Purification of patagonstatin, an inhibitor of collagen-induced platelet aggregation, from Philodryas patagoniensis snake venom

MARÍA ELISA PEICHOTO; LAURA LEIVA; OFELIA ACOSTA; ANITA MITIKO TANAKA-AZEVEDO; MARCELO LARAMI SANTORO

San Pablo (Brasil)

Otro; VIII Annual Scientific Meeting of Instituto Butantan; 2006

Instituto Butantan

Introduction: Venoms of Colubridae snakes are a rich source of novel compounds, which may have applications in medicine and biochemistry. Objective: In the present study we isolated a potent inhibitor of collagen-induced platelet aggregation from Philodryas patagoniensis venom Methodology and Results: Patagonstatin is a single-chain protein, with a molecular mass of 25.3 kDa under non-reducing conditions and 27.4 under reducing conditions. Purification of this protein was accomplished by chromatography on Mono-Q and Phenyl-Sepharose columns. Patagonstatin was homogenous by SDS-PAGE and hydrolyzed neither azocasein nor fibrinogen. Mild hemorrhage developed in mouse skin after i.d. injection of patagonstatin (20 µg of protein produced a hemorrhagic spot of 2.40 mm in diameter). Subcutaneous injection (20 µg) of patagonstatin induced edema, which peaked at 2 h. Patagonstatin showed no platelet pro-aggregating activity per se, but it inhibited collagen-induced platelet aggregation, with an IC50 of 9.1 nM. Ristocetin- and ADP-induced platelet aggregations were not inhibited by patagonstatin. Discussion: This is the first report on the isolation and characterization of a platelet aggregation inhibitor from the venom of a colubrid snake. Due to its potent action on collagen-induced platelet aggregation, patagonstatin may be used on studies of platelet physiology.