Flow cytometric analysis of the surface expression and ligand-binding function of α_IIbβ₃ Osaka-12 platelets.

Washed platelets obtained from Osaka-12 or 3 control subjects were incubated with FITC-AP1 (GPIb-specific mAb), FITC-AP2 (α_IIbβ₃-specific mAb), FITC-AP3 (β3-specific mAb), and FITC-TP80 (α_IIb-specific mAb) at a concentration of 10 μg/mL for 30 minutes at room temperature. FITC-MOPC21 (mouse myeloma IgG₁) was used as a negative control. For OP-G2 binding (activation-independent ligand-mimetic α_IIbβ₃-specific mAb), bound antibodies were detected by FITC-conjugated goat F(ab′)₂ antimouse IgG. Filled and open histograms represent the binding of specific and control antibodies, respectively. For PAC-1 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 was added. Closed and open histograms represent the PAC-1 binding in the absence and presence of 10 μM FK633 (α_IIbβ₃-specific antagonist), respectively. Results are representative of 2 separate experiments.