Soluble fibrinogen binding to mutant β₃integrins.

(A) Dot plots represent FITC-fibrinogen (horizontal) and PT25-2 (vertical) binding to α_IIbβ₃-transfected cells. The α_IIbβ₃-transfected cells were treated with 10 μg/mL PT25-2 (an α_IIbβ₃-activating antibody) for 30 minutes on ice in the presence or absence of 10 μM FK633 (an α_IIbβ₃ antagonist). After washing, cells were incubated with 150 μg/mL FITC-fibrinogen and phycoerythrin-conjugated antimouse IgG for 25 minutes at room temperature. Then, after 5 minutes incubation with propidium iodine, cells were washed and analyzed by flow cytometry. Because the expression levels of β₃ integrins were different in each mutation, we gated and analyzed cells showing the same expression levels of α_IIbβ₃ for fibrinogen binding. (B) Dot plots represent FITC-fibrinogen (horizontal) and LM142 (vertical) binding to α_vβ₃-transfected cells. The α_vβ₃-transfected cells were treated with 1 mM MnCl₂ for 30 minutes on ice in the presence or absence of 1 mM RGDW or 50 μM c(RGDfV). LM142 (10 μg/mL) was added simultaneously to the tubes to monitor expression of α_vβ₃. The following procedures were the same as described above. (C) Fibrinogen binding to α_IIbβ₃ mutants. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type α_IIbβ₃. Two-tailedP values for paired samples were obtained by the Student t test (*P < .01, **P < .05). (D) Fibrinogen binding to α_vβ₃ mutants. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type α_vβ₃. Two-tailed Pvalues for paired samples were obtained by the Student ttest (*P < .01, **P < .05).