Identification of a single peptide containing 27 amino acids of collagen III sequence that specifically binds VWF. (A) The peptides (10 μg/mL) of the Collagen III Toolkit (Table S1) were each immobilized on a 96-well plate, and the adhesion of plasma-derived VWF (1 μg/mL) was determined. (B) Immobilized peptide no. 23 was incubated with plasma-derived VWF (1 μg/mL) in presence or absence of monoclonal antibody RU5 (1 μg/mL), or with recombinant wild-type, delta-A3, or His1023Ala VWF (all at 1 μg/mL). In panels A-B, bound VWF was detected as described in “Solid-phase binding assays,” and the mean ± SD is shown from a representative of 3 independent experiments, each performed in duplicate. (C) Immobilized peptide no. 23 or immobilized collagen III (100 μg/mL) was incubated with increasing concentrations of VWF, detected as in panel A. (D) Peptide no. 23 or vehicle was coated in a 96-well plate (10 μg/mL) or sprayed onto a coverslip (0.5 μg/cm2) and incubated with washed platelets or perfused with whole blood for 5 minutes at a shear rate of 300 s–1 in presence or absence of RU5 (1 μg/mL). Uncoated wells or coverslips served as controls. The adhesion of platelets bound in the static assay (▪) and the surface coverage of platelets (□) were each determined as described in “Static platelet-binding assay” and “Solid-phase binding assays.” Shown is the mean ± SD of 3 independent experiments each performed in triplicate.
