Researchers trying to improve the safety and efficacy of fibrinolytic therapy have been isolating fibrinolytic enzymes from snake venoms. Two fibrinolytic enzymes, colombienases 1 and 2, with significant activity have been isolated from the venom of Bothrops colombiensis. MethodsThe colombienases were characterized for various biological activities which include hemorrhagic, fibrinogenolytic, proteolytic, hemolytic, edematogenic and cytotoxic. ResultsColombienases directly acted on fibrin by degrading fibrinogen Aα and Bβ chains without activating the fibrinolytic system (plasminogen/plasmin), additionally colombienase-2 degraded fibrinogen γ chains as well as the fibronectin molecule. Laminin and type IV collagen were colombienases resistant. Gelatin–zymogram activity was present in B. colombiensis venom (BcV) bands between 64 and 148 kDa. All activities were abolished by metalloproteinases inhibitors. Both enzymes lacked hemorrhagic, hemolytic, cytotoxic, plasminogen activator and coagulant activities. ConclusionsBoth colombienases had direct fibrino(geno)lytic activity without other toxic side effects including in vivo hemorrhaging, which could be promising in terms of therapeutic potential as thrombolytic agents.