Effects of β₃-activating mutant (T562N) on fibrinogen binding.

WT (▪) or Y178Aα_v (■) construct was transiently cotransfected with WT or T562Nβ₃ construct into 293 cells. Fibrinogen binding to transfected α_vβ₃ was determined by flow cytometry. Cells were first incubated with 5 μg/mL LM142 (specific for α_v) for 30 minutes on ice. After washing, cells were incubated with 150 μg/mL FITC-conjugated fibrinogen and PE-conjugated antimouse IgG for 25 minutes at 22°C in the presence or absence of 1 mM RGDW and then incubated with PI for 5 minutes at 22°C. After washing, cells expressing high levels of transfected α_vβ₃ were analyzed. In these experiments, all were performed in the absence of MnCl₂. The results are representative of 3 separate experiments.