Sequence alignment between VWF and peptides inhibiting the vWF-collagen interaction does not result in the identification of a collagen-binding site in VWF

We previously found that two peptides (N- and Q-peptide) selected by phage display for binding to an anti-vWF antibody, were able to inhibit vWF-bindi ng to collagen (1). The sequence of those peptides could be aligned with th e sequence in VWF at position 1129-1136 just outside the A3-domain. As the peptides represent an epitope or mimotope of vWF for binding to collagen we next wanted to study whether the alignment resulted in the identification of a new collagen binding site in vWF, We mutated the 1129-1136 VWTLPDQC se quence in VWF to VATAPAAC. Expressing this mutant VWF (7.8-vWF) in a fur-BH K cell line resulted in well processed 7.8-vWF containing a normal distribu tion of molecular weight multimers. However, binding studies of this mutant VWF to rat tail, human and calf skin collagens type I, to human collagen t ypes III and VI, revealed no decrease in vWF-binding to any of these collag ens. Thus, although the N- and Q-peptides did inhibit the vWF-collagen inte raction, the resulting alignment with the VWF sequence did not identify a c ollagen binding site, pointing out that alignments (although with a high pe rcentage of identity) do not always result in identification of binding epi topes. However, suprisingly removal of the A3-domain or changing the VWF se quence at position 1129-1136 resulted in an increase of vWF-binding to huma n collagen type VI and to rat tail collagen type I, implying that these cha nges result in a different conformation of VWF with an increased binding to these collagens as a consequence.