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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.

Cc -SPase (30 kDa-proteinase; pI 5.98) was isolated from Cerastes cerastes venom. Its sequence of 271 residues yielded from LC-MALDI-TOF showed high degrees of homology when aligned with other proteinases. Cc -SPase cleaved natural and synthetic proteins such as casein and fibrinogen leaving fibrin clots unaffected. Cc -SPase was fully abolished by ion chelators, whereas aprotinin, antithrombin III (Sigma Aldrich, Saint-Louis, Missouri, USA), and heparin were ineffective. Affinity of Cc -SPase to benzamidine indicated the presence of an aspartate residue in the catalytic site as confirmed by three-dimensional structure consisting of 14 β-strands and four α-helices. Molecular mechanisms revealed that Cc -SPase is capable of promoting dysfunctional platelet aggregation via two signaling pathways mediated by the G-coupled protein receptors and αIIbβ3 integrin. Cc -SPase is involved in both extrinsic/intrinsic coagulation pathways in deficient plasmas by replacing defective/lacking factors FII, FVII, and FVIII but not FX. Cc -SPase could substitute missing factors in blood diseases related to plasma factor deficiencies.

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