ABSTRACT
The objective of the present study was an investigation of the crude Bothrops moojeni venom, aiming at the identification of new compounds with platelet-activating or -inhibiting activity. The venom was separated by gel filtration chromatography into 18 fractions, which were tested by means of whole blood aggregometry for their activities affecting the aggregation of blood platelets. In order to eliminate interferences caused by prothrombin activators or thrombin like-enzymes, which are frequently present in snake venoms, a test method for screening protein mixtures was developed. To avoid clotting of the blood samples, the thrombin inhibitor hirudin and the synthetic inhibitor of fibrin polymerization Pefabloc FG were applied. In the present study, a platelet aggregation activator with an activity resembling thrombocytin from B. atrox was identified in one of the examined venom fractions. In addition, a platelet antagonist-most likely a disintegrin-with broad inhibitory activity against aggregation triggered by collagen, adenosine diphosphate and thrombin receptor activating peptide, was identified.
Subject(s)
Biological Assay/methods , Blood Platelets/drug effects , Bothrops , Crotalid Venoms , Animals , Blood Platelets/physiology , Crotalid Venoms/chemistry , Crotalid Venoms/pharmacology , Platelet Activation/drug effects , Platelet Activation/physiology , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/pharmacologyABSTRACT
Among the proteins and peptides already characterized in Bothrops moojeni venom, two novel phospholipases A(2) (PLA(2)) have been purified and fully sequenced by ESI-MS/MS techniques. Both of them belong to the enzymatically non-active Lys49 variants of PLA(2). They consist of 122 amino acids and share a characteristic sequence in their C-terminal region composed of clusters of basic amino acids known to interact with heparin. Thus, as already established, heparin can be used as an antidote to antagonize some myotoxic PLA(2)s from venoms of Bothrops genus. The two PLA(2) variants were shown to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH), neutralizing their anticoagulant properties. Although the influences of PLA(2)s from snake venoms on the blood coagulation system are known, their use to antagonize the anticoagulant effect of heparin in vitro or in vivo has never been proposed. These finding recommend diagnostic and therapeutic applications, which are currently investigated.
Subject(s)
Bothrops/physiology , Crotalid Venoms/chemistry , Heparin Antagonists/chemistry , Phospholipases A/chemistry , Animals , Blood Coagulation/drug effects , Blood Coagulation Tests , Cell Survival/drug effects , Cells, Cultured , Chromatography, Gel , Chromatography, High Pressure Liquid , Crotalid Venoms/pharmacology , Fibroblasts/drug effects , Fibroblasts/pathology , Heparin Antagonists/pharmacology , Humans , Lysine/chemistry , Phospholipases A/pharmacology , Protein Isoforms/chemistry , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
Early studies in the 1930s on the venom of South American Lancehead snakesofthe Bothrops genuslead to the discovery of compounds active in blood coagulation such as batroxobin and botrocetin. The scope of our investigations is to have a deeper look at the crude venom of B. moojeni using state-of-the-art proteomics methods, as well as newly developed bioassays screening for activities in the different fields of application. The proteomics techniques used up to now have included different chromatography methods, mass spectrometry, and bio-computing. The bioassays are focussed on enzymatic and other activities in the field of hemostasis and fibrinolysis. Besides the known activities several new and interesting ones have been found. They still need to be studied and confirmed in more specific supplementary assays.
