Snake venoms are a mixture of hydrolases which produce complex pathogenesis such as bleeding, dermo/ myonecrosis, inflammation and coagulation disorders. The toxicity of venoms cannot be attributed to only one component. It is well known that venom components present antagonist activities, while some of them work synergistically. Binding to their intra-and extra-cellular or molecular targets, leads these components to generate severe disturbances which might concern several systems through complex mechanisms. Some of these mechanisms are still not yet elucidated. Thus, some of these components can act at different steps of blood coagulation by activating or inhibiting several molecular or cellular targets thereby inducing blood disorders. Despite their effects, it is well established that some of components from snake venoms present beneficial effects when acting alone as purified entity. Appropriate treatments of snakebite victims need a complete understanding of the pharmacological roles of the different venom components. Thus, this review emphasizes the toxicological relevance of snake venoms mainly those of Viperidae and their components as pharmacological bioactive tools.

Three-step chromatography and proteomic analysis have been used to purify and characterize a new basic phospholipase A₂ named CC2-PLA₂ from the venom of Cerastes cerastes. This phospholipase A₂ has been isolated to an extent of about 50-folds and its molecular weight was estimated at 13,534 Da. For CC2-PLA₂ identification and LC-MALDI-MS/MS analysis, the protein was reduced, alkylated and double hydrolyzed by lysine-C endopeptidase and trypsin. Tryptic fragments of LC-MS/MS analyzed CC2-PLA₂ showed sequence similarities with other snake venom PLA₂. This presents only 51 % (61/120 amino acid residues) sequence homology with the first PLA₂ (gi |129506|) previously purified from the same venom. The isolated CC2-PLA₂ displayed anti-aggregative effect on platelets and induced an inflammatory response characterized by leukocytosis in the peripheral blood. This inflammatory response is accompanied by a release of inflammatory mediators such as IL-6, eosinophil peroxidase and complement system. Obtained results indicate that CC2-PLA₂ induced a release of high level of pro-inflammatory (IL-6) cytokine and no effect on the level of anti-inflammatory cytokine (IL-10) in blood sera. Furthermore, eosinophil peroxidase activity and hemolytic complement effect increased in peripheral blood. Mononuclear and neutrophil cells were found predominant in the induced leucocytosis following CC2-PLA₂ administration into animals.

A procoagulant metalloproteinase called CCSV-MPase was purified from C. cerastes venom by successive chromatographic methods starting with gel-filtration through Sephadex G-75; ion-exchange DEAE-Cellulose A-50; affinity chromatography on Benzamidine Sepharose 6B and RP-HPLC on a C8 column. CCSV-MPase has been isolated to an extent of about tenfolds and its molecular mass was evaluated at 70 kDa by SDS-PAGE. CCSV-MPase hydrolyzes casein and fibrinogene as natural substrates. Its proteolytic activity was inhibited by EDTA and 1.10-phenanthroline, a chelators of bivalent cation metals and Zn(2+) respectively. CCSV-MPase is therefore a Zn(2+)-metalloproteinase with fibrinogenolytic but not hemorrhagic activity. It greatly decreased levels of plasmatic fibrinogen when administered to rats for 24 h. This fibrinogenase hydrolyzes the Bβ chain of human fibrinogen in vitro releasing fibrinopeptide B only. LC MS/MS analysis of tryptic fragments of CCSV-MPase demonstrated that it showed some sequence similarities with four other venom metalloproteinases. CCSV-MPase could be considered as a potential therapeutic agent as it is a non-hemorrhagic enzyme and could be useful in thrombotic diseases because of its defibrinogenation of blood.

Biomolecules from Cerastes cerastes venom have been purified and characterized. Two phospholipases isolated from Cerastes cerastes venom share 51% of homology. CC2-PLA2 exhibits antiplatelet activity that blocks coagulation. CCSV-MPase, a non-hemorrhagic Zn2+-metalloproteinase, significantly reduced the plasmatic fibrinogen level and hydrolyzes only its Bβ chain. Serine proteinases such as RP34, afaâcytin and CC3-SPase hydrolyze the fibrinogen and are respectively α, αβ and αβ fibrinogenases. In deficient human plasma, afaâcytin replaces the missing factors VIII and IX, and activates purified human factor X into factor Xa. It releases serotonin from platelets and directly aggregates human (but not rabbit) blood platelets. RP34 proteinase also had no effect on both human and rabbit blood platelet aggregation. CC3-SPase revealed a pro-coagulant activity. However, the insolubility of the obtained clot indicates that CC3-SPase does not activate factor XIII. In addition, CC3-SPase clotting activity was carried out with human plasmas from volunteer patients deficient in clotting factors. Results showed that CC3-SPase shortens clotting time of plasma deficient in factors II and VII but with weaker clotting than normal plasma. The clotting time of plasma deficient in factor II is similar to that obtained with normal plasma; suggesting that CC3-SPase is able to replace both factors IIa and VII in the coagulation cascade and thus could be involved in the blood clotting process via an extrinsic pathway. These results imply that CC3-SPase and afaâcytin could repair hemostatic abnormalities and may replace some factors missing in pathological deficiency. Afaâcytin also exhibits α fibrinase property similar to a plasmin-like proteinase. Despite its thrombin-like characteristics, afaâcytin is not inhibited by plasmatic thrombin inhibitors. The procoagulant properties of afaâcytin might have potential clinical applications.

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The present study is the first attempt to report the characterization of a nucleotidase from Cerastes cerastes venom. A 70 kDa 5'-nucleotidase (Cc-5'NTase) was purified to homogeneity. The amino acid sequence of Cc-5'NTase displayed high homology with many nucleotidases. Its activity was optimal at pH 7 with a specific hydrolytic activity toward mono-, di-, and triphosphate adenylated nucleotides. Cc-5'NTase preferentially hydrolyzed ADP and obeyed Michaelis-Menten kinetics. Among the metals and inhibitors tested, Ni and Mg completely potentiated enzyme activity, whereas EGTA, PMSF, iodoacetamide, vanillic acid, vanillyl mandelic acid, and 1,10-phenanthroline partially abolished its activity. Cc-5'NTase was not lethal for mice at 5 mg/kg and exhibited in vivo anticoagulant effect. It also dose-dependently inhibited adenosine diphosphate-induced platelet aggregation by converting adenosine diphosphate to adenosine and prohibited arachidonic acid-induced aggregation but was not effective on fibrinogen-induced aggregation. Cc-5'NTase could be a good tool as pharmacological molecule in thrombosis diagnostic and/or therapy.

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This study aimed to elucidate anticoagulant/antiplatelet mechanisms of two previously purified PLA2s from Cerastes cerastes venom, here, termed Cc1‐PLA2 and Cc2‐PLA2. Both PLA2s present close molecular weights of 13,534 and 13,430 Da and Isoectric pH (pI) 7.38 and 7.86 respectively, for Cc1‐PLA2 and Cc2‐PLA2. These Ca2+‐dependent enzymes showed a high catalytic activity upon phospholipids, inducing indirect hemolysis, since they conserve the catalytic domain of PLA2s 26CYCGWGGKG34. They exhibited dual inhibition of platelet aggregation by targeting P2Y12 and TPα receptors preventing Adenosine diphosphate/arachidonate binding and blood clotting. These effects are due to the interaction of Cc1‐PLA2s/Cc2‐PLA2s with factor FXa through a noncatalytic PL‐independent mechanism leading to nonreleased thrombin. Both proteins consist of 120 amino acid residues and share similar three‐dimensional structures close to other SV‐PLA2s. Structural data of PLA2s allowed the relevant residues involved in binding to FXa and platelet receptors. These findings may lead to the design of novel noncompetitive FXa inhibitors.