The Venomics of Bothrops alternatus is a Pool of Acidic Proteins with Predominant Hemorrhagic and Coagulopathic Activities

Abstract

The venom proteome of Bothrops alternatus, a venomous snake widespread in South America, was analyzed by 2-D electrophoresis followed by mass spectrometric analysis and determination of enzymatic activities. The venomic composition revealed that metallo- and serine proteinases play primary roles in the pathogenesis of the envenomation by this pitviper. The identified 100 venom components with molecular masses from 10 to 100 kDa belong to six protein families: metalloproteinases, serine/thrombin-like proteinases, phospholipases A(2), L-amino acid oxidases, disintegrins and thrombin inhibitors. Metalloproteinases predominate and belong exclusively to the P-III class including the most potent hemorrhagic toxins. They represent 50% of all identified proteins. Two isoforms were identified: homologous to jararhagin, a hemorrhagic toxin, and to beritractivase, a nonhemorrhagic and pro-coagulant metalloproteinase. The B. alternatus venom is a rich source of proteins influencing the blood coagulation system with a potential for medical application. The isoelectric points of the components are distributed in the acidic pH range (the pI values are between 4 and 7) and no basic proteins were detected.

Comparison of Phylogeny, Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

“…One interesting fact was the significant contribution of C-type (true) lectins in the B. jararacussu (8.8%) and B. neuwiedi (3.5%) venoms, in parallel with its absence (<1%) in the other venoms (Figure 1). Comparing these data with previous venomics studies [16], [18]–[20], [23], the major venom protein families as SVMPs, PLA2s and SVSPs were detected in our study in equivalent proportions. However, shotgun nanoESI-LTQ/Orbitrap allowed the detection in all venoms tested of some proteins not yet described as PDIEST, ECTONT, PLB and GLUTCYC.…”

In vitro comparison of enzymatic effects among Brazilian Bothrops spp. venoms

“…The various metalloproteinases identified in B. alternatus venom have molecular masses ranging from 22 to 100 kDa, and are capable of causing hemorrhage, edema, myonecrosis, and coagulation disorders. The venom of B. alternatus also contains many serine proteinases that present coagulant and fibrinogenolytic activities; these enzymes are considered less toxic than metalloproteinases (Ohler et al, 2010). Although venomics studies have revealed that metalloproteinases and serine proteinases are considered the most toxic components , we found that B. alternatus venom showed only moderate proteolytic activity.…”

“…However, an acidic PLA 2 identified in B. alternatus venom was found to be the major compound responsible for the lethality of this venom in mice, producing cardiovascular alterations such as dyspnea, tachycardia, arrhythmia, and circulatory shock, as well as tissue damage, including hemorrhage and necrosis (Nisenbom et al, 1986a(Nisenbom et al, , 1986b. Nevertheless, it is known that the protein content of B. alternatus venom comprises mostly metalloproteinases and serine proteinases, accounting for 43.1% and 24.1%, respectively (Ohler et al, 2010). The various metalloproteinases identified in B. alternatus venom have molecular masses ranging from 22 to 100 kDa, and are capable of causing hemorrhage, edema, myonecrosis, and coagulation disorders.…”

“…B. alternatus showed the lowest LAAO activity. Venomics studies have demonstrated that B. alternatus venom contains five LAAO isoforms, with molecular masses ranging from 50 to 57 kDa (monomeric form), collectively accounting for 6.9% of the crude venom, and that there is a high homology between these LAAOs and those found in B. moojeni venom (Ohler et al, 2010). Nevertheless, in the present study, the activity levels differed between those two species, a fact that might be attributable to the use of crude venom rather than purified enzymes.…”

An alternative method to isolate protease and phospholipase A2 toxins from snake venoms based on partitioning of aqueous two-phase systems

“…Based on literature findings, we are able to assume that B. alternatus venom exhibits a variety of phospholipase A 2 and protease enzymes in its composition (30)(31)(32)(33)(34)(35)(36)(37)(38). At initial extraction steps all protein fractions are present in the system and each enzyme isoform is distributed between the phases, according to its structural properties.…”