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

Abstract

Cerastes cerastes venom contains several bioactive proteins with inhibitory potential of platelet aggregation and blood coagulation. The current study deals with purification, characterization and determination of structural properties of Cc-PDE, the first phosphodiesterase from Cerastes cerastes venom. 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. 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 β-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. 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.