Identification of the minimal collagen sequence required for VWF binding. (A) The truncated and alanine-modified peptides derived from peptide no. 23 (Table S2) were immobilized on a 96-well plate and incubated with plasma-derived VWF (1 μg/mL), and bound VWF was detected as in Figure 1. (B) The same peptides were spray-coated onto Thermanox coverslips (0.5 μg/cm2) and perfused with whole blood at a shear rate of 300 s–1 for 5 minutes; then surface coverage was measured as described for Figure 1. (C) Peptide no. 23 and peptide GPC-(GPP)5-GPRGQOGVMGFO-(GPP)5-GPC-NH2 were immobilized onto a Biacore CM5 sensor chip via their free cysteine residues. The equilibrium binding capacity of the peptides was determined for different concentrations of plasma-derived VWF. (D) Collagen (100 μg/mL) was immobilized on a 96-well plate or a Thermanox coverslip and incubated with purified VWF (1 μg/mL) or perfused with whole blood in the presence or absence of different concentrations of peptide GPC-(GPP)5-GPRGQOGVMGFO-(GPP)5-GPC-NH2. VWF binding and platelet deposition were evaluated as in Figure 1. Shown is a representative of 3 independent experiments, each performed in duplicate (A), triplicate (B,D), or as single determinations (C). Error bars denote SD.
