Fibrin sealant, a widely available tissue adhesive, has been used since 1940 in a variety of clinical applications. Commercially available fibrin sealant products are synthesized from bovine thrombin and human fibrinogen, which may transmit infectious diseases, and recipients may also develop antibodies against bovine thrombin. Bearing these disadvantages in mind, a new fibrin sealant was developed in 1989 by a group of researchers from the Center for the Study of Venoms and Venomous Animals, in Sao Paulo State, Brazil. The main purpose was to produce an adhesive fibrin without using human blood, to avoid transmitting infectious diseases. The components of this novel sealant were extracted from large animals and a serine proteinase extracted from Crotalus durissus terrificus snake venom. The applicability of this sealant was tested in animals and humans with beneficial results. The new fibrin sealant can be a useful tool clinically due to its flexibility and diversity of applications. This sealant is a biological and biodegradable product that (1) does not produce adverse reactions, (1) contains no human blood, (3) has a good adhesive capacity, (4) gives no transmission of infectious diseases, and (5) may be used as an adjuvant in conventional suture procedures. The effectiveness of this new fibrin sealant is reviewed and its development and employment are described.

Hemostatic and adhesive agents date back to World War II, when homologous fibrin sealant came onto scene. Considering that infectious diseases can be transmitted via human blood, a new heterologous fibrin sealant was standardized in the 1990s. Its components were a serine protease (a thrombin-like enzyme) extracted from the venom of Crotalus durissus terrificus snakes and a fibrinogen-rich cryoprecipitate extracted from the blood of Bubalus bubalis buffaloes. This new bioproduct has been used as a coagulant, sealant, adhesive and recently as a candidate scaffold for mesenchymal stem cells and bone and cartilage repair. This review discusses the composition of a new heterologous fibrin sealant, and cites published articles related to its preclinical applications aiming at repairing nervous system traumas and regenerating bone marrow. Finally, we present an innovative safety trial I/II that found the product to be a safe and clinically promising candidate for treating chronic venous ulcers. A multicenter clinical trial, phase II/III, with a larger number of participants will be performed to prove the efficacy of an innovative biopharmaceutical product derived from animal venom.Electronic supplementary materialThe online version of this article (doi:10.1186/s40409-017-0109-8) contains supplementary material, which is available to authorized users.

Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn²+, Cu²+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system

Crotalus durissus terrificus (Cdt) venom major components comprise crotoxin, crotamine, gyroxin and convulxin. Crotamine exerts a myotoxic action, among others, but its expression varies even amid snakes from the same region. Biochemical, enzymatic and pharmacological variations of venoms may be associated with the geography, climate, gender, age, and diet, as well as captivity time and venom extraction intervals. The present study aimed to characterize the Cdt venom from the Botucatu region, (SP, Brazil), by assessing its biochemical, pharmacological and enzymatic properties. Venoms from newly captured snakes and already-captured animals were characterized comparatively to verify the sexual, environmental (length of captivity) and ontogenetic variations that could influence the venom composition. Protein concentration, SDS-PAGE and RP-HPLC were performed and the coagulant, toxic (LD50) and crotamine activities were assayed. Individual SDS-PAGE analyses (315 samples) were performed and the biological activities of the venom of 60 adults (captive and newly captured males and females) and 18 newborns were compared with the Brazilian Reference Venom. Crotamine was found in 39.7% (125/315) of the samples, as determined by SDS-PAGE and RP-HPLC. Protein concentration differed significantly between adults (75%) and newborns (60%). RP-HPLC and SDS-PAGE analyses showed highly variable protein concentration and copious crotoxin isoforms; however, the LD50 values decreased during the captivity time. Cdt venom biological activities were similar among adult groups, but diminished during the captivity period. The current findings demonstrate that venoms vary significantly in terms activity and protein concentration, despite originating from the same specie and region.

BackgroundAmerican visceral leishmaniasis is caused by the intracellular parasite Leishmania (L.) infantum chagasi, and transmitted by the sand fly Lutzomyia longipalpis. Since treatment is based on classical chemotherapeutics with significant side effects, the search for new drugs remains the greatest global challenge. Thus, this in vitro study aimed to evaluate the leishmanicidal effect of Crotalus durissus terrificus venom fractions on promastigote and amastigote forms of Leishmania (L.) infantum chagasi.MethodsPhospholipase A2 (PLA2) and a pool of peptide fraction (<3 kDa) were purified from Crotalus venom. Furthermore, promastigotes and peritoneal macrophages of mice infected by amastigotes were exposed to serial dilutions of the PLA2 and peptides at intervals varying between 1.5625 μg/mL and 200 μg/mL. Both showed activity against promastigotes that varied according to the tested concentration and the time of incubation (24, 48 and 72 h).ResultsMTT assay for promastigotes showed IC50 of 52.07 μg/mL for PLA2 and 16.98 μg/mL for the peptide fraction of the venom. The cytotoxicity assessment in peritoneal macrophages showed IC50 of 98 μg/mL and 16.98 μg/mL for PLA2 and peptide by MTT assay, respectively. In peritoneal macrophages infected by Leishmania (L.) infantum chagasi amastigotes, the PLA2 stimulated growth of parasites, and at higher doses reduced growth by 23 %. The peptide fraction prevented 43 % of the intracellular parasite growth at a dose of 16.98 μg/mL, demonstrating the toxicity of this dose to macrophages. Both fractions stimulated H2O2 production by macrophages but only PLA2 was able to stimulate NO production.ConclusionWe have demonstrated the in vitro leishmanicidal activity of the PLA2 and peptide fraction of Crotalus venom. The results encourage further studies to describe the metabolic pathways involved in cell death, as well as the prospecting of molecules with antiparasitic activity present in the peptide fraction of Crotalus durissus terrificus venom.

BackgroundClassically, Crotalus durissus terrificus (Cdt) venom can be described, according to chromatographic criteria, as a simple venom, composed of four major toxins, namely: gyroxin, crotamine, crotoxin and convulxin. Crotoxin is a non-covalent heterodimeric neurotoxin constituted of two subunits: an active phospholipase A2 and a chaperone protein, termed crotapotin. This molecule is composed of three peptide chains connected by seven disulfide bridges. Naturally occurring variants/isoforms of either crotoxin or crotapotin itself have already been reported.MethodsThe crude Cdt venom was separated by using RP-HPLC and the toxins were identified by mass spectrometry (MS). Crotapotin was purified, reduced and alkylated in order to separate the peptide chains that were further analyzed by mass spectrometry and de novo peptide sequencing.ResultsThe RP-HPLC profile of the isolated crotapotin chains already indicated that the α chain would present isoforms, which was corroborated by the MS and tandem mass spectrometry analyses.ConclusionIt was possible to observe that the Cdt crotapotin displays a preferred amino acid substitution pattern present in the α chain, at positions 31 and 40. Moreover, substitutions could also be observed in β and γ chains (one for each). The combinations of these four different peptides, with the already described chains, would produce ten different crotapotins, which is compatible to our previous observations for the Cdt venom.

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The clinical manifestations of Bothrops atrox envenoming involve local and systemic changes, among which edema requires substantial attention due to its ability to progress to compartmental syndromes and sometimes cause tissue loss and amputations. However, the impact of edema on the poisoned body’s system has not been explored. Thus, the present study aimed to explore the systemic pathological and inflammatory events that are altered by intraplantar injection of B. atrox venom in a mouse model through hematologic, lipidic, and shotgun proteomics analysis. Plasma samples collected showed a greater abundance of proteins related to complement, coagulation, lipid system, platelet and neutrophil degranulation, and pathways related to cell death and ischemic tolerance. Interestingly, some proteins, in particular, Prdx2 (peroxiredoxin 2), Hba (hemoglobin subunit alpha), and F9 (Factor IX), increased according to the amount of venom injected. Our findings support that B. atrox venom activates multiple blood systems that are involved in thromboinflammation, an observation that may have implications for the pathophysiological progression of envenomations. Furthermore, we report for the first time a potential role of Prdx2, Hba, and F9 as potential markers of the severity of edema/inflammation in mice caused by B. atrox.