β₂ GPI interacts with the GpIb-binding conformation of the A1 domain of VWF. (A) β₂ GPI (10 μg/mL) was immobilized onto microtiter wells (1 h at 37°C) and incubated with different concentrations of biotinylated VWF/R1306Q (▴; 0-10 μg/mL) or biotinylated wt-VWF (0-10 μg/mL) in the absence (○) or presence (●) of ristocetin (1 mg/mL). Bound VWF was detected with HRP-conjugated streptavidin. (B) Wells coated with β₂ GPI were incubated with 20 μg/mL wt-VWF, VWF/delta-A1, VWF/delta-A2, or VWF/delta-A3. Bound VWF was detected using HRP-conjugated anti-VWF. **P = .003. (C) Immobilized β₂ GPI was incubated with different concentrations of biotinylated VWF/A1 (0-10 μg/mL; ○) or VWF/A1-R1306Q (0-10 μg/mL; ●). Bound VWF was monitored with streptavidin-HRP. (D) β₂ GPI-coated wells were incubated with biotinylated VWF/A1-R1306Q (1.1 μg/mL) in the presence of different concentrations of plasma-derived VWF (0-100 μg/mL) without (○) or with (●) ristocetin (1 mg/mL). Bound VWF/A1-R1306Q was monitored using HRP-conjugated streptavidin. All data represent the mean (± SD) of 3 experiments.