AAPP specifically inhibits collagen-induced platelet aggregation by binding to collagen. (A) AAPP does not inhibit GPVI agonist-mediated platelet aggregation. PRP was incubated with the indicated concentrations of AAPP for 2 minutes followed by the addition of collagen (2 μg/mL). In the presence of 1000 nM rAAPP, PRPs were stimulated by CRP (0.5 μg/mL) or convulxin (10 ng/mL). As a control, the indicated concentrations of rAAPP were incubated in the absence of CRP or convulxin. Tracings are representative results of a typical experiment. (B) Dose-dependent inhibition of collagen-induced platelet aggregation by AAPP. PRPs were incubated with various amounts of rAAPP for 2 minutes followed by the addition of collagen. Results are means plus or minus SEM from 3 independent experiments. (C) Binding of AAPP to immobilized collagen. Various concentrations of rAAPP or rTrx were incubated in 96-well collagen-coated plates for 1 hour. After washing, bound proteins were detected using the Nickel-HRP. (D) Effect of AAPP on platelet adhesion to collagen. Washed platelet suspension and the indicated amounts of rAAPP were added to 96-well collagen-coated plates and incubated for 1 hour. After washing, adherent platelets were detected by the Dc protein assay kit. Results are means plus or minus SEM from 3 independent experiments. (E) GPVI binds to collagen but not AAPP. The indicated amounts of collagen or rAAPP were coated in 96-well plates. GPVI-expressing Jurkat cells were added to each well and incubated for 1 hour. After washing, adhesion cells were measured by the BCA protein assay. Results are representative of 3 independent experiments and expressed as the mean of tripli-cate reading plus or minus SEM for the indicated concentrations.