Batroxase, a new metalloproteinase of class PI isolated from Bothrops atrox snake venom: functional evaluation

Author(s)

Lanuze Graziele Benato de Toni

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2011-06-03

Examining board members

Suely Vilela; Wilson Roberto Malfará; Auro Nomizo

• Advisor: Suely Vilela

Abstract

Snake venoms are rich in components like proteins, peptides, lipids, carbohydrates and inorganic compounds. Among their main proteins components, the metalloproteinases (SVMPs) are responsible for hemorrhagic and inflammatory effects, edema formation and leucocytes recruitment. Batroxase, a new metalloproteinase isolated from Bothrops atrox snake venom, was purified using three chromatographic steps, namely: two molecular exclusion steps, one as classic chromatography and another as FPLC, and an anionic exchange in HPLC. SVMPs can be classified by specific domains in their structure. Among the various classes, there are SVMP PI, which has weak hemorrhagic activity and low molecular weight. Batroxase showed a molecular mass of 27 kDa (data not shown) and DHM of 10 µg. The proteolytic activity was evaluated on different substrates in the extracellular matrix and some plasma proteins, responsible for activation of coagulation. Batroxase was able to degrade extracellular matrix (ECM) components like, type IV collagen and fibronectin, inducing bleeding process, since these components are found in the ECM of epithelial tissues and blood vessels. The activity of proteins present in plasma was evaluated, demonstrating the ability to degrade fibrinogen and fibrin, especially the chain, which can be inhibited in the presence of metalloproteinase inhibitors as EDTA and EGTA, as well as with a reducing protein agent -mercaptoethanol. Activity on fibrin and fibrinogen interfere with the clotting process, making difficult to interrupt processes like bleeding. After clot formation, Batroxase was able to dissolve it completely, which is related to their fibrinolytic activity. The platelets in the presence of Batroxase were not induced to aggregate, and when, in addition to ADP, was not able to inhibit aggregation. Inflammatory activity has been elucidated, suggesting the participation in edema and hyperalgesia, as well as infiltration of leukocytes. Pharmacological evaluation was observed with participation of pro-inflammatory mediators such as histamine via H1 receptors, serotonin 5-HT1 receptor via, and the synthesis of leukotrienes and histamine on edema via H1 receptors and synthesis of leukotrienes in hyperalgesia. Infiltration of leukocytes was observed mainly mediated by neutrophils in the early hours, followed by the increase of mononuclear cells. Batroxase have absent or very low cytotoxicity on tumor cell lines tested, with significant only for Jurkat analysis, B16-F10, SK-BR3 in high concentrations. In EL-4 cells and PBMC, inducing cell proliferation at low concentrations and inhibiting this growth in higher concentrations as observed in Jurkat cells, A-20 and PBMC. It can stimulate production of proinflammatory and antiinflammatory cytokines, preferentially induces the production of Th1 cytokines such as IFN-, which, after addition of polyclonal anti-CD3 stimulation showed activity in suppressing proliferative capacity of T cells, inhibiting the production of Th1 cytokines such as IL-2 and IFN-, Th2 cytokines such as IL-4 and Th17 cytokine pattern as IL-17A. (AU)

• FAPESP's process: 09/06488-0 - Batroxase, a new class PI metalloproteinase isolated from the venom of Bothrops atrox: evaluation of functional activity
• Grantee: Lanuze Graziele Benato de Toni
• Support Opportunities: Scholarships in Brazil - Master