Systemic toxicity of snake venom metalloproteinases: multi-omics analyses of kidney and blood plasma disturbances in a mouse model

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.

Two-Dimensional Blue Native/SDS Polyacrylamide Gel Electrophoresis for Analysis of Brazilian Bothrops Snake Venoms.

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

Tissue Factor and COVID-19: An Update.

The coronavirus 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Infection with SARS-CoV-2 is associated with acute respiratory distress syndrome, thrombosis and a high rate of mortality. Thrombotic events increase with severity. Tissue factor (TF) expression is increased during viral and bacterial infections. This review summarizes studies that have examined TF expression in response to SARS-CoV-2 infection. SARS-CoV-2 virus and its proteins upregulate TF mRNA, protein and activity in a variety of cells, including bronchial epithelial cells, neutrophils, monocytes, macrophages, endothelial cells and adventitial fibroblasts. COVID-19 patients have increased TF expression in lungs, bronchoalveolar lavage fluid and circulating extracellular vesicles. The increase in TF was associated with coagulation activation markers, thrombosis, inflammatory markers, severity of disease and mortality. Taken together, the studies suggest that TF plays a central role in thrombosis in COVID- 19. TF may be a useful prognostic marker and therapeutic target to reduce thrombosis and inflammation.

The Bioflavonoids Rutin and Rutin Succinate Neutralize the Toxins of B. jararaca Venom and Inhibit its Lethality.

The venom of the Brazilian pit viper Bothrops jararaca (BjV) is a complex mixture of molecules, and snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) are the most abundant protein families found therein. Toxins present in BjV trigger most of the deleterious disturbances in hemostasis observed in snakebites, i.e., thrombocytopenia, hypofibrinogenemia and bleedings. The treatment of patients bitten by snakes still poses challenges and the bioflavonoid rutin has already been shown to improve hemostasis in an experimental model of snakebite envenomation. However, rutin is poorly soluble in water; in this study, it was succinylated to generate its water-soluble form, rutin succinate (RS), which was analyzed comparatively regarding the chemical structure and characteristic features of rutin. Biological activities of rutin and RS were compared on hemostatic parameters, and against toxic activities of crude BjV in vitro. In vivo, C57BL/6 mice were injected i.p. with either BjV alone or pre-incubated with rutin, RS or 1,10-phenanthroline (o-phe, an SVMP inhibitor), and the survival rates and hemostatic parameters were analyzed 48 h after envenomation. RS showed the characteristic activities described for rutin - i.e., antioxidant and inhibitor of protein disulfide isomerase - but also prolonged the clotting time of fibrinogen and plasma in vitro. Differently from rutin, RS inhibited typical proteolytic activities of SVMP, as well as the coagulant activity of BjV. Importantly, both rutin and RS completely abrogated the lethal activity of BjV, in the same degree as o-phe. BjV induced hemorrhages, falls in RBC counts, thrombocytopenia and hypofibrinogenemia in mice. Rutin and RS also improved the recovery of platelet counts and fibrinogen levels, and the development of hemorrhages was totally blocked in mice injected with BjV incubated with RS. In conclusion, RS has anticoagulant properties and is a novel SVMP inhibitor. Rutin and RS showed different mechanisms of action on hemostasis. Only RS inhibited directly BjV biological activities, even though both flavonoids neutralized B. jararaca toxicity in vivo. Our results showed clearly that rutin and RS show a great potential to be used as therapeutic compounds for snakebite envenomation.

Two-dimensional blue native/SDS polyacrylamide gel electrophoresis for analysis of Brazilian Bothrops snake venoms

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

Tissue factor and COVID-19: an update

The coronavirus 2019 [COVID-19] pandemic is caused by severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] virus. Infection with SARS-CoV-2 is associated with an acute respiratory distress syndrome, thrombosis and a high rate of mortality. Thrombotic events increase with severity. Tissue factor [TF] expression is increased during viral and bacterial infections. This review summarizes studies that have examined TF expression in response to SARS-CoV-2 infection. SARS-CoV-2 virus and its proteins upregulate TF mRNA, protein and activity in a variety of cells, including bronchial epithelial cells, neutrophils, monocytes, macrophages, endothelial cells and adventitial fibroblasts. COVID-19 patients have increased TF expression in lungs, bronchoalveolar lavage fluid and circulating extracellular vesicles. The increase in TF was associated with coagulation activation markers, thrombosis, inflammatory markers, severity of disease and mortality. Taken together, the studies suggest that TF plays a central role in thrombosis in COVID-19. TF may be a useful prognostic marker and therapeutic target to reduce thrombosis and inflammation.

The bioflavonoids rutin and rutin succinate neutralize the toxins of B. jararaca venom and inhibit its lethality

The venom of the Brazilian pit viper Bothrops jararaca (BjV) is a complex mixture of molecules, and snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) are the most abundant protein families found therein. Toxins present in BjV trigger most of the deleterious disturbances in hemostasis observed in snakebites, i.e., thrombocytopenia, hypofibrinogenemia and bleedings. The treatment of patients bitten by snakes still poses challenges and the bioflavonoid rutin has already been shown to improve hemostasis in an experimental model of snakebite envenomation. However, rutin is poorly soluble in water; in this study, it was succinylated to generate its water-soluble form, rutin succinate (RS), which was analyzed comparatively regarding the chemical structure and characteristic features of rutin. Biological activities of rutin and RS were compared on hemostatic parameters, and against toxic activities of crude BjV in vitro. In vivo, C57BL/6 mice were injected i.p. with either BjV alone or pre-incubated with rutin, RS or 1,10-phenanthroline (o-phe, an SVMP inhibitor), and the survival rates and hemostatic parameters were analyzed 48 h after envenomation. RS showed the characteristic activities described for rutin – i.e., antioxidant and inhibitor of protein disulfide isomerase – but also prolonged the clotting time of fibrinogen and plasma in vitro. Differently from rutin, RS inhibited typical proteolytic activities of SVMP, as well as the coagulant activity of BjV. Importantly, both rutin and RS completely abrogated the lethal activity of BjV, in the same degree as o-phe. BjV induced hemorrhages, falls in RBC counts, thrombocytopenia and hypofibrinogenemia in mice. Rutin and RS also improved the recovery of platelet counts and fibrinogen levels, and the development of hemorrhages was totally blocked in mice injected with BjV incubated with RS. In conclusion, RS has anticoagulant properties and is a novel SVMP inhibitor. Rutin and RS showed different mechanisms of action on hemostasis. Only RS inhibited directly BjV biological activities, even though both flavonoids neutralized B. jararaca toxicity in vivo. Our results showed clearly that rutin and RS show a great potential to be used as therapeutic compounds for snakebite envenomation.

Involvement of von Willebrand factor and botrocetin in the thrombocytopenia induced by Bothrops jararaca snake venom.

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts.

Involvement of von Willebrand factor and botrocetin in the thrombocytopenia induced by Bothrops jararaca snake venom

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts.

Liver gene regulation of hemostasis-related factors is altered by experimental snake envenomation in mice.

Few studies have addressed gene expression of hemostasis-related factors during acute thrombo-hemorrhagic diseases. Bites by the lanced-headed viper Bothrops jaracaca induce rapid hemostatic disturbances in victims, leading to systemic bleedings, thrombocytopenia and consumption coagulopathy. Although circulating levels of coagulation factors recover rapidly after administration of specific antivenom therapy, it is unclear if B. jararaca venom (BjV) upregulates the mRNA synthesis of hepatic hemostasis-related factors, or if the recovery occurs under basal conditions after the neutralization of venom components by antivenom. Thus, we aimed to investigate if BjV regulates gene expression of important hemostasis-related factors synthetized by the liver. On that account, Swiss mice were injected with saline or BjV (1.6 mg/kg b.w, s.c.), and after 3, 6 and 24 h blood samples and liver fragments were collected to analyze mRNA expression by real-time qPCR. Increased gene expression of fibrinogen chains, haptoglobin and STAT3 was observed during envenomation, particularly at 3 and 6 h. At 24h, mRNA levels of F10 were raised, while those of Serpinc1, Proc and Adamts13 were diminished. Surprisingly, F3 mRNA levels were steadily decreased at 3 h. Gene expression of Thpo, F7, F5 Tfpi, Mug1 was unaltered. mRNA levels of Vwf, P4hb, F8, F2, Plg, and Serpinf2 were minimally altered, but showed important associations with Nfkb1 gene expression. In conclusion, snakebite envenomation upregulates hepatic mRNA synthesis particularly of fibrinogen chains, and acute-phase markers. This response explains the fast recovery of fibrinogen levels after antivenom administration to patients bitten by B. jararaca snakes.

Liver gene regulation of hemostasis-related factors is altered by experimental snake envenomation in mice

Few studies have addressed gene expression of hemostasis-related factors during acute thrombo-hemorrhagic diseases. Bites by the lanced-headed viper Bothrops jaracaca induce rapid hemostatic disturbances in victims, leading to systemic bleedings, thrombocytopenia and consumption coagulopathy. Although circulating levels of coagulation factors recover rapidly after administration of specific antivenom therapy, it is unclear if B. jararaca venom (BjV) upregulates the mRNA synthesis of hepatic hemostasis-related factors, or if the recovery occurs under basal conditions after the neutralization of venom components by antivenom. Thus, we aimed to investigate if BjV regulates gene expression of important hemostasis-related factors synthetized by the liver. On that account, Swiss mice were injected with saline or BjV (1.6 mg/kg b.w, s.c.), and after 3, 6 and 24 h blood samples and liver fragments were collected to analyze mRNA expression by real-time qPCR. Increased gene expression of fibrinogen chains, haptoglobin and STAT3 was observed during envenomation, particularly at 3 and 6 h. At 24h, mRNA levels of F10 were raised, while those of Serpinc1, Proc and Adamts13 were diminished. Surprisingly, F3 mRNA levels were steadily decreased at 3 h. Gene expression of Thpo, F7, F5 Tfpi, Mug1 was unaltered. mRNA levels of Vwf, P4hb, F8, F2, Plg, and Serpinf2 were minimally altered, but showed important associations with Nfkb1 gene expression. In conclusion, snakebite envenomation upregulates hepatic mRNA synthesis particularly of fibrinogen chains, and acute-phase markers. This response explains the fast recovery of fibrinogen levels after antivenom administration to patients bitten by B. jararaca snakes.

Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes.

Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses.

Optimization of von Willebrand factor multimer analysis in vertical mini-gel electrophoresis systems: A rapid procedure.

Von Willebrand disease (VWD) is a common cause of bleeding worldwide. Analysis of von Willebrand factor (VWF) multimer distribution (VWF:MD) is essential to properly classify and treat different types of VWD, and it is performed using a SDS agarose gel electrophoresis followed by Western blotting, a handmade technique that demands days to be completed and requires skillful execution. Aiming both to facilitate gel production and to shorten the preparation time, we developed an uncomplicated technique to provide agility in the analysis of VWF:MD, so that it can be easily accomplished in the routine practice of hemostasis laboratories. On that account, we used a commercial vertical mini-gel electrophoresis system for SDS-PAGE and a semi-dry transfer system, which allowed us to analyze VWF:MD of various samples in a period shorter than 12 h. This technique differentiated VWF:MD in human and animal plasmas under normal, congenital and acquired (experimental envenomation by Bothrops jararaca snake) conditions. This optimized method is cheap, rapid, reproducible, easy to be performed, and uses electrophoresis and Western blotting systems available in most laboratories. All these advantages encourage hemostasis professionals to use it in their routine practices. In order to facilitate the setup and accomplishment of the whole procedure step by step, videos were appended to the article.

Modulation of the mammalian coagulation system by venoms and other proteins from snakes, arthropods, nematodes and insects

The mammalian hemostatic system involves complex interactions between protein components of the coagulation cascade and platelets. The fibrinolytic system removes the hemostatic plug. Dysregulation of coagulation or fibrinolytic systems can induce bleeding or thrombosis. Animals, such as snakes, worms and insects, have evolved to express proteins that modulate the mammalian coagulation and fibrinolytic systems. Many of these proteins have been isolated and characterized. Understanding the mechanisms by which these exogenous factors from venoms and animal saliva modulate the mammalian coagulation and fibrinolytic systems has led to a better understanding of these systems. Furthermore, some of these exogenous proteins are used in diagnostic assays and as therapeutic drugs. This review summarizes our current knowledge of exogenous proteins from venom and saliva that either activate or inhibit the mammalian coagulation and fibrinolytic systems.

Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes

Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial struc-ture/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here,we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. Wevalidated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual(median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably,plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (> median)plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping func-tions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, in-flammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs.PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phe-notype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene ex-pression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such sig-natures translated into functional responses, with PDI-poor plasma promoting impairment of endothelialadhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients withcardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels asreporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes andfunctional responses.

Optimization of von Willebrand factor multimer analysis in vertical mini-gel electrophoresis systems: a rapid procedure

Von Willebrand disease (VWD) is a common cause of bleeding worldwide. Analysis of von Willebrand factor (VWF) multimer distribution (VWF:MD) is essential to properly classify and treat different types of VWD, and it is performed using a SDS agarose gel electrophoresis followed by Western blotting, a handmade technique that demands days to be completed and requires skillful execution. Aiming both to facilitate gel production and to shorten the preparation time, we developed an uncomplicated technique to provide agility in the analysis of VWF:MD, so that it can be easily accomplished in the routine practice of hemostasis laboratories. On that account, we used a commercial vertical mini-gel electrophoresis system for SDS-PAGE and a semi-dry transfer system, which allowed us to analyze VWF:MD of various samples in a period shorter than 12?h. This technique differentiated VWF:MD in human and animal plasmas under normal, congenital and acquired (experimental envenomation by Bothrops jararaca snake) conditions. This optimized method is cheap, rapid, reproducible, easy to be performed, and uses electrophoresis and Western blotting systems available in most laboratories. All these advantages encourage hemostasis professionals to use it in their routine practices. In order to facilitate the setup and accomplishment of the whole procedure step by step, videos were appended to the article.

Rutin (quercetin-3-rutinoside) modulates the hemostatic disturbances and redox imbalance induced by Bothrops jararaca snake venom in mice.

Snakebites are a major Collective Health problem worldwide. In Brazil, Bothrops jararaca snake venom (BjV) evokes hemostatic disturbances, bleeding manifestations, and redox status imbalance. Specific antivenom therapy, although efficacious to revert most snakebite-induced manifestations, is incapable of treating secondary manifestations, such as oxidative/nitrosative stress. Searching for new complementary therapies that could attenuate physiological derangements triggered by envenomation, we elected to test quercetin-3-rutinoside (rutin) by its potential as both a potent antioxidant and a hemostasis modulatory compound. The activity of rutin was evaluated both on the biological activities of crude BjV in vitro, and in vivo by the ability of rutin (14.4 mg/kg b.w.) to modulate hematological, hemostatic and redox status markers altered by BjV injection (1.6 mg/kg b.w., s.c.) in mice. In vitro, rutin failed to inhibit BjV-induced platelet aggregation and biological activities of major BjV enzymes (metalloproteinases, phospholipases A2, serine proteases, and L-amino acid oxidases). On the other hand, rutin attenuated local hemorrhage, and the increase in reactive species, prevented the fall in RBC counts and fibrinogen levels, diminished tail bleeding and shortened prothrombin time (PT) evoked by envenomation. Furthermore, rutin reduced tissue factor (TF) activity and altered the protein expression of TF in liver, lungs, heart and skin. In conclusion, the disturbances in redox status and hemostatic system induced by B. jararaca envenomation were modulated by rutin, suggesting it has a great potential to be used as an ancillary therapeutic agent for snakebites.

Rutin (quercetin-3-rutinoside) modulates the hemostatic disturbances and redox imbalance induced by Bothrops jararaca snake venom in mice

Snakebites are a major Collective Health problem worldwide. In Brazil, Bothrops jararaca snake venom (BjV) evokes hemostatic disturbances, bleeding manifestations, and redox status imbalance. Specific antivenom therapy, although efficacious to revert most snakebite-induced manifestations, is incapable of treating secondary manifestations, such as oxidative/nitrosative stress. Searching for new complementary therapies that could attenuate physiological derangements triggered by envenomation, we elected to test quercetin-3-rutinoside (rutin) by its potential as both a potent antioxidant and a hemostasis modulatory compound. The activity of rutin was evaluated both on the biological activities of crude BjV in vitro, and in vivo by the ability of rutin (14.4 mg/kg b.w.) to modulate hematological, hemostatic and redox status markers altered by BjV injection (1.6 mg/kg b.w., s.c.) in mice. In vitro, rutin failed to inhibit BjV-induced platelet aggregation and biological activities of major BjV enzymes (metalloproteinases, phospholipases A2, serine proteases, and L-amino acid oxidases). On the other hand, rutin attenuated local hemorrhage, and the increase in reactive species, prevented the fall in RBC counts and fibrinogen levels, diminished tail bleeding and shortened prothrombin time (PT) evoked by envenomation. Furthermore, rutin reduced tissue factor (TF) activity and altered the protein expression of TF in liver, lungs, heart and skin. In conclusion, the disturbances in redox status and hemostatic system induced by B. jararaca envenomation were modulated by rutin, suggesting it has a great potential to be used as an ancillary therapeutic agent for snakebites.