Low Von Willebrand Factor Levels Research Articles

Suspected collagen disorders in the bleeding disorder clinic: A case–control study

Disorders of collagen are associated with a mild bleeding tendency because of the potential abnormal interaction of collagen, von Willebrand factor (VWF) and platelets required during primary haemostasis and due to generalized soft tissue fragility. Abnormal collagen may contribute to bleeding in existing mucocutaneous bleeding disorders, but the prevalence in this setting is unknown. Generalized symptomatic joint hypermobility (SJH) is common in collagen disorders and may be objectively measured. To assess the association between symptomatic joint hypermobility and mucocutaneous bleeding disorders, we performed a case-control study in which case subjects were 55 consecutive individuals who had visited our bleeding disorder clinic with a diagnosis of von Willebrand disease, low von Willebrand factor levels, mild platelet function disorder or undefined bleeding disorder. Controls were 50 subjects without a bleeding disorder, and were age and gender matched to the cases. All subjects were assessed with: (i) Beighton score for joint hypermobility, (ii) revised Brighton criteria, (iii) Condensed MCMDM1-VWD bleeding questionnaire, and (iv) haemostasis laboratory studies. The prevalence of SJH/suspected collagen disorder in the bleeding disorder clinic was 24% (13/55) compared with 2% (1/50) in the control population (OR 15, 95% CI 2-121). Seventy-seven per cent of bleeding disorder clinic SJH subjects (10/13) had a prior personal or family history of Ehlers-Danlos, Benign Joint Hypermobility Syndrome or Osteogenesis Imperfecta (OI). Symptomatic joint hypermobility was associated with increased odds of an underlying mucocutaneous bleeding disorder. These findings suggest that a collagen disorder is common and often unrecognized in the bleeding disorder clinic as a potential contributor to the bleeding symptoms.

Laboratory diagnostic approach of the parents–children relationship in differentiating low-level von Willebrand factor from mild type 1 von Willebrand disease

The present study aimed to evaluate the parent-child relationship in differentiating between unaffected healthy individuals and those with von Willebrand disease (VWD). This study was performed on 15 children between the ages of 5 and 15 years and parents with personal and familial evidence of bleeding. Diagnosis of VWD as considered 'low von Willebrand factor (VWF) level or mild type 1 VWD' in the following children: those with low VWF levels (VWF:RCo and VWF:Ag between 30 and 50 U/dl), at least one bleeding symptom and a family member with at least one bleeding symptom. Laboratory values in the parents of families 1-7 were VWF:Ag 65-90, VWF:RCo 54-87, and FVIII:C 74-110, versus VWF:Ag 33-47, VWF:RCo 30-42, and FVIII:C 36-67 in their children. The normal laboratory values in the parents of families 1-7 suggested that their children would probably have low VWF levels. Our findings are that VWF levels are increasing with age. Laboratory values in the parents of families 8-15 were VWF:Ag 30-59, VWF:RCo 32-55, and FVIII:C 44-66, versus VWF:Ag 32-48, VWF:RCo 30-54, and FVIII:C 38-55 in their children. The laboratory values in the children from families 8-15 were close to the minimum range of normal or below normal, which suggested that it was possible that the parents and children in families 8-15 could be diagnosed as having mild type 1 VWD. The present study's findings show that comparison of the VWF levels in parents and their children may be helpful in differentiating children with low VWF levels and mild type 1 VWD from children that only have low VWF levels.

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

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. 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. 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. 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. 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.

Inhibition of Von Willebrand Factor (VWF) by ARC1779 Normalizes Von Willebrand Factor/FVIII Levels and Corrects Thrombocytopenia in VWD Type 2b Patients,

Abstract 3377 Introduction:Infusion of the anti-VWF aptamer ARC1779 effectively elevated platelet counts in some patients with congenital thrombocytopenic purpura, and blunted the platelet drop induced by desmopressin in patients with type 2b von Willebrand disease (VWD). Thus, we hypothesized that ARC1779 may increase VWF levels and correct thrombocytopenia by blocking the hyperactive VWF A1 domain in type 2b VWD. Methods:Thrombocytopenic patients suffering from type 2b VWD received intravenous infusions of ARC1779 (4 mcg/kg/min) for 72 hours in a prospective clinical trial. Patients were monitored using previsouly published assays (Thromb Haemost. 2010;104:563–70). Data are provided as median and the range and were tested for significance by repeated measures analysis of variance. Results:All patients had moderate thrombocytopenia, low levels of VWF:RCo, VWF:CBA, FVIII:C, and collagen/ADP closure times >300s (platelet function analyzer PFA-100). ARC1779 was well tolerated and safe even in a patient who suffered from active chronic gastrointestinal bleeding due to angiodysplasias before start of infusion. Plasma concentrations of ARC1779 increased to 76 mcg/mL (59–130) which suppressed free VWF A1 domains in the REAADS assay to a minimum of <3 to 5%. Consistent with the estimated in vivo half-life of VWF, VWF/FVIII levels increased as early as 12h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. That was best exemplified by VWF:RCo levels which increased from 32% (26–45%) 10-fold to 367% (215–418%) 8h after end of infusion, and decreased again to 33% (20–40%) during follow up. Similarly VWF:CBA levels increased 16-fold (8 to 69-fold), VWF: Ag levels 5.2 fold (3 to 27-fold), and FVIII levels 4.3 fold (3 to 6-fold). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40/nL (38–58/nL) to a maximum of 146/nL (107–248/nL). This was observed although patients were suffering from chronic liver disease due to hepatitis C infection or associated immune thrombocytopenia, which could have blunted the platelet response. All changes were statistically significant (p<0.007). Conclusion:This clinical trial underscores the therapeutic efficacy of the VWF A1 antagonist aptamer ARC1779. ARC1779 significantly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2b patients. This could facilitate (i) the collection of autologous platelets for platelet transfusions pre-operatively or (ii) interferon therapy in patients whose HCV therapy could not be initiated or had to be stopped due to thrombocytopenia. As angiodysplasias may be caused by defective or deficient VWF (Blood 2011;117:1071–80), it may also be tempting to further investigate the potential of chronic anti-VWF aptamer therapy to treat angiodysplasia. Disclosures:Knoebl:Archemix Corp.: Consultancy. Pabinger:Archemix Corp.: Consultancy. Gilbert:Archemix Corp.: previous employment. Schaub:Archemix Corp.: Consultancy, previous employment. Jilma:Archemix Corp.: Consultancy, Research Funding.

Platelet reactive conformation and multimeric pattern of von Willebrand factor in acquired thrombotic thrombocytopenic purpura during acute disease and remission

Binding of von Willebrand factor (VWF) multimers of ultra-large size to platelets is considered the triggering mechanism of microvascular thrombosis in thrombotic thrombocytopenic purpura (TTP). To assess the potential of VWF-related measurements as markers of disease activity and severity in TTP. VWF antigen (VWF:Ag), platelet glycoprotein-Ib-α binding-conformation (GPIb-α/BC) and multimeric pattern were investigated in 74 patients with acquired TTP during acute disease, remission or both and 73 healthy controls. In patients with both acute and remission samples available, VWF ristocetin co-factor activity (VWF:RCo) and collagen binding (VWF:CB) were also measured. The relationships of study measurements with the presence of acute disease and remission and with markers of disease severity were assessed. VWF:Ag and VWF-GPIb-α/BC were higher in TTP patients than controls (P < 0.001 and 0.004). However, there was no statistically significant difference in VWF-GPIb-α/BC between samples obtained during acute TTP and remission. Larger VWF multimers were frequently lacking in acute TTP patients, who displayed ultra-large multimers at remission. The degree of loss of larger VWF multimers correlated with the degree of abnormality of hemoglobin, platelet counts and serum lactate dehydrogenase (LDH) and was associated with low levels of both VWF:RCo/Ag and VWF:CB/Ag ratios. In TTP the platelet-binding conformation of VWF is not exclusively present in acute disease, nor is it associated with its clinical and laboratory severity. The loss of larger VWF multimers, accompanied by low VWF:RCo/Ag and VWF:CB/Ag ratio values, represents an index of disease activity and severity of acute TTP in patients with severe ADAMTS-13 deficiency.

Cross Sectional Study to Evaluate the Utility of Screening Tools to Identify Low Von Willebrand Factor Levels In Women with Menorrhagia (ZPMCB-VWD).

AbstractAbstract 3839Menorrhagia is a common symptom of haemostatic disorders and often triggers an evaluation for a bleeding disorder. Clinical screening tools, to identify women with menorrhagia who have an underlying bleeding disorder, are very desirable. Scoring systems to quantify menstrual blood loss in women, the Pictorial Blood Loss Assessment Chart, (PBAC), and to assess the severity of bleeding symptoms in individuals diagnosed with von Willebrand disease (VWD), European Union Bleeding Score (EU-BS) (MCMDM-1VWD), were evaluated for their ability to predict the presence of low von Willebrand factor (VWF) level in a cohort of 150 women recruited from an outpatient gynecology clinic. One of us (SG or ML) completed a PBAC and an EU Bleeding questionnaire with the participant. Menorrhagia cases were defined by a PBAC ≥185 and controls by a PBAC<185 (n=75 in each group). Complete blood count (CBC), ABO blood group, VWF activity (VWF:RCo) and VWF antigen (VWF:Ag) were measured in all subjects. Due to the variability in the laboratory testing of VWF: RCo and to capture all women with low levels, low VWF level was defined as VWF:RCo or VWF:Ag'60 IU/dl in our study. PBAC score correlated with the presence of menorrhagia defined by the menorrhagia score of MCMDM-1VWD (Spearman's Rank correlation coefficient, r=0.51, p<0.0001). PBAC of ≥185 had a sensitivity and specificity of 86% and 64% for predicting a menorrhagia score≥1 (AUC=0.80, p<0.0001). When women were evaluated for bleeding symptoms using the EU questionnaire, the most common bleeding symptom in the entire cohort was bruising in 42% followed by menorrhagia in 35%. Compared to women without menorrhagia, women with menorrhagia had an increased frequency of other bleeding symptoms including bleeding with invasive procedures (bleeding with tooth extraction 11% vs. 1% and bleeding with surgery 12% vs. 4%), post partum hemorrhage 15% vs. 8% and epistaxis 11% vs. 4%. Of the 150 women, 20 had an overall EU score of ≥4, mean 7.5 (range 4–22) indicating significant bleeding symptoms including menorrhagia and bruising in 85%, bleeding after surgical procedures in 45% followed by post partum hemorrhage and epistaxis in 35 %. Comparing menorrhagia cases to controls, the statistically significant difference between the two groups was in the median overall EU-BS (1 vs. 0, p=0.007) and poor wound healing with frequent scarring (24% vs. 9.3%, p=0.027).We found that 21.3% women with menorrhagia had low VWF level (VWF:RCo or VWF:Ag ≤ 60 IU/dl) compared to 14.6% women without menorrhagia (p =0.28). Comparing the 27 subjects with low VWF level with the 123 subjects with normal VWF level, the median VWF:RCo was 47 vs. 101IU/dl (p<0.0001), median PBAC was 234 vs. 176 (p=0.34), median EU-BS was 0 in both groups. There was no predictive correlation between the PBAC score and VWF:RCo (Spearman's Rank correlation coefficient r=0.05, p=0.54) and between the EU-BS and VWF: RCo level (Spearman's Rank correlation coefficient, r=0.07, p=0.37). The only predictor of low VWF: RCo was blood group O (OR: 8.9, 95% CI 2.9–27.6). The majority (87%) of blood group O subjects with low VWF level did not have a bleeding phenotype (EU-BS ≤3).In our cohort, the predictor of post partum hemorrhage was menorrhagia (OR: 4.1, 95% CI-1.4-11.8, p=0.009) and the only predictor of surgical bleeding and bleeding with tooth extraction was bleeding from minor wounds (OR: 11,95% CI-2.5-47, p=0.001; OR: 34,95% CI-6.9-166, p<0.0001 respectively). We conclude that PBAC is an easy and quick method for assessing the severity of menorrhagia in an outpatient setting. A score of ≥185 can be reliably used to diagnose clinical menorrhagia and as a threshold score to screen for other bleeding symptoms using the EU questionnaire. Women with menorrhagia are more likely to have bleeding with other invasive procedures, post partum bleeding and epistaxis. Menorrhagia could be a surrogate marker for an underlying bleeding disorder or a connective tissue disorder as reflected by the higher EU-BS and symptoms of poor wound healing in women with increased menstrual blood loss. We could not determine a PBAC score or EU-BS that could be used to identify women with low VWF. Low VWF level correlated best with blood group O, however a majority of these women did not have a bleeding phenotype (EU-BS≤3). Studies in a large cohort of women to identify reliable predictors of clinical bleeding are warranted. Disclosures:Gill:CSL Behring: Research Funding.

Genome-Wide Linkage Analysis Reveals Novel Loci Modifying Plasma Von Willebrand Factor Undetected by Genome-Wide Association

AbstractAbstract 2116Plasma levels of von Willebrand Factor (VWF) are highly variable among healthy people and ~70% of this variability is heritable. Elevated levels of VWF are associated with thrombosis while low levels of VWF cause the common bleeding disorder, von Willebrand Disease (VWD). The genome-wide association study (GWAS) is a powerful tool for identifying common genetic variants contributing to complex traits, though less common variants are likely to be missed by this approach. We performed combined GWAS and linkage analysis in a cohort of healthy young college students. The study was approved by the University of Michigan IRB (IRBMED #2005-0080). 1189 healthy individuals (14-35 yrs of age) were recruited, comprising 507 sibships. VWF levels were determined by alphalisa assay (mean 109%, range 26 – 410%). VWF level heritability (h2) was 73% among siblings and 82% among monozygotic twins. Samples were de-identified and DNA was extracted from peripheral blood, quantified and genotyped using the Illumina Omni-1 quad SNP chip at the Broad Institute (MIT, Boston). 1182 individuals passed genotyping quality control with >97% call rates for 979,855 SNPs. Genotyping quality control was performed as part of the NIH's GENEVA program. GWAS on log transformed, age and sex adjusted VWF levels identified a major peak over the ABO locus (p<1×10-36), [Figure 1] confirming the known influence of ABO blood type on plasma VWF. Preliminary genome-wide linkage analysis (MERLIN) performed on plasma VWF levels in the 507 sibships identified 5 novel loci with peak LOD scores greater than 5 (chromosomes 2, 7, and 21), in addition to the ABO locus on chromosome 9 [Figure 2]. Other than ABO, none of these loci were detected in our GWAS analysis, or the previously reported CHARGE Consortium VWF GWAS (Smith, N et al. Circulation, 2010). Our analysis is now being extended to additional hemostasis and other related phenotypes using the same SNP data. In conclusion, linkage analysis identified novel loci contributing to the variation of VWF in healthy siblings that were undetected by GWAS. Though GWAS is an effective tool for the analysis of many complex traits, including plasma VWF variation, the majority of the heritable component of these traits remains unexplained. However, the missing heritability may be due, at least in part, to multiple rare variants at a limited number of loci. Our results suggest that genome-wide linkage analysis in multiple sibships may identify such loci and that this approach may be broadly applicable to hemostasis and other complex traits. Identification of the major loci regulating plasma VWF levels may lead to an improved prediction of bleeding and thrombotic risk and could suggest novel targets for therapeutic intervention. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Reduced Von Willebrand Factor Secretion Is Associated with Loss of Weibel-Palade Formation

AbstractAbstract 541 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 patients with quantitative plasma VWF deficiency with normal VWF structure and function. Aim of the study.We report three different novel type 1 VWF mutations (A1716P, C2190Y and R2663C) which although located in different VWF domains are associated with reduced secretion and lack of formation of Weibel-Palade body-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 to wild-type VWF. Furthermore, expression was also examined in HEK293 cells that form Weibel-Palade body (WPB)-like granules when transfected with wt VWF. Results.The multimer analysis of plasma VWF was compatible with type 1 VWD. The results of 3 different expression experiments showed a slightly reduced VWF synthesis and drastically impaired secretion into the medium with homozygous expression.MutationVWF:Ag Lysate (%)VWF:Ag Medium (%)GpIb binding (%)GpIb binding/VWF:Ag MediumVWF:CB (%)VWF:CB/VWF:Ag MediumA1716P homo62 ± 305 ± 0.22 ± 0.10.480.2 ± 0.010.05A1716P hetero56 ± 145 ± 0.937 ± 0.40.8226 ± 0.20.58C2190Y homo82 ± 1311 ± 0.23 ± 0.10.312 ± 0.10.19C2190Y hetero73 ± 1242 ± 0.539 ± 0.40.9230 ± 0.40.72R2663C homo73 ± 219 ± 0.52 ± 0.10.221 ± 0.10.1R2663C hetero68 ± 145 ± 0.128 ± 0.10.6316 ± 0.80.35In HEK293 cells, homozygous A1716P and C2190Y VWF variants failed to form WPB-like granules, while R2663C was capable of forming granules, but had fewer cells with granules and more with ER-localized VWF. Heterozygous expression of A1716P and C2160Y VWF variants had a negative impact on wild-type VWF and WPB-like granules were observed in transfected cells. Conclusions.Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations can be associated with inability to form endothelial Weibel-Palade-like granules and mutations in different VWF domains can affect the formation of these organelles. Disclosures:No relevant conflicts of interest to declare.

Validation of a rapid test (VWF-LIA) for the quantitative determination of von Willebrand factor antigen in type 1 von Willebrand disease diagnosis within the European multicenter study MCMDM-1VWD

Background Accurate measurement of von Willebrand factor (VWF) is a critical requirement for the diagnosis of von Willebrand disease (VWD). Aim of the study To evaluate the diagnostic efficiency of a rapid quantitative test for the measurement of VWF antigen (VWF:Ag) in type 1 VWD. Patients and methods VWF:Ag was measured with an ELISA in a robotic instrument, as a reference method, and with a fully automated latex-immunoassay (LIA) on an ACL 9000 analyser in 1,716 subjects enrolled within the Molecular and Clinical Markers for Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) Study. Among these subjects, 1,049 were healthy controls, 281 healthy family members and 386 affected members from 127 European families with type 1 VWD. Results The assay linearity range was 10-125 IU/dL for LIA (R 2 = 0.99) and 5-133 IU/dL for ELISA (R 2 = 0.99). The inter-assay CV for low VWF levels (∼ 30 IU/dL) was 2% for the LIA test and 8.7 % for ELISA. The sensitivity for detection of type 1 VWD affected members was 86% and the specificity 91% for LIA, 87% and 90% for ELISA. A receiver-operator (ROC) analysis disclosed only a marginal difference between the two tests, LIA having a slightly greater area under the curve (0.94 vs. 0.93, p = 0.03). Conclusion VWF:Ag LIA compared well to standard ELISA in this large population of patients and controls, showing better CV. However the lower detection limit for the VWF:Ag LIA compared to the VWF:Ag ELISA means that the LIA assay is less good at discriminating between type 3 VWD and moderate type 1 VWD.

The genetic basis of von Willebrand disease

The common autosomally inherited mucocutaneous bleeding disorder, von Willebrand disease (VWD) results from quantitative or qualitative defects in plasma von Willebrand factor (VWF). Mutation can affect VWF quantity or its functions mediating platelet adhesion and aggregation at sites of vascular damage and carrying pro-coagulant factor VIII (FVIII). Phenotype and genotype analysis in patients with the three VWD types has aided understanding of VWF structure and function. Investigation of patients with specific disease types has identified mutations in up to 70% of type 1 and 100% of type 3 VWD cases. Missense mutations predominate in type 1 VWD and act through mechanisms including rapid clearance and intracellular retention. Many mutations are incompletely penetrant and attributing pathogenicity is challenging. Other factors including blood group O contribute to low VWF level. Missense mutations affecting platelet- or FVIII-binding through a number of mechanisms are responsible for the four type 2 subtypes; 2A, 2B, 2M and 2N. In contrast, mutations resulting in a lack of VWF expression predominate in recessive type 3 VWD. This review explores the genetic basis of each VWD type, relating mutations identified to disease mechanism. Additionally, utility of genetic analysis within the different disease types is explored.

Accelerated clearance alone explains ultra-large multimers in von Willebrand disease Vicenza.

von Willebrand disease (VWD) Vicenza is characterized by low plasma von Willebrand factor (VWF) levels, the presence of ultra-large (UL) VWF multimers and less prominent satellite bands on multimer gels, and the heterozygous amino acid substitution R1205H in the VWF gene. The pathogenesis of VWD Vicenza has been elusive. Accelerated clearance is implicated as a cause of low VWF level. We addressed the question, whether the presence of ultra-large multimers is a cause, or a result of accelerated VWF clearance, or whether it is an unrelated phenomenon. We studied the detailed phenotype of three Hungarian patients with VWD Vicenza, expressed the mutant VWF-R1205H in 293T cells and developed a mathematical model to simulate VWF synthesis and catabolism. We found that the half-life of VWF after DDAVP was approximately one-tenth of that after the administration of Haemate P, a source of exogenous wild-type (WT) VWF (0.81 + or - 0.2 vs. 7.25 + or - 2.38 h). An analysis of recombinant mutant VWF-R1205H showed that the biosynthesis and multimer structure of WT and mutant VWF were indistinguishable. A mathematical model of the complex interplay of VWF synthesis, clearance and cleavage showed that decreasing VWF half-life to one-tenth of normal reproduced all features of VWD Vicenza including low VWF level, ultra-large multimers and a decrease of satellite band intensity. We conclude that accelerated clearance alone may explain all features of VWD Vicenza.

Biological and clinical response to desmopressin (DDAVP) in a retrospective cohort study of children with low von Willebrand factor levels and bleeding history

The diagnosis and management of von Willebrand disease (VWD) in paediatrics is challenging. Our aim was to review patient's characteristics related to biological and clinical response to DDAVP in children with low von Willebrand factor (VWF) levels and bleeding history from a single institution. We included a retrospective cohort of 221 children (median age 11 years; 137 females): 27 type 1 (VWF levels within 15-30 IU dL-1) and 194 possible type 1 (VWF levels within 31-49 IU dL-1). The DDAVP infusion-test was performed in 214/221 children, 93.4% of whom showed good response. Patients with type 1 were at higher risk of DDAVP-test failure: 9/26 (34.6%) vs. 18/188 (9.6%) with possible type 1 (RR 3.44, 1.75-6.79; p= 0.002, Fisher's exact test). In 68 children, the clinical response to DDAVP was evaluated 87 times: i) to stop bleeding: menorrhagia (13), mucocutaneous (12), haemarthrosis (1); and ii) to prevent surgical bleeding: adenotonsillectomy (17), major (15) and minor surgery (10); and dental procedures (19). No major adverse events or bleeding were observed. The treatment was effective with one single dose of DDAVP in almost all patients, without antifibrinolytic or local therapy, except in a girl with severe haemorrhage during menarche who required replacement therapy. In conclusion, patients with VWD type 1 were at higher risk of no response to DDAVP infusion-test. In this series, one dose of DDAVP proved effective and safe for children with VWD. Since this is a safe, effective and affordable therapy, we consider that a wider use should be promoted, especially in developing countries.

The Power of a Standardized Bleeding Score in Diagnosing Pediatric Type 1 Von Willebrand Disease.

Abstract 1293Poster Board I-315 INTRODUCTIONType I von Willebrand disease (VWD) is the most common inherited bleeding disorder. Repetitive testing of von Willebrand factor (VWF) levels is necessary before the diagnosis can be safely ruled out, as VWF levels fluctuate in response to genetic and environmental factors. A predictive bleeding score (BS) could reveal individuals that may benefit from repetitive testing and those for whom repetitive testing is unlikely to be of benefit. While a standardized questionnaire (the Vicenza score) was developed to evaluate hemorrhagic symptoms, it was never prospectively validated for a pediatric population in a tertiary care setting. SUBJECTSThe study targeted children, ages 0 to 17 years, referred to the Hemostasis and Thrombosis Center (HTC) of Nationwide Children's Hospital for a coagulation evaluation as a result of bleeding symptoms, family history of a bleeding disorder and/or abnormal coagulation labs found during pre-operative screening. Children were excluded if they had a previously diagnosed bleeding disorder, if their caregiver did not speak English or if the child did not undergo VWF:Ag and VWF:RCo testing. METHODSPrior to the diagnosis or exclusion of a bleeding disorder in the child, caregivers consented to answer the questionnaire over the telephone. Descriptions of the Vicenza score are available online (http://www.euvwd.group.shef.ac.uk/bleed_score.htm). LABORATORY TESTINGA single VWF:Ag or VWF:RCo <30 IU/dL was classified as “Definite Type 1 VWD” while levels from 30-50 IU/dL were classified as “Low VWF” (http://www.nhlbi.nih.gov/guidelines/vwd). Platelet function analysis (PFA-100) screened for platelet function defects, with some patients undergoing follow-up platelet aggregation studies and/or platelet electron microscopy. Laboratory studies from other institutions were excluded from analysis. Patients' medical records were reviewed after hematologic evaluation, and the resultant data was analyzed with STATA 10.1 (Stata Corp., College Station, TX). RESULTSA total of 104 children (52 females and 52 males) with a mean age of 7.53 years (range 1 month to 17 years) were included. At least one hemorrhagic symptom was present in 99 of the 104 children (95%) with the mean number of symptoms being 2.87 (range 0 to 7). The mean Vicenza score was 3.24 (range -1 to 13). Of the 104 children, 8 met criteria for “Definite Type 1 VWD,” 23 met criteria for “Low VWF,” 14 were diagnosed with a “Platelet Function Defect,” and 2 children had bleeding secondary to Ehlers Danlos syndrome. Children with non-bleeding disorders (e.g. Factor XII deficiency) or no laboratory evidence of a bleeding disorder were classified as “No Bleeding Disorder.” In general, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and positive diagnostic likelihood ratio of the bleeding questionnaire demonstrated poor predictive value in our patient population with the exception of high specificity in ruling out “Definite Type 1 VWD” (Table). The NPV was comparably high with both qualitative (>2 bleeding symptoms) and quantitative (BS ≥2) criteria. CONCLUSIONSThe Vicenza score, previously validated in adults and in a pediatric primary care setting, appears to have limited predictive value in a pediatric tertiary care setting when evaluating patients with platelet function defects or low VWF levels. While the Vicenza score has a high NPV to exclude “Definite Type 1 VWD,” the use of simpler qualitative criteria is similarly predictive.TablePredictive power of the Vicenza score in a pediatric tertiary care population. (Sensitivity=SN; Specificity=SP; Positive predictive value=PPV; Negative predictive value=NPV; Positive diagnostic likelihood ratio=DLR+; Bleeding score=BS; Confidence interval=CI)PopulationCriteriaSN(%)SP(%)PPV(%)NPV(%)+95%CIDLR+Definite Type 1 VWD>2 bleeding symptoms62.546.98.993.8 (82.8-98.7)1.17BS ≥287.530.29.596.7 (82.8-99.9)1.25BS >3 or 5 in males and females37.572.910.393.3 (85.1-97.8)1.38Definite Type 1 VWD + Low VWF>2 bleeding symptoms58.147.932.172.9 (58.2-84.7)1.11BS ≥283.934.335.183.3 (65.3-94.4)1.28BS >3 or 5 in males and females41.978.144.876.0 (64.7-85.1)1.91Definite Type 1 VWD + Low VWF + Platelet Function Defect>2 bleeding symptoms59.150.046.462.5 (47.4-76.0)1.18BS ≥281.836.748.773.3 (54.1-87.7)1.29BS >3 or 5 in males and females40.981.762.165.3 (53.5-76.0)2.23 DisclosuresNo relevant conflicts of interest to declare.

Genotype/phenotype association in von Willebrand disease: is the glass half full or empty?

Since the original description of this disease in 1926, major advances have been made in our understanding of the pathogenetic mechanisms responsible for von Willebrand disease (VWD). We now recognize that this disease comprises a collection of diverse quantitative and qualitative abnormalities of the adhesive protein von Willebrand factor (VWF), the key protein involved in platelet adhesion, and the carrier protein for factor VIII (FVIII) in plasma. Since the mid-1970s there has been a growing appreciation of 'variant' forms of the disease and in the 20 years since the cloning of the VWF gene, much progress has been made in characterizing the molecular genetic pathology of type 3 VWD and the various type 2 variants, types 2A, 2B, 2M and 2N VWD. In all of these cases, mechanistic insights into VWF structure/function have been forthcoming. Most recently, preliminary results relating to the mutational landscape of type 1 disease have been published that highlight the complex pathogenic background of this mild/moderate quantitative trait.

Bleeding symptoms and laboratory correlation in patients with severe von Willebrand disease

Type 3 von Willebrand disease (VWD) is a rare bleeding disorder with markedly decreased or absent von Willebrand factor (VWF) protein, accompanied by a parallel decrease in VWF function and factor VIII (FVIII) activity. The goal of this study was to describe the population of patients enrolled in the USA Centers for Disease Control Universal Data Collection (UDC) study with type 3 VWD, defined as a VWF:Ag of <10%, and to correlate bleeding symptoms with VWF and FVIII levels. Data on 150 patients were analysed. Almost all patients experienced bleeding episodes (98%) and required blood and/or factor product treatment (92%). While oral mucosal bleeding (the site of first bleed in 54%) was most common, subsequent muscle and joint bleeds were also seen (28%, 45%, respectively), and intracranial haemorrhage occurred in 8% of individuals. Mean age of first bleed was lower in those with either a FVIII < or =5% or a VWF:Ag <1%. Univariate marginal model analysis showed lower levels of FVIII and VWF:Ag both predicted a higher risk of joint bleeding. Longitudinal multivariate analysis found a lower FVIII level (P = 0.03), increasing age (P < 0.0001), history of joint bleeding (P = 0.001), higher body mass index (BMI) (P < 0.0001), and use of home infusion (P = 0.02) were all negatively associated with joint mobility. Low levels of VWF:Ag (P = 0.003) and male sex (P = 0.007) were also negatively associated with joint function. This study documents the strong bleeding phenotype in severe VWD and provides data to help target therapy, including prophylaxis, for patients most at risk of bleeding complications.

Lower levels of ADAMTS13 are associated with cardiovascular disease in young patients

ADAMTS13 may play a role in arterial thrombosis by cleaving the highly active and thrombogenic ultralarge Von Willebrand Factor (VWF) multimers into less active VWF multimers. The aim was to investigate the relationship between plasma levels of ADAMTS13, VWF and genetic variation in the ADAMTS13 gene with cardiovascular disease. We performed a case–control study in 374 patients with a first-ever arterial thrombosis before the age of 45 years in males and 55 years in women. We included 218 patients with coronary heart disease (CHD), 109 patients with ischemic stroke (IS) and 47 patients with peripheral arterial disease (PAD) and 332 healthy population-based controls. ADAMTS13 and VWF levels were measured 1–3 months after the event. ADAMTS13 levels were associated with cardiovascular disease (OR antigen 5.1 (95% CI 3.1–8.5, p < 0.001) and OR activity 4.4 (95% CI 2.5–7.5, p < 0.001), in the lowest quartiles). VWF levels were associated with cardiovascular disease (OR antigen 2.1 (95% CI 1.3–3.3, p = 0.001) and OR activity 2.0 (95% CI 1.3–3.1, p = 0.003), in the highest quartile). Patients with combined low ADAMTS13 levels and high VWF levels had an odds ratio of 7.7 (95% CI 3.3–17.7) ( p for trend <0.0001). No association was found between genetic variation in the ADAMTS13 gene with levels of ADAMTS13 or with risk of cardiovascular disease. In conclusion, levels of ADAMTS13 and VWF are strongly associated with the risk of cardiovascular disease.

Platelet von Willebrand factor determination does not improve the diagnosis of patients with suspected Type 1 von Willebrand disease

Establishing a laboratory diagnosis of a bleeding disorder can be challenging for some patients who present with mucocutaneous bleeding symptoms. A common clinical scenario is an older patient with a prior diagnosis of von Willebrand disease (VWD) or a family history of VWD, who now has von Willebrand factor (VWF) values repeatedly within the normal range. Plasma VWF antigen levels have been shown to increase with age. Whether platelet VWF increases with age is unknown. We hypothesized that platelet VWF does not increase with age and low platelet VWF levels, despite normal plasma levels, could be a reason for continued bleeding symptoms in some patients. Therefore, we compared the platelet and plasma VWF antigen and activity as well as the platelet function analyzer (PFA)-100 closure times in 35 patients with a history of mucocutaneous bleeding symptoms and consistently normal levels of VWF antigen and activity, despite a prior history of a VWD diagnosis and/or a positive family history of VWD. Overall in our patients (bleeders), the platelet VWF values correlated with the plasma values and only three patients had reduced platelet VWF. In the bleeding group, the PFA-100 results showed an inverse correlation with plasma and platelet values, which was stronger for the plasma values. Therefore, platelet VWF determination was not helpful in the diagnosis of suspected mild type 1 VWD.

Selection on cis-Regulatory Variation at B4galnt2 and Its Influence on von Willebrand Factor in House Mice

The RIIIS/J inbred mouse strain is a model for type 1 von Willebrand disease (VWD), a common human bleeding disorder. Low von Willebrand factor (VWF) levels in RIIIS/J are due to a regulatory mutation, Mvwf1, which directs a tissue-specific switch in expression of a glycosyltransferase, B4GALNT2, from intestine to blood vessel. We recently found that Mvwf1 lies on a founder allele common among laboratory mouse strains. To investigate the evolutionary forces operating at B4galnt2, we conducted a survey of DNA sequence polymorphism and microsatellite variation spanning the B4galnt2 gene region in natural Mus musculus domesticus populations. Two divergent haplotypes segregate in these natural populations, one of which corresponds to the RIIIS/J sequence. Different local populations display dramatic differences in the frequency of these haplotypes, and reduced microsatellite variability near B4galnt2 within the RIIIS/J haplotype is consistent with the recent action of natural selection. The level and pattern of DNA sequence polymorphism in the 5′ flanking region of the gene significantly deviates from the neutral expectation and suggests that variation in B4galnt2 expression may be under balancing selection and/or arose from a recently introgressed allele that subsequently increased in frequency due to natural selection. However, coalescent simulations indicate that the heterogeneity in divergence between haplotypes is greater than expected under an introgression model. Analysis of a population where the RIIIS/J haplotype is in high frequency reveals an association between this haplotype, the B4galnt2 tissue-specific switch, and a significant decrease in plasma VWF levels. Given these observations, we propose that low VWF levels may represent a fitness cost that is offset by a yet unknown benefit of the B4galnt2 tissue-specific switch. Similar mechanisms may account for the variability in VWF levels and high prevalence of VWD in other mammals, including humans.

Alpha 2A adrenergic receptor polymorphism is associated with plasma von Willebrand factor levels in a general population

High levels of plasma von Willebrand factor are a proposed risk factor for atherothrombotic disorders. Previously, ABO blood type and the von Willebrand factor -1793C/G polymorphism were shown to affect interindividual variations in plasma von Willebrand factor levels. We previously reported that polymorphisms of the alpha 2A adrenergic receptor, an epinephrine receptor, were associated with platelet function as assessed by platelet function analyzer-100. The measurement value of platelet function analyzer-100 has been reportedly associated with plasma von Willebrand factor levels. Also, it was demonstrated that epinephrine administration increases plasma von Willebrand factor levels in vivo. Thus, the present study investigated the association between plasma von Willebrand factor levels and genetic polymorphisms as follows: ABO blood type, von Willebrand factor -1051G/A (linked with -1793C/G), alpha 2A adrenergic receptor 1780A/G, and alpha 2A adrenergic receptor 2372A/G. Study subjects were genetically unrelated Japanese men (n = 277) recruited at their regular medical examinations. Genotyping of the polymorphisms was performed using the single nucleotide primer extension-based method. Plasma von Willebrand factor levels were measured as ristocetin-cofactor activities. The O blood type and alpha 2A adrenergic receptor 2372AA genotype were significantly associated with lower von Willebrand factor levels, though von Willebrand factor -1051G/A polymorphism did not affect them. In stratification analysis of the group according to blood type O and non-O, the significant association between the 2372AA genotype and lower plasma von Willebrand factor levels was observed in non-O subjects, but not O subjects. In conclusion, the alpha 2A adrenergic receptor 2372A/G polymorphism is associated with plasma von Willebrand factor levels in a general population.

The Prevalence of Bleeding Disorders Among Healthy Pediatric Patients With Abnormal Preprocedural Coagulation Studies

We evaluated the prevalence of hemostatic disorders among pediatric patients with abnormal screening coagulation tests. We analyzed 48 consecutive referrals for abnormal prothrombin times, partial thromboplastin times, or closure times obtained as preprocedural screens. Patients were evaluated by uniform diagnostic testing. Seventeen patients (35%) had an isolated nonspecific inhibitor (NSI). Six patients (12.5%) presented with mildly low factor activity with a concomitant NSI. These deficiencies were of unclear clinical significance. One patient (2%) had a lupus anticoagulant. Only 9 patients (19%) had a possible or true mild bleeding disorder: 5 patients (10%) had isolated low von Willebrand factor levels, 2 patients (4%) had possible type I von Willebrand disease, and 2 (4%) had platelet aggregation disorders. In all patients, personal and family bleeding history had a positive predictive value of 45% for hemostatic disorders. The most common diagnosis among the patients referred to us for abnormal preoperative coagulation tests was a NSI, which is not associated with an increased risk of operative bleeding complications. Less than 20% had a possible or true mild bleeding disorder. Although certain bleeding disorders can be occult in children and are associated with perioperative bleeding risks, our study demonstrates the inherent limitations in making a laboratory diagnosis of a bleeding disorder in pediatric patients preoperatively. Our findings contribute to existing doubt about the usefulness of prothrombin times, partial thromboplastin times, and closure times to identify occult bleeding disorders in this population.