FIGURE 4.

Large ligands bind less well to clamped αIIbβ3 than normal αIIbβ3; Mn²⁺ and the β3 N339S mutation result in spontaneous ligand binding. A and B, cells expressing either clamped or normal αIIbβ3 were untreated or treated with DTT (5 mm) and incubated with either Alexa488-fibrinogen (200 μg/ml) or FITC-PAC-1 (5 μg/ml) in the presence or absence of activating antibody PT25-2. Nonspecific binding was determined in the presence of the αIIbβ3 inhibitor eptifibatide. Binding was assessed via flow cytometry and expressed as NNFI, in which the geometric mean fluorescence intensity after subtracting nonspecific binding is divided by the relative surface receptor expression as judged by the binding of mAb 10E5. *, p < 0.001 versus normal β3, n = 3; †, p < 0.001 versus normal β3, n = 4. C, cells expressing either clamped or normal αIIbβ3 were incubated with fluorescent fibrinogen in the presence of either Ca²⁺/Mg²⁺ or Mn²⁺. Nonspecific binding was determined in the presence of the αIIbβ3 inhibitor mAb 7E3. Binding was assessed via flow cytometry and expressed as NNFI (calculated as described above) (n = 4).