Evaluation of a mimotope of the Rickettsia outer membrane protein A (OmpA) as an antigen in enzyme-linked immunosorbent assay to detect rickettsiosis in capybaras (Hydrochoerus hydrochaeris), horses (Equus caballus), and opossums (Didelphis sp.).

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever, which is an important tick-borne zoonosis and, in Brazil, it causes Brazilian spotted fever, which has high lethality rate. This study aimed to evaluate a synthetic peptide corresponding to a segment of the outer membrane protein A (OmpA) as an antigen in a serological test for the diagnosis of rickettsial infections. The amino acid sequence of the peptide was selected by predicting B cell epitopes using B Cell Epitope Prediction (Immune Epitope Database and Analysis Resource) and Epitopia and OmpA sequences of Rickettsia rickettsii strain 'Brazil' and Rickettsia parkeri strains 'Maculatum 20' and 'Portsmouth'. A peptide with amino acid sequence common to both Rickettsia species was synthesized and arbitrarily named OmpA-pLMC. To evaluate this peptide in enzyme-linked immunosorbent assay (ELISA), serum samples of capybara (Hydrochoerus hydrochaeris), horse (Equus caballus), and opossum (Didelphis albiventris) that had been previously tested by indirect immunofluorescence assay (IFA) for rickettsial infection were separated into IFA-positive and IFA-negative groups and used in the assay. There were no significant differences in ELISA optical density (OD) values between IFA-positive and IFA-negative groups with horse samples. The mean OD values were significantly higher in the IFA-positive capybara serum samples (IFA-pos vs. IFA-neg = 2.389 ± 0.761 vs. 1.760 ± 0.840). However, receiver operating characteristic (ROC) curve analysis did not show significant diagnostic parameters. On the other hand, 12 out of 14 (85.7%) opossum samples of the IFA-positive group showed reactivity in ELISA, and this was significantly higher than of the IFA-negative group (0.7196 ± 0.440 vs. 0.2318 ± 0.098, respectively; 85.7% sensitivity, 100% specificity). Therefore, our results show that OmpA-pLMC has a potential to be used in immunodiagnostic assays to detect spotted fever group rickettsial infections.

Systemic alterations induced by phospholipase A2 , BmooTX-I, isolated from Bothrops moojeni snake venom.

Ophidic accidents are among the problems of public health in Brazil. The components from bothropic venom are responsible for many systemic clinical complications resulting from envenomation. The present work aimed to analyse the systemic changes induced in mice after intraperitoneal administration of BmooTX-I, a myotoxic acidic phospholipase A2 isolated from Bothrops moojeni venom. Urinalysis was performed and the following plasma biochemical markers were documented: urea, creatinine and uric acid (renal function); glucose and amylase (pancreatic function); alanine aminotransferase, alkaline phosphatase and gamma-GT (intra- and extrahepatic function); creatine kinase and enzymatic lactate (muscle function). Our results showed that after the intraperitoneal injection of BmooTX-I the urine of these animals showed glycosuria, proteinuria, haematuria, bacteriuria, bilirubinuria, polyuria and nitrite. The plasma biochemical analysis showed alterations in levels of urea, creatinine and uric acid. Amylase concentration was not altered significantly, but the plasma glucose increased significantly compared to controls. The plasma levels of alanine aminotransferase and alkaline phosphatase decreased and increased, respectively, in these same animals. On the other hand, the plasma γGT concentration did not undergo significant modification compared to the control group. The plasma concentration of CK increased, while the enzymatic lactate concentration decreased after the injection of the BmooTX-I. Therefore, in mice BmooTX-I is capable of causing systemic alterations which manifest as renal, muscular, hepatic and pancreatic impairment.

Effect of BaltPLA2, a phospholipase A2 from Bothrops alternatus snake venom, on the viability of cells infected with dengue virus.

Dengue fever is considered a major public health problem in tropical and subtropical regions. Our study analyzed the effect of BaltPLA2, a phospholipase A2 from Bothrops alternatus snake venom, on the viability of cells infected with Dengue virus. In presence of BaltPLA2, the viability of infected cells increased significantly in virucidal, post-treatment, and adsorption assays. Although preliminary these results reveal the need for further studies to investigated whether BaltPLA2 has antiviral activity against Dengue virus.

Baltetin: a new C-type lectin-like isolated from Bothrops alternatus snake venom which act as a platelet aggregation inhibiting.

C-type lectin-like proteins found in snake venom, known as snaclecs, have important effects on hemostasis through targeting membrane receptors, coagulation factors and other hemostatic proteins. Here, we present the isolation and functional characterization of a snaclec isolated from Bothrops alternatus venom, designated as Baltetin. We purified the protein in three chromatographic steps (anion-exchange, affinity and reversed-phase chromatography). Baltetin is a dimeric snaclec that is approximately 15 and 25 kDa under reducing and non-reducing conditions, respectively, as estimated by SDS-PAGE. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and Edman degradation sequencing revealed that Baltetin is a heterodimer. The first 40 amino acid residues of the N-terminal region of Baltetin subunits share a high degree of sequence identity with other snaclecs. Baltetin had a specific, dose-dependent inhibitory effect on epinephrine-induced platelet aggregation in human platelet-rich plasma, inhibiting up to 69% of platelet aggregation. Analysis of the infrared spectra suggested that the interaction between Baltetin and platelets can be attributed to the formation of hydrogen bonds between the PO32- groups in the protein and PO2- groups in the platelet membrane. This interaction may lead to membrane lipid peroxidation, which prevents epinephrine from binding to its receptor. The present work suggests that Baltetin, a new C-type lectin-like protein isolated from B. alternatus venom, is the first snaclec to inhibit epinephrine-induced platelet aggregation. This could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

Edema, hyperalgesia and myonecrosis induced by Brazilian bothropic venoms: overview of the last decade.

Snakebite accidents are considered serious public health problems. They are often neglected, and individuals who have received insufficient treatment are subjected to various disabling alterations. Snake venoms are secretions composed of biologically active molecules capable of triggering local and systemic effects in envenomation victims. Bothropic snakes are responsible for most of the ophidian accidents in Brazil; their venoms are mainly related to local manifestations, due to a composition that is especially rich in proteases and phospholipases A2. The most common local damages are inflammation, with consequent cellular activation and release of inflammatory mediators, hemorrhage, edema, pain and (myo)necrosis, which may lead to amputation of the affected areas. Antivenom therapy is the main treatment for snakebites. However, the efficiency is mainly due to the neutralization of the toxins responsible for the systemic alterations. Thus, the local damages can evolve to markedly compromise the tissue. The complexity of these local effects associated with the toxicity of the snake venom components of the genus Bothrops, arouse interest in the study of the biochemical and pathophysiological mechanisms involved with the actions caused by toxins of the venom. Therefore, this review aims to analyze the edematogenic, hyperalgesic and myotoxic effects caused by Brazilian bothropic venoms in order to contribute to the study and elucidation of the mechanisms of action of its components and, consequently, enable discoveries of more effective combined therapies in the treatment of local damages resulting from envenoming.

Bothrops Moojeni Snake Venom: A Source of Potential Therapeutic Agents Against Hemostatic Disorders

Abstract Hemostasis is a complex set of biological processes responsible for blood fluidity within normal vessels and for the physiological interruption of bleeding in cases of vascular injury. Bothrops moojeni snake venom is rich in bioactive compounds of pharmacological and clinical interest since its protein components are capable of interfering with many points of the hemostatic process. Here, we present the B. moojeni venom proteins that affect hemostasis and discuss their pharmacological and clinical potential. This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol. Data were obtained from the CAPES Journal Portal database, using the terms "Bothrops" AND "hemostasis", in a search for scientific articles made available in the last 20 years. Many components isolated from B. moojeni snake venom are characterized for their effect on hemostasis and possible application in the diagnosis and treatment of hemostatic disorders.

BaltPLA2: A New Phospholipase A2 from Bothrops Alternatus Snake Venom with Antiplatelet Aggregation Activity.

BACKGROUND: In last decades, snake venoms have aroused great interest of the medicine due to the pathophysiological effects caused by their toxins. These include the phospholipases A2, low molecular weight proteins capable of causing haemorrhagic, myotoxic, inflammatory and neurotoxic effects after an ophidian accident. The present work describes the isolation and biochemical characterization of a new PLA2 isolated from the B. alternatus snake venom, which was named BaltPLA2. METHOD: The rapid and efficient purification of this toxin was performed using only two chromatography steps (anion exchange and hydrophobic chromatography). RESULTS: BaltPLA2 is an acidic protein (pI 4.4) with an apparent molecular mass of 17000 (SDSPAGE) and 14074.74 Da (MALDI TOF/TOF). Analysis of fragments ion by MS / MS showed the following internal amino acid sequence SGVIICGEGTPCEK, which did not exhibit homology with other PLA2 from the same venom. BaltPLA2 is a catalytically active, which displayed an anticoagulant action, inhibition of platelet aggregation induced by epinephrine (~ 80%) and ADP (24%). BaltPLA2 also was able to induce myonecrosis and the release of cytokines (IL-10, IL-12 and TNF- α) in macrophages culture. CONCLUSION: The results presented in this work greatly contribute to a better understanding of the mechanism of enzymatic and pharmacological actions of PLA2s from snake venoms and they may contribute to its application in medical research.

The role of platelets in hemostasis and the effects of snake venom toxins on platelet function.

The human body has a set of physiological processes, known as hemostasis, which keeps the blood fluid and free of clots in normal vessels; in the case of vascular injury, this process induces the local formation of a hemostatic plug, preventing hemorrhage. The hemostatic system in humans presents complex physiological interactions that involve platelets, plasma proteins, endothelial and subendothelial structures. Disequilibrium in the regulatory mechanisms that control the growth and the size of the thrombus is one of the factors that favors the development of diseases related to vascular disorders such as myocardial infarction and stroke, which are among the leading causes of death in the western world. Interfering with platelet function is a strategy for the treatment of thrombotic diseases. Antiplatelet drugs are used mainly in cases related to arterial thrombosis and interfere in the formation of the platelet plug by different mechanisms. Aspirin (acetylsalicylic acid) is the oldest and most widely used antithrombotic drug. Although highly effective in most cases, aspirin has limitations compared to other drugs used in the treatment of homeostatic disorders. For this reason, research related to molecules that interfere with platelet aggregation are of great relevance. In this regard, snake venoms are known to contain a number of molecules that interfere with hemostasis, including platelet function. The mechanisms by which snake venom components inhibit or activate platelet aggregation are varied and can be used as tools for the diagnosis and the treatment of several hemostatic disorders. The aim of this review is to present the role of platelets in hemostasis and the mechanisms by which snake venom toxins interfere with platelet function.

A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom.

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom.

BACKGROUND: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. METHODS: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. RESULTS: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO32- groups, present in BaltDC, form hydrogen bonds with the PO2- groups present in the non-lipid portion of the membrane platelets. CONCLUSIONS: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

Biochemical and functional characterization of BmooSP, a new serine protease from Bothrops moojeni snake venom.

In this work, we describe the purification and characterization of a new serine protease enzyme from Bothrops moojeni snake venom (BmooSP). On SDS-PAGE, BmooSP was found to be a single-chain protein with an apparent molecular mass of 36,000 and 32,000 under reduced and non-reduced conditions, respectively. Mass spectrometry analysis showed that the BmooSP is composed by two isoforms with molecular mass of 30,363 and 30,070, respectively. The purified enzyme consists of 277 amino acid residues, disregarding the cysteine and tryptophan residues that have been degraded by acid hydrolysis, and its N-terminal sequence showed similarity with other serine protease enzymes. BmooSP induced blood-clotting in vitro, defibrination in vivo, caseinolytic and fibrin(ogen)olytic activities. The enzyme is stable at high temperatures (up to 100 °C) and shows maximum activity at pH around 7.0. Preliminary results show that BmooSP can induce the formation of a stable fibrin clot for more than 10 days. BmooSP presents medical interest because it can be used as biodegradable fibrin glue and for the treatment and prevention of cardiovascular disorders because of its ability to promote the defibrination in vivo, decreasing blood viscosity and improving blood circulation.

Comparative analysis of local effects caused by Bothrops alternatus and Bothrops moojeni snake venoms: enzymatic contributions and inflammatory modulations.

Bothropic envenomation is characterised by severe local damage caused by the toxic action of venom components and aggravated by induced inflammation. In this comparative study, the local inflammatory effects caused by the venoms of Bothrops alternatus and Bothrops moojeni, two snakes of epidemiological importance in Brazil, were investigated. The toxic action of venom components induced by bothropic venom was also characterised. Herein, the oedema, hyperalgesia and myotoxicity induced by bothropic venom were monitored for various lengths of time after venom injection in experimental animals. The intensity of the local effects caused by B. moojeni venom is considerably more potent than B. alternatus venom. Our results also indicate that metalloproteases and phospholipases A2 have a central role in the local damage induced by bothropic venoms, but serine proteases also contribute to the effects of these venoms. Furthermore, we observed that specific anti-inflammatory drugs were able to considerably reduce the oedema, the pain and the muscle damage caused by both venoms. The inflammatory reaction induced by B. moojeni venom is mediated by eicosanoid action, histamine and nitric oxide, with significant participation of bradykinin on the hyperalgesic and myotoxic effects of this venom. These mediators also participate to inflammation caused by B. alternatus venom. However, the inefficient anti-inflammatory effects of some local modulation suggest that histamine, leukotrienes and nitric oxide have little role in the oedema or myotoxicity caused by B. alternatus venom.

Histological and ultrastructural analyses of muscle damage induced by a myotoxin isolated from Bothrops alternatus snake venom.

Muscular necrosis is a serious consequence of Bothrops snake bites that may lead to permanent loss of tissue or function. Myonecrosis may be due to injury to blood vessels, destabilization and/or rupture of plasma membrane, and inflammatory mechanisms triggered by different proteins from the snake venom. In this work we describe the isolation and partial functional characterization of a myotoxin from B. alternatus snake venom. The myotoxin was isolated by a combination of ion exchange and gel filtration chromatography and displayed a molecular weight of approximately 15,000, as estimated by SDS-PAGE under reducing conditions. In non-reducing conditions a protein band of approximately 25,000 was also observed, suggesting that its native form is a homodimer. The myotoxin induced myonecrosis, but had no proteolytic and phospholipase A2 activities. The myotoxic activity was assessed on the basis of the histological and ultrastructural alterations induced by the toxin in the gastrocnemius skeletal muscle of Swiss mice. The toxin led to a series of drastic degenerative events characterized by extensive cellular destruction, loss of the arrangements of skeletal fibers, intense infiltration of inflammatory cells, fatty degeneration and hemorrhage. Electron microscopy analyses revealed that the myotoxin caused cell swelling, mitochondrial alterations and dilation of the sarcoplasmic reticulum, but did not affect the integrity of the muscle cell membranes. The myonecrosis caused by this toxin was related to the perturbation in the membrane permeability, intracellular alterations and inflammatory reaction.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Abstract Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 groups, present in BaltDC, form hydrogen bonds with the PO 2 groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.(AU)