Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities

Fernanda C V Portaro,Fábio Carlos Magnoli,Leo Kei Iwai,Ângela Alice Amadeu Megale,Alexandre Kazuo Kuniyoshi,Wilmar Dias Da Silva,Denise V Tambourgi

doi:10.1186/s40409-018-0176-5

Fernanda C V Portaro, Fábio Carlos Magnoli + Show 5 more

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https://doi.org/10.1186/s40409-018-0176-5

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Abstract

BackgroundBitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP).MethodsSolubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically.ResultsA serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity.ConclusionsThe in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO’s recommendation of immunotherapy in cases of human accidents with venomous snakes.

Highlights

Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia
Fluorescent Resonance Energy Transfer (FRET) substrate screening B. arietans venom presented proteolytic activity on both tested FRET substrates: O-aminobenzoic acid (Abz)-FRSSR-EDDnp and Abz-RPPGFSPFR-EDDnp, which contain a sequence recognized by proteases of different catalytic natures or bradykinin amino acid sequence, respectively (Fig. 1)
To conclude, this study described the purification and characterization of a novel bifunctional serine protease, from B. arietans venom, that acts upon human fibrinogen and presents kinin-releasing activity in in vitro studies

Summary

Introduction

In order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). In sub-Saharan Africa an estimated 90,000–400,000 envenoming snakebites occur every year, resulting in up to 32,000 deaths [1] and 14,000 victims suffering amputations, local tissue damage and chronic disabilities [2]. These data are probably underestimated because they are based on just a few case reports or on the epidemiological literature. SVSPs exhibit about 51–98% identity with each other, 26–33% with human thrombin and 34– 40% with human plasma kallikrein [7]

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