Vitiligo in equine: a four-year case study of a roan horse - case report / [Vitiligo em equinos: um estudo de caso de quatro anos de um cavalo roan - relato de caso]

Vitiligo is a dermatological disease affecting both animals and humans. It is characterized by depigmented macules of varying shape and size, originated from melanocyte destruction. Even though there are some theories tackling causation, disease etiopathology is not yet certain. Moreover, lesion areas can either increase or diminish over time, and therefore, available treatment alternatives tend to prove inconsistencies. No epidemiological data or registered cases were found for equines in Brazil. The horse in this case description displayed depigmentation areas in facial regions, including upper lip, nose and lips. However, the individual did not happen to develop any systemic alteration. Through clinical evaluation, backed by a histopathological exam, a definitive vitiligo diagnosis was obtained. However, no therapeutic plan was stipulated. The animal was accompanied for four years, during which period some affected areas diminished while others increased in size. In addition, emergence of new skin lesions was also observed during the time the animal was studied. Overall, this disease does not display alterations to organism functionality, only aesthetic changes. Therefore, treatment plans may vary from case to case, occasionally being even ruled out.(AU)

O vitiligo é uma doença dermatológica que pode afetar animais e humanos. Caracteriza-se por áreas despigmentadas, de formas e tamanhos variáveis, que surgem devido a destruição dos melanócitos. Existem algumas teorias que tentam explicar a etiopatogenia da doença, entretanto ainda não é totalmente esclarecida. As lesões podem aumentar ou diminuir com o tempo, por isso os tratamentos disponíveis são inconsistentes. Não foram encontrados dados epidemiológicos ou relatos de vitiligo em cavalos no Brasil. O equino deste relato apresentava lesões despigmentadas na região da face, incluindo pálpebras, narina e lábios, sem alterações sistêmicas. Por meio da avaliação clínica em conjunto com o exame histopatológico obteve-se o diagnóstico definitivo de vitiligo. Não foi instituído nenhuma terapia, e o equino foi acompanhado durante quatro anos. Durante esse período algumas lesões diminuíram e outras aumentaram de tamanho sendo também observado o aparecimento de novas lesões. O vitiligo não traz alterações sistêmicas, apenas mudanças estéticas, por isso a escolha pelo tratamento dependerá de cada caso.(AU)

Idiopathic seasonal alopecia in horse: case report / Alopecia sazonal idiopática em equino: relato de caso

This report describes the first case of idiopathic seasonal alopecia in a horse in Brazil. The disease is of unknown etiology, characterized by alopecic processes in the thoracic and lateral abdominal regions, in a bilaterally symmetrical way. An eight-year-old male grade horse was treated presenting hair loss in a bilaterally symmetrical manner in the arm and abdomen areas, without any other associated clinical signs. The areas with alopecia showed no pruritus, inflammation or scaling. On the epidermis, the histological evaluation presented irregular hyperplasia, hyperpigmentation, compact orthokeratosis, edema and an inflammatory infiltrate. The hair follicles were active and containing hair shaft. The case was monitored with photographic records for two consecutive years (2012 to 2014), in which the hair fall occurred at the end of autumn with spontaneous hair growth in the middle of the summer. The diagnosis was based on the history, histopathology and photographic follow-up performed. Although mentioned in the literature, this is the first clinical and pathological description of such disorder affecting an equine in Brazil.(AU)

Relata-se o primeiro caso de alopecia sazonal idiopática em um equino no Brasil, doença de etiologia desconhecida, caracterizada por processos alopécicos, nas regiões torácicas e abdominais laterais, de forma simétrica bilateralmente. Um equino mestiço, macho, de oito anos de idade, foi atendido sob queixa de perda dos pelos em regiões do tórax e do abdômen, simétrica bilateralmente, sem qualquer outro sinal clínico associado. As regiões alopécicas não apresentavam prurido, inflamação nem descamação. A avaliação histológica revelou, na epiderme, hiperplasia irregular, hiperpigmentação e ortoqueratose compacta, edema e infiltrado inflamatório. Os folículos pilosos estavam ativos e contendo hastes de pelos. O caso foi acompanhado com registros fotográficos durante dois anos consecutivos (2012 a 2014), com a queda do pelo acontecendo no final do outono e com retorno espontâneo em meados do verão. O diagnóstico baseou-se no histórico, na histopatologia e no acompanhamento fotográfico. Mesmo sendo mencionada na literatura, esta é a primeira descrição clínico-patológica de tal distúrbio acometendo um equino no Brasil.(AU)

Stephanofilariasis in beef cattle - case report / Estefanofilariose em bovinos de corte - relato de caso

Stephanofilariasis is an ulcerative dermatitis caused by nematodes that affect cattle in several countries in the world. However, it has not been described in beef cattle in Brazil. The objective of this study is to describe three cases of stephanofilariasis, which occurred in beef cows in the municipality of Ipê, RS, Brazil. The disease was characterized by pruritic, ulcerated and crusty seasonal lesions present in the cranial region of the udder. The diagnosis was confirmed by analyses of secretions stained smears and by direct optical microscopic examination of the sediment and the treatment was effectively carried out with topical trichlorphon. This report indicates that stephanofilariasis should be included as a differential diagnosis for dermatopathies in beef cattle in Brazil.(AU)

A estefanofilariose é uma dermatite ulcerativa causada por nematódeos que acometem bovinos em vários países do mundo, no entanto não tem sido descrita em bovinos de corte no Brasil. O objetivo do trabalho é a descrição de três casos de estefanofilariose em vacas de corte ocorridos no município de Ipê, RS. A enfermidade foi caracterizada por lesões sazonais pruriginosas, ulceradas e crostosas, presentes na região cranial do úbere. O diagnóstico foi confirmado pela análise dos esfregaços corados das secreções e por exame direto do sedimento em microscopia óptica, e o tratamento foi realizado de maneira eficaz com triclorfon tópico. Este relato demonstra que a estefanofilariose deve ser incluída como diagnóstico diferencial nas dermatopatias em bovinos de corte no Brasil.(AU)

Systemic toxic reaction due to multiple honeybee stings in equine: Case report / Reação tóxica sistêmica devido a múltiplas ferroadas de abelha em equino: relato de caso

Accidents caused by insects of the Hymenoptera are rarely described in large animals. The attacks caused by honeybee (Apis mellifera) may cause severe consequences and its intensity changes according to the number of stings. Local and systemic reactions can occur, including progression to death. This report describes a case of honeybee attack on an equine, which took place in the city of Lages, in the state of Santa Catarina, Brazil. In the clinical assessment the horse showed apathy, anorexia, head and pectoral edemas, dyspnea, icteric mucosa, increased mandibular lymph nodes and darkened urine. The blood count showed anemia and serum biochemical tests suggested, muscular and hepatic lesions. The urinalysis test indicated hemoglobinuria and increased clotting time. Treatment included lactate Ringer's solution fluid therapy, furosemide, promethazine, corticosteroids and 20% mannitol solution. Hot and cold compresses were applied alternately on areas with edema. There was a satisfactory response to treatment and the animal was discharged after 30 days in veterinary hospital. The description of honeybee sting accidents in large animals is important because of the evolution that can lead to death. The early approach associated with appropriate treatment, avoiding the worsening of the lesions is fundamental for the recovery of the patient.(AU)

Os acidentes causados por insetos da ordem Hymenoptera são raramente descritos em grandes animais. Os ataques provocados por abelhas (Apis mellifera) causam consequências graves, e sua intensidade varia de acordo com a quantidade de ferroadas. Reações locais a sistêmicas podem ocorrer, incluindo a evolução para a morte. Este relato descreve um caso de ataque por abelhas em equino, ocorrido no município de Lages, SC. No exame clínico, o equino apresentava apatia, anorexia, edema de cabeça e região peitoral, dispneia inspiratória, mucosas ictéricas, linfonodos mandibulares aumentados e urina de coloração marrom-escura. O hemograma evidenciou anemia hemolítica, e os exames de bioquímica sérica sugeriram lesão muscular e lesão hepática. A urinálise demonstrou hemoglobinúria, e o tempo de coagulação apresentava-se aumentado. Como tratamento, foram administrados solução de ringer com lactato, furosemida, prometazina, corticosteroides e solução de manitol a 20%. Compressas quentes e frias foram aplicadas alternadamente sobre as áreas de edema. Houve adequada resposta ao tratamento instituído e o animal recebeu alta hospitalar após 30 dias de internamento. A descrição de casos de acidentes por picadas de abelhas em grandes animais é importante devido à evolução, que pode levar à morte. A abordagem precoce associada ao tratamento adequado, evitando o agravamento das lesões, é fundamental para a recuperação do paciente.(AU)

Characterization of Neuwiedin, a new disintegrin from Bothrops neuwiedi venom gland with distinct cysteine pattern.

Disintegrins are cysteine-rich toxins containing the RGD motif exposed in a loop that binds integrins such as αIIbß3, α5ß1 and αvß3. The flexibility of the RGD loop, controlled by the profile of the cysteine pairs and the residues flanking the RGD sequence, are key structural features for the functional activity of these molecules. Recently, our group reported a transcript in the venom gland of Bothrops neuwiedi corresponding to a new P-II SVMP precursor, BnMPIIx, in which the RGD-binding loop includes many substituted residues and unique cysteine residues at the C-terminal. In this paper, we obtained the recombinant disintegrin domain of BnMPIIx, Neuwiedin, which inhibited ADP-induced platelet aggregation, endothelial cell adhesion to fibrinogen and tube formation in Matrigel with no particular selectivity to αIIbß3 or endothelial cell integrins. This value was also comparable to the inhibition observed with other recombinant disintegrins with conserved cysteine positions and residues in RGD loop. In this regard, Neuwiedin is an important component to understand the functional relevance of the diversity generated by accelerated evolution of venom toxins as well as to find out eventual new disintegrin-dependent targets that may be approached with disintegrins.

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.

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP.

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.

Collagen binding is a key factor for the hemorrhagic activity of snake venom metalloproteinases.

Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.

Importance of jararhagin disintegrin-like and cysteine-rich domains in the early events of local inflammatory response.

Jararhagin is a multi-domain SVMP from Bothrops jararaca venom comprising catalytic, disintegrin-like and cysteine-rich domains, which cause a local reaction manifested by hemorrhage, edema, cytokine release and inflammatory cell recruitment. In this study, the importance of disintegrin-like/cysteine-rich domains of jararhagin was addressed by analyzing the effects of jararhagin-C, which lacks the catalytic domain, in induction of leukocyte rolling and release of pro-inflammatory cytokines. Jararhagin-C was isolated from B. jararaca venom conserving the same ability of complete jararhagin molecule in inhibiting collagen-induced platelet-aggregation. Treatment of trans-illuminated cremaster muscle in vivo with jararhagin-C increased number of rolling leukocytes (approximately 250%) in post-capillary venules in all periods analyzed, without interfering with microvasculature haemodynamic, like vessel diameter, the erythrocyte speed or the blood flow rate. The release of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 was significantly enhanced in the local of jararhagin-C injection, showing the maximum levels in periods between 2 and 4 h after treatment. Besides the action of jararhagin-C, the presence of the inactivated catalytic domain in o-phenanthrolin-treated jararhagin was related to a higher increase in the number of rolling leukocytes. Moreover, the levels of IL-6 and IL-1beta induced by catalytically active jararhagin were higher than those induced by jararhagin-C. In conclusion, our findings suggest that the disintegrin-like/cysteine-rich domains of jararhagin are sufficient to locally activate the early events of an acute inflammatory response as leukocyte rolling and pro-inflammatory cytokine release and this action may add to the effect of catalysis, which enhances the primary cell activation.

Jararhagin, a snake venom metalloproteinase, induces a specialized form of apoptosis (anoikis) selective to endothelial cells.

Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40microg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-xL. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability.

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin A factor contributing to snake venom variability

A subfamília reprolisina de metaloproteinases inclui metaloproteinases veneno de serpente (SMVP) e desintegrina mamíferos / metaloproteinase. Estas proteínas são sintetizadas como zimogénios e submetidos a processamento proteolítico, resultando em uma variedade de proteínas multifuncionais.

Cytochemical analysis of acid phosphatase activity in the venom secretory cells of Bothrops jararaca.

A study of the histochemical reaction for acid phosphatase (AcPase) in venom gland secretory cells from Bothrops jararaca was done to investigate the distribution of lysosomes and related structures in stages of high- and low-protein synthesis. From this analysis, it was expected to gain insight into the cellular pathway by which AcPase is secreted into the venom. Two subtypes of AcPase reactivities were detected in the venom gland secretory cells: one was found in lysosomes and related structures and in some trans-Golgi network (TGN) elements and reacts with beta-glycerophosphate (betaGP) as substrate; the other was found in secretory vesicles, apical plasmalemma, lysosomes and related structures, and in some TGN elements, and reacts with cytidine monophosphate (CMP). The results are compatible with the possibility that there is a secretory via for AcPase in the venom gland of B. jararaca and that the elements composing this pathway are noted only when CMP is used as substrate. Large autophagosomes reactive to both betaGP and to CMP were commonly observed in the basal region of the secretory cells, and they were more abundant in the glands during the stage of low activity of protein synthesis.

Crystallization and preliminary X-ray analysis of jararhagin, a metalloproteinase/disintegrin from Bothrops jararaca snake venom.

Jararhagin is a toxic protein, isolated from the venom of the snake Bothrops jararaca, which is composed of a metalloprotease domain coupled to a disintegrin/cysteine-rich domain. It induces local haemorrhage owing to the proteolytic digestion of the basement membrane of capillaries. Jararhagin also cleaves the alpha(2)beta(1) integrin on the surface of platelets, thereby acting as a potent inhibitor of collagen-induced platelet aggregation. Crystals of jararhagin were obtained by the vapour-diffusion technique at 273 K in 200 mM sodium acetate, 100 mM cacodylate buffer pH 6.5 and 30% PEG 8000. Diffraction data have been obtained to a resolution of 2.8 A from a single frozen crystal, which belonged to space group P2(1)2(1)2(1) with unit-cell parameters a = 73.7, b = 100.3, c = 133.4 A. The asymmetric unit contains two jararhagin molecules and has a solvent content of 45%. A molecular-replacement solution has been obtained using a homology-built model based on the crystal structure of acutolysin, a haemorrhagic zinc metalloproteinase from the venom of the snake Agkistrodon acutus; attempts are under way to locate the remaining domains.

Jararhagin ECD-containing disintegrin domain: expression in escherichia coli and inhibition of the platelet-collagen interaction.

Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound A Disintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet-collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet-collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin-disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC(50) of 8.5 microg/ml).