R6H8 demonstrates effects on platelet aggregation that vary based on concentration and whether the intact R6H8 IgG or fragments of R6H8 are tested. (A) Washed platelets (2 × 108 platelets per mL) were treated with the indicated dose of the mAb for 20 minutes at room temperature. Stirring at 37°C was initiated in an aggregometer, and changes in light transmission were continuously measured. After 5 minutes, nonaggregated platelets were activated with 25-μM T6. Data shown are representative of at least 3 experiments. (B-C) Neither mAb R6H8 F(ab')2 nor Fab induced agonist-independent platelet aggregation, but they did block T6-induced platelet aggregation in a concentration-dependent fashion. Washed platelets (2 × 108 platelets per mL) were treated with the indicated dose of R6H8 F(ab')2 (B) or Fab (C) for 20 minutes at room temperature. Platelets were then transferred to an aggregometer and stirring at 37°C was initiated. After ∼5 minutes, platelets were activated with 25-μM T6. Data shown are representative of at least 3 experiments. (D) Washed platelets in HEPES-buffered modified Tyrode's solution (1 × 108 platelets per mL) were treated with the indicated concentrations of R6H8 for 20 minutes at room temperature and then incubated with PE-labeled anti–P-selectin antibody for another 20 minutes. Samples were then diluted and analyzed by flow cytometry. Control platelets were used to set a gate at ∼1% to 2% positive events. P-selectin binding percentage (%) represents positive events. (E) Washed platelets were incubated with 20 μg/mL R6H8, 40 μg/mL R6H8-Fab, or 1-μM eptifibatide for 15 minutes at 22°C, and then 10 μg/mL Alexa488-labeled AP5 was added for 15 minutes and the binding of AP5 was detected by flow cytometry. FITC-H, fluorescence.
