Purification and preliminary characterization of "Bothrops moojeni" venom components active on haemostasis : ("Botmo thesis")

For almost every factor involved in haemostasis there exists a venom protein that can mimic, activate or deactivate it. Many of these components are insensitive to the physiological and therapeutically used coagulation inhibitors and, because of their unique features, are applied as molecular tools for diagnosis and therapy of haemostatic disorders. Batroxobin, purified from the venoms of Bothrops moojeni and B. atrox snakes, commonly known under its trade names Reptilaseâ and Defibraseâ is still widely used in pharmaceutical and diagnostic applications. However, the venom material needed for its production is not further analyzed, although it still contains a large number of potentially active compounds. The goal of this Botmo Thesis was the investigation of the crude B. moojeni venom for the identification of new active ingredients affecting haemostasis. Various protein purification methods, mass spectrometry and biocomputing, as well as newly developed coagulation screening tools were applied.
B. moojeni crude venom and its fractions were used as model substances for the development of different screening tools applying mass spectrometry (MS) technology. The complexity of the venom provides a good selectivity assessment for the developed methods. By means of the novel MS approaches, 15 new sequences of Bradykinin-potentiating peptides (BPPs) were revealed, as well as the presence of acetylcholinesterase inhibiting venom component. To our knowledge, it is the first time acetylcholinesterase inhibiting properties were described in the venom of B. moojeni snake.
Further, two novel Lys49 phospholipases A2 (PLA2) have been purified and fully sequenced, showing a characteristic sequence in the C-terminal region known in the literature to interact with heparin. Both PLA2s were able to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH) neutralizing their anticoagulant properties. The revealed sequences, possessing the LMWH neutralization properties, may be of medical interest, because there is no LMWH antidote used in clinical applications.
Moreover, a procoagulant protein resembling the coagulation FVIIa could be identified in the B. moojeni venom. Such activity has never been reported for any snake venom until now and further investigations would be of medical interest.
In addition a new method was developed for the identification of active ingredients acting on blood platelets. It can be used for screening complex protein mixtures, such as B. moojeni venom, containing platelet activating prothrombin activators or thrombin like-enzymes. By means of this methodology, a platelet aggregation activator and platelet antagonist with broad inhibitory activity, which is most likely a disintegrin, could be identified.
Up to now, it could be shown that B. moojeni venom is a highly complex mixture of active ingredients and still remains a huge “source of undiscovered potential”. Further purifications and characterization steps are needed in order to get a complete activity profile of the venom, also in fields other than haemostasis. Nevertheless, in the framework of this Botmo Thesis, some active ingredients, also such of medical interest, were identified.