Figure 4. Botrocetin-induced binding of VWF to GPIb-IX reconstituted in Nanodiscs.

(A) Botrocetin-induced VWF binding to GPIb-IX in Nanodiscs measured by ELISA. Increasing concentrations of VWF was incubated with immobilized GPIb-IX/ND (●) or empty Nanodiscs (■) or BSA (▲) in the presence of 0.2 U/ml botrocetin. The binding of VWF was determined by rabbit anti-VWF antibody and HRP-conjugated goat anti-rabbit antibody. (B) The effect of monoclonal antibodies against individual subunits of GPIb-IX complex on VWF binding. 10 μg/ml antibodies were pre-incubated with immobilized GPIb-IX/ND before 0.8 nM VWF with 0.2 U/ml botrocetin was added. The bound VWF was detected by HRP-conjugated anti-VWF antibody. Absorbance at 450 nm was measured from 3 independent experiments and presented as the mean ± SD. For some data points, their error bars are smaller than the symbols. One hundred percent in (B) is defined as the binding of VWF to GPIb-IX/ND in the absence of inhibitory antibodies after subtracting the binding to empty Nanodiscs. Groups were compared using the Student’t test. **P < 0.01 v.s. control.