Cell adhesion to immobilized fibrinogen.

Transfected CHO cells expressing different forms of GPIIb-IIIa complexes were labeled with calcein-am at 37°C for 30 minutes. After washing, the cells were allowed to attach at 37°C for 60 minutes to wells coated with the indicated concentrations of fibrinogen, or BSA as the control. Nonadherent cells were removed by washing; the adherent cells were examined by phase microscopy and quantified with a fluorescence plate reader. To calculate the percentage of the bound cells, nonspecific cell adhesion on BSA-coated wells has been subtracted. (A) Phase contrast microscopic analysis of cell adhesion to immobilized fibrinogen. The right panel showed that CHO cells expressing either of GPIIb-IIIa forms bound normally to immobilized fibrinogen coated at high concentration (10 μg/mL). The left panel showed that only CHO cells expressing either GPIIb-Ala5IIIa or GPIIb-Ala435IIIa complex bound wells coated with low concentration of immobilized fibrinogen (2.5 μg/mL), and CHO cells expressing WT GPIIb-IIIa failed to adhere to immobilized fibrinogen at this low concentration. Magnification × 20. (B) Quantitative analysis of the cell adhesion. CHO cells expressing WT GPIIb-IIIa, GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa bound to immobilized fibrinogen in a dose-dependent manner, whereas the nontransfected CHO cells failed to adhere to immobilized fibrinogen. (C) Cell adhesion to immobilized fibrinogen (2.5 μg/mL) performed in the presence of LIBS antibodies. CHO cells were pretreated with LIBS antibodies D3, AP5, or 7G2 (40 μg/mL). After washing, cell adhesion was performed as described above.