In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victim’s local effects and time to heal.

In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victims local effects and time to heal.(AU)

Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Background:Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods:The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results:The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion:These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Abstract Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

South American rattlesnake (Crotalus durissus spp) envenomation in dogs in the semiarid region of Brazil

Background: South American rattlesnake (Crotalus durissus spp.) envenomation is rarely reported in small animals andlivestock in Brazil. Minor swelling at the snakebite site, skeletal muscle, and renal damage, and severe neurological signscharacterize the crotalic envenomation. This case report aims to present epidemiological, clinical, and pathological dataof two cases of Crotalus durissus spp envenomation in dogs in the Northeast of Brazil.Cases: Envenomation by Crotalus durissus spp. was recorded in two dogs in Patos, State of Paraíba, Brazil. In Case 1,the dog presented flaccid paralysis, hyporeflexia, a deficit of cranial nerves, epistaxis, and gingival hemorrhages. Laboratory assay showed proteinuria, myoglobinuria, regenerative thrombocytopenia, and increased serum activities of creatinekinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). The dogwas medicated with crotalic antivenom and wholly recovered from local and systemic clinical signs. In Case 2, the dogdied and was detected fang marks at the ventral region of the left mandible (two small parallel perforations spaced 2.0 cmapart) at the snakebite site. Cyanosis of the oral cavity, congestion, and hemorrhages in several organs were observed atnecropsy. Tubular nephrosis, muscular necrosis, hepatocytes swelling were observed. The owners witnessed snakebites,and the rattlesnakes (Crotalus durissus spp.) identified by the rattle at the end portion of the tail in both cases.Discussion: Natural South American rattlesnake envenomation presents complex clinical signs that makes diagnosis achallenge for veterinary practitioners. The criteria for the correct diagnosis and observed in the two dogs include witness ofthe snakebite, identification of the snake, detection of fang marks, clinical-pathological findings, and therapeutic responseto treatment with specific anti-venom....

South American rattlesnake (Crotalus durissus spp) envenomation in dogs in the semiarid region of Brazil

Background: South American rattlesnake (Crotalus durissus spp.) envenomation is rarely reported in small animals andlivestock in Brazil. Minor swelling at the snakebite site, skeletal muscle, and renal damage, and severe neurological signscharacterize the crotalic envenomation. This case report aims to present epidemiological, clinical, and pathological dataof two cases of Crotalus durissus spp envenomation in dogs in the Northeast of Brazil.Cases: Envenomation by Crotalus durissus spp. was recorded in two dogs in Patos, State of Paraíba, Brazil. In Case 1,the dog presented flaccid paralysis, hyporeflexia, a deficit of cranial nerves, epistaxis, and gingival hemorrhages. Laboratory assay showed proteinuria, myoglobinuria, regenerative thrombocytopenia, and increased serum activities of creatinekinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). The dogwas medicated with crotalic antivenom and wholly recovered from local and systemic clinical signs. In Case 2, the dogdied and was detected fang marks at the ventral region of the left mandible (two small parallel perforations spaced 2.0 cmapart) at the snakebite site. Cyanosis of the oral cavity, congestion, and hemorrhages in several organs were observed atnecropsy. Tubular nephrosis, muscular necrosis, hepatocytes swelling were observed. The owners witnessed snakebites,and the rattlesnakes (Crotalus durissus spp.) identified by the rattle at the end portion of the tail in both cases.Discussion: Natural South American rattlesnake envenomation presents complex clinical signs that makes diagnosis achallenge for veterinary practitioners. The criteria for the correct diagnosis and observed in the two dogs include witness ofthe snakebite, identification of the snake, detection of fang marks, clinical-pathological findings, and therapeutic responseto treatment with specific anti-venom....(AU)

Non-Avian Reptiles of the state of Rio de Janeiro, Brazil: status of knowledge and commented list

Abstract We assessed the current knowledge on non-avian reptile species composition in the state of Rio de Janeiro (RJ), southeastern Brazil. We used published data in indexed journals and verified voucher specimens housed in the herpetological collections of the Museu Nacional (UFRJ), and the Laboratory of Reptiles (ZUFRJ). We also confirmed vouchers from Instituto Vital Brazil (IVB) and from the Museum of Comparative Zoology (MCZ). We compiled a list containing 149 species of reptiles, distributed among Testudines (N = 15), Crocodylia (N = 1) and Squamata (N = 133; six amphisbaenians, 38 lizards and 89 snakes). Our results add 20 species to the previous list known for the state. Four species recorded are endemic to the state of Rio de Janeiro (Anolis neglectus, Glaucomastix littoralis, Leposternon scutigerum, and Liolaemus lutzae). We identified that 21 reptile species recorded in RJ state (nearly 15% of the total) are included in some threat category either in the IUCN, Brazilian (ICMBio) or state lists of endangered species. We also report that seven of the reptiles recorded are non-indigenous to the state: Cnemidophorus aff. lemniscatus (= Cnemidophorus cryptus), Anolis sagrei, Trachemys scripta, Trachemys dorbigni, Hemidactylus mabouia, Crotalus durissus terrificus, and Rhinoclemmys punctularia. The checklist presented here provides a comprehensive database for further research on the herpetofauna of the Brazilian Atlantic Forest.

Non-Avian Reptiles of the state of Rio de Janeiro, Brazil: status of knowledge and commented list

We assessed the current knowledge on non-avian reptile species composition in the state of Rio de Janeiro (RJ), southeastern Brazil. We used published data in indexed journals and verified voucher specimens housed in the herpetological collections of the Museu Nacional (UFRJ), and the Laboratory of Reptiles (ZUFRJ). We also confirmed vouchers from Instituto Vital Brazil (IVB) and from the Museum of Comparative Zoology (MCZ). We compiled a list containing 149 species of reptiles, distributed among Testudines (N = 15), Crocodylia (N = 1) and Squamata (N = 133; six amphisbaenians, 38 lizards and 89 snakes). Our results add 20 species to the previous list known for the state. Four species recorded are endemic to the state of Rio de Janeiro (Anolis neglectus, Glaucomastix littoralis, Leposternon scutigerum, and Liolaemus lutzae). We identified that 21 reptile species recorded in RJ state (nearly 15% of the total) are included in some threat category either in the IUCN, Brazilian (ICMBio) or state lists of endangered species. We also report that seven of the reptiles recorded are non-indigenous to the state: Cnemidophorus aff. lemniscatus (= Cnemidophorus cryptus), Anolis sagrei, Trachemys scripta, Trachemys dorbigni, Hemidactylus mabouia, Crotalus durissus terrificus, and Rhinoclemmys punctularia. The checklist presented here provides a comprehensive database for further research on the herpetofauna of the Brazilian Atlantic Forest.

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

Abstract Background: Endogenous phospholipase A2 inhibitors from snake blood (sbPLIs) have been isolated from several species around the world, with the primary function of self-protection against the action of toxic phospholipases A2. In American snakes, sbPLIs were solely described in pit vipers, in which the natural protection role is justified. In this study, we described a sbPLI in Boa constrictor (popularly known as jiboia), a non-venomous snake species from America. Methods: PLA2 inhibitory activity was tested in the blood plasma of B. constrictor using C. d. terrificus venom as the enzyme source. Antibodies developed against CNF, a sbγPLI from Crotalus durissus terrificus, were used to investigate the presence of homologues in the blood plasma of B. constrictor. A CNF-like molecule with a PLA2 inhibitory activity was purified by column chromatography. The encoding gene for the inhibitor was cloned from B. constrictor liver tissue. The DNA fragment was cloned, purified and sequenced. The deduced primary sequence of interest was aligned with known sbγPLIs from the literature. Results: The blood plasma of B. constrictor displayed PLA2 inhibitory activity. A CNF-like molecule (named BcNF) was identified and purified from the blood plasma of B. constrictor. Basic properties such as molecular mass, composing amino acids, and pI were comparable, but BcNF displayed reduced specific activity in PLA2 inhibition. BcNF showed highest identity scores (ISs) with sbγPLIs from pit vipers from Latin America (90-100%), followed by gamma inhibitors from Asian viperid (80-90%). ISs below 70% were obtained for BcNF and non-venomous species from Asia. Conclusion: A functional sbγPLI (BcNF) was described in the blood plasma of B. constrictor. BcNF displayed higher primary identity with sbγPLIs from Viperidae than to sbγPLIs from non-venomous species from Asia. The physiological role played by sbγPLIs in non-venomous snake species remains to be understood. Further investigation is needed.(AU)

Non-Avian Reptiles of the state of Rio de Janeiro, Brazil: status of knowledge and commented list

We assessed the current knowledge on non-avian reptile species composition in the state of Rio de Janeiro (RJ), southeastern Brazil. We used published data in indexed journals and verified voucher specimens housed in the herpetological collections of the Museu Nacional (UFRJ), and the Laboratory of Reptiles (ZUFRJ). We also confirmed vouchers from Instituto Vital Brazil (IVB) and from the Museum of Comparative Zoology (MCZ). We compiled a list containing 149 species of reptiles, distributed among Testudines (N = 15), Crocodylia (N = 1) and Squamata (N = 133; six amphisbaenians, 38 lizards and 89 snakes). Our results add 20 species to the previous list known for the state. Four species recorded are endemic to the state of Rio de Janeiro (Anolis neglectus, Glaucomastix littoralis, Leposternon scutigerum, and Liolaemus lutzae). We identified that 21 reptile species recorded in RJ state (nearly 15% of the total) are included in some threat category either in the IUCN, Brazilian (ICMBio) or state lists of endangered species. We also report that seven of the reptiles recorded are non-indigenous to the state: Cnemidophorus aff. lemniscatus (= Cnemidophorus cryptus), Anolis sagrei, Trachemys scripta, Trachemys dorbigni, Hemidactylus mabouia, Crotalus durissus terrificus, and Rhinoclemmys punctularia. The checklist presented here provides a comprehensive database for further research on the herpetofauna of the Brazilian Atlantic Forest.(AU)

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

Background:Endogenous phospholipase A2 inhibitors from snake blood (sbPLIs) have been isolated from several species around the world, with the primary function of self-protection against the action of toxic phospholipases A2. In American snakes, sbPLIs were solely described in pit vipers, in which the natural protection role is justified. In this study, we described a sbPLI in Boa constrictor (popularly known as jiboia), a non-venomous snake species from America.Methods:PLA2 inhibitory activity was tested in the blood plasma of B. constrictor using C. d. terrificus venom as the enzyme source. Antibodies developed against CNF, a sbγPLI from Crotalus durissus terrificus, were used to investigate the presence of homologues in the blood plasma of B. constrictor. A CNF-like molecule with a PLA2 inhibitory activity was purified by column chromatography. The encoding gene for the inhibitor was cloned from B. constrictor liver tissue. The DNA fragment was cloned, purified and sequenced. The deduced primary sequence of interest was aligned with known sbγPLIs from the literature.Results:The blood plasma of B. constrictor displayed PLA2 inhibitory activity. A CNF-like molecule (named BcNF) was identified and purified from the blood plasma of B. constrictor. Basic properties such as molecular mass, composing amino acids, and pI were comparable, but BcNF displayed reduced specific activity in PLA2 inhibition. BcNF showed highest identity scores (ISs) with sbγPLIs from pit vipers from Latin America (90-100%), followed by gamma inhibitors from Asian viperid (80-90%). ISs below 70% were obtained for BcNF and non-venomous species from Asia.Conclusion:A functional sbγPLI (BcNF) was described in the blood plasma of B. constrictor. BcNF displayed higher primary identity with sbγPLIs from Viperidae than...(AU)

Fractal dimension in liver histological findings of Wistar rats experimentally intoxicated with venom of Crotalus durissus terrificus

Background: Accidents caused by venom of Crotalus durissus snakes, popularly known in Brazil as rattlesnake, aresecond in relation to the occurrence and first place in deaths in humans and animals, mainly due to the great neurotoxic,myotoxic, coagulant, nephrotoxic and hepatotoxic potential of their venom. The effects observed are due to the action ofthe main poison fractions and among them we can mention crotoxin (representing 50% of the total poison), crotamine,gyroxine and conxulxin. The present study aimed to analyze the liver of experimentally poisoned Wistar rats with venomof Crotalus durissus terrificus by means of histological and fractal analysis. The hypothesis is that the venom of Crotalusdurissus terrificus is can induce hepatic damage at the dose recommended in this study, that its alterations can be quantifiedby the fractal dimension and that the antiofidic serum botropic crotalic be able to minimize the hepatic lesions inducedby the venom.Materials, Methods & Results: Ninety rats were distributed into different groups and treated with: control group (GC, n= 30) 0.9% sodium chloride solution; venom group (GV, n = 30) crotalic venom at the dose of 1 mg/kg; (GVS, n = 30)crotalic venom at the dose of 1 mg/Kg and antiofidic serum 6 h after the application of the venom at the dose recommendedby the manufacturer. Liver samples were collected at 2 h (M1), 8 h (M2) and 24 h (M3) after venom administration andsubmitted to histological analysis and fractal dimension (DF) using the ImageJ® software and box-counting method. Procedures for collecting, processing and analyzing samples were standardized. For statistical analyzes, after the normalitywas verified by the Shapiro-Wilk test and homogeneity by the Bartlett test, the data were submitted to analysis of variance(ANOVA) with Duncan test contrast with a significance level of 5%. No significant lesions were observed in GC, howevernecrosis...(AU)

Fractal dimension in liver histological findings of Wistar rats experimentally intoxicated with venom of Crotalus durissus terrificus

Background: Accidents caused by venom of Crotalus durissus snakes, popularly known in Brazil as rattlesnake, aresecond in relation to the occurrence and first place in deaths in humans and animals, mainly due to the great neurotoxic,myotoxic, coagulant, nephrotoxic and hepatotoxic potential of their venom. The effects observed are due to the action ofthe main poison fractions and among them we can mention crotoxin (representing 50% of the total poison), crotamine,gyroxine and conxulxin. The present study aimed to analyze the liver of experimentally poisoned Wistar rats with venomof Crotalus durissus terrificus by means of histological and fractal analysis. The hypothesis is that the venom of Crotalusdurissus terrificus is can induce hepatic damage at the dose recommended in this study, that its alterations can be quantifiedby the fractal dimension and that the antiofidic serum botropic crotalic be able to minimize the hepatic lesions inducedby the venom.Materials, Methods & Results: Ninety rats were distributed into different groups and treated with: control group (GC, n= 30) 0.9% sodium chloride solution; venom group (GV, n = 30) crotalic venom at the dose of 1 mg/kg; (GVS, n = 30)crotalic venom at the dose of 1 mg/Kg and antiofidic serum 6 h after the application of the venom at the dose recommendedby the manufacturer. Liver samples were collected at 2 h (M1), 8 h (M2) and 24 h (M3) after venom administration andsubmitted to histological analysis and fractal dimension (DF) using the ImageJ® software and box-counting method. Procedures for collecting, processing and analyzing samples were standardized. For statistical analyzes, after the normalitywas verified by the Shapiro-Wilk test and homogeneity by the Bartlett test, the data were submitted to analysis of variance(ANOVA) with Duncan test contrast with a significance level of 5%. No significant lesions were observed in GC, howevernecrosis...

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1. Methods: Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins. Results: When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis Conclusion: These results suggest that the recombinant enzyme has no potential as a thrombolytic agent but can be applied in the prevention of thrombus formation in some pathological processes and as molecular tools in studies related to hemostasis.(AU)

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

Background:The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1.Methods:Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins.Results:When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis...(AU)

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)

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)

Hormonal and ultrasonographic characterization of the seasonal reproductive cycle of male and female Crotalus durissus terrificus

Research concerning to characterize seasonal reproductive cycle in males and females of Crotalus durissus terrificus by ultrasound and hormonal measurement. Reproductive aspects (follicular and testicular cycles, and pregnancy) from 28 adult snakes (14 males and 14 females) during different months of the years were studied. Snakes housed individually in cages in an environment with controlled luminosity and humidity, and fed monthly. Females were examined by ultrasound during quiescence and active follicular phase, and pregnancy for follicular and embryo/fetal development. Males were evaluated to testicular echotexture and measurements during reproductive and non-reproductive season. The blood samples were collected from males and females for serum testosterone and progesterone determination, respectively. In 77% males the testes were identified by ultrasound and found increased size during summer, decreased serum testosterone in winter, and positive correlation between serum testosterone and testes size. There was no change in testicular echotexture in according to season. Testosterone concentration was decreased during winter and it was positively correlated with testes size. In 71% females, were observed follicular development (vitellogenesis) and gestation since winter to spring by ultrasonography. Parturition occurred mainly in summer. Pregnancy length was 123.0 ± 11.4 days, with mean 6.9 ± 1.5 newborns/female, and there was gradual increase of serum progesterone during this period. There was no variation in progesterone concentration in nongravid females. Males and females Tropical Rattlesnake show seasonal variation of reproductive cycle and was clear a biennial cycle in female. The ultrasonography can be considered an essential tool to accomplish the follicular development, pregnancy and testicular alterations in Tropical Rattlesnake.(AU)