title: Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor.
abstract: Investigation 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.
authors.affiliation: Katharine Dormandy Haemophilia Centre and Thrombosis Unit, The Royal Free and University College Medical School, London, United Kingdom.
authors.forename: Anne F
authors.initials: AF
authors.lastname: Riddell
authors.forename: Keith
authors.initials: K
authors.lastname: Gomez
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Millar
authors.forename: Gillian
authors.initials: G
authors.lastname: Mellars
authors.forename: Saher
authors.initials: S
authors.lastname: Gill
authors.forename: Simon A
authors.initials: SA
authors.lastname: Brown
authors.forename: Megan
authors.initials: M
authors.lastname: Sutherland
authors.forename: Mike A
authors.initials: MA
authors.lastname: Laffan
authors.forename: Thomas A J
authors.initials: TA
authors.lastname: McKinnon
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism*; D005190:Family; D005260:Female; D015870:Gene Expression; D006470:Hemorrhage / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000191:economics / Q000378:metabolism; D014842:von Willebrand Diseases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II; D011994:Recombinant Proteins; D014841:von Willebrand Factor