A 49-residue protein, echistatin, which inhibits platelet aggregation, was purified from the venom of the saw-scaled viper Echis carinatus. The purification procedure included gel filtration on Sephadex G-50, cation-exchange chromatography on Mono S, and C18 reverse-phase high pressure liquid chromatography. The purified protein was homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, reverse-phase high pressure liquid chromatography, and NH2-terminal sequence analysis. Echistatin is a single-chain polypeptide with a molecular weight of 5400 and a native isoelectric point of 8.3. The most abundant amino acid, cysteine, accounts for 8 of the 49 residues in the protein. A 10-residue segment of echistatin shows 90% identity to a portion of the sequence of trigramin, a platelet aggregation inhibitor from the green tree viper Trimereserus gramineus (Huang, T.-F., Holt, J. C., Lukasiewicz, H., and Niewiarowski, S. (1987) J. Biol. Chem. 262, 16157-16163). Echistatin contains the sequence arginine-glycine-aspartic acid, which is common to proteins which bind to the glycoprotein IIb/IIIa complex. It also contains the sequence proline-arginine-asparagine-proline, which is found in the A alpha chain of human fibrinogen at position 267-270. The purified protein inhibits fibrinogen-dependent platelet aggregation initiated by ADP with an IC50 of 3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of echistatin abolished its inhibitory activity.