Etudes structurales et fonctionnelles de l'activateur du plasminogène du venin de serpent Trimeresurus stejnegeri

PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Cerastatin, a new potent inhibitor of platelet aggregation from the venom of the Tunisian viper, Cerastes cerastes.

International audienceCerastatin, a potent platelet aggregation inhibitor, was purified by gel nitration on Sephadex G-75, followed by two ion exchange chromatographies on Mono-S columns. Cerastatin is a neutral glycoprotein (pI = 6.2) of 32 kDa, made up of at least three subunits. It is devoid of phospholipase A2, esterase, fibrinogenolytic and amidolytic activities. It inhibits aggregation of washed platelets, induced by either collagen, PAF acether or thrombin, with similar ic50 of 2.3 nM. Cerastatin also inhibits the thrombin-induced clot retraction of platelet-rich plasma. It does not inhibit the amidolytic or the procoagulant activities of thrombin. Cerastatin caused no lytic effect on platelet membranes since it did not cause release of lactate dehydrogenase. Pretreatment of platelets with cerastatin irreversibly inhibits the aggregation induced by thrombin. Also, cerastatin completely inhibits the fibrinogen-induced aggregation of a chymotrypsin-treated platelets. Cerastatin therefore inhibits platelet aggregation by interfering with the interaction of fibrinogen with fibrinogen receptors

Toxines et douleurs

Malgré les progrès récents réalisés au cours de la dernière décennie, les mécanismes cellulaires et les structures moléculaires impliqués dans la perception, la transmission et l'intégration des sensations douloureuses restent encore largement inconnus. D'un autre point de vue, des observations récentes ont démontré que des toxines issues d'organismes variés, et pour certaines connues depuis longtemps pour agir sur la propagation ou la transmission des influx nerveux, possèdent des propriétés antalgiques. L'identification de ces toxines actives contre la douleur est loin d'être achevée et leur inventaire se poursuit avec beaucoup de succès. Par ailleurs, les cibles moléculaires et les mécanismes d'action de ces toxines analgésiantes commencent à être identifiés. Elles apparaissent ainsi comme des outils précieux pour analyser et caractériser les structures moléculaires impliquées dans la perception et la transmission de la douleur. Certaines de ces toxines analgésiantes sont même à l'origine de médicaments puissants et originaux utilisables pour atténuer voire supprimer la sensibilité à la douleur. Sans prétendre pour autant couvrir la totalité de ce vaste sujet, le cinquième volume des Rencontres en Toxinologie illustre, au moyen de quelques exemples précis, l'ensemble de cette problématique et témoigne de la vitalité de la recherche, notamment française, dans ce domain

Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes

International audienceCerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1–10 μM) also cleaved prothrombin and Factor X

Toxines et recherches biomédicales

Les toxines sont devenues un outil indispensable dans l'étude de la physiologie cellulaire. L'identification de nouveaux récepteurs membranaires de haute spécificité par des toxines de divers venins ouvre la voie à la sélection d'effecteurs d'intérêt thérapeutique. Les toxines peuvent aussi servir de modèle structural pour la synthèse de molécules organiques affranchies de toute propriété antigénique. Et les domaines constitutifs de certaines toxines microbiennes peuvent être isolés et utilisés dans une perspective de thérapie cellulaire. Les toxines offrent d'immenses perspectives biomédicales

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization

International audienceWe analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410

Purification And Characterization Of A Hemorrhagic Metalloproteinase From Bothrops Lanceolatus (fer-de-lance) Snake Venom.

Bothrops snake venoms contain metalloproteinases that contribute to the local effects seen after envenoming. In this work, a hemorrhagic metalloproteinase (BlaH1) was purified from the venom of the snake Bothrops lanceolatus by a combination of gel filtration, affinity (metal chelating) and hydrophobic interaction chromatographies. The hemorrhagin was homogeneous by SDS-PAGE and had a molecular mass of 28 kDa that was unaltered by treatment with beta-mercaptoethanol. BlaH1 gave a single band in immunoelectrophoresis and immunoblotting using commercial bothropic antivenom. BlaH1 had hemorrhagic, caseinolytic, fibrinogenolytic, collagenolytic and elastinolytic activities, but no phospholipase A(2) activity. The hemorrhagic and caseinolytic activities were inhibited by EDTA, indicating that they were metal ion-dependent. In contrast, aprotinin, benzamidine and PMSF did not affect these activities. The caseinolytic activity of BlaH1 had a pH optimum of 8.0 and was stable in solution at up to 40 degrees C; activity was completely lost at > or =70 degrees C. The hemorrhagic activity was neutralized by commercial bothropic antivenom. These properties suggest that this new hemorrhagin belongs to class P-I snake venom metalloproteinases.45411-2