title: Binding of factor VIII to von willebrand factor is enabled by cleavage of the von Willebrand factor propeptide and enhanced by formation of disulfide-linked multimers.
abstract: von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.
authors.affiliation: Brockton/West Roxbury VAMC, West Roxbury, MA, USA.
authors.forename: A V
authors.initials: AV
authors.lastname: Bendetowicz
authors.forename: J A
authors.initials: JA
authors.lastname: Morris
authors.forename: R J
authors.initials: RJ
authors.lastname: Wise
authors.forename: G E
authors.initials: GE
authors.lastname: Gilbert
authors.forename: R J
authors.initials: RJ
authors.lastname: Kaufman
mesh_terms: D001665:Binding Sites; D019281:Dimerization; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D017510:Protein Folding; D013440:Sulfides; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
chemical_list: D013440:Sulfides; D014841:von Willebrand Factor; D005169:Factor VIII