Studies on Thrombin Inhibitor and Fibrinolytic Protease from Moringa oleifera Lam and Evaluation of their Potential to Control Thrombosis

Abstract

Thrombosis is a blood disorder characterized by the formation of abnormal blood clots newlinein the vessels. Available treatment strategies involve drugs that either prevent blood clot newlineformation or dissolve the clots that have been formed. Endogenous antithrombin-III newlinedeficiency contributes to excessive blood clot formation. Thus, supplementing with newlineantithrombin-III like activity may be used as anticoagulant therapy. An antithrombin-III newlinelike protein from Punica granatum (ALPP) has been identified and characterized using newlinean in-silico approach. Based on sequence homology, an ALPP was selected depending newlineon its highest binding affinity of 41.28 kcal/mol with thrombin. Thrombin structure newlinecomplexed with ALPP was docked with substrate TAME using AutoDock Vina. No newlinebinding of TAME was observed at active site residue Ser195 of thrombin. MD simulation newline(50ns) was performed to evaluate the flexibility and stability of Thrombin-TAME and newlineALPP-Thrombin-TAME docked complexes. MD simulation of ALPP-Thrombin newlinecomplex docked with TAME resulted in greater RMSD of ALPP-Thrombin and caused newlinestructural changes by occupying the binding site of TAME on thrombin protein newlinesupporting the inhibitory role of ALPP. In-vitro assays with crude protein from leaves newlineof Punica granatum showed 78% thrombin inhibition at 5and#61549;g and IC50 value was 0.188 newlineµg. The presence of thrombin inhibitors in crude protein was also confirmed by reverse newlinezymography. Thus, it is very likely that the protein identified from P. granatum may newlineact as a thrombin inhibitor. Most of the factors involved in blood clotting are serine newlineproteases which are essential for retaining the fluidity of blood to maintain normal newlinehaemostasis... newline