Assessment of VWF-GPIIb/IIIa and GPIbα binding under static conditions. (A) rVWF variants were incubated in microtiter wells with immunoadsorbed GPIIb/IIIa purified from platelets. Bound VWF was detected using an HRP-conjugated anti-VWF antibody. Presented is the ratio relative to Phe2561. (B) Immunoadsorbed rVWF variants were incubated with HEK293 cells stably expressing a constitutively active mutant of GPIIb/IIIa. Bound cells were detected using mouse anti-GPIIb/IIIa and an HRP-coupled secondary antibody polyclonal goat anti-mouse/HRP. (C) rVWF variants were incubated in microtiter wells with immunoadsorbed recombinant wild-type GPIbα fragment (amino acids 1-285) at indicated Ristocetin concentrations. Detection of bound VWF was performed employing a rabbit anti-human VWF antibody and an HRP-coupled secondary antibody polyclonal goat anti-rabbit/HRP. The data (A-C) represent the mean values ±SD of 3 independent measurements. Unpaired t-test, *P < .05; n.s., not significant.