Different binding sites for GpIbα and AU/VWFa-11. (A-C) Whole blood was perfused over coverslips coated with collagen type III (30 μg/cm2 in 0.05 M/L acetic acid) in the absence (A) or presence (B) of the control nanobody (125 nM) or AU/VWFa-11 (625 nM, C) at a shear rate of 1600 s–1 (D-E) Reconstituted blood was perfused over coverslips coated with pd VWF (15 μg/mL) in the absence (D) or presence of 625 nM AU/VWFa-11 (E) at a shear rate of 1600 s–1. After perfusion, adhered platelets were fixed in 0.5% glutaraldehyde in PBS, dehydrated in methanol, and stained with May-Grünwald-Giemsa. Platelet aggregates are represented by the dark regions. (F) Platelet adhesion was evaluated using computer-assisted analysis and was expressed as the percentage of surface covered with platelets (n = 3). Adhered platelets were visualized using light microscopy (Leitz Diaplan; Leica, Rijswijk, the Netherlands) and computer-assisted analysis (AMS 40-10; Saffron, Walden, United Kingdom). Original magnification was 400 × (40 ×/1.00 NA objective lens).
