Patagonfibrase, an a-fibrinogenase isolated from Philodryas patagoniensis colubrid snake venom: multiple effects on platelets and blood coagulation

MARÍA ELISA PEICHOTO; PAMELA TEIBLER; LAURA LEIVA; OFELIA ACOSTA; LUIS ROBERTO CAMARGO GONÇALVES; ANITA MITIKO TANAKA-AZEVEDO; MARCELO LARAMI SANTORO

Ma’ale Hachamisha (Israel)

Simposio; Platelets 2006 Symposium; 2006

Venoms of Colubridae snakes are a rich source of novel compounds, which may have applications in medicine and biochemistry. Patagonfibrase is a 57.5-kDa metalloproteinase isolated from Philodryas patagoniensis colubrid snake venom. Purification of this enzyme was accomplished by chromatography on FPLC/Mono-Q and ÄKTA/HiTrap Blue HP columns, and it was homogenous by SDS-PAGE and C8 reversed-phase HPLC. It possessed proteolytic activity toward azocasein, with a specific activity of 4.84 units/mg, and hydrolyzed selectively the Aα-chain of fibrinogen, leaving the Bβ- and γ-chains unaffected. When patagonfibrase was incubated with fibrinogen or plasma, the mixture was hardly clottable by thrombin. Prominent hemorrhage developed in mouse skin after intradermal injection of patagonfibrase, and its minimum hemorrhagic dose was 0.27 μg. When administered into mouse gastrocnemius muscle, patagonfibrase induced hemorrhage and necrosis at the site of injection, and systemic bleeding in lungs. Patagonfibrase showed no platelet pro-aggregating activity per se, but it inhibited collagen-induced platelet aggregation, with an IC50 of 129 nM, and exhibited 64% inhibition of ADP-induced aggregation at a final concentration of 174 nM. Thrombin-induced platelet aggregation was not inhibited by this enzyme. In conclusion, the capacity of patagonfibrase to induce hemorrhage after its injection in experimental animals likely depends on its ability to degrade various components of basement membrane, as well as fibrinogen, and to inhibit platelet aggregation. Therefore, patagonfibrase may be used as a biological tool to explore many facets of hemostasis.