified from a panel of 42 families with type 1 VWD enrolled by 10 expert centers. OC were identified by the presence of an offspring and another first degree relative with type 1 VWD (affected subjects, AFF). A standardized questionnaire was administered to evaluate hemorrhagic symptoms at the time of first examination, using a bleeding score ranging from 0 (no symptom) to 3 (hospitalization, replacement therapy, blood transfusion). Sensitivity, specificity, diagnostic likelihood ratios, positive and negative predictive values for the diagnosis of type 1 VWD were calculated from the data collected in OC and in 215 controls. RESULTS: Having at least three hemorrhagic symptoms or a bleeding score of 3 in males and 5 in females was very specific (98.6%) for the bleeding history of type 1 VWD, although less sensitive (69.1%). None of the misclassified OC had life-threatening bleeding episodes after diagnosis. CONCLUSIONS: We suggest that the use of a standardized questionnaire and bleeding score may be useful for the identification of subjects requiring laboratory evaluation for VWD.     

Reduced von Willebrand factor secretion is associated with loss of Weibel–Palade body formation

Background:  von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. Objectives:  We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel–Palade body (WPB)‐like granules. Methods:  Transient expression of recombinant mutant full‐length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild‐type VWF. Expression was also examined in HEK293 cells that form WPB‐like granules when transfected with wild‐type VWF. Results:  Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB‐like granules, while R2663C was capable of WPB‐like granules. Heterozygous expression of VWF variants had a negative impact on wild‐type VWF with a reduction in elongated WPB‐like granules in co‐transfected cells. Conclusions:  Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB‐like granules.     

Factor VIII and von Willebrand factor changes after desmopressin and during pregnancy in type 2M von Willebrand disease Vicenza: a prospective study comparing patients with single (R1205H) and double (R1205H-M740I) defect

BACKGROUND: Type 2M von Willebrand disease (VWD) Vicenza is characterized by the presence of ultra-large von Willebrand factor (VWF) multimers in plasma and very low factor VIII (FVIII)/VWF measurements. So far, R1205H mutation, alone or associated with M740I defect, has been constantly detected in these patients. No data on FVIII/VWF changes after desmopressin and during pregnancy in patients with phenotypic VWD Vicenza has been reported. OBJECTIVE: To evaluate biological responsiveness to desmopressin, the FVIII/VWF changes during pregnancy and the clinical outcome in pregnancies and deliveries of six primipara with type 2M VWD Vicenza prospectively followed. PATIENTS AND METHODS: Three women with single (R1205H) and three with double (R1205H and M740I) mutation in the VWF gene were enrolled in the study. Prior to pregnancy, all patients had undergone desmopressin test-infusion to assess biological responsiveness and its possible clinical usefulness. RESULTS: The results of test-infusion with desmopressin showed the full normalization of FVIII/VWF measurements, with rapid clearance of all moieties postinfusion. However, FVIII/VWF measurements in patients with double defect remained greater after 4 h than those of patients with single defect. The severely reduced basal FVIII/VWF measurements did not change during pregnancy, although somewhat higher VWF levels were observed in patients with double defect. Five out of six women underwent successful delivery under desmopressin prophylaxis, without immediate or delayed bleeding and only one was given a FVIII/VWF concentrate because of a cesarean section. CONCLUSIONS: Delivery in women with VWD type 2M Vicenza is safely managed by using desmopressin, despite the fact that basal low FVIII/VWF is not significantly increased during the pregnancy.     

Different bleeding risk in type 2A and 2M von Willebrand disease: a 2‐year prospective study in 107 patients: a rebuttal

To cite this article: Favaloro EJ, Bonar R, Marsden K. Different bleeding risk in type 2A and 2M von Willebrand disease: a 2‐year prospective study in 107 patients: a rebuttal. J Thromb Haemost 2012; 10: 1455–8.See also Castaman G, Baronciani L, Canciani MT, Federici AB. Different bleeding risk in type 2A and 2M von Willebrand disease: a 2‐year prospective study in 107 patients: a reply to a rebuttal. This issue, pp 1458–60.     

Treatment of severe von Willebrand disease with a high-purity von Willebrand factor concentrate (Wilfactin®): a prospective study of 50 patients

BACKGROUND AND OBJECTIVES: A plasma-derived von Willebrand factor (VWF) concentrate with low factor VIII (FVIII) content was specifically developed to treat von Willebrand disease (VWD). Efficacy and safety were investigated by merging the results of two comparable protocols conducted prospectively in 5 European and 12 French centers. METHODS AND RESULTS: Fifty patients with clinically severe VWD (72% had VWF ristocetin cofactor activity less than 10 IU dL(-1) and 46% had FVIII < 20 IU dL(-1)) were treated with the concentrate as the only therapy, except for clinical situations requiring a priming dose of FVIII to rapidly correct an intrinsic coagulation defect. A total of 139 spontaneous bleeding episodes were treated; only 53 (38%) needed a concomitant FVIII dose. Outcome was excellent or good in 89% of the episodes. Forty-four patients underwent 108 surgical or invasive procedures. Outcome was excellent or good in 95 scheduled procedures (only VWF was infused) and 13 emergency procedures (a priming FVIII dose was co-administered with the first VWF infusion). There were no thrombotic complications and none of the 18 patients with type 3 VWD developed anti-VWF or anti-FVIII antibodies. CONCLUSIONS: This concentrate safely and effectively provides hemostasis in patients with clinically severe VWD.     

An association of candidate gene haplotypes and bleeding severity in von Willebrand disease type 2A, 2B, and 2M pedigrees

Summary.  We analyzed the association of bleeding severity with candidate gene haplotypes within pedigrees of 11 index cases of von Willebrand disease (VWD) type 2 (two type 2A, three type 2B and six type 2M), using the QTL Association model (MENDEL 5.5). In addition to the 11 index cases, these pedigrees included 47 affected and 49 unaffected relatives, as defined by VWF mutations and/or phenotype. A bleeding severity score was derived from a detailed history and adjusted for age. Donors were genotyped using a primer extension method, and eight candidate genes were selected for analysis. VWF antigen (or ristocetin cofactor activity) levels had the strongest influence on bleeding severity score. After Bonferroni correction for multiple testing, only ITGA2 promoter haplotype -52T was associated with an increased bleeding severity score ( P  < 0.01). This association remained statistically significant when the three type 2B pedigrees were excluded ( P  = 0.012) or when gender-specific bleeding categories were excluded ( P  < 0.01). The major haplotypes of seven other candidate genes, GP1BA , ITGA2B, ITGB3 , GP6 , VWF , FGB , and IL6 , were not associated with bleeding severity. These results establish that genetic differences in the expression of the integrin subunit α ₂ can influence the bleeding phenotype of VWD type 2 and complement our previous findings in VWD type 1. Genetically controlled attenuation of platelet collagen receptor expression can influence risk for morbidity in clinical settings where hemostasis is compromised.     

一例2N型遗传性血管性血友病家系的表型诊断和基因型分析

目的：探讨1个遗传性血管性血友病（von Willebrand disease,vWD）2N型家系的表型诊断和基因型分析结果,明确患者的发病机制。方法：对该家系的先证者和家系成员进行出血时间、活化部分凝血活酶时间、瑞斯托霉素诱导的血小板聚集（ristocetin induced platelet aggregation,RIPA）试验、血管性血友病因子（von Willebrand factor,vWF）瑞斯托霉素辅因子、vWF抗原、vWF活性测定、vWF胶原结合试验、凝血因子Ⅷ（coagulation factor Ⅷ,FⅧ）活性、vWF与FⅧ结合试验检测,并作出表型诊断。提取先证者的外周血基因组DNA,用PCR法扩增VWF基因和F8基因的所有外显子及侧翼序列,通过直接测序分析VWF基因和F8基因变异。结果：vWD家系先证者的活化部分凝血活酶时间和出血时间明显延长,血浆瑞RIPA试验、vWF瑞斯托霉素辅因子、vWF抗原、vWF活性和vWF胶原结合试验检测结果均正常,FⅧ活性下降,vWF与FⅧ的结合能力降低。对先证者进行基因测序,发现其VWF基因19号外显子存在c.2446C>T（p.Arg816Trp）错义突变,其儿子在该位点为杂合突变,而先证者及家系成员的F8基因未发现突变。结论：c.2446C>T（p.Arg816Trp）错义突变是导致该家系先证者发生2N型遗传性vWD的原因。     

Different bleeding risk in type 2A and 2M von Willebrand disease: a 2‐year prospective study in 107 patients: a reply to a rebuttal

To cite this article : Castaman G, Baronciani L, Canciani MT, Federici AB. Different bleeding risk in type 2A and 2M von Willebrand disease: a 2‐year prospective study in 107 patients: a reply to a rebuttal. J Thromb Haemost 2012; 10 : 1458–60.See also Favaloro EJ, Bonar R, Marsden K. Different bleeding risk in type 2A and 2M von Willebrand disease: a 2‐year prospective study in 107 patients: a rebuttal. This issue, pp 1455–8.     

Effect of von Willebrand disease type 2B and type 2M mutations on the susceptibility of von Willebrand factor to ADAMTS-13

BACKGROUND: von Willebrand disease (VWD) type 2 is associated with mutations in von Willebrand factor (VWF) that affect its secretion, multimeric pattern, affinity for platelet receptors and clearance of the protein. While increased proteolysis by a disintegrin-like and metalloprotease with thrombospondin type 1 motifs-13 (ADAMTS-13) has been clearly established for VWF type 2A, only little is known about VWF types 2B and 2M in this regard. OBJECTIVES: Sensitivity of wild-type (WT) and mutated recombinant (r) VWF to proteolysis by ADAMTS-13 was investigated to better understand the role of this process in the pathophysiology of VWD. METHODS: We used human rADAMTS-13-WT to digest 11 full-length recombinant forms of VWF carrying molecular abnormalities identified in patients with VWD type 2A (E1638K and P1648S), type 2B (InsM1303, R1306W, R1308P and V1314F) and type 2M (G1324A, E1359K, K1362T, R1374H and I1425F). RESULTS: Using low ionic strength conditions, all mutations induced increased proteolysis of rVWF by rADAMTS-13 as compared with rVWF-WT. The susceptibility of mutants decreased in the following order: type 2A > type 2B > type 2M > rVWF-WT. At physiological salt concentration (150 mm NaCl) the sensitivity of all rVWF to rADAMTS-13 was significantly decreased. However, type 2A and type 2B mutants still exhibited a significantly higher susceptibility to rADAMTS-13 than rVWF-WT, whereas type 2M mutants normalized. CONCLUSIONS: Type 2M mutants and rVWF-WT exhibit a similar sensitivity to rADAMTS-13-mediated proteolysis, in agreement with the normal multimeric pattern in vivo. In VWD type 2B, the spontaneous binding to platelets and excessive degradation by ADAMTS-13 of VWF high-molecular-weight multimers may account for their clearance from plasma.     

Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD)

Summary.  Background:  Type 1 von Willebrand disease (VWD) is a congenital bleeding disorder characterized by a partial quantitative deficiency of plasma von Willebrand factor (VWF) in the absence of structural and/or functional VWF defects. Accurate assessment of the quantity and quality of plasma VWF is difficult but is a prerequisite for correct classification. Objective:  To evaluate the proportion of misclassification of patients historically diagnosed with type 1 VWD using detailed analysis of the VWF multimer structure. Patients and methods:  Previously diagnosed type 1 VWD families and healthy controls were recruited by 12 expert centers in nine European countries. Phenotypic characterization comprised plasma VWF parameters and multimer analysis using low- and intermediate-resolution gels combined with an optimized visualization system. VWF genotyping was performed in all index cases (ICs). Results:  Abnormal multimers were present in 57 out of 150 ICs; however, only 29 out of these 57 (51%) had VWF ristocetin cofactor to antigen ratio below 0.7. In most cases multimer abnormalities were subtle, and only two cases had a significant loss of the largest multimers. Conclusions:  Of the cases previously diagnosed as type 1 VWD, 38% showed abnormal multimers. Depending on the classification criteria used, 22 out of these 57 cases (15% of the total cohort) may be reclassified as type 2, emphasizing the requirement for multimer analysis compared with a mere ratio of VWF functional parameters and VWF:Ag. This is further supported by the finding that even slightly aberrant multimers are highly predictive for the presence of VWF mutations.     

Critical von Willebrand factor A1 domain residues influence type VI collagen binding

Summary. Background: von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen. Objectives: We report here on several mutations that exclusively alter binding to type VI collagen. Patients/methods: Healthy controls and index cases from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen. VWF gene sequencing was performed for all subjects. Results: Two healthy controls and one type 1 VWD subject were heterozygous for an A1 domain sequence variation, R1399H, and displayed a selective decreased binding to type VI collagen but not types I and III. Expression of recombinant 1399H VWF resulted in absent binding to type VI collagen. Two other VWF A1 domain mutations, S1387I and Q1402P, displayed diminished binding to type VI collagen. An 11 amino acid deletion in the A1 domain also abrogated binding to type VI collagen. Conclusions: VWF:CB may be useful in diagnosis of VWD, as a decreased VWF:CB/VWF:Ag ratio may reflect specific loss of collagen binding ability. Mutations that exclusively affect type VI collagen binding may be associated with bleeding, yet missed by current VWF testing.