Adhesion ability of transfectants to immobilized fibrinogen.

(A) Binding of transfected CHO cells to immobilized fibrinogen. Transfected cells expressing Pro₃₃ (solid line) and Pro₃₃ΔLys₆₁₁ (broken line) of GPIIIa were labeled with calcein-am. Labeled cells were allowed to adhere to microtiter wells coated with fibrinogen, BSA, or mAb Gi5 specific for GPIIb-IIIa complex. After washing, adherent cells were measured in fluorescence microtiter plate reader. Nonspecific cell adhesion on BSA was subtracted. Cell adhesion was normalized to cell binding on Gi5 (Fg/Gi5 ratio). (B) Tyrosine phosphorylation of pp125^FAK following cell adhesion to immobilized fibrinogen. Stable transfectants expressing GPIIb-IIIa Pro₃₃ (lanes 1) and GPIIb-IIIa Pro₃₃Δ Lys₆₁₁ (lanes 2) were allowed to bind fibrinogen-coated wells. Adherent cells were lysed and precipitated with rabbit polyclonal anti-pp125^FAK. Immunoprecipitates were separated on 7.5% SDS-PAGE, blotted, and probed subsequently with mAbs against phosphotyrosine (pTyr) and pp125^FAK and visualized using peroxidase-labeled secondary antibody and chemiluminescence substrate.