The p.Arg127Gln variant increases the binding of VWF to GPIbα. (A) and (B) Surface expression of GPIbα in CHO β/IX cells transfected with WT, p.Arg127Gln or p.Gly249Val as assessed by flow cytometry using the LJ-P19 antibody and a FITC-conjugated goat anti-mouse IgG shown as percentage of positive cells (A) and mean fluorescence intensity (MFI) (B) (n = 10, * P < .05 vs CHO β/IX, one-way ANOVA). (C) Binding of VWF to CHO β/IX cells transfected with WT, p.Arg127Gln, or p.Gly249Val as assessed by flow cytometry using a mouse anti-human VWF antibody, clone 4f9, and a FITC-conjugated goat anti-mouse IgG after incubation with 8 μg/ml of purified human VWF and different concentrations of ristocetin for 5 minutes at 37°C (n = 10, * P < .05 vs 0 mg/ml; § P < .05 vs WT; # P < .05 vs Gly249Val; two-way ANOVA). (D) Average rolling speed of CHO β/IX cells transfected with WT, p.Arg127Gln, or p.Gly249Val on a VWF-coated surface under flow conditions assessed in a laminar-flow perfusion chamber. Cell rolling was continuously recorded using a Zeiss, ObserverZ.1 (Carl Zeiss, Jena, Germany) inverted microscope equipped with AxioCam MRm (Carl Zeiss). Images were captured at 1-second intervals for 10 minutes and then analyzed offline with the ImageJ v. 1.52 TrackMate (v5.2.0) software (n = 4, * P < .05 vs 0mg/ml; § P < .05 vs WT, # P < .05 vs p.Gly249Val, one-way ANOVA). CHO β/IX cells transfected with the empty vector did not tether to the VWF-coated surface and flowed away without rolling, as shown in supplemental Movie 4.