Platelet adhesion to collagen type III in the presence of wtVWF and VWF glycosylation variants under high shear stress. (A) Platelets resuspended in plasma-free blood supplemented with 10 μg/mL wtVWF or VWF OLG variants were perfused over a collagen type III–coated surface at 1500 s⁻¹ for 5 minutes. Videos were processed off-line, and platelets that remained attached over the duration of 3 subsequent frames (0.15 seconds) were counted. The number of bound platelets is expressed as the percentage of surface coverage and is plotted against time after the start of perfusion. To ensure the specificity of the interaction, platelets without VWF were used. Values shown are mean ± SEM of minimum 3 independent experiments performed in duplicate. (B) wtVWF and VWF variants were perfused over collagen type III–coated surface at a shear rate of 1500 s⁻¹ for 5 minutes. Bound proteins were recovered from the slides using 2× SDS-PAGE reducing buffer and heating at 60°C for 30 minutes and then analyzed by SDS-PAGE followed by Western blotting with polyclonal anti–VWF-HRP–conjugated antibodies. (C) Intensity of the bands corresponding to VWF (∼ 260 kDa) was determined using ImageJ and expressed as a proportion of wtVWF (mean ± SEM of 3 independent experiments). (D) wtVWF and OLG variants at a concentration of 10 μg/mL were bound under static conditions to flow slides coated with collagen type III. Next, platelets resuspended in plasma-free blood were perfused over at 1500 s⁻¹ for 5 minutes, and videos were processed off-line as described in panel A. The number of bound platelets is expressed as the percentage of surface coverage after the 5 minutes from initiation of perfusion. Values shown are mean ± SEM of minimum 3 independent experiments. One-way ANOVA was used to determine differences, and the P value was calculated to be .62.