Compound 1 inhibits fibrinogen binding to purified αIIbβ3; priming with eptifibatide or RGDS peptide, but not compound 1, enhances fibrinogen binding. (A) Purified αIIbβ3 was allowed to adhere to wells coated with the anti-β3 mAb 7H2 for 2 hours at 37°C. Fibrinogen binding to αIIbβ3 took place in the presence of buffer alone; DMSO (1%); compound 1 (Cmpd 1, 100 μM); an analog of compound 1 in which the piperazine group is disrupted (100 μM); tirofiban (10 μM); or mAb 10E5 (20 μg/mL), with or without AP5 (60 μg/mL) for 2 hours at 37°C. The extent of fibrinogen binding was determined using HRP-conjugated antifibrinogen polyclonal antibodies and a peroxidase substrate as described in “Fibrinogen binding to purified αIIbβ3.” (B) For priming experiments, the same procedure was followed with the following exceptions: priming agents were added along with αIIbβ3, and wells were washed 10 times following fibrinogen addition. Binding is presented as mean plus or minus SD for 4 separate experiments, calculated as the percentage of “control” (fibrinogen binding in the presence of buffer alone without AP5) (*P < .001, †P < .02 both vs control).