VWF-GpIbα interaction is unaffected by AU/VWFa-11 in static adhesion. (A) Microtiter wells coated with 5 μg/mL AU/VWFa-11 (•) or control nanobody (○) were blocked 30 minutes at 37°C with PBS/3% BSA/0.1% Tween-20 and incubated with 3.7 nM VWF/R1306Q. After washing, wells were incubated with GpIbα (0.12-31.1 nM) and subsequently with a monoclonal anti-GpIb antibody. Wells were washed and incubated with an HRP-conjugated rabbit anti–mouse antibody. Binding was detected by measuring the peroxidase activity. (B) Microtiter wells were coated with wt VWF (37 nM in 50 mM NaHCO3 buffer), overnight at 4°C. After blocking wells 1 hour at room temperature with 0.5% polyvinylpyrrolidone in PBS, wells were incubated with AU/VWFa-11 or control nanobody (1.25 μM in PBS, 1 hour, room temperature). After washing with PBS, CHO cells expressing the GpIb/IX/V complex (1 × 105 cells in DMEM containing 0.1% BSA) were allowed to bind in the presence or absence of the control nanobody or AU/VWFa-11 (1.25 μM). Binding of these cells was monitored by measuring the intrinsic alkaline phosphatase activity of the cells and set to be 100% in the absence of nanobodies. Data represent the mean ± SEM of 3 experiments.
