scholarly journals Collagen Binding Provides a Sensitive Screen for Variant von Willebrand Disease

2013 ◽  
Vol 59 (4) ◽  
pp. 684-691 ◽  
Author(s):  
Veronica H Flood ◽  
Joan Cox Gill ◽  
Kenneth D Friedman ◽  
Pamela A Christopherson ◽  
Paula M Jacobi ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Reference Intervals ◽  
Von Willebrand Disease ◽  
Type Iii Collagen ◽  
Healthy Controls ◽  
Collagen Binding ◽  
Vessel Injury ◽  
Clinical Biology ◽  
Von Willebrand

BACKGROUND von Willebrand factor (VWF) is a multimeric protein that binds platelets and collagen, facilitating hemostasis at sites of vessel injury. Measurement of VWF multimer distribution is critical for diagnosis of variant von Willebrand disease (VWD), particularly types 2A and 2B, but the typical measurement by gel electrophoresis is technically difficult and time-consuming. A comparison of VWF collagen binding (VWF:CB) and VWF multimer distribution was performed to evaluate the utility of VWF:CB as a diagnostic test. METHODS Participants were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. VWF:CB was analyzed with type III collagen and multimer distribution by agarose gel electrophoresis. The study population included 146 healthy controls, 351 individuals with type 1 VWD, and 77 with type 2 VWD. Differences between individuals with multimer group results within (controls) and outside the reference intervals were assessed with Mann–Whitney tests. RESULTS The mean VWF:CB/VWF antigen ratio was 1.10 for individuals with multimer distribution within the reference intervals and 0.51 for those with multimer distribution outside the reference intervals (P < 0.001). Sensitivity of VWF:CB for multimer abnormalities was 100% for healthy controls, 99% for patients with type 1, and 100% for patients with type 2A and type 2B VWD using a VWF:CB/VWF antigen cutoff ratio of 0.6, and decreased to 99% for all patients with a ratio of 0.7. With the exception of individuals with novel or unclassified mutations, the VWF:CB was able to correctly categorize participants with variant VWD. CONCLUSIONS These findings suggest that VWF:CB may substitute for multimer distribution in initial VWD testing, although further studies are needed to validate the clinical utility of VWF:CB.

A Novel VWF A3 Domain Mutation with Absent Binding to Type I and Type III Collagen.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 541-541
Author(s):  
Veronica H. Flood ◽  
Carol A. Lederman ◽  
Jeffrey S. Wren ◽  
Pamela A. Christopherson ◽  
Kenneth D. Friedman ◽  
...  
Keyword(s):  
Index Case ◽  
Von Willebrand Disease ◽  
Type Iii Collagen ◽  
Type I ◽  
Healthy Controls ◽  
Wild Type ◽  
Collagen Binding ◽  
Type Iii ◽  
The Mean ◽  
Von Willebrand

Abstract Abstract 541 Von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Laboratory testing for von Willebrand disease (VWD) utilizes various assays of VWF protein and function, but collagen binding (VWF:CB) has not always been performed. Samples obtained from a large, multi-center US study of von Willebrand disease (VWD), the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD), were evaluated for VWF:CB. Whole blood was collected for DNA sequencing and plasma samples were collected for VWF testing. VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), and VWF:CB were performed in the BloodCenter of Wisconsin hemostasis laboratory. A normal range for the VWF:CB was established using 233 healthy controls enrolled in the ZPMCB-VWD. The mean VWF:Ag was 105 IU/dL and the mean VWF:CB was 123 U/dL, with a mean VWF:CB/VWF:Ag ratio of 1.19. This yields a normal range of 0.86-1.51 (mean ± 2 SD). No sequence variations in the VWF A3 domain affecting VWF:CB were observed in the healthy controls. Collagen binding was then examined in subjects enrolled with a diagnosis of VWD. The index case in one family was noted to have a low VWF:CB/VWF:Ag ratio of 0.54. The VWF:Ag was 41 IU/dL, VWF:RCo was 44 IU/dL, VWF:CB was 22 U/dL, and multimer distribution was normal. Decreased VWF:CB/VWF:Ag was found in the father and sister of the index case, while the mother had normal VWF:CB/VWF:Ag. The index case initially presented with epistaxis and easy bruising. Her bleeding score was 8, as determined by the revised European Union questionnaire. DNA sequencing of exons 28-32 was performed and revealed a heterozygous mutation (5356C>G) predicting replacement of wild-type histidine 1786 by aspartic acid in all affected family members. This amino acid is located in a critical face of the A3 loop. The 1786D mutation was cloned into a full-length VWF expression vector and expressed, as was a previously reported collagen-binding mutation, 1731T. Collagen binding assays were performed using type I human placental collagen at 5 μg/mL or type III human placental collagen at 1 μg/mL to capture either plasma or recombinant VWF (rVWF). Both mutations had normal expression, at 97% and 87% of the expression seen with wild-type VWF, respectively. Multimer distribution was also normal, including presence of high molecular weight multimers. Plasma from family members heterozygous for the H1786D mutation showed a mean VWF:CB/VWF:Ag ratio of 0.23 ± 0.05 (mean ± SD) for type I collagen and 0.50 ± 0.11 for type III collagen. When the rVWF constructs were examined, the novel 1786D mutation showed essentially no binding to either type I or type III collagen, at <1% of wild-type rVWF. In contrast, the previously reported collagen binding mutation 1731T showed VWF:CB/VWF:Ag ratios of 45% for type I and 50% for type III collagen when compared to wild-type rVWF. As the proband had a minimally decreased VWF:Ag, we then examined collagen binding in ZPMCB-VWD subjects with VWF:Ag between 30 and 50 IU/dL. The mean VWF:CB/VWF:Ag ratio in this group was 1.02, with a range of 0.62 to 1.28. These results, taken together with the data in the healthy controls, suggest that decreased VWF:CB/VWF:Ag ratios may identify a specific defect in VWF function. In particular, the H1786D mutation appears to result in a complete loss of VWF's ability to bind either type I or type III collagen. The subjects with this mutation were all heterozygous, which may account for reduction, rather than absence, of in vitro VWF:CB. The VWF:CB may be useful in diagnosis of VWD, and a decreased VWF:CB/VWF:Ag ratio in the face of normal VWF multimer distribution may reflect specific loss of collagen binding ability leading to bleeding symptoms. Disclosures: Montgomery: GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

VWF Binding to Types I, III or VI Collagen In the ZPMCB-VWD with Identification of Subjects with Selective Reduced Plasma VWF:CB

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 235-235
Author(s):  
Veronica H Flood ◽  
Sandra L Haberichter ◽  
Kenneth D Friedman ◽  
Steven R Lentz ◽  
Joan Cox Gill ◽  
...  
Keyword(s):  
Amino Acid ◽  
Type I ◽  
Healthy Controls ◽  
Type Vi Collagen ◽  
Collagen Binding ◽  
Type Iii ◽  
Type 3 ◽  
Von Willebrand

Abstract 235 Von Willebrand Disease (VWD) is a common bleeding disorder caused by quantitative (types 1 and 3) and qualitative (type 2) abnormalities in von Willebrand factor (VWF). Defective VWF binding to collagen (VWF:CB) has been identified in VWD patients, but the type(s) and amount of collagen vary between recommended assays. We measured VWF:CB using separate assays for types I, III, and VI collagen in plasmas from 233 healthy controls and 315 VWD index cases (261 type 1, 37 type 2 and 17 type 3) recruited into the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD). Additional studies included VWF antigen (Ag), VWF ristocetin-cofactor (RCo), VWF multimer, and VWF gene sequence analysis. VWF:CB was tested using ELISA assays with type I human placental collagen (5 ug/ml), type III human placental collagen (1 ug/ml), and by a commercial human placental type VI VWF:CB assay (Technoclone, Austria). Normal ranges were established for VWF:CB and VWF:CB/Ag ratios with the ZPMCB-VWD healthy control population. Three subjects (2 healthy controls and 1 type 1 VWD subject) were identified with reduced VWF:CB and reduced VWF:CB/Ag ratios to type VI collagen, despite normal results with type I and type III collagen. The two control subjects had VWF:CB/Ag ratios of 0.43 and 0.51 for type VI collagen. The patient with type 1 VWD had absent type VI VWF:CB and a VWF:Ag of 54 IU/dL with a history of clinical epistaxis and an EU Bleeding Score of 8 (normal less than or equal to 3). VWF sequencing demonstrated that these three individuals had an A1 loop polymorphism, R1399H, which has been previously reported as a VWF polymorphism. No other individuals in our study had this sequence change. Of particular note was that the VWF:CB/Ag ratios for types I and III collagen were each normal – suggesting a selective abnormality. As anticipated, collagen binding with all collagens was undetectable in the type 3 VWD subjects. Type 2A and 2B VWD subjects demonstrated reduced VWF:CB and VWF:CB/Ag ratios to all three types of collagen. Type 2M and 2N VWD subjects exhibited normal VWF:CB/Ag ratios to all three types of collagen, but one type 2M patient with an I1425F A1 loop mutation had a reduced VWF:CB/Ag ratio of 0.58. In the VWF A1 domain crystal structure, amino acid 1425 is in close proximity to amino acid 1399, suggesting the conformation of that region may be critical to type VI collagen binding to VWF. Since the frequency of the R1399H polymorphism is estimated to be 2% of the population (Sadler and Ginsburg, 1993), defective binding to type VI collagen may be an important contributor to the variability in the bleeding phenotype of VWD. Disclosures: Lentz: Novo Nordisk: Consultancy. Montgomery:GTI Diagnostics, Inc: Consultancy.

Fourteen Percent of Healthy African Americans Participating In the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB VWD) Are Heterozygous for the VWF Gene Mutation H817Q Associated with Type 2N Von Willebrand Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 239-239
Author(s):  
Kenneth D Friedman ◽  
Daniel B. Bellissimo ◽  
Pamela A. Christopherson ◽  
Veronica H Flood ◽  
Joan Cox Gill ◽  
...  
Keyword(s):  
African American ◽  
African Americans ◽  
Racial Differences ◽  
Healthy Subjects ◽  
Von Willebrand Disease ◽  
Compound Heterozygous ◽  
Healthy Controls ◽  
Clinical Biology ◽  
A1 Domain ◽  
Von Willebrand

Abstract Abstract 239 Von Willebrand disease (VWD) is a common hereditary bleeding disorder caused by reduced concentration or abnormal structure/function of von Willebrand Factor (VWF). Most published studies of normal VWF have been carried out in European or North American subjects without regard to racial differences. In the process of studying healthy controls in the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD), we identified a common polymorphism (D1472H) in the VWF A1-domain in African Americans that affects the measurement of VWF function by ristocetin cofactor (VWF:RCo) but does not appear associated with increased bleeding risk. We therefore explored whether other polymorphisms or mutations were identified more frequently in African Americans. VWF sequencing was performed on 191 healthy controls including 66 that were self-identified as African American. European Bleeding Score was obtained and normal in all healthy subjects. Among the African Americans, 9 individuals were heterozygous for the reported type 2N H817Q mutation and one was homozygous. Factor VIII binding to VWF (VWF:F8B) was determined with a standard FVIII binding assay using the subject's plasma VWF and recombinant FVIII. The VWF:F8B was significantly reduced in H817Q heterozygotes when compared to 10 healthy study subjects without the H817Q mutation (65 ± 11 versus 108 ± 11, p=0.003). The VWF:F8B was further reduced to 37 using the plasma VWF from the homozygous H817Q subject. However, the observed VWF:F8B in these individuals with H817Q are still considerably higher that that observed in patients enrolled in ZPMCB-VWD that are either homozygous or compound heterozygous with the common R854Q type 2N VWD (VWF:F8B < 13). Of the 116 self-identified Caucasian healthy subjects, none had the H817Q mutation, but 3 were heterozygous for the R854Q mutation; their mean plasma VWF:F8B was reduced to 51. While the homozygous R854Q patients had reduced plasma FVIII levels (mean FVIII=24 IU/dL), none of the sequenced healthy control subjects had plasma FVIII levels below 53 IU/dL, Some have advocated FVIII/VWF:Ag ratios as a screen for type 2N VWD. The subject with homozygous H817Q had only a mildly reduced FVIII/VWF:Ag ratio (0.59), while the heterozygous H817Q were not reduced (mean=0.90), thereby demonstrating that the VWF:F8B assay has greater sensitivity for type 2N VWF binding defects than the FVIII/VWF:Ag ratio. Since the previously reported A1-domain D1472H polymorphism was common in African Americans, we explored the prevalence of this polymorphism in the healthy subjects with the H817Q mutation. All H817Q heterozygous subjects were either homozygous (4) or heterozygous (5) for the D1472H polymorphism. The one individual who was H817Q homozygous was also D1472H homozygous, suggesting that there may be an extended haplotype present in African Americans. In summary, an H817Q type 2N mutation is commonly found in healthy African American subjects with an allele frequency of 0.083, predicting that approximately 7 in 1,000 African Americans would be homozygous for the H817Q type 2N mutation. Our data, and the rarity of diagnosis of type 2N VWD in African Americans suggests that while mutation H817Q may interfere with the interaction of FVIII with VWF, this mutation appears to confer little or no clinical symptoms. Disclosures: No relevant conflicts of interest to declare.

Characterisation of W1745C and S1783A, Two Novel Collagen Binding Defects in the A3 Domain of Von Willebrand Factor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 424-424 ◽  
Author(s):  
Anne Riddell ◽  
Keith Gomez ◽  
Carolyn Millar ◽  
G. Mellars ◽  
Simon A Brown ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Clinical Samples ◽  
Type Iii Collagen ◽  
Type I ◽  
Collagen Binding ◽  
Type Iii ◽  
Von Willebrand

Abstract Investigation of three families with von Willebrand disease showed that haemorrhagic symptoms were associated with disproportionately reduced collagen binding activity whilst Ristocetin co-factor activity was commensurate with antigen and multimeric analysis was normal. Genetic analysis revealed heterozygosity for two novel mutations in two of the families: W1745C in exon 30 and S1783A in exon 31. In the third family the affected individuals were heterozygous for a previously-described mutation: S1731T in exon 30 but two unaffected individuals also carried this mutation. All three mutations lie in the A3 domain containing the main collagen binding site in VWF. In patients’ samples VWF:CB activity was measured using human type I and type III collagen. Patients heterozygous for W1745C and S1731T showed a reduction in binding to both collagens but more marked reduction in binding to type III collagen. Heterozygosity for S1731T resulted in mild impairment of type I collagen binding but normal binding to type III collagen. Site-directed mutagenesis was used to generate vectors containing the three mutations (S1731T, W1745C and S1783A) and also one containing a W1745A mutation. Mutated VWF was expressed in HEK293T cells both singly and in co-transfection with a wild-type VWF (wtVWF) vector. All VWF mutants were expressed at a similar rate to wtVWF. Multimeric analysis demonstrated that all the mutants had a similar multimeric structure compared to recombinant wtVWF. However recombinant-wtVWF (wtVWF) had a lower collagen binding to VWF antigen ratio (CB:Ag) compared to plasma VWF (0.39 type I collagen and 0.45 type III collagen vs &gt;0.7 for plasma VWF). This is most likely due to the slight shift towards lower molecule weight multimers seen with recombinant VWF. CB:Ag ratios for the recombinant VWF showed the same pattern of binding to collagen type I and III as the clinical samples. The W1745A mutant demonstrated a similar CB:Ag ratio to W1745C. Kinetic analysis of binding to type I collagen demonstrated that W1745C, W1745A and S1783A did not bind and that S1731T bound with significantly less affinity compared to wtVWF (KD,app 27.1 ± 0.5nM and 7.3 ± 0.8nM respectively). Analysis of binding to type III collagen demonstrated that W1745C and W1745A both bound with ~ 8-fold reduced affinity (KD,app 16 ± 2.6nM and 21.3 ± 6.3nM) but wtVWF and S1731T bound with similar affinity, (KD,app 2.0 ± 0.1nM and 3.7 ± 0.85nM respectively). Analysis of the crystal structure of the VWF A3 domain showed that W1745 may interact with Y1780 and we noted the mutation Y1780A has also been shown to significantly reduce collagen binding. Measurement of free thiols present in VWF demonstrated that the new cysteine residue in W1745C is not involved in disulphide bond formation. These results indicate that it is the loss of W1745 rather than the creation of a new cysteine residue that is responsible for the loss of collagen binding activity. We therefore hypothesised that W1745 and Y1780 participate in an internal aromatic interaction that helps to maintain the structural configuration of A3. We sought confirmation by expressing another mutant; W1745F, replacing the tryptophan with another aromatic amino acid. As predicted this did not significantly affect collagen binding. In conclusion, our findings demonstrate that type 2 VWD may be arise from mutations in A3 causing abnormal collagen binding without other functional defects or abnormalities in multimer formation. This type of VWD may be under-recognised unless laboratories measure binding to both types I and III collagen. Mutations in A3 yield insights into the structural requirements for collagen binding may have differential effects on binding to collagen types I and III and can result in variable clinical phenotypes. Some mutations may not be consistently associated with bleeding symptoms.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 379-379
Author(s):  
Veronica H Flood ◽  
Joan Cox Gill ◽  
Kenneth D Friedman ◽  
Pamela A Christopherson ◽  
Paula M. Jacobi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Disease ◽  
Collagen Binding ◽  
The Mean ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Normal Controls ◽  
High Molecular Weight Multimers

Abstract Abstract 379FN2 Collagen 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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1215-1215 ◽  
Author(s):  
Sandra L Haberichter ◽  
Paula M Jacobi ◽  
Veronica H Flood ◽  
Pamela A. Christopherson ◽  
Joan Cox Gill ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Quantitative Analysis ◽  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Similar Distribution ◽  
Image Analyzer ◽  
Healthy Controls ◽  
Essentially Normal ◽  
Von Willebrand

Abstract Abstract 1215 The 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.

Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor

Blood ◽  
2009 ◽  
Vol 114 (16) ◽  
pp. 3489-3496 ◽  
Author(s):  
Anne F. Riddell ◽  
Keith Gomez ◽  
Carolyn M. Millar ◽  
Gillian Mellars ◽  
Saher Gill ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Type I Collagen ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Site Directed Mutagenesis ◽  
Type Iii Collagen ◽  
Type I ◽  
Collagen Binding ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractInvestigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.

von Willebrand Disease in a Pediatric-based Population - Comparison of Type 1 Diagnostic Criteria and Use of the PFA-100® and a von Willebrand Factor/Collagen-binding Assay

2000 ◽  
Vol 84 (09) ◽  
pp. 401-409 ◽  
Author(s):  
J.A. Dean ◽  
V. S. Blanchette ◽  
M. D. Carcao ◽  
A. M. Stain ◽  
C. R. Sparling ◽  
...  
Keyword(s):  
Diagnostic Criteria ◽  
Von Willebrand Factor ◽  
Screening Test ◽  
Binding Assay ◽  
Von Willebrand Disease ◽  
Consensus Guidelines ◽  
Collagen Binding ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryDefinitive diagnosis of type 1 von Willebrand Disease (VWD) remains a problem. Provisional consensus guidelines for the diagnosis of definite and possible type 1 VWD were prepared by the Scientific Subcommittee on von Willebrand factor (VWF) of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) during the 1996 annual meeting for the specific purpose of further evaluation in retrospective and prospective studies by a Working Party on Diagnostic Criteria (1996 Annual Report of the SSC/ISTH Subcommittee on VWF). In the first phase of this study, we compared 2 definitions of type 1 VWD, each with 3 criteria: significant bleeding history, laboratory investigations, and family history. Using the ISTH consensus guidelines for type 1 VWD definition, significantly fewer patients were diagnosed with definite type 1 disease as compared to our “in house” Hospital for Sick Children (HSC) criteria (4 vs. 31). While we recognize that the provisional ISTH consensus guidelines were not intended for clinical use, we believe that the results of our studies are of interest and will assist in any future refinements to the ISTH guidelines.In the second phase of this study, we investigated the utility of 2 new tests, a laboratory screening test and a functional test, for VWD in our well characterized, pediatric-based population. The Platelet Function Analyzer (PFA-100®) provides an in vitro measure of primary hemostasis under conditions of high shear, using disposable cartridges containing collagen and either epinephrine or ADP. All tested subjects with types 2 or 3 VWD had prolonged PFA-100 closure times (CTs) with both cartridge types (n = 17) and prolonged bleeding times (n = 14). In subjects with definite type 1 VWD, 20/24 (83%) had prolonged CTs with the collagen/ADP cartridge (19/24 (79%) with collagen/epinephrine), compared with 7/26 (27%) with prolonged bleeding times. In subjects with definite types 1, 2, or 3 VWD, collagen/ADP CTs were abnormal in 37/41 subjects, giving an overall sensitivity of 90%. With this high sensitivity, the PFA-100 is a better screening test for VWD than the bleeding time.We also tested a VWF collagen-binding assay (VWF:CBA) as a functional test for VWF, in comparison with the more routinely-used ristocetin cofactor assay (VWF:RCo). The VWF:CBA is based on an ELISA technique, which has the potential to be more reproducible than the VWF:RCo. We found that the VWF:CBA detected 43/49 (88%) subjects with definite types 1, 2, or 3 VWD, performing as well as the VWF:RCo, that detected 42/48 (88%). We also showed that, used in conjunction with VWF antigen levels, the VWF:CBA may be useful in classification of VWD subtypes.

Evaluation of commercial von Willebrand factor collagen binding assays to assist the discrimination of types 1 and 2 von Willebrand disease

2010 ◽  
Vol 104 (11) ◽  
pp. 1009-1021 ◽  
Author(s):  
Emmanuel Favaloro
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Binding Assays ◽  
Collagen Binding ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Control Samples

SummaryThis study reports on the evaluation of seven commercial von Wille-brand factor (VWF) collagen binding (VWF:CB) assays to potentially assist the discrimination of types 1 and 2 von Willebrand disease (VWD). Samples from 25 patients with type 1 VWD, of varying severity, were co-tested with 16 samples from patients with types 2A or 2B VWD, plus various control samples, using each commercial VWF:CB assay assessed against our standard (reference) in-house VWF:CB assay, as well as our in-house VWF antigen (VWF:Ag) and ristocetin cofactor (VWF:RCo) assays. Commercial VWF:CB assays varied in their ability to discriminate types 1 and 2A/2B VWD. The optimal VWF:CB/VWF:Ag ratio at which optimal discrimination occurred also differed between assays, with some improvements observed with some (but not all) as-says following a harmonisation process that aimed to correct for different calibrator effects. Assay variability also compromised assay utility in some test occasions. Future standardisation and improvements in some commercial VWF:CB assays are needed before the VWF:CB assay can be more fully and globally utilised for discrimination of VWD types in diagnostic laboratories.

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