RESUMO
BACKGROUND: When feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding. METHODS: Ticks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species. RESULTS: Changes in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as "sialome switching." This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva. CONCLUSIONS: Overall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.
Assuntos
Rhipicephalus , Animais , Feminino , Bovinos , Rhipicephalus/fisiologia , Saliva/química , Proteômica , Proteínas de Artrópodes/metabolismo , Proteínas e Peptídeos Salivares/metabolismoRESUMO
Here we present the proteomic profile datasets of two Fasciola hepatica NEJ isolates derived from different snail hosts: Lymnaea viatrix and Pseudosuccinea columella. The data used in the analysis are related to the article 'A proteomic comparison of excretion/secretion products in Fasciola hepatica newly excysted juveniles (NEJ) derived from Lymnaea viatrix or Pseudosuccinea columella' (Di Maggio et al., 2019).
RESUMO
In parasites, cathepsins are implicated in mechanisms related to organism surveillance and host evasion. Some parasite cathepsins have fibrinogenolytic and fibrinolytic activity, suggesting that they may contribute to maintain blood meal fluidity for extended feeding periods. Here, it is shown that BmGTI (Rhipicephalus [Boophilus] microplus Gut Thrombin Inhibitor), a protein previously described as an inhibitor of fibrinogen hydrolysis and platelet aggregation by thrombin, and BmCL1 (Rhipicephalus [Boophilus] microplus Cathepsin-L like 1) are the same protein, hereinafter referred to using the earliest name (BmCL1). To further characterize BmCL1, Rhipicephalus microplus native and recombinant (rBmCL1) proteins were obtained. Native BmCL1 was isolated using thrombin-affinity chromatography, and it displays thrombin inhibition activity. We subsequently investigated rBmCL1 interaction with thrombin. We show that rBmCL1 and thrombin have a dissociation constant (ΚD) of 130.2⯱â¯11.2â¯nM, and this interaction likely occurs due to a more electronegative surface of BmCL1 at pH 7.5 than at pH 5.0, which may favor an electrostatic binding to positively charged thrombin exosites. During BmCL1-thrombin interaction, thrombin is not degraded or inhibited. rBmCL1 impairs thrombin-induced fibrinogen clotting via a fibrinogenolytic activity. Fibrinogen degradation by BmCL1 occurs by the hydrolysis of Aα- and Bß-chains, generating products similar to those produced by fibrinogenolytic cathepsins from other organisms. In conclusion, BmCL1 likely has an additional role in R. microplus blood digestion, besides its role in hemoglobin degradation at acid pH. BmCL1 fibrinogenolytic activity indicates a proteolytic activity in the neutral lumen of tick midgut, contributing to maintain the fluidity of the ingested blood, which remains to be confirmed in vivo.
Assuntos
Catepsina L/metabolismo , Rhipicephalus/enzimologia , Trombina/metabolismo , Sequência de Aminoácidos , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/metabolismo , Catepsina L/química , Catepsina L/isolamento & purificação , Bovinos , Cinética , Modelos Moleculares , ProteóliseRESUMO
The characteristics of parasitic infections are often tied to host behavior. Although most studies have investigated definitive hosts, intermediate hosts can also play a role in shaping the distribution and accumulation of parasites. This is particularly relevant in larval stages, where intermediate host's behavior could potentially interfere in the molecules secreted by the parasite into the next host during infection. To investigate this hypothesis, we used a proteomic approach to analyze excretion/secretion products (ESP) from Fasciola hepatica newly excysted juveniles (NEJ) derived from two intermediate host species, Lymnaea viatrix and Pseudosuccinea columella. The two analyzed proteomes showed differences in identity, abundance, and functional classification of the proteins. This observation could be due to differences in the biological cycle of the parasite in the host, environmental aspects, and/or host-dependent factors. Categories such as protein modification machinery, protease inhibitors, signal transduction, and cysteine-rich proteins showed different abundance between samples. More specifically, differences in abundance of individual proteins such as peptidyl-prolyl cis-trans isomerase, thioredoxin, cathepsin B, cathepsin L, and Kunitz-type inhibitors were identified. Based on the differences identified between NEJ ESP samples, we can conclude that the intermediate host is a factor influencing the proteomic profile of ESP in F. hepatica.
Assuntos
Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Lymnaea/parasitologia , Proteômica , Caramujos/parasitologia , Animais , Anidrases Carbônicas/classificação , Anidrases Carbônicas/metabolismo , Proteínas de Helminto/classificação , Larva/metabolismo , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/metabolismo , Peroxirredoxinas/classificação , Peroxirredoxinas/metabolismo , Inibidores de Proteases/classificação , Inibidores de Proteases/metabolismo , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/metabolismoRESUMO
Lipids play key roles in arthropod metabolism. In ticks, these biomolecules are transported from fat body to other organs, such as ovary and Gené's organ. Gené's organ, an apparatus found exclusively in female ticks, secretes a protective wax coat onto the egg surface, increasing egg viability in the environment due to waterproof, cohesive, and antimicrobial properties. In this work, a combined transcriptomic and proteomic approach shows that Gené's organ not solely secrets compounds taken up from the hemolymph, but is actively engaged in synthesis, modification, and oxidation of lipids. Gené's organ was analyzed at two distinct stages: 1) when ticks detach from host by the end of hematophagous phase, and 2) during egg-laying. Data show that Gené's organ undergoes a maturation process before the onset of oviposition, in preparation for its role during egg-laying. Because it deals with a wax-secreting organ, the study focused on lipid metabolism, examining a full machinery to synthesize, modify, and oxidize fatty acids. Proteins involved in sterol modification, transport, and degradation were also addressed. In addition to highlighting Gené's organ importance in tick reproductive physiology, the results reveal proteins and pathways crucial to egg wax secretion, and consequently, egg development in the environment. Tools targeting these molecules and pathways would impair egg viability in the environment, and therefore have the potential to be developed into novel tick control methods.
Assuntos
Metabolismo dos Lipídeos , Proteoma , Carrapatos/anatomia & histologia , Carrapatos/metabolismo , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Oviposição , Óvulo , Proteômica , Controle de Ácaros e Carrapatos , Carrapatos/genética , CerasRESUMO
Ticks are arthropod ectoparasites of importance for public and veterinary health. The understanding of tick oogenesis and embryogenesis could contribute to the development of novel control methods. However, to date, studies on the temporal dynamics of proteins during ovary development were not reported. In the present study we followed protein profile during ovary maturation. Proteomic analysis of ovary extracts was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using shotgun strategy, in addition to dimethyl labelling-based protein quantification. A total of 3,756 proteins were identified, which were functionally annotated into 30 categories. Circa 80% of the annotated proteins belong to categories related to basal metabolism, such as protein synthesis and modification machineries, nuclear regulation, cytoskeleton, proteasome machinery, transcriptional machinery, energetic metabolism, extracellular matrix/cell adhesion, immunity, oxidation/detoxification metabolism, signal transduction, and storage. The abundance of selected proteins involved in yolk uptake and degradation, as well as vitellin accumulation during ovary maturation, was assessed using dimethyl-labelling quantification. In conclusion, proteins identified in this study provide a framework for future studies to elucidate tick development and validate candidate targets for novel control methods.
Assuntos
Proteínas de Artrópodes/metabolismo , Ovário/crescimento & desenvolvimento , Proteoma/análise , Carrapatos/crescimento & desenvolvimento , Vitelogênese , Animais , Feminino , Ovário/metabolismo , Proteoma/metabolismo , Carrapatos/metabolismoRESUMO
Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite.
Assuntos
Fasciola hepatica/crescimento & desenvolvimento , Proteínas de Helminto/metabolismo , Fígado/parasitologia , Proteômica/métodos , Animais , Fasciola hepatica/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Interações Hospedeiro-ParasitaRESUMO
In recent years, phenotypic screening has assumed a leading role in drug discovery efforts. However, development of new drugs from bioactive compounds obtained in screening campaigns requires identification of the cellular targets responsible for their biological activities. A new energetics-based method for target identification is presented: pulse proteolysis and precipitation for target identification (PePTID). In this method, proteins incubated with or without a ligand and submitted to a brief proteolytic pulse are directly analyzed and compared using a label-free semiquantitative mass spectrometry strategy, dispensing the SDS-PAGE readout and greatly improving the throughput. As a proof-of-concept, we applied the PePTID method to identify ATP-binding proteins in Mycobacterium smegmatis, a model system for Mycobacterium tuberculosis, the etiological agent of tuberculosis.
Assuntos
Proteínas de Bactérias/análise , Precipitação Química , Descoberta de Drogas/métodos , Proteólise , Proteínas de Transporte/análise , Ligantes , Mycobacterium smegmatis/química , Mycobacterium tuberculosis/químicaRESUMO
Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity. BIOLOGICAL SIGNIFICANCE: Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout-vent length and weight, with females being larger than males. This sexual size dimorphism suggests the tendency for female specimens to feed on larger prey, and for male specimens to go on a diet similar to that of juveniles. Variation in the snake venom proteome is a ubiquitous phenomenon occurring at all taxonomic levels. At the intraspecific variation level, the individual contribution to the venom proteome is important but effects contributed by age and feeding habits may also affect the proteome phenotype. Whether sex-based factors play a role in venom variation of a species that shows sexual size dimorphism is poorly known. The use of proteomic strategies supported by transcriptomic data allows a more comprehensive assessment of venom proteomes uncovering components that are gender-specific.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/metabolismo , Caracteres Sexuais , Animais , Biomarcadores/metabolismo , Feminino , MasculinoRESUMO
BACKGROUND: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. METHODS: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. RESULTS: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. CONCLUSIONS: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.
Assuntos
Proteínas de Artrópodes/química , Ixodidae/crescimento & desenvolvimento , Ixodidae/metabolismo , Proteoma/química , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Feminino , Ixodidae/química , Ixodidae/genética , Masculino , Ninfa/química , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica , Coelhos , Saliva/química , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismoRESUMO
The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.
Assuntos
Bothrops/crescimento & desenvolvimento , Venenos de Crotalídeos/metabolismo , Proteoma/metabolismo , Proteínas de Répteis/metabolismo , Animais , Bothrops/metabolismo , Glicosilação , Espectrometria de Massas , ProteômicaRESUMO
The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2‐DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N‐glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P‐III‐rich to a P‐I‐rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in‐depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.
RESUMO
Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.
Assuntos
Artrópodes/microbiologia , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Metarhizium/química , Metarhizium/fisiologia , Proteômica , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional , Feminino , Proteínas Fúngicas/genética , Metarhizium/genética , Metarhizium/patogenicidade , Dados de Sequência MolecularRESUMO
The caterpillar Lonomia obliqua is a venomous animal that causes numerous accidents, especially in southern Brazil, where it is considered a public health problem. The clinical manifestations include several haemostatic disturbances that lead to a hemorrhagic syndrome. Considering that platelets play a central role in hemostasis, in this work we investigate the effects of L. obliqua venomous secretion upon blood platelets responses in vitro. Results obtained shows that L. obliqua venom directly induces aggregation and ATP secretion in human washed platelets in a dose-dependent manner. Electron microscopy studies clearly showed that the venomous bristle extract was also able to produce direct platelets shape change and adhesion as well as activation and formation of platelet aggregates. Differently from other enzyme inhibitors, the venom-induced platelet aggregation was significatively inhibited by p-bromophenacyl bromide, a specific inhibitor of phospholipases A2. Additional experiments with different pharmacological antagonists indicate that the aggregation response triggered by the venom active components occurs through a calcium-dependent mechanism involving arachidonic acid metabolite(s) of the cyclooxygenase pathway and activation of phosphodiesterase 3A, an enzyme that leads to the consumption of intracellular cAMP content. It was additionally found that L. obliqua-induced platelet aggregation was independent of ADP release. Altogether, these findings are in line with the need for a better understanding of the complex hemorrhagic syndrome resulting from the envenomation caused by L. obliqua caterpillars, and can also give new insights into the management of its clinical profile.
Assuntos
Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Acetofenonas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Hemorragia , Humanos , Inibidores de Fosfolipase A2 , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
Caterpillar envenomation has been an emergent health issue. Lonomia obliqua is a medically important animal that causes a hemorrhagic syndrome that can progress to acute renal failure, intracranial hemorrhage and death. In the past few years the molecular characterization of L. obliqua venom in addition to experimental models has provided fundamental information to the understanding of the envenomation syndrome. Herein studies from several authors which characterized the complex toxic-pharmacological actions of whole venom are reviewed.
Assuntos
Venenos de Artrópodes/toxicidade , Transtornos Hemorrágicos/etiologia , Mariposas/química , Animais , Antivenenos/uso terapêutico , Venenos de Artrópodes/química , Coagulação Sanguínea/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Transtornos Hemorrágicos/sangue , Transtornos Hemorrágicos/terapia , Humanos , Mordeduras e Picadas de Insetos/tratamento farmacológico , Mordeduras e Picadas de Insetos/patologia , Rim/efeitos dos fármacos , Larva/química , Larva/genética , Modelos Biológicos , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Agregação Plaquetária/efeitos dos fármacos , SíndromeRESUMO
Envenomation caused by Lonomia obliqua is a public health hazard in Southern Brazil. Envenomed victims present severe hemorrhagic syndrome that can progress to intracranial hemorrhage and death. To understand the mechanisms that lead to hemorrhage, we investigated the platelet dysfunction and blood coagulation disturbances following experimental envenomation in rats. L. obliqua bristle extract was injected (s.c.) and blood collected at different times post-venom administration for determination of platelet response and analysis of blood coagulation. Rats presented hypofibrinogenemia and platelet hypoaggregation in platelet rich plasma (PRP). After addition of exogenous fibrinogen to PRP, platelet hypoaggregation was not corrected. Interestingly, normoaggregation was observed when platelets were separated from plasma. In addition, incubation of plasma from envenomed rats inhibits aggregation response of normal washed platelets. These results indicate that an aggregation inhibitor is generated in plasma during envenomation. Moreover, rats presented an increase in nitric oxide plasmatic levels which coincided with maximum inhibition in platelet aggregation. Animals also showed blood incoagulability and a significant increase in thrombin, plasmin and urokinase plasmatic activities. Despite this intravascular thrombin generation, only a slight decrease in platelet numbers was detected. Certainly, the platelet hypoaggregation and blood incoagulability described herein contribute to systemic bleeding observed in patients.
Assuntos
Venenos de Artrópodes/toxicidade , Coagulação Sanguínea/efeitos dos fármacos , Mordeduras e Picadas de Insetos/complicações , Mariposas/química , Agregação Plaquetária/efeitos dos fármacos , Animais , Venenos de Artrópodes/isolamento & purificação , Brasil , Modelos Animais de Doenças , Fibrinogênio/análise , Hemorragia/etiologia , Hemorragia/mortalidade , Mordeduras e Picadas de Insetos/sangue , Mordeduras e Picadas de Insetos/mortalidade , Larva/química , Masculino , Nitratos/sangue , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Nitritos/sangue , Inibidores da Agregação Plaquetária/sangue , Ratos , Ratos Wistar , Fatores de TempoRESUMO
In light of the complexity of wound tissue, proteomic analysis may not clearly reveal the nature of the wound or the processes involved in healing. However, exudate associated with wounds may provide a "window" on cellular events leading to the development of the wound and/or its healing. In this investigation we performed proteomic analysis on wound exudates from muscular wounds in mice caused by two very different types of snake venom toxins: BaP1, a snake venom metalloproteinase and Mtx-I, a snake venom phospholipase A2. Proteomic analysis of the exudates associated with these wounds clearly differentiated them and offered new perspectives on functional mechanisms by which these toxins cause tissue damage. In the case of wounds caused by the metalloproteinase, there was evidence of degradation of nonfibrillar collagens whereas the phospholipase wound exudate was noted by the presence of fibrillar collagen type I, apolipoproteins A-I, A-IV, and E, and fibronectin. These results suggest that the hemorrhage caused by snake venom metalloproteinases may be associated with the degradation of specific extracellular matrix proteins which play a role in matrix/capillary stabilization and that release of apolipoproteins from their complexes may be involved with the dysfunctional hemostasis observed following snake envenoming.
Assuntos
Exsudatos e Transudatos/química , Metaloendopeptidases , Músculo Esquelético , Fosfolipases A2 , Proteômica/métodos , Venenos de Serpentes , Cicatrização/fisiologia , Animais , Bothrops , Cromatografia Líquida/métodos , Proteínas da Matriz Extracelular/metabolismo , Queratinas/metabolismo , Espectrometria de Massas/métodos , Metaloendopeptidases/farmacologia , Metaloendopeptidases/toxicidade , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Fosfolipases A2/farmacologia , Fosfolipases A2/toxicidade , Pele/citologia , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Mordeduras de Serpentes , Venenos de Serpentes/farmacologia , Venenos de Serpentes/toxicidade , Espectrometria de Massas em Tandem/métodosRESUMO
Bleeding at the site of bite and/or systemic hemorrhage are symptoms frequently observed in envenomation by Bothrops jararaca snakes. In this study, we purified and characterized a prothrombin activator from B. jararaca that is probably involved in these clinical manifestations. The enzyme was isolated by a combination of gel filtration and ion exchange chromatographies and named bothrojaractivase. It has a single polypeptide chain with a molecular weight of 22,829 Da as measured by mass spectroscopy. Bothrojaractivase generates active thrombin from prothrombin, independently of cofactors. SDS-PAGE analysis of the prothrombin activation products shows that bothrojaractivase converts prothrombin into meizothrombin producing similar fragments to those generated by group A prothrombin's activators. In addition, bothrojaractivase degraded fibrinogen and fibrin. Chelating agents completely inhibited the enzymatic activity, whereas inhibitors of serine and cysteine proteinases had no effect. Amino acid sequence of four peptides demonstrated high similarity of bothrojaractivase with P-I class of snake venom metalloproteinases. Thus, our results indicate that bothrojaractivase is a new metalloproteinase that acts on different protein factors of the clotting cascade especially displaying a key and most relevant functional action in the generation of thrombin through prothrombin activation in a similar mode of action as that of group A activators.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Metaloproteases/metabolismo , Protrombina/metabolismo , Sequência de Aminoácidos , Animais , Cátions Bivalentes , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Metaloproteases/isolamento & purificação , Dados de Sequência Molecular , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/metabolismoRESUMO
The hemorrhagic syndrome caused by Lonomia obliqua caterpillars is an increasing problem in Southern Brazil. The clinical profile is characterized by both hemorrhagic and pro-coagulant symptoms, constituting a paradoxical action of the venom. The effects upon blood coagulation and fibrin(ogen)olysis have been shown to result from the combined action of several active principles found mostly in the bristle extract. The present study reports quali-quantitative differences among L. obliqua secretions: Cryosecretion, hemolymph, bristle extract and tegument extract. Cryosecretion and hemolymph displayed strong amidolytic activity upon several substrates, presented moderated procoagulant activity and high fibrinogen degrading ability. Bristle and tegument extracts presented low amidolytic activity, but bristle extract showed the most potent procoagulant activity and both extracts presented low fibrinogen degrading ability. The differential involvement of these secretions during the accidents with L. obliqua can elucidate the different symptoms presented after envenomation.
Assuntos
Venenos de Artrópodes/enzimologia , Venenos de Artrópodes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Amidoidrolases/metabolismo , Animais , Venenos de Artrópodes/metabolismo , Testes de Coagulação Sanguínea , Brasil , Fibrinogênio/metabolismo , Lepidópteros , Fatores de TempoRESUMO
The hemorrhagic syndrome caused by accidents with caterpillars of the genus Lonomia has been the focus of several clinical and biochemical studies, since its venom is composed of many active principles that interfere with the hemostatic system. Whereas a fibrinolytic agent has been characterized in the venom of Lonomia achelous, in Lonomia obliqua, only a prothrombin activator activity has been reported so far, even though both species cause similar bleeding disorders, characterized by hemorrhage, disseminated intravascular coagulation (DIC), and acute renal failure. Considering the possibility that the hemorrhagic syndrome resulting from envenoming by L. obliqua may be due to fibrinolytic and procoagulant activities acting together, we decided to investigate the effects of bristle extract (BE) of this species upon blood coagulation and fibrin(ogen)olysis. This study shows that besides a procoagulant activity related to the activation of prothrombin, the venom contains at least one fibrin(ogen)olytic activity, as shown by fibrinolysis in a fibrin (F) plate assay, by interference in thrombin-catalyzed fibrinocoagulation, and by polyacrylamide gel electrophoresis profile of fibrin and fibrinogen (Fg) degradation. Considering that a recombinant prothrombin activator from L. obliqua has been suggested in other studies to be used as an anti-thrombotic agent, it is important in the first place to better characterize the different active principles of this venom.