BackgroundEnvenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption.Methodology/Principal FindingsIn this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues.Conclusions/SignificanceThese results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to alpha(2)beta(1) integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to alpha(2)beta(1) integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant alpha(2)beta(1) integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant alpha(2)beta(1) integrin nor to cell-surface-exposed alpha(2)beta(1) integrin (alpha(2)-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and alpha(2)beta(1) integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.

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Jararhagin is a metalloproteinase isolated from Bothrops jararaca snake venom, which has been extensively studied. These studies showed its involvement on most of the systemic and local damaging effects of snakebite envenomings. In this review we comment on the major targets of jararhagin as the vascular endothelium, platelets and coagulation factors and also its action on other cell systems as inflammatory cells and their mediators, cancer and cell signaling. The mechanisms of jararhagin action are discussed together with structural features essential for the expression of its biological activities. The studies reviewed here denote jararhagin as a prototype for studies of snake venom metalloproteinases, bringing new insights into cellular-matrix interactions and adding for the improvement of snakebite treatment.

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The Snake Venom Metalloproteinases (SVMPs) play a relevant role in the multifactorial inflammatory response induced by Bothrops envenomations. Neuwiedase, an SVMP isolated from Bothrops neuwiedi venom, is devoid of hemorrhagic activity on skin tests, but is able to induce myonecrosis and degrade fibrinogen, fibrin, type I collagen, fibronectin and laminin. In this study, we analyzed the inflammatory reaction induced by neuwiedase in gastrocnemius muscle, with special focus on cytokines release. Our results showed clear evidence of inflammatory infiltrate in the gastrocnemius muscle and an increase of MMP-9, and the cytokines KC, IL-1 beta and IL-6 in the early periods after toxin injection. The cytokine release was also evaluated in inflammatory and muscular cell culture. Both murine peritoneal adherent cells (MPACs) and muscle cells (C2C12) released pro-inflammatory cytokines after stimulus with neuwiedase. MPACs showed increased production of KC, IL-1 beta and IL-6 in the cell culture supernatant while in C2C12, the predominant chemokine expressed was KC. These data reinforce the importance of SVMPs in the inflammatory response caused by envenomation and point out the role of muscle cells in this event by releasing pro-inflammatory mediators able to attract leukocytes to the muscle, thus starting and amplifying the setting of the inflammatory reaction.