Ligand-binding function of α_vβ₃ mutants.

(A) The binding of soluble fibrinogen and a ligand-mimetic mAb, WOW-1 Fab, to WT or mutant α_vβ₃ were examined in the presence or absence of 0.5 mM MnCl₂ by flow cytometry. For fibrinogen binding, washed cells were first incubated with 5 μg/mL mAb LM142 (specific for α_v) for 30 minutes on ice. After washing, cells with 0.5 mM MnCl₂ were incubated with 150 μg/mL FITC-conjugated fibrinogen and PE-conjugated antimouse IgG (1:5 dilution) for 25 minutes at 22°C and then incubated with PI for 5 minutes at 22°C. After washing, fibrinogen binding (FL1) was analyzed on the gated subset of single, high α_vβ₃ expression (FL2) and live cells (PI-negative, FL3) as indicated. (B) Relative amounts of fibrinogen binding are normalized to a 100% value for the binding to cells expressing WT α_vβ₃ (% of WT). Fibrinogen binding in the presence of 1 mM RGDW was used as a negative control. Data represent the mean ± SE of 3 experiments. (C) WOW-1 Fab binding. For WOW-1 binding, cells with 0.5 mM MnCl₂ were first incubated with 5 μg/ml WOW-1 Fab for 30 minutes at 22°C. After washing, cells were incubated with 5 μg/mL Alexa-conjugated antimouse IgG for 25 minutes on ice and then incubated with PI for 5 minutes at 22°C. After washing, bound antibodies were analyzed. WOW-1 binding to 293 cells was used as a negative control. Relative amounts of WOW-1 Fab binding are expressed by the following formula: % binding of WOW-1 Fab to WT α_vβ₃/% binding of LM609 to WT α_vβ₃. The α_vα_IIb mutant represents a chimera in which the C142-C155 loop in α_v was swapped with the corresponding sequence of α_IIb C146-C167. Data represent the mean ± SE of 3 experiments.