RESUMO
BACKGROUND: The aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (µm2), the area of the fibrin fibers (µm2), and the width of the fibrin fibers (µm) were determined for the dog clots. The platelet area (µm2), the area of fibrin fibers (µm2), the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots. RESULTS: Cat platelets displayed full activation. Dog platelets displayed lysis with loss of normal architecture. In both species, a statistically significant difference was found (P < 0.01) between the fibrin fiber measurements in the PC clots activated with CG and CGB. CONCLUSIONS: The findings suggest that activation with CG caused platelet alpha granules to release their contents. In cats, fibrin production was greater when the PC was activated with CG. In dogs, activation with CG produced thick fibrin fibers.
Assuntos
Batroxobina/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/ultraestrutura , Gluconato de Cálcio/farmacologia , Fibrina/ultraestrutura , Fibrinolíticos/farmacologia , Animais , Batroxobina/administração & dosagem , Plaquetas/efeitos dos fármacos , Gluconato de Cálcio/administração & dosagem , Gatos/sangue , Cães/sangue , Quimioterapia Combinada , Espaço Extracelular/efeitos dos fármacos , Fibrina/efeitos dos fármacos , Fibrinolíticos/administração & dosagem , Masculino , Microscopia Eletrônica de Transmissão/veterinária , Trombose/veterináriaRESUMO
In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of A alpha and B beta chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of A alpha and B beta chains at 5 min and of gamma chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm(2)/microg, respectively using 25 microg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD(50) of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 microg protein, showed that B. isabelae venom contained higher specific activity (50 mm(2)/microg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Bothrops/fisiologia , Venenos de Crotalídeos/farmacologia , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Hemostasia/efeitos dos fármacos , Amidoidrolases/sangue , Animais , Venenos de Crotalídeos/química , Eletroforese em Gel de Poliacrilamida , Fibrina/efeitos dos fármacos , Fibrinolíticos/química , Gelatina/efeitos dos fármacos , Gelatina/metabolismo , Hemorragia/induzido quimicamente , Hemorragia/patologia , Dose Letal Mediana , Masculino , Camundongos , Especificidade da Espécie , VenezuelaRESUMO
OBJECTIVE: To compare the adhesion and maturation of blood components on chemically conditioned root surfaces. METHOD AND MATERIALS: Clinical root samples of human teeth were obtained (n = 150) and manually scaled. Five groups of 30 samples were treated as follows: (1) saline solution irrigation (control); (2) 24% EDTA gel; (3) 25% citric acid solution; (4) tetracycline solution (50 mg/mL); and (5) 30% sodium citrate solution. After these treatments, 15 samples of each group received a blood drop and were analyzed by SEM. The remaining 15 had their surface morphology evaluated for collagen fibrils exposure by SEM. Photomicrographs were analyzed according to the score of adhesion of blood components. Kruskal-Wallis and Dunn multiple comparison tests were employed. RESULTS: The control group was characterized by the absence of blood elements on the surface. The best result was observed in the citric acid group, which had a dense fibrin network with blood elements adhered. The EDTA group showed a moderate fibrin network formation. In contrast, a scarce fibrin network and a few cells were present in the tetracycline samples, and an absence of blood elements was found on sodium citrate specimens. The citric acid group was statistically different from the control group (P < .01). No differences were found among the control, EDTA, tetracycline, and sodium citrate groups (P > .05). CONCLUSION: Under these experimental conditions, citric acid is indicated to stabilize clots on the root surface, which act as a scaffold for connective tissue cell development.
Assuntos
Condicionamento Ácido do Dente/métodos , Coagulação Sanguínea/efeitos dos fármacos , Quelantes/uso terapêutico , Dentina/efeitos dos fármacos , Raiz Dentária/efeitos dos fármacos , Células Sanguíneas/ultraestrutura , Adesão Celular/efeitos dos fármacos , Citratos/uso terapêutico , Ácido Cítrico/uso terapêutico , Colágeno/ultraestrutura , Cemento Dentário/efeitos dos fármacos , Cemento Dentário/ultraestrutura , Raspagem Dentária , Dentina/ultraestrutura , Ácido Edético/uso terapêutico , Fibrina/efeitos dos fármacos , Fibrina/ultraestrutura , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Aplainamento Radicular , Método Simples-Cego , Camada de Esfregaço , Cloreto de Sódio , Citrato de Sódio , Tetraciclina/uso terapêutico , Raiz Dentária/ultraestruturaRESUMO
Mutalysin II, a zinc endopeptidase possessing direct-acting fibrinolytic activity has been previously purified from bushmaster (Lachesis muta muta) snake venom. We now report a method to isolate two isoforms of natural mutalysin II (mut IIa and mut IIb) using chromatographies on Sephacryl S-200, CM Sepharose CL 6B and Sephadex G-50. The two proteins are monomeric non-glycosylated proteinases with similar molecular masses of 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Both isoforms showed high similarity in all enzymatic properties using fibrinogen, fibrin and dimethylcasein as substrates. Thus, the specific fibrinolytic activity was estimated as 12+/-1.04 and 11.5+/-1.02 U/microg for mut IIa and mut IIb, respectively. The antigenic cross-reactivity of both isoforms was examined using rabbit hyperimmune serum or immunoglobulin G anti-mut IIa assays on immunodiffusion microscope slides, indirect enzyme-linked immunoabsorbent assay and western blots. From these experiments it was concluded that the two metalloproteinases mut IIa and mut IIb share identical antigenic structures. Since the stability of mutalysin II is dependent upon the presence of zinc, we examined the EDTA sensitivity of the isoforms of mutalysin II. Thus, the IC(50) values (concentration of EDTA to produce 50% inhibition of dimethylcasein hydrolysis) for mut IIa is 180 microM and 165 microM for mut IIb.
Assuntos
Glicoproteínas/metabolismo , Metaloendopeptidases/metabolismo , Venenos de Serpentes/isolamento & purificação , Venenos de Serpentes/metabolismo , Viperidae/metabolismo , Animais , Caseínas/efeitos dos fármacos , Caseínas/metabolismo , Cromatografia Líquida de Alta Pressão , Fibrina/efeitos dos fármacos , Fibrina/metabolismo , Fibrinogênio/efeitos dos fármacos , Fibrinogênio/metabolismo , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Imuno-Histoquímica , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Mapeamento de Peptídeos , Coelhos , Venenos de Serpentes/química , Venenos de Serpentes/enzimologia , Análise Espectral , Zinco/farmacologiaRESUMO
Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picado, Costa Rica, were assessed for their ability to neutralize various toxic activities of the venoms of North American snakes of the genera Crotalus, Agkistrodon and Micrurus, in assays involving preincubation of venom and antivenom. When the intraperitoneal route of injection was utilized, polyvalent (Crotalinae) antivenom was effective in the neutralization of the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus viridis viridis, Crotalus horridus atricaudatus, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus, whereas the venom of Crotalus scutulatus was not neutralized. When the intravenous route was used, results differed depending on the "challenge dose" of venom employed. Polyvalent antivenom neutralized all venoms when mice were challenged with 2 LD(50)s of venom. When 5 LD(50)s were used, antivenom neutralized the venoms of C. atrox, C. adamanteus, C. v. viridis and C. h. atricaudatus, being ineffective in the neutralization of C. scutulatus, A. c. contortrix and A. p. piscivorus. Polyvalent antivenom was effective in the neutralization of hemorrhagic and myotoxic activities of all venoms studied. It also neutralized coagulant activity of C. adamanteus venom, whereas most of the venoms were devoid of clotting activity on plasma in vitro. Moreover, it neutralized defibrinating activity of the only three venoms that induced this effect (i.e. C. adamanteus, A. c. contortrix and A. p. piscivorus). Anticoral (Elapidae) antivenom neutralized lethality induced by the venom of Micrurus fulvius, using either the intravenous or the intraperitoneal routes of injection. Moreover, it neutralized myotoxic effect of this venom as well. It is concluded that polyvalent antivenom neutralizes lethality and other activities of most of the crotaline venoms tested. However, since it is ineffective in neutralizing the lethal effect of C. scutulatus venom, it is suggested that a venom containing presynaptically-active neurotoxic phospholipases A(2) related to "mojave toxin" needs to be introduced in the immunizing mixture in order to increase the neutralizing scope of this product in North America. Anticoral antivenom is highly effective in the neutralization of the venom of M. fulvius.
Assuntos
Antivenenos/farmacologia , Venenos de Crotalídeos/antagonistas & inibidores , Venenos Elapídicos/antagonistas & inibidores , Hemólise/efeitos dos fármacos , Animais , Antivenenos/administração & dosagem , Antivenenos/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Costa Rica , Venenos de Crotalídeos/toxicidade , Venenos Elapídicos/toxicidade , Fibrina/efeitos dos fármacos , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Infusões Intravenosas , Injeções Intradérmicas , Injeções Intramusculares , Injeções Intraperitoneais , Dose Letal Mediana , Camundongos , Testes de Neutralização , Serpentes , Estados UnidosRESUMO
The extract of Marsypianthes chamaedrys, a plant used against snakebites, in the present study was shown to inhibit fibrinoclotting induced by several Brazilian snake venoms or thrombin. These data indicate that this extract affected thrombin-like enzymes. In this first report we determine some features of the components present in the extract regarding the antifibrinoclotting action. Our results show that active components responsible for those effects are thermo-resistant and are concentrated in the methanolic fraction.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Lamiaceae , Fitoterapia , Extratos Vegetais/farmacologia , Venenos de Serpentes/farmacologia , Animais , Anticoagulantes/administração & dosagem , Brasil , Relação Dose-Resposta a Droga , Fibrina/efeitos dos fármacos , Concentração Inibidora 50 , Componentes Aéreos da Planta , Extratos Vegetais/administração & dosagem , Venenos de Serpentes/antagonistas & inibidores , Serpentes , Trombina/farmacologiaRESUMO
A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature not observed for most of the snake venom metalloproteinases, including the group A prothrombin activators.
Assuntos
Venenos de Crotalídeos/química , Ativadores de Enzimas/isolamento & purificação , Protrombina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bothrops , Moléculas de Adesão Celular/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular , DNA Complementar , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Fibrina/efeitos dos fármacos , Fibrinogênio/efeitos dos fármacos , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fator de von Willebrand/metabolismoRESUMO
On the basis of growing clinical evidence, it is well known that elevated plasma homocysteine (Hcy) levels are associated with higher risk of venous and arterial thrombosis. Several experimental studies have been carried out in order to elucidate the mechanisms involved that still remain unclear. The aim of our study was to evaluate the homocysteine effects on formation and structure of plasmatic fibrin network. We also assayed homocystine and cysteine to determinate possible participation of thiol group in the tested activity. Aliquots of a pool of plasma incubated separately with sulfur compounds were clotting with thrombin. Fibringeneration and fibrin networks were evaluated by kinetic studies and scanning electronic microscopy, respectively. No significant differences were observed on fibrin generation of the substances assayed in comparison to control. The scanning electronic microscopy showed that Hcy-associated networks were different from control, with shorter, thicker and more branched fibers, resulting in a more compact structure and probably more resistant to fibrinolysis. The thiol group would be involved in this effect. Our findings would be a new contribution to elucidate the mechanisms involved in harmful effects associated to hyperhomocysteinemia.
Assuntos
Fibrina/efeitos dos fármacos , Homocisteína/farmacologia , Compostos de Sulfidrila/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fibrina/ultraestrutura , Humanos , Cinética , Microscopia Eletrônica de Varredura , Trombina/farmacologia , Trombose/etiologiaRESUMO
This investigation compared the ability of six Latin American antivenoms (monovalent antibothropic INS, Santafé de Bogotá; polyvalent INS; polyvalent probiol, Santafé de Bogotá; antibothropic Instituto Butantan, IB, São Paulo, Brazil; polyvalent Instituto Clodomiro Picado, ICP, San José, Costa Rica; polyvalent MYN, Mexico) to neutralize various pharmacological and enzymatic effects of Bothrops atrox venom from Antioquia and Chocó, north-west of Colombia. Our results demonstrated conspicuous differences in the ability of the six antivenoms. In terms of neutralization of lethality, the highest efficacy was observed in the polyvalent INS and the lowest in the polyvalent MYN antivenom. All antivenoms were highly effective in the neutralization of hemorrhage, polyvalent INS and probiol being the highest. In the neutralization of edema-forming activity, the most effective antivenom was the polyvalent (ICP); monovalent (INS) and polyvalent (MYN) were the least effective. All antivenoms were effective in the neutralization of the myotoxic activity of B. atrox venom, the most effective being the polyvalent (INS) and antibothropic (IB). Defibrinating activity was neutralized by all antivenoms; polyvalent (MYN) showed the lowest efficiency. Polyvalent (ICP) antivenom had the highest neutralizing ability against the indirect hemolytic effect of B. atrox venom; polyvalent (MYN) did not neutralize this enzymatic activity. Overall, the polyvalent antivenom (INS) showed the highest neutralizing ability.