Ruthenium Antivenom Inhibits the Defibrinogenating Activity of Crotalus adamanteus Venom in Rabbits.

Eastern Diamondback Rattlesnake (Crotalus adamanteus) envenomation is a medical emergency encountered in the Southeastern United States. The venom contains a snake venom thrombin-like enzyme (SVTLE) that is defibrinogenating, causing coagulopathy without effects on platelets in humans. This investigation utilized thrombelastographic methods to document this coagulopathy kinetically on the molecular level in a rabbit model of envenomation via the analyses of whole blood samples without and with platelet inhibition. Subsequently, the administration of a novel ruthenium compound containing site-directed antivenom abrogated the coagulopathic effects of envenomation in whole blood without platelet inhibition and significantly diminished loss of coagulation in platelet-inhibited samples. This investigation provides coagulation kinetic insights into the molecular interactions and results of SVTLE on fibrinogen-dependent coagulation and confirmation of the efficacy of a ruthenium antivenom. These results serve as a rationale to investigate the coagulopathic effects of other venoms with this model and assess the efficacy of this site-directed antivenom.

Biochemical and hemostatic description of a thrombin-like enzyme TLBro from Bothrops roedingeri snake venom.

Objective: The current study's objective is to characterize a new throm-bin-like enzyme called TLBro that was obtained from Bothrops roedingeris snake from a biochemical and hemostatic perspective. Methodology: One chromatographic step was used to purify it, producing the serine protease TLBro. Molecular mass was estimated by SDS-PAGE to be between reduced and unreduced by 35 kDa. Tryptic peptide sequencing using Swiss Prot provided the complete amino acid sequence. Expasy.org by conducting a search that is limited to Crotalinae snake serine proteases and displaying a high degree of amino acid sequence. Results: Ser (182) is inhibited by phenylmethylsulfonyl fluoride (PMSF), and TLBro demonstrated the presence of Asp (88) residues. It also deduced the positions of His (43) and Ser (182) in the set of three coordinated amino acids in serine proteases. It was discovered that this substrate had high specificity for BANA, Michaelis-Menten behavior with KM 0 point85 mM and Vmax 1 point89 nmoles -NA/L/min, and high stability between temperatures (15 to 70°C) and pHs (2 point0 to 10 point0). According to doses and incubation times, TLBro degraded fibrin preferentially on the B-chain; additionally, its activities were significantly diminished after preincubation with divalent ions (Zn2 and Cd2). When incubated with PMSF, a particular serine protease inhibitor, enzymatic activities and platelet aggregation were inhibited. Conclusion: The findings revealed distinct structural and functional differences between the serine proteases, adding to the information and assisting in the improvement of the structure-function relationship.

Towards toxin PEGylation: The example of rCollinein-1, a snake venom thrombin-like enzyme, as a PEGylated biopharmaceutical prototype.

PEGylation was firstly described around 50 years ago and has been used for more than 30 years as a strategy to improve the drugability of biopharmaceuticals. However, it remains poorly employed in toxinology, even though it may be a promising strategy to empower these compounds in therapeutics. This work reports the PEGylation of rCollinein-1, a recombinant snake venom serine protease (SVSP), able to degrade fibrinogen and inhibit the hEAG1 potassium channel. We compared the functional, structural, and immunogenic properties of the non-PEGylated (rCollinein-1) and PEGylated (PEG-rCollinein-1) forms. PEG-rCollinein-1 shares similar kinetic parameters with rCollinein-1, maintaining its capability of degrading fibrinogen, but with reduced activity on hEAG1 channel. CD analysis revealed the maintenance of protein conformation after PEGylation, and thermal shift assays demonstrated similar thermostability. Both forms of the enzyme showed to be non-toxic to peripheral blood mononuclear cells (PBMC). In silico epitope prediction indicated three putative immunogenic peptides. However, immune response on mice showed PEG-rCollinein-1 was devoid of immunogenicity. PEGylation directed rCollinein-1 activity towards hemostasis control, broadening its possibilities to be employed as a defibrinogenant agent.

Hypnale coagulopathy: snake envenomation of a different kind.

Hypnale hypnale is a small pit viper that is known as the hump-nosed pit viper (HNV) or 'Merrem's hump-nosed pit viper' and is endemic to Sri Lanka and the Western Ghats of South India. Previously, it was thought that this viper bite would result in mild systemic or local envenomation, however, we now know that HNV bites could cause severe systemic toxicity and mortality. The most common systemic toxicity of this viper envenomation is coagulopathy, which is known as 'Hypnale coagulopathy'. Hypnale coagulopathy is quite different from the haemotoxicity caused by other vipers. In this paper, an effort has been made to discuss the unique and unpredictable nature of this coagulopathy due to Hypnale envenomation.

Thrombin- and plasmin-like and platelet-aggregation-inducing activities of Plumeria alba L. latex: Action of cysteine protease.

ETHNOPHARMACOLOGICAL RELEVANCE: In folk medicine, parts of Plumeria alba L. are used for the treatment of many diseases, with its latex being used for curing skin diseases and promoting wound healing. AIM OF THE STUDY: This study aimed to study the role of P. alba L. latex in hemostasis and platelet aggregation. MATERIALS AND METHODS: The latex of P. alba L. was processed to remove waxes and enrich protein content, and the final extract was named Plumeria alba L. natant latex (PaNL). PaNL was analyzed for protease activity against casein. The type of protease in PaNL was identified by using protease inhibitors such as E-64, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, and pepstatin A. Human fibrinogen, fibrin, and collagen types I and IV were subjected to hydrolysis with different concentrations of PaNL. The thrombin-like activity of PaNL was determined by analyzing its fibrinogen-clotting and procoagulant activities. The role of PaNL in platelet aggregation was also investigated. Its hemorrhagic and edema-inducing activities were evaluated in a mouse model. Phytochemical compounds were identified by gas chromatography-mass spectroscopy. RESULTS: The findings of casein/gelatin zymography confirmed that PaNL possesses protease activity. The results of the protease inhibition study indicated the presence of a cysteine-type protease(s) in PaNL. PaNL hydrolyzed the subunits of fibrinogen, fibrin, and collagen types I and IV. Its fibrin-degradation activity indicated that PaNL possesses plasmin-like activity. PaNL induced clotting of citrated human plasma within 3 min of incubation in the absence of CaCl2, indicating the presence of thrombin-like activity, which was further confirmed by the results of the fibrinogen-clotting assay. PaNL induced platelet aggregation in the absence of agonists. There was no hemolytic activity. Mice injected with PaNL did not show edema/ hemorrhagic activity. CONCLUSION: PaNL possesses procoagulant, fibrino(geno)lytic, thrombin- and plasmin-like activities and induces platelet aggregation, which could explain its usage for wound treatment in folk medicine.

Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization.

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold ß/ß hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.

Unique heterologous fibrin biopolymer with hemostatic, adhesive, sealant, scaffold and drug delivery properties: a systematic review.

Fibrin biopolymers, previously referred as "fibrin glue" or "fibrin sealants", are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was "heterologous fibrin sealant". The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.

Unique heterologous fibrin biopolymer with hemostatic, adhesive, sealant, scaffold and drug delivery properties: a systematic review

Fibrin biopolymers, previously referred as "fibrin glue" or "fibrin sealants", are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was "heterologous fibrin sealant". The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.(AU)

New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom.

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80⁻6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bß chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

Cloning, expression, purification and bioactivity evaluation of a thrombin-like enzyme from Deinagkistrodon acutus venom gland library.

OBJECTIVES: To identify a new member of serine proteases from Deinagkistrodon acutus via phage display technique and appraise its biocatalytic activities. RESULTS: A novel thrombin-like enzyme gene was cloned by screening the phage display library of D. acutus venom gland. The gene has a 783 bp ORF encoding 260 amino acids. A recombinant enzyme expression vector was constructed and the fused protein was expressed in Escherichia coli. The protein was purified showing a single band of approx. 49.4 kDa after SDS-PAGE. The recombinant enzyme was capable of congealing normal human plasma in vitro with the minimum coagulant dose of 6 µg in 57 s. It exhibited fibrinogenolytic activity by hydrolyzing the Aα-chain of human fibrinogen. It was most active at pH 7.5-8.0 and 35-40 °C with the highest clotting activity of 120 NIH units/mg. It was completely inhibited by PMSF but not by EDTA. Multiple sequence alignments demonstrate that this protein shares high identity with other thrombin-like enzymes from snake venoms. CONCLUSIONS: A novel thrombin-like protein from D. acutus venom was identified, expressed and biologically characterized in vitro. Its fibrinogenolytic properties make the enzyme applicable for biochemical research and drug development on thrombolytic therapy.

Expression, purification and characterization of a novel recombinant SVTLE, r-agkihpin-2, from Gloydius halys Pallas venom gland in Escherichia coli.

In our previous work, a thrombin-like enzyme (TLE), agkihpin, was successfully isolated, purified, cloned and named from the venom of Gloydius halys Pallas, having fibrinolytic, fibrinogenolytic and thrombosis-reduced activities, attenuating migration of liver cancer cell, and without bleeding risk. To explore the possibility of agkihpin as a thrombolytic and/or anti-metastasis agent in the future, in this study recombinant agkihpin was expressed and purified in Escherichia coli, and its biological activities investigated. Thus, r-agkihpin-2 was successfully expressed and purified and confirmed by Western blot and peptide mass fingerprinting. After purification and renaturation, 46 mg (399 U) of active r-agkihpin-2 was obtained from 1 L bacterial culture. The results of the arginine esterase activity assay, fibrin plate test fibrinogenolytic activity assay, thrombin-induced venous thrombosis assay, Scratch-Wound assay and bleeding assay showed that active r-agkihpin-2 had slightly lower TAME hydrolytic, fibrinolytic, fibrinogenolytic, thrombus-reduced and migration-attenuated activities than those of native agkihpin, and had no bleeding risk. These findings confirmed that, active r-agkihpin-2 could be further investigated for thrombolytic and/or anti-metastasis drug discovery in the future.

Heterologous fibrin sealant derived from snake venom: from bench to bedside - an overview.

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.

Acute and repeated dose (28 days) toxicity studies in rats and dogs of recombinant batroxobin, a snake venom thrombin-like enzyme expressed from Pichia pastoris.

Recombinant batroxobin is a thrombin-like enzyme of Bothrops atrox moojeni venom. To evaluate its toxicological effect, it was highly expressed in Pichia pastorisand successfully purified to homogeneity from culture broth supernatant following Good Manufacturing Practice (GMP). The maximum tolerated dose of the recombinant batroxobin was examined in Sprague-Dawley (SD) rat and Beagle dogs following Good Laboratory Practice (GLP) regulations. The approximate lethal dose of recombinant batroxobin was 10 National Institute of Health (NIH) u/kg in male and female rats. Slight test substance-related effects were clearly in male and female dogs at more than 10 NIH u/kg. The maximum tolerated dose (MTD) was considered to be greater than 30 NIH u/kg in dogs. To investigate the repeated dose toxicity of batroxobin, the test item was intravenously administered to groups of SD rat and Beagle dog every day for 4 weeks. We observed that all animals survived the duration of the study without any effects on their mortality. There were no effects in both rats and dogs regarding their clinical signs, body weight, food consumption, ophthalmological examination, urinalysis, hematology, clinical chemistry, organ weightand gross post mortem examinations. The no adverse effect level (NOAEL) of recombinant batroxobin for both males and females is considered to be greater than 2.5 NIH u/kgin rats and 1 NIH u/kg in dogs, respectively. No toxic effects were noted in target organs. In conclusion, these results show a favorable preclinical profile and may contribute clinical development of recombinant batroxobin.

Heterologous fibrin sealant derived from snake venom: from bench to bedside an overview

Abstract 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.

Heterologous fibrin sealant derived from snake venom: from bench to bedside - an overview

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.(AU)

Agkihpin, a novel SVTLE from Gloydius halys Pallas, promotes platelet aggregation in vitro and inhibits thrombus formation in vivo in murine models of thrombosis.

In previous work, a snake venom arginine esterase (SVAE), agkihpin from the venom of Gloydius halys Pallas, was isolated and its biochemical data including Mr, PI, amino acid components and sugar content was collected. Here, the agkihpin was cloned and further characterized and we found that agkihpin could promote ADP-induced platelets aggregation, hydrolyze fibrin, cleave Aα and Bß chains of fibrinogen and reduce the thrombosis induced by thrombin. Moreover, agkihpin hydrolyzed TAME with optimum temperatures at 30 °C-45 °C, and the hydrolysis was inhibited by EDTA, PMSF, DTT and promoted by Ca2+, Fe3+, Mg2+, Zn2+. The sequence features of agkihpin were detected as follows: the N-terminal residues was determined as I(V)L(Y)GDDECNINE by protein sequencing; the ORF was determined as 705 bp, and the deduced amino acid sequence was identified by peptide mass fingerprinting; the cysteines, cleavage sites, active sites and substrate binding sites of snake venom thrombin-like enzyme (SVTLE), were all conserved in amino acid sequence of agkihpin; 2 Leu(Tyr), 4 Asn and 121 Ile in amino acid sequence of agkihpin were first found in the amino acid sequences of SVTLEs. These findings indicated that agkihpin is a novel SVTLE. What's more, due to its several advantages of fibrino(gen)olytic and thrombosis-reduced activities, and devoid of bleeding risk, agkihpin may be developed into a thrombolytic drug in the future.

The structure-function relationship of thrombin-like enzymes from the green pit viper (Trimeresurus albolabris).

Pit viper venoms can decrease fibrinogen levels in snakebite patients. Studies have shown that the hypofibrinogenemia is a consequence of snake venom thrombin-like enzymes (TLEs), the serine proteases that have the potential to be both diagnostic and therapeutic agents. Exosites of thrombin are the molecular regions that determine the substrate specificities, but its presence and significance in TLEs are unclear. Therefore, the putative exosites of recombinant TLEs derived from Green pit viper (Trimeresurus albolabris), GPV-TL1 and GPV-TL2, were mutated in a Pichia pastoris system. In a previous report, GPV-TL1 showed a strong fibrinogenolytic activity on the Aα and Bß chains of fibrinogen, as well as a plasma clotting activity. Compared with GPV-TL1, the GPV-TL1m mutated in the putative exosite (TRN to RRR at residues 60-62) showed a weaker fibrinogenolytic activity with a similar clotting activity of 207.1 thrombin units/mg. GPV-TL2 contained two-residue differences from GPV-TL1 in the putative exosite (N73M and V74Y). GPV-TL2 selectively cleaved only the Aα chain of fibrinogen without detectable clotting activity. The mutated GPV-TL2 (GPV-TL2m) showed a weaker fibrinogenolytic activity compared with that of the wild type. These results support the important roles of the putative exosite in snake venom TLE activities. This information is helpful for future protein engineering.

Effects of single N-glycosylation site knockout on folding and defibrinogenating activities of acutobin recombinants from HEK293T.

Acutobin, the α-fibrinogenase from Deinagkistrodon acutus venom, contains four N-glycosylation sites with disialylated complex-typed glycans. Here, we explore the functional roles of each of the N-glycan by site-directed mutagenesis. The wild-type (ATB-wt) and single glycan-knockout mutants of recombinant acutobin were prepared from HEK293T, demonstrating that mutations at Asn(77), Asn(81) and Asn(100) impaired the folding while the S79A mutant and various Asn(229)-deglycosylated mutants were correctly folded. Based on homology modeling of acutobin and multiple sequence alignment with various venom thrombin-like enzymes, the importance of a hydrophilic environment at each glycosylation site to the enzyme folding could be rationalized. Remarkably, all the mutants showed similar catalytic activities for the chromogenic substrate and similar thermal stabilities as ATB-wt, suggesting that the glycan knockout did not affect the gross conformation and stability of the active sites. Although SDS-PAGE analyses revealed that ATB-wt and the D229-mutant degraded all human fibrinogen subunits faster but less specifically in vitro as compared with other mutants that cleaved only the α-subunit, ATB-wt and D229-mutant were not able to release fibrinogen-peptide A and thus coagulated human plasma slower than the other mutants did. In the mice model, the defibrinogenating effect of ATB-wt was stronger and lasting-longer than those of all the mutants. Taken together, all the glycans contribute to the pharmacokinetics of acutobin and ATB-wt in vivo, and the microenvironment around the Asn(229)-glycan appears to regulate the fibrinogen-chain specificity of acutobin while the N-glycans at positions 77, 81 and 100 are crucial for its folding.

Cross-neutralization of the coagulant activity of Crotalus durissus terrificus venom from the northeast of Argentina by bivalent bothropic antivenom

Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.

Serum kinetics of Calloselasma rhodostoma (Malayan pit viper) venom components in a rabbit

The serum kinetics of Calloselasma rhodostoma (Malayan pit viper) venom - specifically two of its components, the major hemorrhagin (rhodostoxin) and a thrombin-like enzyme - was examined in a rabbit by double-sandwich enzyme-linked immunosorbent assay (ELISA). The animal received intramuscularly a 1.0-mg/kg dose of C. rhodostoma venom. The venom level in serum peaked 12 hours after the injection, followed by a gradual decline and finally reached low rates 72 hours after administration. The serum kinetic profile of venom components, however, did not correspond to the profile of the whole C. rhodostoma venom. The serum levels of the C. rhodostoma thrombin-like enzyme increased slowly and peaked only 48 hours post-injection. Then both thrombin-like enzyme and rhodostoxin remained at relatively high levels 72 hours after administration. Data suggest that various venom components bind to tissue at the injection site with different affinities and that conjugated venom components were continuously released into circulation at different rates. The prolonged high serum levels of both thrombin-like enzyme and hemorrhagin are consistent with the clinical picture of prolonged clotting deficiency in severe cases of C. rhodostoma envenomation. Our results also suggest that since venom components are being released into and eliminated from the circulation at different rates, the "average composition" of the venom antigen in the circulation changes over time. This implies that data from ELISA quantification of antigen levels from serum venom employing "whole venom" as reagent must be interpreted with care.(AU)