Effect of the LIBS mAb AP5 on the binding of R21D10 to αIIbβ3 and vice versa. Effect of AP5 on R21D10 binding to platelets. Untreated washed platelets (A) or eptifibatide-primed platelets (B) were preincubated with 40 μg/mL AP5 or unlabeled R21D10 for 15 minutes at room temperature, and then 10 μg/mL Alexa Fluor 488–labeled R21D10 was added for 15 minutes. The binding of Alexa Fluor 488–labeled R21D10 was detected using flow cytometry. (C) Washed platelets were preincubated with 40 μg/mL R21D10 or unlabeled AP5 for 15 minutes at room temperature, 10 μg/mL Alexa Fluor 488–labeled AP5 was added for 15 minutes, and the binding of AP5 was detected by flow cytometry. (D) Washed platelets were preincubated with 1 μM eptifibatide for 15 minutes at room temperature and then 40 μg/mL R21D10 or unlabeled AP5 was added to the platelets for 15 minutes, followed by the addition of Alexa Fluor 488–labeled AP5 for another 15 minutes. (E) Washed platelets were incubated with R21D10 for 15 minutes and then 1 μM eptifibatide was added. After 15 minutes, Alexa Fluor 488–labeled AP5 was added. The binding of AP5 was then detected using flow cytometry.