Anti-platelet aggregation activity of two novel acidic Asp49-phospholipases A2 from Bothrops brazili snake venom

Abstract

Phospholipases A (PLAs) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLAs from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLAs was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ​​of 13,894 and 13,869Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLAs using acid PLABthA-I-PLA from B. jararacussu template showed high quality. Both acidic PLAs showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100μg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10μg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLAs biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads.

Venomics and antivenomics of the poorly studied Brazil’s lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará

“…MS/MS analysis confirmed the lack of identity of the PLA 2 molecules of B. brazili (Pará) with conspecific PLA 2 sequences reported in the literature. PLA 2 molecules eluted in RP-HPLC peaks 11-14 were identified as homologs of basic BrTx-II [4K09] and MTx-II [I6L8L6, 4K06, 4DCF] [25,54], and the tryptic peptide sequences derived from PLA 2 in RP-HPLC peak 15, 24, 25, 27 and 28 showed high similarity with homologue internal sequences of acidic PLA 2 s Braziliase-I and Braziliase-II [26]. Clearly, the extent of geographic venom variability of B. brazili across its wide distribution range requires detailed future studies.…”

“…In the murine model, Peruvian B. brazili exhibited minimum hemorrhagic dose (MHD) of 7.40 μg/ mouse), minimum dermonecrotic dose (MND) of 152.15 μg/ mouse, minimum coagulant dose against plasma (MCD-P) and fibrinogen (MCD-F) of 19.20 and 1020.0 μg/mL, respectively, and minimum defibrinogenating dose (MDD) of 7.0 μg/mouse [11]. Although described as a new Bothrops from Brazil 65 years ago [15], very few studies have been reported on the toxin arsenal of the Brazil's lancehead venom, and these were mainly focused on the pharmacological effects and possible biotechnological applications of isolated toxins [21][22][23][24][25][26][27][28][29][30][31], including acidic and basic phospholipase A 2 (PLA 2 ) molecules (myotoxic Braziliase I and II, MTX I and II, brazilitoxins II and III) [23][24][25][26]; a PI-snake venom metaloproteinase (SVMP), with in vitro antiplasmodial properties [27]; coagulant thrombin-like and pro-angiogenic snake venom serine proteinase (SVSP) [28,29]; and a hyaluronidase [30].…”

The allergic response mediated by fire ant venom proteins

“…The reaction was triggered by the addition of 20.0 µM of the substrate 12NBD PC; after which the release of fatty acids was measured for 1 h in a plate-reading fluorimeter (Victor™ X5 Multilabel-Perkin Elmer) set for excitation and emission wavelengths of 460 nm and 534 nm, respectively. The negative control was reaction medium without added venom, and the positive control sample contained 1.0 μl (∼13.0 µg/µl) of Bothrops jararaca venom 36 .…”

New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

“…The Bothropstoxin-I (BthTX-I) and Bothropstoxin-II (BthTX-II) from Bothrops jararacussu were obtained from the Venom Bank at CEBio (Centro de Estudos de Biomoléculas Aplicadas a Saúde/Fiocruz Rondônia/UNIR), located in Porto Velho, RO. PLA 2 LmtTX from Lachesis muta provided by Diniz-Sousa et al [46], PLA 2 BnuTX-I from Bothrops urutu provided by Corrêa et al [47], PLA 2 Braziliase-I and Braziliase-II from Bothrops brazili provided by Kayano et al [48] and Sobrinho et al [49].…”