Assessment of affinity state of _IIbβ₃ by PAC1 (A) and fibrinogen (B) binding. In (A), 24/12 cells (a through c) or AM-1 cells (d through f) were preincubated with 10 μmol/L FK633 (a and d), 10 μg/mL PT25-2 (c and f), or buffer (b and e) for 30 minutes on ice. Then, 250× diluted PAC1 ascites were added and incubated for another 30 minutes at room temperature. After washing, cells were incubated with FITC-conjugated antimouse IgM for 25 minutes on ice. To exclude dead cells, PI were added to the cells and incubated for 5 minutes. After washing, cells were resuspended in buffer and flow cytometric analysis was performed. In (B), cells were preincubated with 10 μmol/L FK633 (dotted lines), 10 μg/mL PT25-2 (solid lines), or buffer (bold lines) for 30 minutes on ice. Cells were then incubated with 150 μg/mL of FITC-labeled fibrinogen for 25 minutes at room temperature and then with PI for 5 minutes. After washing, flow cytometric analysis was performed.