Title : Identification of thrombin inhibiting antithrombin-III like protein from Punica granatum using in-silico approach and in-vitro validation of thrombin inhibition activity in crude protein
Abstract:
Thrombin, a multifunctional serine protease, plays a pivotal role in the blood coagulation cascade by mediating the conversion of fibrinogen to fibrin and activating clotting factors (V, VIII, and XI). Thrombosis, a condition characterized by the formation of clots in blood vessels, is often associated with a deficiency of antithrombin-III (AT-III), a vital anticoagulant protein. Deficiencies in AT-III increases the risk of venous thrombosis, highlighting the importance of AT-III as a critical physiological inhibitor of coagulation. In this study, an antithrombin-III-like protein (ALP) from Punica granatum has been identified and characterized through in-silico approach, followed by experimental validation of its thrombin inhibition activity. Plant protein sequences homologous to antithrombin-III were retrieved using BLAST bioinformatics program developed by NCBI. Sequences with over 32% homology were shortlisted, and their three-dimensional structure models were generated using the SWISS MODEL web server due to the unavailability of crystal structures in the Protein Data Bank (PDB). These models were validated through the SAVES web server, and docked with thrombin using HawkDock web server. Antithrombin-III like protein from Punica granatum (ALPP) was selected for its highest binding affinity at -41.28 kcal/mol. To investigate the inhibitory mechanism, the thrombin-ALPP complex was docked with TAME (Tosyl-L- Arginine Methyl Ester) using AutoDock Vina. No interaction was observed for TAME at thrombin’s active site residue Ser195, indicating that ALPP binding disrupts thrombin’s affinity for TAME. Molecular dynamics (MD) simulation over 50 ns revealed stable interactions, with RMSD values of 0.35 nm for the Thrombin- TAME complex and 0.4 nm for the ALPP-Thrombin-TAME complex. In vitro assays with crude protein extracts from P. granatum showed 78% thrombin inhibition at 5 mg concentration, with an IC50 of 0.188 mg. Reverse zymography confirmed the presence of thrombin inhibitors in the crude extract. These findings suggest that ALPP may serve as a promising plant-derived thrombin inhibitor, offering a novel therapeutic approach for anticoagulant therapy. This presentation will provide an insight on the identification, structure modelling, docking, and simulation studies performed to characterize the ALPP. Additionally, these findings suggest the potential application of ALPP as an alternative phytotherapeutic agent for the treatment of thrombosis. Attendees will gain insights into the integration of computational biology and experimental techniques to discover novel therapeutic proteins from plant sources.
Key Words: Thrombosis, Antithrombin-III, Plant thrombin inhibitor, Molecular dynamic simulations, Punica granatum.
