The association of either Ala5GPIIIa or Ala435GPIIIa with GPIIb and the cell surface expression of GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes.

(A) Immunoprecipitation analysis of the association of Ala435GPIIIa with GPIIb. Nontransfected CHO cells (mock) and transfected CHO cells expressing WT GPIIb-IIIa (WT) or GPIIb-Ala435IIIa (Ala435) were surface-labeled with biotin, and solubilized in detergent lysis buffer (see “Materials and methods”). The recombinant cell surface proteins were immunoprecipitated with mAbs specific either for GPIIIa subunit (AP3, left), or for the GPIIb-IIIa complex (AP2, right). (B) Flow cytometric analysis of GPIIb-Ala5IIIa and GPIIb-Ala435IIIa expressed on the cell surface. Nontransfected (left) and transfected CHO cells were stained with mAbs specific for GPIIIa (AP3, lower), GPIIb-IIIa complex (AP2, upper), or normal mouse IgG (NMIgG) as negative control at 40 μg/mL, and then subjected to flow cytometric analysis using a FACScan. These 2 antibodies specifically and positively stained CHO cells expressing WT GPIIb-IIIa (left), GPIIb-Ala5IIIa (middle), and GPIIb-Ala435IIIa (right).