Name: GUSTAVO BAPTISTA NAUMANN

Publication date: 05/04/2018
Advisor:

Name	Role
SUELY GOMES DE FIGUEIREDO	Advisor *

Examining board:

Name	Role
NAZARE SOUZA BISSOLI	Internal Examiner *
RITA GOMES WANDERLEY PIRES	External Examiner *
SONIA ALVES GOUVEA	Internal Examiner *
SUELY GOMES DE FIGUEIREDO	Advisor *

Summary: Bothrops leucurus (white tail jararaca) is the main responsible for ophidian accidents in the northeastern region of Brazil and northern Espírito Santo. Several studies have evaluated the biochemical features of this snake’s venom, as well as its local effects. However, systemic effects – in particular cardiovascular effects – remain rather poorly
explored. In the present study, we sought to investigate the acute cardiovascular activities induced by B. leucurus venom (VB) in vivo and in vitro. In anaesthetized rats it was demonstrated that BlV (10-100μg/kg) induces immediate and transient hypotension,
while maximum response was observed in 5 min and a return to baseline was observed in ≈ 20 minutes. No change in the heart rate of the animals was observed. In vitro effects were evaluated on pre-contracted mesenteric artery rings with phenylephrine, employing
a resistance myograph. BlV (1-20 μg/mL) has been shown to cause dose-dependent vasodilation in these vessels, reaching the maximum response in ≈ 10 minutes and progressively recovering after 15 minutes. Lower levels of relaxation were observed in vessels without endothelium, implying that, in addition to vascular smooth muscle, the
endothelium participates in this response. Pretreatment of the animals and/or the vessels with inhibitors of pathways involved in these responses - L-NAME (nitric oxide synthase), heparin (thrombin inhibitor), atropine (muscarinic antagonist), PMSF (serinoprotease inhibitor) and EDTA (metaloprotease inhibitor) - revealed that only indomethacin was
able to reduce the cardiovascular response induced by BlV (50 μg / kg). This inhibitor was able to block the changes in systolic and diastolic pressures (≈ 45% and ≈ 77%) and ≈ 30% of vascular relaxation induced by BlV. These data point to the participation of
arachidonic acid metabolites in these responses. A protein component that reproduces the observed cardiovascular effects was purified through two chromatographic steps (ion exchange and reverse phase). Mass spectrometric analysis - MALDI-ToF and MALDIISD
- revealed that the protein presenting cardiovascular activity had a MW of 13.889,975 Da, along with identifying an internal sequence of 27 amino acid residues. This MW corresponds to that of the blD-PLA2, already purified from the B. leucurus venom, with the identified sequence showing 100 % identity with residues 10 to 36 - 22%
coverage. These data allow us to infer that the purified protein is blD-PLA2. In this study it was shown that B. leucurus venom induces vasorelaxation and, as a consequence, immediate hypotension after the envenomation, with some of these effects being elicited by the increased release of vasodilator prostanoids by the esterase activity of blD-PLA2.

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