Von Willebrand Disease Subjects Research Articles

Quantitative Analysis of Von Willebrand Factor (VWF) Multimers in Von Willebrand Disease (VWD) Patients Recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD

Quantitative Analysis of Von Willebrand Factor (VWF) Multimers in Von Willebrand Disease (VWD) Patients Recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD

Contribution of Select ISTH-BAT Subscores to the Total Score in Type 1 VWD

Background:von Willebrand Disease (VWD) is the most common inherited bleeding disorder with partial quantitative deficiency in von Willebrand Factor (VWF); Type 1 comprises 80% of VWD. Clinical manifestations include mucocutaneous bleeding, prolonged bleeding after surgery or injury and excessive menorrhagia in females which have also been reported in normal subjects. A diagnosis of Type 1 VWD is based upon reduced plasma VWF, abnormal mucocutaneous bleeding symptoms and usually a family history of bleeding and VWD. Evaluating abnormal bleeding symptoms involves a detailed bleeding history, which can be quite subjective and time consuming. To address these challenges, the Vicenza-based Bleeding Assessment Tool (BAT) was developed as a validated score and has evolved into the current International Society on Thrombosis and Haemostasis-Bleeding Assessment Tool (ISTH-BAT). A large multi-center NIH supported study (The Zimmerman Program for the Molecular and Clinical Biology of VWD), began in 2005 to correlate clinical laboratory testing, bleeding severity determined by the ISTH-BAT and genetic variability in VWD. In this current study, we investigated which bleeding symptoms were most sensitive in applying the ISTH-BAT to the diagnosis of Type I VWD.Methods:Subjects were enrolled in the Zimmerman Program . Each symptom in the validated ISTH-BAT questionnaire was scored from 0-4 (no symptoms to symptom intervention) and included epistaxis, bruising, oral bleeding, hematuria, gastrointestinal (GI) bleeding, head bleeding, hemarthrosis, hematoma and prolonged bleeding with minor wounds, tooth extraction, surgery, menstruation and child birth. Other scored bleeding symptoms included bleeding at circumcision, excessive umbilical stump bleeding, venipuncture bleeding, vacuum extraction delivery bleeding, ovulatory bleeding and cephalohematoma. The scores of each symptom were analyzed in both healthy controls and subjects diagnosed with Type 1 VWD. Only subjects with VWF antigen (VWF:Ag) and/or VWF ristocetin cofactor activity (VWF:RCo) <30 IU/dL were considered in the comparative analysis. VWF levels were performed by a central laboratory, including VWF:Ag, VWF:RCo, VWF propeptide (VWFpp) and VWF collagen binding with type III collagen (VWF:CB). Statistical analysis included Mann-Whitney test, Chi-square or a Fisher's exact test, Pearson correlations and Spearman correlations. Results were considered significant for a two-sided p-value <0.05. A regression analysis was performed to examine which subscores contributed the most to the total score of the ISTH-BAT.Results:Analysis included 383 subjects (Female=262, Male=121) enrolled in the Zimmerman Program . Of these subjects, 244 were healthy controls (mean age of 38.93 [range 10-73 years of age]) and 139 had Type 1 VWD (mean age of 19.46 [range 1-76 years of age]). Within the Type 1 subjects, there were more females >12 years of age (no gender difference < 12 years of age) and greater number of Caucasians and younger subjects. Type 1 VWD subjects scored significantly higher than the healthy control group in both ISTH-BAT total scores (p<0.0001) as well as all subscores (p<0.001). In comparing bleeding symptoms from healthy controls with Type 1 VWD subjects: epistaxis 4% vs 46%, bruising 16% vs 64%, minor wound bleeding 1% vs 42%, oral bleeding 3% vs 35%, tooth extraction bleeding 3% vs 18%, GI bleeding 3% vs 11%, hematuria 0% vs 11%, surgical bleeding 4% vs 23%, menstrual bleeding 60% vs 85%, bleeding after delivery 2% vs 12%, other bleeding 0% vs 11% (47% were circumcision bleeding), head bleeding 0% vs 4%, hemarthrosis 0% vs 11%, and hematoma 0% vs 7%. Regression analysis revealed that epistaxis, bruising, excessive bleeding with minor wounds, oral bleeding, and menstrual bleeding subscores contributed the most to the ISTH-BAT total score (p<0.016).Conclusion:The ISTH-BAT currently is strongly predictive of bleeding symptoms in the diagnosis of Type 1 VWD. Though all ISTH-BAT subscores correlated with a diagnosis of Type 1 VWD, some subscores contributed more significantly to the total score than others and were more prevalent than in normal subjects. Accordingly, the sensitivity and specificity of ISTH-BAT subscores are critical to the assessment of bleeding in the diagnosis of Type 1 VWD. DisclosuresFlood:Shire: Consultancy; CSL Behring: Consultancy. Gill:Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. James:CSL Behring: Research Funding; Shire: Research Funding; Bayer: Research Funding.

Characterization of the Pathogenicity of Novel Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program for the Molecular and Clinical Biology Study

Von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Von Willebrand factor's (VWF) complex intracellular processing may result in variable VWF expression which requires exploring the underlying mechanism related to individual variants. The mechanisms underlying type 1 VWD are not fully understood, although reduced secretion and increased clearance have been implicated. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program in order to (1) define the underlying mechanism, (2) explore if mutations in a particular domain are mechanistically similar, and (3) correlate expression study data to patient phenotype.Homozygous and heterozygous expression in mammalian cells was used to study the effect of SV on VWF secretion, intracellular retention, function, and multimerization. Novel SV identified throughout the entire VWF protein were introduced into VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Variant VWF cDNA was transfected homozygously and cotransfected heterozygously 1:1 with wild type (WT) VWF pCIneo into Human Embryonic Kidney cells (HEK293T). Supernatants (sup) and cell lysates from 3 separate transfections were collected and analyzed by ELISA for VWF:Ag, VWF binding to platelet glycoprotein 1b (VWF:GPIbM), VWF binding to collagen type III (VWF:CB). Typically VWF:GP1bM/VWF:Ag > 0.7 indicates normal platelet binding and VWF:CB/VWF:Ag > 0.7 indicates normal multimer structure. VWF multimer structure is analyzed by SDS-agarose electrophoresis and western blotting with differential staining of the VWF secreted from variant and WT alleles.V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, and A2569E demonstrated secretion of VWF in sup comparable to that of WT-VWF both when transfected homozygously and hetereozygously, normal VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and normal multimer structure. Two of the stop codon SV W199X and E1660X were not expressed homozgyously (as expected) but demonstrated normal secretion, function, and multimerization when cotransfected 1:1 with WT. Homozygous expression of R960P and C2693F demonstrated reduced VWF secretion, VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and abnormal multimerization. Heterozygous transfection with WT improved VWF secretion, but not multimer defects,reflected by a reduced VWF:CB/VWF:Ag. Homozygous expression of C524Y, G994D, C996W, Q1353X, C2199Y, C2701Y, and C2754Y resulted in absent or greatly reduced secretion with increased intracellular retention. Heterozygous expression resulted in reduced VWF secretion (<50% of WT) for all 7 variants. Q1353X, C2199Y, C2701Y, and C2754Y had normal VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag, and multimer structure, but consisted primarily of secreted WT-VWF. G994D had VWF:CB/VWF:Ag ˂ 0.7 and reduced high molecular weight (HMW) multimers. Co-expression of C524Y and C996W with WT-VWF demonstrated nearly absent VWF secretion, indicating a strong dominant negative effect. The very low VWF:Ag levels precluded assessment of VWF function and multimerization.The VWF:CB/VWF:Ag ratio < 0.7 proved a reliable indicator of multimer structure. No clear domain-specific correlation of VWF secretion or multimer abnormality was observed. Co-expression results of Q1353X, C2199Y, C2701Y, C2754Y, C524Y, G994D, and C996W are consistent with the patient's phenotype, except that patients with C2701Y, and C524Y had slight defect in multimers. In contrast, the in-vitro G994D showed low VWF:CB/VWF:Ag and abnormal multimers but were normal in the patient. C2693F showed consistently abnormal multimer structure in both in vitro and in the patient while R960P had abnormal multimer structure in vitro only.In sum, 52.6% of the 19 SV theoretically are not causative of the VWD phenotype observed in the patients. These subjects had normal VWF propeptide/VWF:Ag suggesting reduced plasma survival is unlikely to be the causative mechanism. The VWD mechanism in these subjects remains undefined. Variants R960P, C2693F, and G994D (15.7%) behaved like Type 2 VWD SV with loss of HMW multimers. We identified 31.6% of the 19 VWF SV as likely to cause the type 1 VWD phenotype with reduced secretion. DisclosuresNo relevant conflicts of interest to declare.

Prospective Diagnosis of VWD in a Large Cohort of Patients with Bleeding Symptoms through the Zimmerman Program

Prospective Diagnosis of VWD in a Large Cohort of Patients with Bleeding Symptoms through the Zimmerman Program

Common VWF sequence variants associated with higher VWF and FVIII are less frequent in subjects diagnosed with type 1 VWD

Unlabelled Box BackgroundGenetic variation in the VWF gene is associated with von Willebrand factor (VWF) and factor VIII (FVIII) levels in healthy individuals. ObjectivesWe hypothesized that VWF sequence variants associated with higher VWF or FVIII could impact the diagnosis of type 1 von Willebrand disease (VWD). MethodsWe examined VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF propeptide (VWFpp), and FVIII levels along with VWF gene sequencing in 256 healthy control and 97 type 1 VWD subjects as part of a cross‐sectional study. ResultsWe found several VWF sequence variants (VWF c.2880G>A and VWF c.2365A>G(;)c.2385T>C, found in linkage disequilibrium) associated with higher VWF and FVIII levels in healthy controls (P<.001 for both variants). In addition, these variants were significantly more common in controls than in subjects diagnosed with type 1 VWD and VWF:Ag <30 (P<.005). The decreased variant frequencies in type 1 VWD was not seen in other VWD types. VWF:Ag, VWF:RCo, and FVIII were not statistically different in type 1 VWD subjects who had these VWF variants compared to type 1 VWD patients without them. There was no difference in ABO blood group, VWF propeptide levels (excluding subjects with known VWF clearance defects), or bleeding score using the ISTH bleeding assessment tool. ConclusionsThese data suggest that certain VWF sequence variants associated with elevated FVIII and VWF levels may protect against reduced VWF levels. These findings were independent of other pathogenic sequence variants in VWF, suggesting a possible independent effect of c.2880G>A and c.2365A>G(;)c.2385T>C on VWF levels.

Expression of Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program Study Reveals Defects in VWF Secretion and Multimerization

von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Type 1 is the most common form of VWD and results in a partial quantitative deficiency of von Willebrand Factor (VWF). The mechanisms underlying type 1 VWD are still not very well understood although reduced VWF secretion and increased VWF clearance have been implicated in causing VWD. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD in order to define the underlying mechanism and explore if SV in a particular domain are mechanistically similar.We utilized homozygous expression in human embryonic kidney cells (HEK-293T) to study the effect of VWF SV on VWF secretion, intracellular retention, multimerization, and function. Novel SV have been identified throughout the entire VWF protein. We introduced the following variants into a VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Sequence variants were confirmed by Sanger sequencing. Variant VWF cDNA is transfected homozygously into HEK-293T cells. The supernatants and cell lysates from 3 independent transfections are collected and analyzed by ELISA for VWF:Ag and VWF binding to collagen type III (VWF:CB). VWF multimer structure is analyzed by SDS-agarose gel electrophoresis and western blotting. The genotype-phenotype patient data is correlated with the data from expression studies to explore a model to predict the impact of the SV on the VWD phenotype.Variants V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F demonstrated secretion comparable to that of wild type (WT)-VWF. In contrast, VWF variants R960P and C2701Y showed reduced VWF secretion (<50% of WT) with increased VWF in the cell lysate. VWF variants W199X, C524Y, G994D, C996W, Q1353X, E1660X, C2199Y, and C2754Y demonstrated a complete absence of secreted VWF. Not unexpectedly, homozygous expression of stop codon variants W199X, Q1353X, and E1660X demonstrated no VWF in the cell lysate. However, non-secreted VWF variants C524Y, G994D, C996W, C2199Y, and C2754Y showed intracellular retention with detectable VWF in the cell lysate. SV occurring at cysteine residues (C524Y, C996W, C2199Y, C2701Y, and C2754Y) all had reduced secretion and increased intracellular retention, consistent with altered conformation leading to increased intracellular chaperone interaction and proteasomal degradation. VWF binding to collagen is dependent on the presence of high molecular weight multimers (HMWM). VWF:CB/VWF:Ag is used to predict multimer structure with VWF:CB/VWF:Ag < 0.7 indicative of loss of the HMWM. VWF variants V86M, M947V, R1763Q, Q2256H, P2524L, C2701Y had VWF:CB/VWF:Ag ≥ 0.7 consistent with normal multimer structure, while variants R960P, R1204W, T2282I, A2569E, and C2693F had VWF:CB/VWF:Ag < 0.7 indicating abnormal multimer structure.47.3% of the 19 VWF variants studied had normal VWF secretion, 10.5% had reduced secretion with increased intracellular retention, and 26.3% revealed absent secretion with intracellular retention. Variants with a premature stop codon did not synthesize VWF at all. Some SV had normal secretion and multimerization (V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F) implying that the VWD phenotype in these patients results from yet unidentified mechanisms and may not be associated with these SV. Reduced plasma survival is unlikely as these patients had normal VWFpp/VWF:Ag level consistent with normal VWF clearance. Among the VWF variants with normal or decreased secretion, 45.4% had reduced VWF:CB/VWF:Ag consistent with abnormal multimer structure. Heterozygous expression, as observed in the patient, is expected to normalize these multimerization defects. The decreased or absent secretion observed for 52.7% of the variants studied correlates with the patient phenotype, indicating reduced secretion is the mechanism underlying these patients' type 1 VWD phenotype. No domain-specific correlation of VWF secretion or multimer abnormality was observed.In summary, VWF expression studies confirmed the causative nature of many, but not all of the novel sequence variants identified in type 1 VWD subjects in the Zimmerman Program. DisclosuresNo relevant conflicts of interest to declare.

Von Willebrand Factor (VWF) Propeptide and Factor VIII (FVIII) Levels Identify the Contribution of Decreased Synthesis and/or Increased Clearance Mechanisms in the Pathogenesis of Type 1 Von Willebrand Disease (VWD) in the Zimmerman Program

Laboratory diagnosis of VWD is challenging, with multiple tests required to obtain an accurate assessment. Plasma VWF level represents a balance between synthesis, secretion, and clearance. Although synthesized together, VWFpp and VWF circulate in plasma independently with differing half-lives. Plasma VWFpp level is used to assess synthesis/secretion, VWFpp/VWF:Ag ratio to indicate clearance of VWF, and FVIII/VWF:Ag ratio to assess VWF synthesis and clearance.We sought to identify the underlying VWD pathophysiology in subjects enrolled in the Zimmerman Program including 245 healthy controls, 175 "low VWF" (VWF:Ag 30-50), 69 type 1 (VWF:Ag<30), 57 type 1C (VWF:Ag<30 and VWFpp/VWF:Ag>3), and 9 type 1-severe (1S) (VWF:Ag<5). FVIII levels were significantly reduced (p < 0.0001) in VWD subjects compared to controls. The mean FVIII/VWF:Ag ratio in "low VWF" (1.5), types 1 (2.4), 1C (2.9), and 1S (8.7) were significantly different from controls (p < 0.0001). VWFpp levels in all subjects were significantly lower than controls with type 1S subjects demonstrating the most reduced levels. The mean VWFpp/VWF:Ag was significantly increased compared to controls in type 1 (1.9) and 1C (8.4) subjects.VWFpp level, VWFpp/VWF:Ag and FVIII/VWF:Ag were used to define the underlying pathophysiology in VWD subtypes. A VWFpp/VWF:Ag > 3.0 indicates increased VWF clearance. VWFpp level < 50 IU/dL or FVIII/VWF:Ag > 2.0 indicate reduced VWF synthesis/secretion. A combination of increased clearance/reduced secretion may be identified or neither mechanism. Only 9% of "low VWF" subjects had increased FVIII/VWF:Ag, while 15% had decreased VWFpp suggesting reduced secretion. Other unidentified mechanisms may explain the majority of these "low VWF" cases. In type 1, 38 had increased FVIII/VWF:Ag and 48 had reduced VWFpp indicating reduced secretion in 55-70% of cases. In type 1C, all had increased VWFpp/VWF:Ag, 44 had increased FVIII/VWF:Ag and 19 reduced VWFpp. Reduced secretion may play a role in 33 -77% of these subjects, in addition to increased VWF clearance. All 9 type 1S had reduced VWFpp and 8/9 had increased FVIII/VWF, indicating a reduced secretion mechanism in nearly all cases.To assess the influence of sequence variant (SV) location on VWF synthesis/secretion, levels were analyzed by presence or absence of SV and SV location by VWF domain. 62% of the VWD cohort had SV identified (23% with >1 SV) while 38% had no SV. Of those with SV, 52% had increased FVIII/VWF:Ag and 48% had decreased VWFpp, suggesting reduced secretion; 31% had increased VWFpp/VWF:Ag indicative of increased clearance; and 32% had neither mechanism. In the group with no SV, only 7% had increased FVIII/VWF:Ag and 17% had decreased VWFpp while 77% had neither mechanism identified.Of those subjects with reduced secretion as indicated by increased FVIII/VWF:Ag, the majority of SV were found in A1 (29%), D3 (22%) and D1 (13%) which was similar to those with reduced secretion indicated by decreased VWFpp level in A1 (25%), D3 (21%) and D1 (15%). An increased VWFpp/VWF:Ag ratio predicting increased clearance was found in patients with SV in D3, A1 and D4 domains. Patients with both increased clearance and reduced secretion had SV in A1 (53%), D3 (19%) and D4 (11%). Subjects who had neither mechanism identified had SV in A2 (25%), C1-C6 (18%) and D2 (16%), suggesting these SV are associated with other, yet unidentified mechanisms.Although VWFpp level and FVIII/VWF:Ag are both thought to indicate VWF synthesis/secretion, some discrepancies were observed. VWFpp level may be the more specific marker, as VWFpp and VWF share a common precursor protein. Reduced secretion plays a role in nearly all type 1S, 70% of type 1, and 15% of "low VWF" subjects. Additionally, SV in A1, D3, and D4 may be associated with decreased secretion and/or increased clearance while those in D1 may be associated with decreased secretion alone. No SV were found in 85% of "low VWF" subjects that is consistent with the observation that the majority of these cases (81%) had neither decreased secretion nor increased clearance mechanisms identified. The mechanistic cause of bleeding in these patients remains undefined. Assay of VWFpp and corresponding VWF:Ag ratios may help to define the underlying mechanism in VWD subjects and identify true type 1C VWD patients, which is clinically important for therapeutic treatment. DisclosuresFlood:CSL Behring: Consultancy; Baxalta: Consultancy. Friedman:Shire: Consultancy; NovoNordisk: Consultancy; CSL Behring: Consultancy; Alexion: Speakers Bureau.

Spectrum of Type 2 Von Willebrand Disease in the Zimmerman Program

▪While von Willebrand disease (VWD) is the most common inherited bleeding disorder, most patients have quantitative defects in von Willebrand factor (VWF). The qualitative variants, collectively termed type 2 VWD, are less common, but also in general more severe than type 1 VWD. However, despite a common laboratory phenotype of decreased VWF:RCo/VWF:Ag ratio for types 2A, 2B, and 2M VWD, the clinical phenotype is highly variable. We examined index cases and affected family members enrolled in the Zimmerman Program with a phenotypic diagnosis of type 2 VWD. All subjects had factor VIII (FVIII), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and multimer distribution analyzed in a central laboratory. For calculation of mean VWF:RCo values, a level of 5 was assigned to subjects with VWF:RCo below the laboratory lower limit of detection of 10 IU/dL. A platelet binding assay was also performed using a gain of function GPIb containing 2 mutations that enable spontaneous binding to VWF in the absence of ristocetin (VWF:GPIbM). Full length VWF gene sequencing was performed for all index cases. Targeted sequencing was performed for family members to ascertain the presence or absence of sequence variations found in the index case. Bleeding symptoms were quantified using the ISTH bleeding assessment tool and reported as bleeding scores (BS). Mean FVIII, VWF:Ag, VWF:RCo, and BS are listed in the table below for each type 2 variant.For type 2A VWD, 113 subjects have been enrolled to date. All had an abnormal multimer distribution with loss of high molecular weight multimers. 6 type 2A subjects had a VWF:RCo/VWF:Ag ratio of ≤0.7. The lowest VWF:RCo levels were seen in the type 2A cohort with 60% <10. 98% of type 2A subjects had an identified sequence variation on full length sequencing. 25% had the p.R1597W sequence variation and an additional 4 subjects had p.R1597Q. The mean bleeding score for the subjects with sequence variations at 1597 was 10.6. 11% of subjects had p.R1374H, which correlated with a higher mean bleeding score of 12.4. Mean bleeding score for the remainder of the type 2A subjects was lower, at 6.6, suggesting that differences in VWF genetics may account for differences in phenotype, despite the common type 2A laboratory presentation of reduced VWF:RCo and loss of high molecular weight multimers.44 type 2B subjects have been enrolled to date, all with abnormal multimer distribution and either documented abnormal VWF-platelet binding or a presence of a known type 2B sequence variation. Sequence variations were found in 100% of subjects. The most common sequence variations were p.V1316M (20%), p.R1306W (18%), p.R1341Q (11%), and p.H1268Y (9%). Mean VWF:RCo/VWF:Ag ratios ranged from 0.32-1.12, suggesting that a normal VWF:RCo/VWF:Ag ratio cannot completely exclude the possibility of type 2B VWD. Most (94%) had increased VWF:GPIbM. Subjects with p.V1316M and p.R1306W/Q sequence variations had lower VWF:RCo compared to subjects with p.R1341Q/W but mean bleeding scores did not differ.59 type 2M subjects have been enrolled to date. Mean VWF:RCo/VWF:Ag ratio was 0.46 (range 0.14-0.7). Sequence variations were found in 93% of subjects. R1374C was found in 13 members from one family. While mean VWF levels were similar to the entire 2M group, a wide range in VWF:Ag and VWF:RCo/VWF:Ag ratio was observed, accompanied by a corresponding range in BS from 0-8. This suggests that other modifiers of phenotype may be present aside from the VWF sequence variation.11 type 2N subjects have been enrolled to date, all with low VWF binding to FVIII. Sequence variations were found in 100% of this cohort. R854Q was present in 89% of subjects. Bleeding scores were highest for homozygous 2N sequence variations.Overall, the mean BS for type 1 VWD subjects was 6.3, the mean BS for type 2 VWD subjects was 7.5, and the mean BS for type 3 VWD subjects was 16.8. Types 2A and 2N had higher bleeding scores on average than type 2B, and type 2M subjects had on average the lowest bleeding scores. Although heterogeneity was seen across all the type 2 variants, both laboratory testing and genetic testing are useful in categorizing and phenotyping type 2 VWD.TableFVIII (mean)VWF:Ag (mean)VWF:RCo (mean)BS (mean)Type 2A4734128.7Type 2B4536237.1Type 2M6254215.4Type 2N3069768.3 DisclosuresMontgomery:Immucor: Patents & Royalties.

Validation Study Of The Composite Score To Identify Von Willebrand Disease in Children

BackgroundThe diagnosis of type 1 von Willebrand disease (VWD), the most common inherited bleeding disorder, presents a diagnostic challenge in children. In the absence of a prior hemostatic challenge in children, a VWD diagnosis may be missed by the Tosetto bleeding score, as it is based, in part, on bleeding symptoms, i.e. with surgery, menses, or childbirth, that may not occur until after childhood. In fact, 25% or more of children with VWD may be diagnosed only after they experience postoperative bleeding. Other bleeding scores, such as the James score, which assesses early life bleeding, e.g. cephalohematoma, macroscopic hematuria, and umbilical stump, post-circumcision, post-venipuncture bleeding, has been validated in children with VWD, but early life bleeding events are uncommon in VWD. We previously described a 4-variable Composite Score that has 92.5% sensitivity and 95% specificity for diagnosing VWD in children < 11 years of age, when at least two of four criteria are positive: 1) Tosetto bleeding score ≥ 1; 2) family history of VWD or bleeding; 3) personal history of iron deficiency anemia; and/or 4) positive James early bleeding score. The purpose of this study was to validate a Composite Score of ≥ 2 (i.e. at least 2 of 4 variables positive) for identifying children with VWD. MethodsWe designed and conducted a prospective validation study to determine the accuracy of the Composite Score for identification of children (<11 years of age) with VWD. Following parental consent, children without a prior history of VWD undergoing VWD testing were enrolled, and survey data were collected including personal bleeding history (including Tosetto and James scores), history of iron deficiency anemia, and family history of VWD or bleeding. Results of VWD testing were collected, including VWF:RCo, VWF:Ag, and VIII:C in addition to VWF multimers, if performed. Survey data and bleeding scores were compared between those with and without VWD, defined by VWF:RCo <30 (NIH 2008 criteria), or <40, or <50 IU/dL. Prior to study, it was determined that a Composite Score ≥ 2 would have 90% power to identify VWD defined by VWF:RCo <0.30 IU/dL, and 80% power to identify VWD by VWF:RCo <40 IU/dL. Categorical variables were compared using chi-square or Fischer's exact test. Continuous variables were compared using two-sample t-test or Wilcoxon's rank sum test. Sensitivity, specificity, positive and negative predictive value of the Composite Score of ≥ 2 to diagnose VWD were determined. ResultsA total of 195 subjects were enrolled from 12 participating centers from 2010-2013, of whom 193 patients with no missing data were included in the analysis. A total of 81 (41.9%) children had type 1 VWD, including 11 (5.7%) with VWF:RCo <30 IU/dL (Group A), 24 (12.4%) with VWF:RCo <40 IU/dL (Group B), and 46 (23.8%) with VWF:RCo <50 IU/dL (Group C); the remainder with VWF:RCo ≥50 IU/dL were unaffected. There were no significant demographic differences between groups: overall 54.9% were male, 82.4% were Caucasian, and the mean age was 4.7 years. A composite score of ≥ 2 had 63.6%, 75%, and 67.4% sensitivity in diagnosing type 1 VWD in Groups A, B, and C respectively; 33.5%, 34.9%, and 34% specificity in diagnosing type 1 VWD, respectively; and 93.8%, 90.8%, and 76.9% negative predictive value, respectively. Based on the performance of the composite, with VWD being defined by a VWF:RCo of <30, <40, or <50 IU/dL, 31.6%, 30.6%, and 25.9% of subjects, respectively, had a negative composite score and therefore would not have required VWD testing. DiscussionThis is the first study to prospectively validate the Composite Score to identify children with type 1 VWD. Although the sensitivity and specificity of the Composite Score were lower than in the original study (above), the latter included only VWD subjects, while this validation study included all, VWD and non-VWD, undergoing testing. The negative predictive value of the Composite Score was robust, especially at lower VWF:RCo, and indicates that VWD testing could be eliminated in nearly a third of children referred for VWD testing. Given the difficulty of diagnosing VWD in children, the Composite Score may be an important screening tool for diagnosis of type 1 VWD in children. Disclosures:Kerlin:Bayer Healthcare US: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk A/S: Consultancy, Honoraria.

Binding Of VWF To Type IV Collagen: An Additional Collagen Binding Mechanism Beyond Types I, III, and VI Collagen?

Von Willebrand disease (VWD) is a common bleeding disorder characterized by defects in the interaction of von Willebrand factor (VWF) with its ligands factor VIII, platelet glycoprotein Ib, and/or collagen. Nearly all assessment of collagen binding is performed with types I or III collagen. Type VI collagen, which is formed as a tight triple helix, has also been reported to interact with VWF. In contrast, type IV collagen lacks this tight helical structure and is located in the basal lamina. Type IV collagen may therefore serve as another important ligand for VWF. We examined this interaction in samples from the Zimmerman Program for the Molecular and Clinical Biology of VWD, a large US study of subjects with all types of VWD.Subjects were enrolled based on a pre-existing diagnosis of VWD and compared to a cohort of healthy controls. VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and type III collagen binding using 1 mcg/mL human placental type III collagen (VWF:CB3) were performed in the clinical laboratory at the BloodCenter of Wisconsin. Type IV and type VI collagen binding (VWF:CB4 and VWF:CB6) were performed in the research laboratory by ELISA using 1 mcg/mL human placental type IV or type VI collagen captured on amine binding plates. DNA sequencing was performed on all subjects to identify VWF sequence variations. Bleeding scores were calculated according to the scoring system used in the MCMDM-1VWD study.For this analysis, 251 healthy controls, 535 type 1, 49 type 2A, 16 type 2B, 18 type 2M, 11 type 2N, and 18 type 3 VWD were included. The mean VWF:CB4/VWF:Ag ratio for the healthy controls was 0.96, with a standard deviation of 0.24. This was similar to the mean VWF:CB6/VWF:Ag ratio (mean 0.95, p=NS), but slightly lower than the mean VWF:CB3/VWF:Ag ratio (mean 1.05, p<0.01). The mean VWF:CB4/VWF:Ag ratio was 0.87 for the type 1 VWD subjects. The mean VWF:CB4/VWF:Ag ratio was significantly lower in type 1 subjects as compared to healthy controls (p<0.01). This was attributed in part to the presence of several A1 domain sequence variations, including p.R1399H, p.F1369I, and p.L1382P. While the latter sequence variations were isolated findings in single subjects, p.R1399H was present in 1% of the healthy controls, 1% of the type 1, and 6% of the type 2M VWD subjects.Mean VWF:CB4/VWF:Ag ratios were low for both type 2A and type 2B VWD subjects, at 0.54 and 0.52 respectively. This was not significantly different from the VWF:CB3/VWF:Ag data (p=NS for both type 2A and 2B VWD), consistent with the loss of high molecular weight multimers in these subjects and the concomitant reduction in collagen binding. As expected, VWF:CB4/VWF:Ag ratios were normal in the type 2N subjects, with a mean of 1.04. The type 2M subjects, however, displayed a significant reduction in type IV collagen binding, with a mean VWF:CB4/VWF:Ag ratio of 0.79. One subject with an 11 amino acid deletion (from p.R1392 through p.Q1402) had absent binding to type IV collagen yet normal binding to type III collagen with a VWF:CB3/VWF:Ag ratio of 0.82. Another subject with p.I1425F and p.R1399H had a VWF:CB4/VWF:Ag ratio of 0.48, and VWF:CB3/VWF:Ag ratio of 1.10. Type IV collagen binding did not correlate with VWF:RCo (R squared less than 0.1). The type 2M subjects with a mean VWF:CB4/VWF:Ag ratio less than 0.7 had higher bleeding scores, with a mean bleeding score of 9 compared to a mean of 6 for type 2M subjects with a mean VWF:CB4/VWF:Ag ratio greater than 0.7. Not surprisingly, the subjects with type 3 VWD and undetectable VWF:Ag also had undetectable VWF:CB4.These data suggest that type IV collagen binding reflects an independent function of VWF apart from platelet binding, and distinct from binding to type III collagen. In our study, more variation was seen between type IV and type III collagen than between type IV and type VI collagen. Neither VWF:CB4 nor VWF:CB6 are currently tested as part of the clinical workup of VWD. Measurement of this function may assist in identification of variant VWD, particularly in the subgroup of patients with type 2M VWD. Disclosures:No relevant conflicts of interest to declare.

No increase in bleeding identified in type 1 VWD subjects with D1472H sequence variation

AbstractThe diagnosis of von Willebrand disease (VWD) is complicated by issues with current laboratory testing, particularly the ristocetin cofactor activity assay (VWF:RCo). We have recently reported a sequence variation in the von Willebrand factor (VWF) A1 domain, p.D1472H (D1472H), associated with a decrease in the VWF:RCo/VWF antigen (VWF:Ag) ratio but not associated with bleeding in healthy control subjects. This report expands the previous study to include subjects with symptoms leading to the diagnosis of type 1 VWD. Type 1 VWD subjects with D1472H had a significant decrease in the VWF:RCo/VWF:Ag ratio compared with those without D1472H, similar to the findings in the healthy control population. No increase in bleeding score was observed, however, for VWD subjects with D1472H compared with those without D1472H. These results suggest that the presence of the D1472H sequence variation is not associated with a significant increase in bleeding symptoms, even in type 1 VWD subjects.

Similarity in joint function limitation in Type 3 von Willebrand's disease and moderate haemophilia A

Type 3 von Willebrand's disease (VWD) is a rare bleeding diathesis with complete or near complete deficiency of von Willebrand factor (VWF) and low factor VIII (FVIII) levels. In contrast, only FVIII is decreased in haemophilia A (HA). Both disorders are complicated by arthropathy. The purpose of this study was to further clarify the roles of FVIII and VWF: Antigen (VWF:Ag) in joint range of motion (ROM) loss over time. We compared joint ROM loss and other bleeding manifestations in 100 Type 3 VWD subjects (FVIII<5%) and 1814 moderate HA subjects (FVIII 1-5%) within the U.S. Universal Data Collection (UDC) database. High rates of bleeding were reported at baseline. During follow-up, moderate HA patients reported a joint (46% vs. 34%, P < 0.0001) or muscle bleed (27% vs. 16%, P < 0.0001) in a higher proportion of visits than VWD patients. Other bleeds, including mucosal, were reported in a greater proportion of visits among patients with Type 3 VWD than among those with HA (49% vs. 32%, P < 0.0001). Multivariate analysis revealed no difference in joint ROM loss over time in the Type 3 VWD vs. moderate HA populations. A higher FVIII level was protective in both VWD and HA (P < 0.001). Our findings support the hypothesis of primacy of the FVIII level in determining risk of joint haemorrhage, and may help target therapy in Type 3 VWD and moderate HA to prevent joint disability.

Variability in platelet‐ and collagen‐binding defects in type 2M von Willebrand disease

Type 2M von Willebrand disease (VWD) includes qualitative defects in von Willebrand factor (VWF) function, with normal multimer distribution but a defect in VWF activity with respect to platelet or collagen binding. We characterized novel VWF gene mutations found in type 2M VWD subjects enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. Subjects were enrolled based on a pre-existing diagnosis of type 2M VWD. Testing included full-length gene sequencing, VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding and multimer distribution. Recombinant VWF variants were synthesized using site-directed mutagenesis and expressed in HEK293T cells. Platelet binding was measured by flow cytometry with fixed platelets and ELISA with recombinant glycoprotein Ibα (GPIbα). Four novel VWF A1 domain mutations were found in individuals with type 2M VWD: S1358N, S1387I, S1394F and Q1402P. All subjects had a history of bleeding, VWF:RCo < 40 IU dL(-1) , VWF:RCo/VWF:Ag ratios <0.6 and normal multimer distribution. No defect in expression, secretion, or multimerization was found for any of the mutations. All showed decreased binding to intact platelets, and decreased or absent binding to a mutant GPIbα construct with spontaneous VWF binding. 1387I had decreased binding to all collagen types tested. 1402P had reduced binding exclusively to type VI collagen. Type 2M VWD is a heterogeneous category comprised of both collagen- and platelet-binding defects. Understanding the precise defect for each mutation may ultimately lead to better diagnosis and treatment.

Comparison of type I, type III and type VI collagen binding assays in diagnosis of von Willebrand disease

von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Collagen binding assays (VWF:CB), however, are not part of the routine work-up for von Willebrand disease (VWD). This study presents data on collagen binding for healthy controls and VWD subjects to compare three different collagens. VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen were examined for samples obtained from the Zimmerman Program. Mean VWF:CB in healthy controls was similar and highly correlated for types I, III and VI collagen. The mean VWF:CB/VWF:Ag ratios for types I, III and VI collagen were 1.31, 1.19 and 1.21, respectively. In type 1 VWD subjects, VWF:CB was similar to VWF:Ag with mean VWF:CB/VWF:Ag ratios for types I, III and VI collagen of 1.32, 1.08 and 1.1, respectively. For type 2A and 2B subjects, VWF:CB was uniformly low, with mean ratios of 0.62 and 0.7 for type I collagen, 0.38 and 0.4 for type III collagen, and 0.5 and 0.47 for type VI collagen. Normal ranges for type I, III and VI collagen are correlated, but higher values were obtained with type I collagen as compared with types III and VI. The low VWF:CB in type 2A and 2B subjects suggests that VWF:CB may also supplement analysis of multimer distribution. However, these results reflect only one set of assay conditions per collagen type and therefore may not be generalizable to all collagen assays.

Von Willebrand Factor Collagen Binding Provides a Sensitive Screen for Identification of Type 2A and 2B Von Willebrand Disease

Abstract 379▪FN2▪This icon denotes a clinically relevant abstractCollagen binding is an easily performed test of von Willebrand factor (VWF) function but its role in clinical evaluation is still debated. Analysis of multimer distribution, on the other hand, is time-consuming and technically challenging. We hypothesized that VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), and collagen binding (VWF:CB) could identify the subset of von Willebrand disease (VWD) cases in which multimer analysis would be informative. Subjects from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF:Ag, VWF:RCo, VWF:CB (with type III human placental collagen), multimer distribution, and full VWF exon sequencing. Normal controls as well as patients with type 1, 2A, 2B, 2M, and 2N VWD were analyzed. The mean VWF:CB/VWF:Ag ratio for subjects with normal multimers was 1.10, while the mean ratio for subjects with abnormal multimers was 0.51 (p<0.001). When results were restricted to those subjects with confirmed type 2A or type 2B mutations, however, the mean ratio for subjects with abnormal multimers decreased to 0.41 (p<0.001 compared to those with normal multimers). For the 146 normal controls with multimer results available, 2 had absence of the highest molecular weight multimers, but normal collagen binding, normal bleeding scores, and no evidence of a VWF gene mutation, suggesting that the multimer results represented assay artifact. 354 type 1 subjects were examined; of those, 12 had abnormal multimer patterns. 7 had loss of the high molecular weight multimers. Of these, 5 had known type 1 VWD mutations and normal VWF:CB/VWF:Ag ratios, possibly representing sample artifacts rather than a true multimer abnormality, as no multimer issues have been previously reported for these mutations. One had no mutation found and one had a type 2A mutation. 2 had a full spectrum of multimers with relatively increased staining of the lower molecular weight bands; both with novel A1 domain mutations that are currently under investigation. 3 had larger than normal multimers observed, all with normal VWF:CB/VWF:Ag ratios. Of the 342 type 1 subjects with normal multimers, only one had a VWF:CB/VWF:Ag ratio of <0.7, likely due to very low values (VWF:CB of 2 and VWF:Ag of 4). There were 36 type 2A subjects available for analysis. 27 had loss of high molecular weight multimers. Only 3 of those had VWF:CB/VWF:Ag ratios >0.7, but none of those subjects had VWF mutations consistent with type 2A VWD. 7 subjects had a shift from high to low molecular weight multimers, 4 with VWF:CB/VWF:Ag ratios >0.7 and either known type 1 mutations or novel VWF gene mutations. 2 subjects had normal multimer distribution, one with a type1 VWD mutation and one with a novel mutation. Characterization of these novel mutations is in progress. All the 17 type 2B subjects had loss of high molecular weight multimers and abnormal collagen binding, with a VWF:CB/VWF:Ag ratio <0.7. Interestingly, however, not all had a reduced VWF:RCo/VWF:Ag ratio, suggesting VWF:CB would be required in addition to VWF:RCo if multimer distribution was omitted in initial evaluation of this type of VWD. Of 18 type 2M subjects, only one had an abnormal multimer distribution. That subject had no mutations in the VWF coding sequence and normal VWF:CB, although the VWF:RCo/VWF:Ag ratio was low at 0.53. Repeat analysis of a new sample from this subject is pending. All 7 type 2N VWD subjects had normal multimers and VWF:CB/VWF:Ag ratios >0.7. In our population, with the exception of mutations that are yet to be characterized, the combination of VWF:Ag, VWF:RCo and VWF:CB was sufficient to categorize patients as normal, type 1, type 2A, 2B or 2M in the before multimer analysis. These findings suggest that VWF:CB is a sensitive screen for detection of an abnormal multimer distribution. Collagen binding is technically much easier to perform, allowing multimer analysis to be reserved for those cases with low VWF:RCo/VWF:Ag or low VWF:CB/VWF:Ag ratios. Disclosures:No relevant conflicts of interest to declare.

Quantitative Analysis of VWF Multimer Structure: Discrimination Between VWD Subtypes

Abstract 1215The diagnosis of von Willebrand disease (VWD) and discrimination between its subtypes includes analysis of VWF:Ag, VWF:RCo, and VWF multimer structure. VWF multimer analysis is qualitative, and therefore a subjective assessment open to interpretation. It is often difficult to assess subtle differences in multimer structure. To address these shortcomings we have developed a quantitative method for analysis of VWF multimers. We have analyzed multimer structure for VWD patients and healthy controls recruited through the Zimmerman Program for the Molecular and Clinical Biology of von Willebrand Disease (ZPMCB-VWD). The patient population includes type 1 and type 2 VWD with well-defined genotypes and phenotypes. Multimer analysis was performed using a 0.65% LiDS-agarose gel electrophoresis system and western blotting with chemilumiscent detection using the Fujifilm LAS-3000 luminescent image analyzer. Densitometry was performed and area-under-the-curve calculated using MultiGauge analysis software. We calculated the percentage of low molecular weight (LMW) multimers defined as bands 1 – 5, mid-molecular weight (MMW) multimers (bands 6 – 10) and high molecular weight (HMW) multimers (bands >10). For healthy controls, the distribution of multimer density (mean ± standard deviation) was 25.3 ± 2.7% HMW, 56.1 ± 4.9% MMW, and 18.6 ± 3.4% LMW. Type 1 VWD (including type 1C) patients had a similar distribution of multimers (22.5 ± 7.6% HMW, 48.5 ± 6.7% MMW, 29.0 ± 7.2 % LMW), although there was a slight shift in distribution to increased LMW. For some type 1C patients with mutations including C1130Y and W1144G, we observed a small loss of HMW multimers (14.2 ± 0.8% HMW, 51.1 ± 1.4% MMW, 34.7 ± 2.3% LMW), as has been previously reported in patients with a C1130F variation. In contrast, some patients with the type 1C “Vicenza” mutation, R1205H, demonstrated increased HMW multimers (32.6 ± 1.0% HMW, 42.2 ± 4.0% MMW, 25.2 ± 3.0% LMW) as previously reported. Although the multimers in the type 1 patients are essentially normal, quantitative analysis reveals subtle abnormalities in structure. In type 2B VWD patients with mutations including V1316M, R1306W, and R1341W, a loss of HMW and MMW multimers was observed (7.1 ± 3.2% HMW, 40.4 ± 8.3% MMW, and 52.5 ± 11.4% LMW). A greater loss of HMW and MMW multimers was observed in patients with type 2A VWD with mutations including Y1349C, R1597W, G1609R, I1628T, G1631D, and G1670S (3.5 ± 6.2% HMW, 19.7 ± 20.4% MMW, and 76.9 ± 26.3% LMW). The type 2A subjects consisted of two groups: those with a virtually complete loss of HMW and MMW (0.0 ± 0% HMW, 4.0 ± 1.0% MMW, and 96.0 ± 1.0% LMW), and those with loss of HMW and decreased MMW (8.7 ± 7.5% HMW, 41.0 ± 14.7% MMW, and 50.3 ± 20.9% LMW). The latter group had a similar multimer distribution to that of type 2B VWD subjects. While most type 2A patients with mutations associated with increased susceptibility to ADAMTS13 proteolysis had severe multimer abnormalities (>95% LMW), some had only moderate abnormalities. Our study demonstrates that quantitative analysis of VWF multimer patterns more clearly distinguishes patients with various subtypes of VWD than subjective analysis. Although one of the two groups of type 2A patients is similar to the type 2B group, the other group is clearly different and is associated with specific genotypes, perhaps eliminating the need for DNA sequence analysis to make a definitive diagnosis for this group. This technique provides an objective measure of VWF structure to better characterize subtle changes observed in the subtypes of VWD and may help to determine the nature of any additional clinical laboratory testing to reach a clear-cut diagnosis. Disclosures:No relevant conflicts of interest to declare.

Bleeding Symptoms and Laboratory Correlation in 150 Patients with Severe von Willebrand Disease.

Severe von Willebrand disease (VWD) is a rare bleeding disorder with markedly decreased or absent von Willebrand factor (VWF) protein, with a parallel decrease in VWF function and FVIII activity. Patients experience excessive mucosal bleeding, characteristic of VWD, but may also experience joint bleeding, a phenotype more typical of hemophilia A. The CDC sponsored Universal Data Collection (UDC) project collects joint range of motion (ROM) as well as other clinical data on patients with blood disorders followed at Hemophilia Treatment Centers (HTCs) in the U.S. As of 1/07, 253 patients in the UDC were classified as having severe VWD. The goal of this study was to further characterize these patients and assess relations between levels of FVIII/VWF and joint bleeding and function.Methods: To validate the diagnosis of severe VWD, HTCs were contacted to obtain baseline laboratory values. Patients with VWF:Ag < 10%, and consistent VWF:RCo and FVIII levels, were considered to have severe VWD. Of 253 patients, laboratory values were obtained in 201, 162 were confirmed to have severe VWD and 150 had joint ROM data. For the analyses, joint bleeding was categorized as either present or absent at each visit. In addition, the joint function measure was calculated for each patient as the overall proportion of normal joint ROM based on measures taken of 10 joints.Results: The 150 severe VWD subjects consisted of 73 females (49%) and 77 males (51%), with a mean age of 25 yrs (2–75). Racial distribution was Caucasian: 121; African-American: 8; Hispanic: 8, and other: 13. 86/150 gave a family history of a bleeding disorder. 147/150 subjects reported bleeding episodes. Mean age of first documented bleed was 3, but first use of an HTC was not until a mean age of 8. Of the first bleeding episodes, 79 were mucosal, 10 during circumcision, 12 joint, 6 cranial, 7 were from I.M. injections, 21 were other sites, and 12 were unknown. 137/150 subjects had received blood/factor products and 75 had received them at home. 12 subjects had experienced an ICH. Patients with FVIII levels ≤ 5% (n=105) had a lower mean age of first bleed than those with FVIII > 5% (1.5 vs. 6.1 yrs, p=0.0005), but there was no significant association with VWF:Ag level (p=0.08). Lower FVIII and VWF levels were associated with higher risk of joint bleeding [OR 2.95 (p=0.006) and OR 3 (p=0.02), respectively] but not with non-muscle other bleeding. In a longitudinal multivariate analysis, lower FVIII level (p=0.02), increasing age (p<0.0001), joint bleeding (p<0.0001), and higher BMI (p<0.0001) were all significantly negatively associated with joint function. In a separate analysis of 88 patients with complete information on VWF:Ag, low levels were negatively associated with joint function after adjusting for age, BMI, gender and joint bleeding (p=0.002).Conclusion: Patients with severe VWD in the U.S. have bleeding symptoms beginning at an early age, with mucosal bleeding most common, but also joint and intracranial bleeding, consistent with a severe bleeding phenotype. Our results verify the strong associations between low levels of FVIII and VWF and both joint bleeding and joint function in this disorder. Further analyses will examine whether levels of these proteins are responsible for the phenotypic similarity between severe VWD and hemophilia A.

Platelet adhesion to collagen in normal and Von Willebrand's disease subjects

A rapid and sensitive method for quantitation of platelet adhesion was developed, utilizing a combination of optical density monitoring and interval platelet counting after addition of partially purified rat tail tendon collagen in a stirred cell system. EDTA inhibited aggregation but did not affect adhesion. The rate of platelet adhesion was significantly diminished in von Willebrand's disease and by decreasing normal plasma concentrations to less than 20%. Addition of dipyridamole to normal platelet rich plasma resulted in inhibition of adhesion, whereas acetylsalicylic acid and sulfinpyrazone had no significant effects on adhesion.