Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom.

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

Toxinological characterization of venom from Leptodeira annulata (Banded cat-eyed snake; Dipsadidae, Imantodini).

We investigated the histology of Duvernoy's venom gland and the biochemical and biological activities of Leptodeira annulata snake venom. The venom gland had a lobular organization, with secretory tubules formed by serous epithelial cells surrounding each lobular duct. The latter drained into a common lobular duct and subsequently into a central cistern. In contrast, the supralabial gland was mucous in nature. SDS-PAGE revealed a profile of venom components that differed from pitviper (Bothrops spp.) venoms. RP-HPLC also revealed greater complexity of this venom compared to Bothrops venoms. The venom had no esterase, l-amino acid oxidase or thrombin-like activity, but was proteolytic towards elastin-Congo red, fibrin, fibrinogen, gelatin and hide powder azure. The venom showed strong α-fibrinogenase and fibrinolytic activities and reduced the rate and extent of plasma recalcification. The proteolytic activity was inhibited by EDTA and 1,10-phenanthroline (metalloproteinase inhibitors) but not by AEBSF and PMSF (serine proteinase inhibitors). The venom had phospholipase A2 (PLA2) activity that was inhibited by varespladib. The venom cross-reacted with antivenoms to lancehead (Bothrops spp.), coralsnake (Micrurus spp.) and rattlesnake (Crotalus durissus terrificus) venoms. The venom did not aggregate rat platelets or inhibit collagen-induced aggregation, but partially inhibited thrombin-induced aggregation. The venom was hemorrhagic (inhibited by EDTA) and increased the vascular permeability (inhibited by varespladib) in rat dorsal skin. In gastrocnemius muscle, the venom caused myonecrosis and increased serum creatine kinase concentrations. In conclusion, L. annulata venom has various enzymatic and biological activities, with the local effects being mediated primarily by metalloproteinases and PLA2.

Edematic and coagulant effects caused by the venom of Bothrops rhombeatus neutralized by the ethanolic extract of Piper auritum.

ETHNOPHARMACOLOGICAL RELEVANCE: In Colombia, the only authorized treatment to cure snakebite envenomation is with the use of antivenom. The antivenom neutralizes the systemic effects properly, but is not very effective at neutralizing local effects, thus several cases have lead to complications. On the other hand, rural communities turn to the use of plants that are easily accessible and available for basic health care. One of these plants is named Piper auritum (PA), which is traditionally highlighted in some indigenous communities of Antioquia and Chocó. AIM OF THE STUDY: The main objective of this work was to characterize the venom's toxicity by determining the Minimum Edema Dose (MED), the Minimum Coagulant Dose-Plasma (MCD-P), the Minimum Hemorrhagic Dose (MHD) and to determine the neutralizing power of the Total Ethanolic Extract (TEE) from leaves of PA on the localized and systemic effects caused by the Bothrops rhombeatus venom. MATERIALS AND METHODS: To begin, the minimum dose that causes edema-forming, coagulant and hemorrhagic activities was determined. The protocols investigated include coagulant and edematic activities caused by the venom of Bothrops rhombeatus which were neutralized by the TEE of PA. RESULTS: The MCD-P was found to be 0.206 ±â€¯0.026 µg, the MED is the same at 0.768 ±â€¯0.065 µg, and the MHD is 3.553 ±â€¯0.292 µg, which are different from the reports for Bothrops asper and Bothrops ayerbei. Next, a phytochemical screening was done to the TEE where mainly triterpenes, steroids, coumarins, saponins, and lignans were identified. Also present were 43,733 ±â€¯2106 mg AG/g ES of phenols, which are secondary metabolites that are probably responsible for the neutralization of coagulant and edematic activities at rates of 2363.870 µL and 1787.708 µL of extract/mg of venom, respectively. As a comparative parameter, the National Institute Health's (NHI) effective dose of the antivenom was used as a comparative parameter. In addition, we determined the toxicity of the TEE of PA on to Artemia salina, being moderately toxic at 6 and 24 h, while the essential oil of PA at the same observation hours is in the extremely toxic range. CONCLUSIONS: The results reflect that the extract of P. auritum has an anti-inflammatory effect similar to that of the NIH serum. It could be used as a complement of NIH antivenom, using them together so it contributes to effectively reduce inflammation and the socio-economic impact generated by the permanence of a patient victim of snakebite in health centers. CLASSIFICATIONS: Immunological products and vaccines.

Utility of Milk Coagulant Enzyme of Moringa oleifera Seed in Cheese Production from Soy and Skim Milks.

In this study, the potential use of Moringa oleifera as a clotting agent of different types of milk (whole, skim, and soy milk) was investigated. M. oleifera seed extract showed high milk-clotting activity followed by flower extract. Specific clotting activity of seed extract was 200 times higher than that of flower extract. Seed extract is composed by four main protein bands (43.6, 32.2, 19.4, and 16.3 kDa). Caseinolytic activity assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and tyrosine quantification, showed a high extent of casein degradation using M. oleifera seed extract. Milk soy cheese was soft and creamy, while skim milk cheese was hard and crumbly. According to these results, it is concluded that seed extract of M. oleifera generates suitable milk clotting activity for cheesemaking. To our knowledge, this study is the first to report comparative data of M. oleifera milk clotting activity between different types of soy milk.

Coagulant Activity of Water-Soluble Moringa oleifera Lectin Is Linked to Lowering of Electrical Resistance and Inhibited by Monosaccharides and Magnesium Ions.

Moringa oleifera seeds contain a water-soluble lectin [water-soluble M. oleifera lectin (WSMoL)] that has shown coagulant activity. Magnesium ions are able to interfere with the ability of this lectin to bind carbohydrates. In this study, we performed structural characterization of WSMoL and analyzed its effect on the electrical resistance of a kaolin clay suspension in both presence and absence of monosaccharides (N-acetylglucosamine, glucose, or fructose) and magnesium ions. The coagulant activity of WSMoL was monitored by measuring optical density and electrical resistance over a period of 60 min. Native WSMoL had a molecular mass of 60 kDa and exhibited anionic nature (pI 5.5). In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it appeared as three polypeptide bands of 30, 20, and 10 kDa. WSMoL reduced the optical density and electrical resistance of the kaolin suspension, which suggests that suspended particles are destabilized and that this is followed by formation of complexes. The coagulant activity of lectin decreased in the presence of Mg2+ ions and carbohydrates at concentrations that also inhibited hemagglutinating activity. This was most likely due to conformational changes in lectin structure. Our findings suggest that the coagulant activity of WSMoL is enhanced by lowering of electrical resistance of the medium and is impaired by lectin-carbohydrate and lectin-Mg2+ interactions.

Influence of chymosin type and curd scalding temperature on proteolysis of hard cooked cheeses.

In this work, we studied the influence of the type of coagulant enzyme and the curd scalding temperature on the proteolysis and residual coagulant and plasmin activities of a cooked cheese, Reggianito, in the interest of reducing ripening time. A two-factor experimental design was applied in two levels: type of coagulant enzyme, bovine chymosin or camel chymosin, and curd scalding temperature, 50 or 56 °C. The experimental treatments were applied in Reggianito cheese making experiments, and the samples were ripened for 90 d at 12 °C. Scalding temperature influenced residual coagulant activity; the cheeses cooked at 50 °C had significantly higher activity than those treated at 56 °C. In contrast, scalding temperature did not modify plasmin activity. Proteolysis was primarily affected by curd cooking temperature because chymosin-mediated hydrolysis of αs1 casein was slower in cheeses treated at 56 °C. Additionally, the nitrogen content in the cheese soluble fractions was consistently lower in the cheeses scalded at 56 °C than those cooked at 50 °C. A significant influence of the type of coagulant enzyme was observed, especially in the nitrogen fractions and peptide profiles, which demonstrated that camel chymosin was slightly less proteolytic; however, these differences were lower than those caused by the scalding temperature.

Biochemical and biological evaluation of gyroxin isolated from Crotalus durissus terrificus venom

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.(AU)

Biochemical and biological evaluation of gyroxin isolated from Crotalus durissus terrificus venom

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.

Purification and partial characterization of a coagulant serine protease from the venom of the Iranian snake Agkistrodon halys

Agkistrodon halys is one of several dangerous snake species in Iran. Among the most important signs and symptoms in patients envenomated by this snake is disseminated intravascular coagulation. A thrombin-like enzyme, called AH143, was isolated from Agkistrodon halys venom by gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a DEAE-Sepharose and high performance liquid chromatography (HPLC) on a C18 column. In the final stage of purification, 0.82 mg of purified enzyme was obtained from 182.5 mg of venom. The purified enzyme showed a single protein band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), under reducing conditions, and its molecular mass was found to be about 30 kDa. AH143 revealed clotting activity in human plasma, which was not inhibited by EDTA or heparin. This enzyme still demonstrated coagulation activity when exposed to variations in temperature and pH ranging, respectively, from 30 to 40°C and from 7.0 to 8.0. It also displayed proteolytic activities on synthetic substrate. The purified enzyme did not show any effect on casein. We concluded that the venom of the Iranian snake Agkistrodon halys contains about 0.45% single procoagulant protein which appears to be a thrombin-like enzyme.

Purification and partial characterization of a coagulant serine protease from the venom of the Iranian snake Agkistrodon halys

Agkistrodon halys is one of several dangerous snake species in Iran. Among the most important signs and symptoms in patients envenomated by this snake is disseminated intravascular coagulation. A thrombin-like enzyme, called AH143, was isolated from Agkistrodon halys venom by gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a DEAE-Sepharose and high performance liquid chromatography (HPLC) on a C18 column. In the final stage of purification, 0.82 mg of purified enzyme was obtained from 182.5 mg of venom. The purified enzyme showed a single protein band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), under reducing conditions, and its molecular mass was found to be about 30 kDa. AH143 revealed clotting activity in human plasma, which was not inhibited by EDTA or heparin. This enzyme still demonstrated coagulation activity when exposed to variations in temperature and pH ranging, respectively, from 30 to 40°C and from 7.0 to 8.0. It also displayed proteolytic activities on synthetic substrate. The purified enzyme did not show any effect on casein. We concluded that the venom of the Iranian snake Agkistrodon halys contains about 0.45 percent single procoagulant protein which appears to be a thrombin-like enzyme.(AU)