Identification of the von Willebrand Factor Binding Site in Collagen Using Triple Helical Peptides.
Authors: Lisman Ton , Raynal Nicholas , Groeneveld Dafna , Maddox Ben , Peachey Tony , Huizinga Eric G. , de Groot Philip G. , Farndale Richard W. 
Published: October 2019

Abstract
The interaction of the plasma protein von Willebrand factor (VWF) with subendothelial collagen initiates adhesion of blood platelets to the damaged vessel wall or ruptured atherosclerotic plaque. A detailed molecular description of the VWF-collagen interaction may facilitate development of a novel class of antithrombotic drugs that inhibits this vital step in platelet thrombus formation. We have previously used site-directed mutagenesis to map the collagen-binding site in the VWF A3 domain. Here, we report the identification of a 9-aminoacid sequence in collagen type III which mediates VWF binding. We have synthesized a set of 57 peptides, each containing 27 amino acids of native collagen sequence flanked at each end by five GPP (standard amino acid nomenclature) triplets which support the triple helical structure that is essential for ligand recognition by collagen. The sequence of each peptide overlaps by 9 amino acids with that of each adjacent peptide. A single peptide from this set (#23) was shown to bind VWF in a solid-phase binding assay. The affinity of peptide #23 for VWF was comparable to that of native collagen type III. The peptide #23-VWF interaction was abolished by a monoclonal antibody directed against the collagen-binding site on the VWF A3 domain. Furthermore, recombinant VWF variants that were previously shown to lack collagen-binding capacity (delta A3, His1023Ala) were not able to bind to the peptide. Using surface plasmon resonance, we showed that the peptide bound strongly to the isolated VWF A3 domain (Kd 300 nM). Immobilized peptide #23 also supported platelet adhesion from whole blood under flow conditions and washed platelet adhesion under static conditions. Platelet adhesion to peptide #23 could be abrogated by a monoclonal antibody directed against the VWF A3 domain, which inhibits the interaction of VWF with full-length collagen. We subsequently synthesized a set of truncated and alanine-modified triple helical peptides based on the sequence of #23, which were all tested for VWF and platelet binding from whole blood under flow conditions. Modified peptides either strongly interacted with both VWF and platelets, or lacked both VWF and platelet binding. Based on these experiments, we identified the sequence RGQOGVMGF (O is hydroxyproline) as the minimal VWF binding sequence in collagen type III. Mutation of either Q or M to alanine (A) did not affect VWF binding, whereas replacement of R, O, V, and F by A completely abolished VWF binding. Glycine residues were not replaced, as they are essential for triple helix formation. A model of the VWF A3 domain with this nonapeptide collagen sequence was constructed to give detailed insight into the VWF-collagen interaction. In conclusion, we have identified a 9 amino acid sequence in collagen type III that is entirely responsible for high affinity binding to VWF. The detailed molecular description of the VWF-collagen interaction described here may facilitate development of agents disrupting this interaction, which may have potential as antithrombotic drugs.