Ligand binding and LIBS exposure. (A) Soluble fibrinogen (Fg) or PAC-1 binding of CHO-K1 stable transfectants in the presence of either 1 mM Ca²⁺/1 mM Mg²⁺, 1 mM Mn²⁺, 10 μg/mL PT25–2 plus 1 mM Ca²⁺/1 mM Mg²⁺, 10 μg/mL PT25–2 plus 1 mM Mn²⁺, or 1 mM DTT plus 1 mM Ca²⁺/1 mM Mg²⁺ as indicated. Binding activity was determined by flow cytometry and expressed as described in “Soluble ligand binding.” (B) Adhesion of CHO-K1 transfectants (preincubated with 10 μg/mL mAb LM609) in the presence of 1 mM Ca²⁺/1 mM Mg²⁺ or 1 mM Ca²⁺/1 mM Mg²⁺ plus 1 mM DTT to surfaces coated with fibrinogen at indicated concentrations. The amount of bound cells was determined by measuring the lactate dehydrogenase (LDH) activity as described in “Cell adhesion.” Data are representative of 3 independent experiments, each in triplicate. (C) LIBS exposure. Cells were stained with 2 anti-LIBS antibodies, D3 and LIBS1, in the presence or absence of 50 μM RGD peptides (GRGDSP) or 3 mg/mL human fibrinogen in 1 mM Ca²⁺/1 mM Mg²⁺ condition. LIBS epitope expression was expressed as the percentage of mean fluorescence intensity of anti-LIBS antibody relative to the conformational-independent anti-β3 mAb AP3. Error bars are standard deviation (SD).