Figure 3.
mAb R6H8 activates platelets via simultaneously binding αIIbβ3 and engaging FcγRIIa on nearby platelets and inhibits platelet aggregation by blocking ligand binding to integrin αIIbβ3. (A-B) 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 FITC-labeled anti–PAC-1 antibody (25 μg/mL) (A) or 200 μg/mL Alexa488-human fibrinogen with or without T6 (25 μM) (B) for another 20 minutes. Samples were then diluted and analyzed by flow cytometry. Control platelets were used to set a gate at ∼1% to 4% positive events. PAC-1 binding percentage (%) represents PAC-1 positive events inside the gate. Results are of 3 independent experiments, reported as mean ± standard deviation (SD). (C) Washed platelets (2 × 108 platelets per mL) were treated with 10 μg/mL of anti- FcγRIIa (mAb VI.3) for 20 minutes at room temperature, followed by a 20-minute treatment with the indicated dose of mAb R6H8. Platelets were then transferred to the aggregometer, and stirring was initiated. After ∼8 minutes, nonaggregated platelets were activated with 25-μM T6. Data shown are representative of at least 3 similar experiments. (D) Washed platelets (1 × 108 platelets per mL) were treated with 10 μg/mL of anti-FcγRIIa mAb IV.3 for 20 minutes at room temperature, followed by a 20-minute treatment with the indicated dose of mAb R6H8. Platelets were then treated with PE-labeled anti–P-selectin antibody for another 20 minutes. Samples were then diluted and analyzed by flow cytometry. (E) Washed platelets were diluted in HEPES-buffered modified Tyrode's solution to the indicated concentration and incubated for 20 minutes with 5 μg/mL of mAb R6H8. Platelets samples were then incubated for another 20 minutes with PE–P-selectin antibody. Control platelets were used to set a gate at ∼1% to 2% positive events. P-selecting binding percentage (%) represent positive events with fluorescence values ± SD. MFI, mean fluorescence intensity.

mAb R6H8 activates platelets via simultaneously binding αIIbβ3 and engaging FcγRIIa on nearby platelets and inhibits platelet aggregation by blocking ligand binding to integrin αIIbβ3. (A-B) 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 FITC-labeled anti–PAC-1 antibody (25 μg/mL) (A) or 200 μg/mL Alexa488-human fibrinogen with or without T6 (25 μM) (B) for another 20 minutes. Samples were then diluted and analyzed by flow cytometry. Control platelets were used to set a gate at ∼1% to 4% positive events. PAC-1 binding percentage (%) represents PAC-1 positive events inside the gate. Results are of 3 independent experiments, reported as mean ± standard deviation (SD). (C) Washed platelets (2 × 108 platelets per mL) were treated with 10 μg/mL of anti- FcγRIIa (mAb VI.3) for 20 minutes at room temperature, followed by a 20-minute treatment with the indicated dose of mAb R6H8. Platelets were then transferred to the aggregometer, and stirring was initiated. After ∼8 minutes, nonaggregated platelets were activated with 25-μM T6. Data shown are representative of at least 3 similar experiments. (D) Washed platelets (1 × 108 platelets per mL) were treated with 10 μg/mL of anti-FcγRIIa mAb IV.3 for 20 minutes at room temperature, followed by a 20-minute treatment with the indicated dose of mAb R6H8. Platelets were then treated with PE-labeled anti–P-selectin antibody for another 20 minutes. Samples were then diluted and analyzed by flow cytometry. (E) Washed platelets were diluted in HEPES-buffered modified Tyrode's solution to the indicated concentration and incubated for 20 minutes with 5 μg/mL of mAb R6H8. Platelets samples were then incubated for another 20 minutes with PE–P-selectin antibody. Control platelets were used to set a gate at ∼1% to 2% positive events. P-selecting binding percentage (%) represent positive events with fluorescence values ± SD. MFI, mean fluorescence intensity.

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