Effect of ristocetin, botrocetin, and the R1306Q mutation on binding to AU/VWFa-11. (A) Microtiter wells were coated overnight at 37°C with AU/VWFa-11 (5 μg/mL in 50 mM NaHCO3, pH 9.6) and blocked 30 minutes at 37°C with 3% BSA, 0.1% Tween-20 in PBS. After washing, microtiter wells were incubated with medium containing different concentrations of wt VWF (squares, triangles) or VWF/R1306Q (circles; 0-3.7 nM). Binding was allowed for 1 hour at 37°C in the absence of modulators (▪, •), or in the presence of 1 mg/mL ristocetin (□, •) or 0.2 U/mL botrocetin (▴). Microtiter wells were washed using 0.1% Tween-20 in PBS and incubated with HRP-conjugated polyclonal anti-VWF antibody. Bound VWF was detected by measuring peroxidase activity. An enlargement of the linear part of the binding curves is provided in the inset. Data represent the mean ± SD of 3 experiments. (B) AU/VWFa-11–coated microtiter wells were incubated with wt VWF in the presence or absence of various concentrations of ristocetin (0.08-1 mg/mL). After washing, wells were incubated with an HRP-conjugated polyclonal anti-VWF antibody. Bound VWF was detected by measuring the peroxidase activity.
