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

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

Elevated plasma levels of hepatocyte growth factor in rats experimentally envenomated with Bothrops jararaca venom: Role of snake venom metalloproteases.

The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500 µL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ±â€¯91) compared with control values (68 ±â€¯18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na2-EDTA) strongly reduced the ability of Bjv to process proHGFA and generated one active band similar to that of thrombin. Since Bjv contains prothrombin and factor X activators, increased intravascular thrombin formation might partly explain the increased HGF levels after bothropic envenomation. In conclusion, these findings suggest that snake venom metalloproteases may be determinant for elevation of plasma levels of HGF in rats experimentally envenomated with Bjv.

Involvement of circulating platelets on the hyperalgesic response evoked by carrageenan and Bothrops jararaca snake venom.

BACKGROUND: The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. OBJECTIVE: To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. METHODS: Rats were (i) rendered thrombocytopenic by administration of polyclonal anti-rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. RESULTS: Platelet counts were markedly diminished in rats administered with either ARPI (± 88%) or busulfan (± 96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre-treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre-incubation with alkaline phosphatase. CONCLUSIONS: Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.

Involvement of circulating platelets on the hyperalgesic response evoked by carrageenan and Bothrops jararaca snake venom

Background:The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. Objective:To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. Methods:Rats were (i) rendered thrombocytopenic by administration of polyclonal anti-rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. Results:Platelet counts were markedly diminished in rats administered with either ARPI (±88%) or busulfan (±96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre-treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre-incubation with alkaline phosphatase. Conclusions:Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.

Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions.

BACKGROUND AND PURPOSE: The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process. EXPERIMENTAL APPROACH: We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa. KEY RESULTS: Pretreatment of the animals with BbCI (2.5 mg·kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1ß or tumour necrosis factor-α, however, did not change. CONCLUSIONS AND IMPLICATIONS: Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms.

Species of the genus Bothrops induce the vast majority of snakebite envenomings in Latin America. A preclinical study was performed in the context of a regional network of public laboratories involved in the production, quality control and development of antivenoms in Latin America. The ability of seven polyspecific antivenoms, produced in Argentina, Brazil, Peru, Bolivia, Colombia and Costa Rica, to neutralize lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities of the venoms of Bothrops neuwiedi (diporus) (Argentina), Bothrops jararaca (Brazil), B. neuwiedi (mattogrossensis) (Bolivia), Bothrops atrox (Peru and Colombia) and Bothrops asper (Costa Rica) was assessed using standard laboratory tests. Despite differences in the venom mixtures used in the immunization of animals for the production of these antivenoms, a pattern of extensive cross-neutralization was observed between these antivenoms and all the venoms tested, with quantitative differences in the values of effective doses. This study reveals the capacity of these antivenoms to neutralize, in preclinical tests, homologous and heterologous Bothrops venoms in Central and South America, and also highlight quantitative differences in the values of Median Effective Doses (ED50s) between the various antivenoms.

Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions

The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (Kiapp 5.3 nM) and cathepsin G (Kiapp 160.0 nM), and the cysteine peptidase cathepsin L (Kiapp 0.2 nM). These three peptidases play important roles in the inflammatory process. We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte–endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.Pretreatment of the animals with BbCI (2.5 mg·kg−1), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1â or tumour necrosis factor-á, however, did not change.Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Crotoxin is responsible for the long-lasting anti-inflammatory effect of Crotalus durissus terrificus snake venom: involvement of formyl peptide receptors

In the present study, it was investigated which components are responsible for the antiinflammatory properties of Crotalus durissus terrificus venom (CdtV). The effect of crotoxin,as well as of other CdtV components was evaluated on edema, cell migration and alterations in leukocyte–endothelium interactions induced by carrageenan. Crotoxin (40 mg kg 1) was injected at different time periods before or after the injection of carrageenan (15 mg kg 1)into the mouse hind paw, peritoneum or scrotum. Results showed that crotoxin, but not other CdtV components, significantly inhibited inflammatory edema and cell migration when administered before or after carrageenan injection in mice. This toxin also prevented the occurrence of alterations in leukocyte–endothelium interactions induced by carrageenaninjection, such as the increase in adhered cells. In animals pretreated with Boc2 (a selective antagonist of formyl peptide receptors), crotoxin showed neither inhibitoryeffects on edema and cell migration, nor prevented alterations in leukocyte–endothelium interactions induced by carrageenan. These findings demonstrate that crotoxin is thecomponent responsible for the long-lasting anti-inflammatory activity of crude C. durissus terrificus venom, and activation of formyl peptide receptors seems to play a major role inthis effect.

Crotoxin is responsible for the long-lasting anti-inflammatory effect of Crotalus durissus terrificus snake venom: involvement of formyl peptide receptors.

In the present study, it was investigated which components are responsible for the anti-inflammatory properties of Crotalus durissus terrificus venom (CdtV). The effect of crotoxin, as well as of other CdtV components was evaluated on edema, cell migration and alterations in leukocyte-endothelium interactions induced by carrageenan. Crotoxin (40 microg kg(-1)) was injected at different time periods before or after the injection of carrageenan (15 mg kg(-1)) into the mouse hind paw, peritoneum or scrotum. Results showed that crotoxin, but not other CdtV components, significantly inhibited inflammatory edema and cell migration when administered before or after carrageenan injection in mice. This toxin also prevented the occurrence of alterations in leukocyte-endothelium interactions induced by carrageenan injection, such as the increase in adhered cells. In animals pretreated with Boc2 (a selective antagonist of formyl peptide receptors), crotoxin showed neither inhibitory effects on edema and cell migration, nor prevented alterations in leukocyte-endothelium interactions induced by carrageenan. These findings demonstrate that crotoxin is the component responsible for the long-lasting anti-inflammatory activity of crude C. durissus terrificus venom, and activation of formyl peptide receptors seems to play a major role in this effect.

Effect of dexamethasone associated with serum therapy on treatment of Bothrops jararaca venom-induced paw edema in mice.

OBJECTIVE AND DESIGN: The present study investigates the supposed advantage of using an association of anti-inflammatory drugs and serum therapy to treat mouse paw edema induced by injection of Bothrops jararaca snake venom (BjV). MATERIAL AND METHODS: Edema was induced by injecting BjV (2 microg) into the footpad of male Swiss mice (20-25 g) and measured by plethysmography. Groups of mice were treated 15, 30 or 45 min after BjV injection with Bothrops antivenom or anti-inflammatory drugs (dexamethasone, indomethacin or zileuton), or with the association of antivenom and each one of these drugs. RESULTS: Antivenom, dexamethasone and indomethacin were effective in reducing the paw edema when used up to 30 min after BjV injection. Zileuton had the same effect, but only if used up to 15 min after BjV injection. The association of antivenom and dexamethasone showed the greatest inhibitory effect when used up to 45 min after BjV injection. At this time, antivenom or anti-inflammatory drugs administered alone were ineffective. CONCLUSION: Results suggest that dexamethasone combination with serum therapy can be beneficial for treatment of inflammatory edema caused by B. jararaca envenomation.

Purification and characterization of patagonfibrase, a metalloproteinase showing alpha-fibrinogenolytic and hemorrhagic activities, from Philodryas patagoniensis snake venom.

Venoms of Colubridae snakes are a rich source of novel compounds, which may have applications in medicine and biochemistry. In the present study, we describe the purification and characterization of a metalloproteinase (patagonfibrase), the first protein to be isolated from Philodryas patagoniensis (Colubridae) snake venom. Patagonfibrase is a single-chain protein, showing a molecular mass of 53,224 Da and an acidic isoelectric point (5.8). It hydrolyzed selectively the Aalpha-chain of fibrinogen and when incubated with fibrinogen or plasma, the thrombin clotting time was prolonged. Prominent hemorrhage developed in mouse skin after intradermal injection of patagonfibrase. When administered into mouse gastrocnemius muscle, it induced local hemorrhage and necrosis, and systemic bleeding in lungs. Patagonfibrase showed proteolytic activity toward azocasein, which was enhanced by Ca(2+) and inhibited by Zn(2+), cysteine, dithiothreitol and Na(2)EDTA. Patagonfibrase impaired platelet aggregation induced by collagen and ADP. Thus, patagonfibrase may play a key role in the pathogenesis of disturbances that occur in P. patagoniensis envenomation, and may be used as a biological tool to explore many facets of hemostasis.

Effects of Lonomia obliqua (lepidoptera, saturniidae) toxin on clotting, inflammatory and antibody responsiveness in genetically selected lines of mice.

Lines of mice genetically selected for high (H) or low (L) antibody response and for maximal (AIRMAX) or minimal (AIRMIN) acute inflammatory reaction, in which the opposite extreme potentialities have been clearly defined, offer an appropriate model for investigating the environmental and genetic factors acting on innate and adaptative immunobiological functions. This model has been successfully employed to study the resistance or susceptibility against pathogens and/or toxins. It had been demonstrated that the skin contact with Lonomia obliqua caterpillar bristles induces local inflammation and may elicit severe hemorrhagic disorders. In the present study, blood coagulation time, and the acute inflammatory reaction were scored 24 h after injection of the Lonomia bristles crude extract in a subcutaneous dorsal air pouch. The acute inflammation was determined by the leukocyte concentration in the local exudates. The highest interline differences were observed between the AIRMAX (10(6) cells/ml) and AIRMIN (2 x 10(5) cells/ml) and this distinct expression involves the number of monocytes, eosinophils and mainly neutrophils. Regarding coagulation, the highest interline difference was observed between the HIII and LIII mice, and the F1)[LIII x HIII] hybrids showed the overdominance of the fast clotting character. The adaptative immune response was evaluated by comparing the anti-Lonomia bristle extract IgG titer among the lines: the antibody titers were higher in the H lines than in the L ones and equivalent in the AIRMAX and AIRMIN mice, in accordance to the phenotype profiles generated by the distinct selective processes. The genetically selected mice lines-AIRMAX, AIRMIN, HI, HIII, HG, LIII and LG-showed an almost continuous distributions for inflammation, coagulation time and IgG antibody titers, being the interline variances always higher than the intraline ones for the individually measured phenotypes. Altogether, these results suggest the independent polygenic regulation of these traits, being indicative of the genetic control to Lonomia toxin innate and adaptative sensitivity in humans.