Isolation and Characterization of CD39-like Phosphodiesterase (Cc-PDE) from Cerastes cerastes Venom: Molecular Inhibitory Mechanism of Antiaggregation and Anticoagulation.

BACKGROUND: Cerastes cerastes venom contains several bioactive proteins with inhibitory potential of platelet aggregation and blood coagulation. OBJECTIVE: The current study deals with purification, characterization and determination of structural properties of Cc-PDE, the first phosphodiesterase from Cerastes cerastes venom. MATERIAL AND METHODS: The purification process consists of three successive chromatographies including G75-Sephadex size exclusion, DEAE exchange chromatography and affinity using Sildenafil as a main PDEs' specific inhibitor. The amino acid sequence of purified Cc-PDE was determined by liquid chromatography coupled off line to MALDI-TOF/TOF. Modeling and structural features were obtained using several bioinformatics tools. In vivo and in vitro antiplatelet aggregation and anticoagulant assays were performed. RESULTS: Cc-PDE (73 506.42 Da) is a 654-residue single polypeptide with 1-22 signal peptide and it is characterized by the presence of predominant basic amino acids suitable to alkaline pI (8.17). Cc-PDE structure is composed of ß-strands (17%) and α-helices (24%) and it shares a high identity with homologous snake venom PDEs. Cc-PDE hydrolyzes both Bis-p-nitrophenyl phosphate (Km = 2.60 ± 0.95 mM, Vmax = 0.017 ± 0.002569 µmol.min-1) and p-nitrophenyl phosphate (Km = 7.13 mM ± 0.04490 mM, Vmax = 0.053 ±0.012 µmol.min-1). Cc-PDE prevents ADP- and ATP-induced platelet aggregation by hydrolyzing ADP and ATP, reducing surface P-selectin expression and attenuating platelet function. In addition, Cc-PDE inhibits coagulation factors involved in the intrinsic pathway demonstrated by a significant prolongation of activated partial thromboplastin time and in vivo long-lasting anticoagulation. CONCLUSION: The obtained results revealed that Cc-PDE may have a therapeutic potential and could be a remedy for thromboembolic diseases as an alternative of anticoagulant and antiplatelet aggregation chemical origins.

Isolation and Functional Identification of an Antiplatelet RGD-Containing Disintegrin from Cerastes cerastes Venom.

The current report focuses on purification, structural and functional characterization of Cerastategrin from Cerastes cerastes venom, a novel basic disintegrin (pI 8.36) with 128 amino acid residues and a molecular weight of 13 835.25 Da measured by MALDI-MSMS. The 3D structure of Cerastategrin is organized as α-helix (13%), ß-strand (15%) and disordered structure (30%) and presents homologies with several snake venom disintegrins. Structural modeling shows that Cerastategrin presents an RGD motif that connects specifically to integrin receptors. Cerastategrin exhibits the inhibition of ADP induced platelets with an IC50 of 0.88 µg/mL and shows in vivo long stable anticoagulation effect 24 h post-injection of increasing doses ranging from 0.2 to 1 mg/kg, therefore, Cerastategrin maintained irreversibly the blood incoagulable. Moreover, Cerastategrin decreases the amount of bounded αIIbß3 and reduced significantly the quantity of externalized P-Selectin. Cerastategrin acts as a molecule targeting specifically the receptor αIIbß3; therefore, it behaves as a potent platelet activation inhibitor. As a new peptide with promising pharmacological properties, Cerastategrin could have a potential therapeutical effect in the vascular pathologies and may be a new effective treatment approach.

Molecular modeling, biochemical characterization, and pharmacological properties of Cc3 -SPase: A platelet-aggregating thrombin-like enzyme purified from Cerastes cerastes venom.

Cc3 -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. Cc3 -SPase cleaved natural and synthetic proteins such as casein and fibrinogen leaving fibrin clots unaffected. Cc3 -SPase was fully abolished by ion chelators, whereas aprotinin, antithrombin III (Sigma Aldrich, Saint-Louis, Missouri, USA), and heparin were ineffective. Affinity of Cc3 -SPase to benzamidine indicated the presence of an aspartate residue in the catalytic site as confirmed by three-dimensional structure consisting of 14 ß-strands and four α-helices. Molecular mechanisms revealed that Cc3 -SPase is capable of promoting dysfunctional platelet aggregation via two signaling pathways mediated by the G-coupled protein receptors and αIIbß3 integrin. Cc3 -SPase is involved in both extrinsic/intrinsic coagulation pathways in deficient plasmas by replacing defective/lacking factors FII, FVII, and FVIII but not FX. Cc3 -SPase could substitute missing factors in blood diseases related to plasma factor deficiencies.

Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73-like, from Cerastes cerastes venom.

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, Ni2+ and Mg2+ 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.