Higher rates of bleeding and use of treatment products among young boys compared to girls with von Willebrand disease.

There are limited observational studies among children diagnosed with von Willebrand Disease (VWD). We analyzed differences in bleeding characteristics by sex and type of VWD using the largest reported surveillance database of children with VWD (n = 2712), ages 2 to 12 years old. We found that the mean ages of first bleed and diagnosis were lowest among children with type 3 VWD. It was even lower among boys than girls among all VWD types, with statistically significant difference among children with type 1 or type 3 VWD. Children with type 3 VWD also reported higher proportions of ever having a bleed compared to other VWD types, with statistically higher proportions of boys compared to girls reporting ever having a bleed with type 1 and type 2 VWD. A similar pattern was observed with the use of treatment product, showing higher usage among type 3 VWD, and among boys than girls with type 1 and type 2 VWD. While there were no differences in life quality or in well-being status by sex, children with type 3 VWD showed a greater need for mobility assistance compared to children with type 1 and type 2 VWD. In an adjusted analysis among children with type 1 VWD, boys showed a significant association of ever bleeding [hazard ratio 1.4; P-value <.001)] compared to girls. Understanding phenotypic bleeding characteristics, well-being status, treatment, and higher risk groups for bleeding among pre-adolescent children with VWD will aid physicians in efforts to educate families about bleeding symptoms.

Gingival bleeding and mild type 1 von Willebrand disease.

Oral bleeding is a frequently reported symptom of von Willebrand disease (VWD) and evaluated by several VWD bleeding scores. Gingival bleeding is also known to be a leading symptom of plaque-induced gingivitis and untreated periodontal disease. This study evaluates whether mild type 1 VWD is associated with an increased susceptibility to gingival bleeding. In this case-control study, 50 patients and 40 matched controls were periodontally examined, answered a questionnaire, and underwent professional teeth cleaning. Compared with the controls, the VWD patients more frequently reported periodontal symptoms, such as bleeding (34 vs. 20%, P = 0.141) and swelling (54 vs. 32.5%, P = 0.538) of the gingiva, and these patients had their teeth professionally cleaned less often (58 vs. 77.5%, P = 0.051). However, there was less gingival bleeding, as determined by the gingival bleeding index, in the VWD patients compared with the controls (10 vs. 12.2%, P = 0.047); the amount of bleeding on probing was similar between the VWD patients and the controls (17 vs. 17.2%, P = 0.723). Gingival bleeding in VWD patients may be triggered by gingival inflammation, but it is not a genuine symptom of mild type 1 VWD.

Baseline factor VIII plasma levels and age at first bleeding in patients with severe forms of von Willebrand disease.

INTRODUCTION: von Willebrand disease (VWD) is the most common inherited bleeding disorder. The age of bleeding onset is highly variable, also in patients with similar degree of severity. AIM: The primary aim of this study was to evaluate whether baseline factor VIII (FVIII) plasma levels correlate with age at first bleeding in patients with extremely low levels of VWF:RCo (<6 IU dL(-1) ). METHODS: One hundred and three patients with VWF:RCo <6 IU dL(-1) (6 VWD1, 73 VWD2 and 24 VWD3) undergoing a medical examination between September 2010 and September 2013 were included. The relationship between baseline FVIII levels and age at first bleeding was tested in a multivariable linear regression model, adjusting for sex. RESULTS: The median age at first bleeding was lower in patients with VWD3 than in those with severe forms of VWD1 or VWD2 (1 year vs. 7 and 8 years, respectively, P < 0.0001). A positive non-linear relationship between FVIII levels and age at first bleeding was found, the latter increasing by 5 years for every 10 IU dL(-1) increase of FVIII (ß = 4.95 [95% CI: 2.02-7.87]) until levels of 30 IU dL(-1) , after which the age increased slowly. This relationship was not found in VWD 2A and 2B. In 65 patients (63%) there was a more than 6-month delay between bleeding onset and VWD diagnosis, with no difference over decades. CONCLUSIONS: Baseline FVIII plasma levels influence the age at bleeding onset in VWD patients with extremely low levels of VWF:RCo, except in those with types 2A and 2B.

Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease.

Patients with von Willebrand disease (VWD) are often heterozygous for a missense mutation in the von Willebrand factor (VWF) gene. Investigating the pathogenic features of VWF mutations in cells directly derived from patients has been challenging. Here, we have used blood outgrowth endothelial cells (BOECs) isolated from human peripheral blood to analyze the storage and secretion of VWF. BOECs showed full endothelial characteristics and responded to Weibel-Palade body (WPB) secretagogues except desmopressin. We examined BOECs derived from a single subject heterozygous for a type 2N mutation (p.Arg854Gln) and from 4 patients with type 1 VWD who were, respectively, heterozygous for p.Ser1285Pro, p.Leu1307Pro, p.Tyr1584Cys, and p.Cys2693Tyr. Compared with normal BOECs, BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr showed morphologically abnormal WPB and retention of VWF in the endoplasmic reticulum, whereas BOECs heterozygous for p.Arg854Gln or p.Tyr1584Cys showed normal WPB. The agonist-induced exocytosis of WPB from BOECs and formation of VWF strings on BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr, but not for p.Arg854Gln or p.Tyr1584Cys, were reduced. In conclusion, VWD phenotype can be recapitulated in BOECs, and thus BOECs provide a feasible bona fide cell model to study the pathogenic effects of VWF mutations.

Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells.

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

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.