Effects of Tyr143Hisα_IIb, Tyr143Alaα_IIb, Asp163Alaα_IIb, or KO mutant on the expression and ligand-binding function of α_IIbβ₃ on 293 cells.

Wild-type α_IIb or each mutant α_IIb cDNA was transiently cotransfected with wild-type β₃ cDNA into 293 cells. CD36 expression vector was cotransfected with α_IIband β₃ constructs, and CD36 expression was measured by FITC–anti-CD36 mAb to monitor transfection efficiency. The binding of FITC-AP2, FITC-AP3, FITC-TP80, OP-G2, and PT25-2 was analyzed by flow cytometry 2 days after transfection (filled histograms). FITC-MOPC21 was used as a negative control (open histograms). For OP-G2 or PT25-2 binding, bound antibodies were detected by FITC-conjugated goat F(ab′)₂ antimouse IgG. For PAC-1 or fibrinogen binding, washed platelets were preincubated with 10 μg/mL PT25-2 (activating α_IIbβ₃-specific mAb) for 30 minutes, and then 10 μg/mL FITC-labeled PAC-1 or 150 μg/mL FITC-labeled fibrinogen was added. Closed and open histograms represent PAC-1 or fibrinogen binding in the absence and presence of 10 μM FK633 (α_IIbβ₃-specific antagonist), respectively. Results are representative of 3 separate experiments.