ABSTRACT
Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn²+, Cu²+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system.
Subject(s)
Animals , Rats , Crotalid Venoms , Thrombin/isolation & purification , Thrombin/toxicityABSTRACT
Snakebites comprise a serious health problem in several countries due to their global incidence, which exceeds 2.5 million per year, and the elevated number of victim fatalities. To counteract envenomations, antivenoms have been used regularly for more than a century. Apart from side effects including anaphylactic reactions, antivenoms are not able to efficiently neutralize local tissue damage, which contributes to increasing the severity and morbidity observed in patients. This fact, in turn, may be responsible for economic hardship, particularly in rural populations of developing countries. In the present work, we evaluated the antiophidian properties of 12 Brazilian plant extracts against the hemolytic, coagulant, hemorrhagic and proteolytic effects of Lachesis muta venom. Taken together, our data revealed that most of these aqueous products were capable of inhibiting those activities at different levels, except for Sapindus saponaria extract. In contrast, Stryphnodendron barbatiman extract completely neutralized all the analyzed biological activities. Thus, we may conclude that Brazilian flora may also be useful against L. muta accidents.
Subject(s)
Animals , Rats , Antivenins , Lachesis muta , Plant Extracts , PhytotherapyABSTRACT
An acidic phospholipase A2 isolated from Lachesis muta snake venom denoted LM-PLA2, showed neither toxic nor anticoagulant activities in contrast to a potent inhibitory effect of collagen-induced platelet aggregation [Fuly, A.L., Machado. O.L.T., Alves, E.W. and Carlini, C.R., 1997. Thromb. Haemost 78, 1372-1380.]. Now, the myotoxicity induced by LM-PLA2 was investigated by using both in vivo and in vitro experiments. LM-PLA2 induced in vitro a dose- and time-dependent release of creatine-kinase (CK) from mouse Extensor Digitorium Longus (EDL) muscles and also increased the plasma CK activity in treated animals. Histopathological studies confirm myonecrosis of mouse skeletal muscles as a major effect. Edema could also be seen in muscle tissue. The amino-terminal sequence of LM-PLA2 (previously reported) indicates an aspartic acid residue located at position 49, together with other conserved amino acids present in the Asp-49 phospholipases, such as Tyr-28, Gly-30, Gly-32, His-48. Chemical modification of the protein moiety was also performed. Histidine alkylation with p-bromophenacyl bromide and lysine acetylation with acetic anhydride, abolished both indirect hemolytic and myotoxic activities of LM-PLA2. On the other hand, contrarily to what has been observed with several basic myotoxic phospholipases, the myotoxic effect induced by LM-PLA2 was not abolished by heparin.
Subject(s)
Muscles/drug effects , Mycotoxins/toxicity , Phospholipases A/toxicity , Viper Venoms/enzymology , Viperidae , Animals , Creatine Kinase/blood , Creatine Kinase/metabolism , Mice , Muscles/pathology , Mycotoxins/chemistry , Mycotoxins/isolation & purification , Phospholipases A/chemistry , Phospholipases A/isolation & purification , Phospholipases A2 , RabbitsABSTRACT
Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources. LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercaptoethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lysophosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet's response to several agonists.
Subject(s)
Phospholipases A/pharmacology , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Viper Venoms/pharmacology , Amino Acid Sequence , Animals , Blood Platelets/drug effects , Cell Separation , Hemolysis/drug effects , Hot Temperature , Lysophosphatidylcholines/pharmacology , Mice , Molecular Sequence Data , Phospholipases A/isolation & purification , Phospholipases A/toxicity , Phospholipases A2 , Platelet Aggregation/drug effects , Rabbits , Viper Venoms/chemistry , Viper Venoms/toxicityABSTRACT
Screening of the biochemical-pharmacological properties of the crude venom from the snake Lachesis muta indicated the presence of phospholipase A2 (PLA2; 5260 U/mg protein), procoagulant (2630 U/mg protein), platelet aggregating (43 U/mg protein) and caseinolytic activities (6670 U/mg protein). These activities were separated by filtration of the crude venom on Sephacryl S-200. The material containing PLA2 activity was further fractioned by DEAE-cellulose ion exchange chromatography into four active fractions (F-I to F-IV, containing 1.7, 1.2, 0.3, and 0.05% of the crude venom protein, respectively) by stepwise elution with buffers of increasing ionic strength. All fractions presented a molecular weight of approximately 15,000 and isoelectric points in the range pH 4.6-6.0. In addition to their indirect hemolytic activity, the partially purified fractions inhibited platelet aggregation induced either by collagen or thrombin. p-Bromophenacyl bromide-treated fractions lost both phospholipase A2 activity and their inhibitory effect on collagen-induced platelet aggregation.
Subject(s)
Phospholipases A/isolation & purification , Viper Venoms/chemistry , Animals , Chromatography, Gel , Chromatography, Ion Exchange , Phospholipases A/pharmacology , Phospholipases A2 , Platelet Aggregation , Viper Venoms/enzymology , Viper Venoms/metabolism , ViperidaeABSTRACT
Screening of the biochemical-pharmacological properties of the crude venom from the snake Lachesis muta indicated the presence of phospholipase A2 (PLA2; 5260 U/mg protein), procoagulant (2630 U/mg protein), platelet aggregating (43 U/mg protein) and caseinolytic activities (6670 U/mg protein). These activities were separated by filtration of the crude venom on Sephacryl S-200. The material containing PLA2 activity was further fractioned by DEAE-cellulose ion exchange chromatography into four active fractions (F-I to F-IV, containing 1.7, 1.2, 0.3, and 0.05 per cent of the crude venom protein, respectively) by stepwise elution with buffers of increasing ionic strength. All fractions presented a molecular weight of approximately 15,000 and isoelectric points in the range pH 4.6-6.0. In addition to their indirect hemolytic activity, the partially purified fractions inhibited platelet aggregation induced either by collagen or thrombin. p-Bromophenacyl bromide-treated fractions lost both phospholipase A2 activity and their inhibitory effect on collagen-induced platelet aggregation
