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

[ Vol. 28 , Issue. 4 ]

Author(s):

Hamida Kiheli, Fatah Chérifi, Meriem Ameziani, Samah Saoud, Ghania Hariti and Fatima Laraba-Djebari*   Pages 426 - 441 ( 16 )

Abstract:


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 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.

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.

Keywords:

Cc-PDE, intrincis pathway blocker, anticoagulant, antiplatelet aggregation, ADP, thromboembolic diseases.

Affiliation:

Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, University of Science and Technology Houari Boumediene (USTHB), BP 32 El-Alia, Bab Ezzouar, Algiers, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, University of Science and Technology Houari Boumediene (USTHB), BP 32 El-Alia, Bab Ezzouar, Algiers, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, University of Science and Technology Houari Boumediene (USTHB), BP 32 El-Alia, Bab Ezzouar, Algiers, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, University of Science and Technology Houari Boumediene (USTHB), BP 32 El-Alia, Bab Ezzouar, Algiers, Unverisity of Benyoucef Benkheda Algiers 1, Faculty of Medicine, Centre de Transfusion Sanguine, CHU Bab El-Oued, Algiers, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, University of Science and Technology Houari Boumediene (USTHB), BP 32 El-Alia, Bab Ezzouar, Algiers

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