AAP, a tripeptide that inhibited rabbit platelet aggregation, was isolated from Agkistrodon acutus venom by ion-exchange, gel filtration and reverse-phase chromatography. Amino acid sequences which determined mainly by amino acid analyses and NMR spectroscopy indicated it was a tripeptide including pyroglutamic acid, asparagine and tryptophane residues. The ESMS experiment assigned a molecular weight of 429 Da. AAP inhibited rabbit platelet aggregation induced by ADP, PAF-acether, collagen and thrombin, the IC(50)s were 178 microM, 332 microM, 179 microM and 203 microM, respectively. AAP also inhibited thrombus formation in vivo thrombosis model and prevented the combination between fibrinogen and GP IIb/IIIa. Besides, AAP was not toxic after intravenous injection into mice at a higher dose. Those studies might be helpful to delineate unknown mechanisms involved in platelet aggregation and serve as a model for developing antithrombotic agents.