Development of a collagen-binding activity assay as a screening test for type II von Willebrand disease in dogs

2006 ◽  
Vol 228 (4) ◽  
pp. 567-567
Author(s):  
Elizabeth Peet Sabino ◽  
Hollis N. Erb ◽  
James L. Catalfamo
Keyword(s):  
Screening Test ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Type Ii ◽  
Activity Assay ◽  
Collagen Binding ◽  
Von Willebrand

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 >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.

Prospective study of low-dose ristocetin-induced platelet aggregation to identify type 2B von Willebrand disease (VWD) and platelet-type VWD in children

2010 ◽  
Vol 104 (12) ◽  
pp. 1158-1165 ◽  
Author(s):  
Juan Pablo Frontroth ◽  
Mirta Hepner ◽  
Gabriela Sciuccati ◽  
Aurora Feliú Torres ◽  
Graciela Pieroni ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Low Dose ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Bleeding Disorders ◽  
Phenotypic Data ◽  
Collagen Binding ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Ristocetin Cofactor

SummaryType 2B von Willebrand disease (VWD2B) and platelet-type von Wille-brand disease (PT-VWD) are rare bleeding disorders characterised by an increased ristocetin-induced platelet aggregation (RIPA) at low dose of ristocetin. It was the objective of this study to detect children with VWD2B and PT-VWD using RIPA at low dose of ristocetin (0.5 mg/ml) in the screening evaluation of bleeding disorders, and to analyse the phenotypic data along with the molecular findings. Over a 14-year period, 641 children with personal and family bleeding symptoms or bleeding from birth with previously uncharacterised haemostatic disorders were prospectively studied. Six unrelated patients (0.93%) showed RIPA at low dose of ristocetin. RIPA-based mixing studies identified that the plasma of the six probands and at least one parent from five unrelated families induced aggregation of normal platelets with the addition of low-dose ristocetin. None of the probands’ platelets showed aggre-gation with cryoprecipitate. Low ristocetin cofactor activity/VWF antigen ratio with absent collagen binding activity or thrombocytopenia were detected respectively in only two patients. Molecular analysis of exon 28 of the VWF gene identified mutations in only three patients. No mutation in the GP1BA gene was found. In this large prospective paediatric study, the screening approach including RIPA at low dose of ristocetin permitted the detection of patients with VWD2B that would otherwise have been missed. No patient with phenotype or genotype of PT-VWD was identified. Heterogeneity of bleeding symptoms and phenotypic parameters were found among members of the same family.

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.

Laboratory diagnosis of von Willebrand disease

2010 ◽  
Vol 30 (04) ◽  
pp. 203-206 ◽  
Author(s):  
R. Schneppenheim ◽  
J. Patzke
Keyword(s):  
Laboratory Diagnosis ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Activity Assay ◽  
Binding Assays ◽  
New Type ◽  
Von Willebrand ◽  
Made In

SummaryOver the last decade, considerable progress has been made in the laboratory diagnosis of VWD. Precise, sensitive and automated VWF : Ag assays became widely available. The VWF : RCo performance was improved to a certain degree. However, the sensitivity, precision and general availability of automated applications is not yet optimal. Nevertheless, this type of assay is still recognized as superior to other activity assays, e. g. VWF : CBA assays and antibody-binding “activity” assays, for the detection of defects in VWF function.A decision limit of either 30 or 40 IU dl-1 VWF (VWF:RCo or VWF:Ag) is recommended for a diagnosis of type 1 VWD. Type 2 VWD can be differentiated from type 1 by calculating the VWF:RCo/VWF:Ag ratio.Improved and easier to perform multimer analysis and genetic testing are beginning to facilitate the diagnosis of the VWD type 1, 2A, 2B, 2N, 2M or 3. Within type 1 or 2, a decreased VWF survival can be detected by the VWFpp assay and its ratio to VWF : Ag.A new type of VWF activity assay, based on the binding of VWF to a GPIb〈-fragment, has been developed. One assay variant does not need ristocetin as a cofactor anymore. The performance investigations presented so far are very promising. It is probable that these GPIb〈-binding assays will detect functional VWF defects as the VWF : RCo assay, but are much more sensitive and precise. Fully automated applications on routine analyzers are expected to be commercialized soon.

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.

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