Pharmacological and histopathological characterization of Bothrops lanceolatus (Fer de lance) venom-induced edema.

OBJECTIVE: Bothrops venoms cause local edema, pain, hemorrhage and necrosis. In this study, we investigated the ability of Bothrops lanceolatus venom to cause edema in rat hind paws and examined the mediators involved. MATERIALS AND METHODS: Hind paw edema was induced in male Wister rats by the subplantar injection of venom (12.5-100 microg/paw) in the absence and presence of antagonists. Edema was quantified by hydroplethysmometry at 0.25, 0.5, 2, 4, 6 and 24 h post-injection and was expressed as the percentage increase relative to the contralateral (control) paw. The ability of the venom to release histamine from rat peritoneal mast cells was also assessed. RESULTS: Venom caused dose- and time-dependent edema that was maximal within 15 min but disappeared after 24 h and was accompanied by hemorrhage. Dexamethasone (1 mg/kg, s.c.), methysergide (6 mg/kg, i.p.), HOE 140 (0.6 mg/kg, i.v.) and mepyramine (6 mg/kg, i.p.) significantly ( p < 0.05) reduced edema formation, whereas indomethacin (10 mg/kg, i.p.) was ineffective. Dialysis did not affect venom-induced edema. Venom (1, 10 and 30 microg/ml) caused a concentration-dependent release of histamine (13 +/- 1%, 61.9 +/- 4.6% and 73.6 +/- 2.4%, respectively; n = 5) from rat peritoneal mast cells in vitro. Histological analysis confirmed the presence of edema, hemorrhage and neutrophil infiltration. Pretreating the venom with EDTA partially inhibited the edema and hemorrhage, but did not affect the migration of neutrophils. CONCLUSIONS: B. lanceolatus venom produced dose- and time-dependent edema in rat paws. This edema was not dependent on low molecular weight substances in the venom, but was partially dependent on a hemorrhagin and also involved the release of arachidonic acid metabolites, bradykinin, histamine and serotonin.

Coagulant and anticoagulant activities of Bothrops lanceolatus (Fer de lance) venom.

Bothrops lanceolatus venom contains caseinolytic, phospholipase, esterase and haemorrhagic activities. We have investigated the coagulant and anticoagulant actions of B. lanceolatus venom on human citrated plasma and on purified plasma components. Although B. lanceolatus venom up to 50 microg/ml was unable to clot citrated plasma, at concentrations > or = 5 microg/ml the venom dose-dependently clotted purified human fibrinogen, indicating the presence of a thrombin-like enzyme. Human plasma (final concentration > or = 12.5%) dose-dependently inhibited the venom-induced fibrinogen clotting. This finding suggested that endogenous plasma protease inhibitors can affect the venom's action on fibrinogen. To investigate this possibility, B. lanceolatus venom was incubated with different plasma protease inhibitors and the activity on fibrinogen tested. alpha(2)-Macroglobulin and alpha(1)-antitrypsin did not interfere with the coagulant activity of the venom whereas the antithrombin-III/heparin complex partially inhibited this activity. A non-toxic, acidic phospholipase A(2) purified from B. lanceolatus venom prolonged the activated partial thromboplastin time in human plasma from 39.7+/-0.5 s (control with saline) to 60.2+/-0.9 s with 50 microg of PLA(2) (p<0.001), suggesting an anticoagulant activity associated with this enzyme. This anticoagulant activity may account for some of the effects of the venom on blood coagulation.

Purification from Bothrops lanceolatus (fer de lance) venom of a fibrino(geno)lytic enzyme with esterolytic activity.

Bothrops lanceolatus venom has high caseinolytic, phospholipasic, esterolytic and hemorrhagic activities. In spite of having no coagulant effect on plasma, this venom contains a thrombin-like enzyme. Using gel filtration and ion-exchange chromatographies, we have purified an esterolytic fraction (F-II-1a) from this venom with a protein yield of 4% and a 58% recovery in enzyme activity. SDS-PAGE in the presence of beta-mercaptoethanol showed that the enzyme is a single chain polypeptide with a MW=38,100. Immunodiffusion and immunoelectrophoresis of fraction F-II-1a against serum from horses immunized with B. lanceolatus venom and against rabbit antiserum prepared using fraction F-II-1a both showed a single immunoprecipitin line. The Km and Vmax values for TAME hydrolysis were 0.85 mM and 38.6 micromol/min/mg, respectively. The esterolytic activity was completely inhibited by PMSF (10 mM) but not by EDTA (20 mM). Fraction F-II-1a hydrolyzed the alpha and beta chains of fibrinogen. Degradation of the alpha chain occurred within 10 min while that of the beta-chain was slower. The enzyme had no effect on the gamma-chain even after 4 h of hydrolysis.

Determination of phospholipase A2 activity by a colorimetric assay using a pH indicator.

We have set up an assay of phospholipase A2 by a spectrophotometric method, based on the pH change due to the liberation of fatty acids. Among the pH indicators used, phenol red was found to be one of the most sensitive. The activities of different phospholipases A2 from venom and from porcine pancreas were measured by this assay. The results are comparable to those obtained by the pH stat method. This very simple test is rapid, sensitive and especially useful for assaying numerous samples. For quantitative results in absolute units it must be considered that the pH indicator may inhibit some phospholipases.