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A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.

Incubation of human blood in saline solution of 0-36% (v/v) ethanol for 30 min produces lysis or stabilization of erythrocytes depending on the ethanol concentration. Under less elevated concentrations of ethanol, erythrocytes are present in expanded shapes (R state) that present lower stability and suffer lysis with increase in the ethanol concentration. Under more elevated concentrations of ethanol, erythrocytes are present in contracted shapes (T state) that have higher stability and suffer lysis at even more elevated ethanol concentrations. This work evaluated the effects of glycerol (0 to 2.0 M) and temperature (7 to 47 degrees C) on the stability of the R erythrocytes, characterized by the ethanol concentration at the mid-transition point (D (50R )) of the hemolysis curve (D (50R )). D (50R ) declined sigmoidally with increase in the glycerol concentration or temperature, due to transition of the R to the T state erythrocytes. In 1.5 M glycerol, the erythrocytes stability decreased below 32 but increased above 37 degrees C. The combination of temperature, glycerol and ethanol actions generates a critical value of osmotic pressure below which the R state predominates and above which the T state predominates. At 7 degrees C 1.5 M glycerol decreased the erythrocytes stability against ethanol but increased the erythrocytes stability against hypotonic shock. Those conditions favor the R state, which has a lower stability against ethanol; however, in the absence of ethanol, glycerol determines less water entrance in the erythrocytes, making more difficult its lysis by hypotonicity.

This work describes classification, functions, location, inhibition, activation, and therapeutic applications of proteases from snake venoms and vegetables. Snake venoms and vegetables can present toxins that unchain necrosis or proteolysis due to the direct cytotoxic action of venom proteases. These proteases are potential tools in the development of drugs for the prevention and treatment of several illnesses. We report herein mainly fibrinogenolytic metallo proteases and serine proteases ("thrombin-like"). These enzymes are extensively used in the treatment and prevention of thrombotic disorders, since they serve as defibrinogenating agents. The therapeutic uses of fibrin(ogen)olytic metallo proteases hold promise for clinical application due to potential in reversing the effects of thrombosis; this has been shown to be an alternative approach to the prevention and treatment of cardiovascular disorders, which are among the most prominent causes of mortality around the world. Plant proteases can be utilized for many cellular and molecular activities, in antibacterial and anticancer therapies, and in the treatment of snakebites, inhibiting snake venom activities such as blood-clotting, defibrinogenation, and fibrin(ogen)olytic and hemorrhagic actions. These toxins also display potential for clinical use in the treatment of hemostatic disorders.