Flow cytometric analysis of the conformational change of the surface-expressed GPIIb-Ala5IIIa or GPIIb-Ala435IIIa.

(A) The binding of LIBS antibodies AP5 and D3 to the WT GPIIb-IIIa, GPIIb-Ala5IIIa, and GPIIb-Ala435IIIa complexes. Transfected CHO cells were incubated with AP5 or D3, followed by FITC-conjugated goat-anti-mouse IgG. Preimmune normal mouse IgG (NMIgG) was used to establish background binding. (B) Activation index of WT GPIIb-IIIa, GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa with different LIBS antibodies. LIBS antibody binding was expressed as an activation index by standardizing the mean fluorescent intensity (MFI) for each LIBS mAb to that obtained using AP2 whose binding was not affected by the mutations. Activation index = LIBS mAb MFI/AP2 MFI for the patient and for the control separately. Note that the epitope of CRC54 is located within the first 100 N-terminal residues of GPIIIa,28 and to date no information on the epitope of 7C7 is available.