Flow cytometric analysis and quantitative ELISA for platelet β₃ integrins on control and Osaka-5 platelets.

(A) Expression of α_IIbβ₃. Washed platelets obtained from a control subject and patient Osaka-5 were incubated with 10 μg/mL AP1 (specific for GPIb), 10 μg/mL AP2 (specific for α_IIbβ₃ complex), 10 μg/mL AP3 (specific for β₃), or 10 μg/mL TP80 (specific for αIIb) for 30 minutes at 22°C. After washing, bound MoAbs were detected by FITC-conjugated goat F(ab′)₂ antimouse IgG. MOPC21 (mouse IgG₁) was used as a negative control (dotted line). (B) Expression of α_vβ₃. Control and Osaka-5 platelets were incubated with 5 μg/mL LM609 (specific for α_vβ₃ complex) for 30 minutes at 22°C. After washing, bound MoAbs were detected by Alexa-conjugated goat F(ab′)₂ antimouse IgG. MOPC21 (mouse IgG₁) was used as a negative control (dotted line). The amounts of bound LM609 were expressed as MFI from 11 control subjects (mean ± SD) and MFI from patient Osaka-5 (mean of duplicate). (C) The amounts of α_vβ₃ measured by quantitative ELISA; 100 μL platelet lysate (1 × 10⁶ platelets/μL) was applied to a sandwich ELISA. Standard curve was obtained using purified α_vβ₃. Data represents the mean ± SD from 11 control subjects.