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Thakur R.,Tezpur University | Kumar A.,Indian Institute of Technology Guwahati | Bose B.,Indian Institute of Technology Guwahati | Panda D.,Indian Institute of Technology Bombay | And 3 more authors.
Biochimie | Year: 2014

Compounds showing dual inhibition of thrombin and factor Xa (FXa) are the subject of great interest owing to their broader specificity for effective anticoagulation therapy against cardiovascular disorders. This is the first report on the functional characterization and assessment of therapeutic potential of a 4423.6 Da inhibitory peptide (Ruviprase) purified from Daboia russelii russelii venom. The secondary structure of Ruviprase is composed of α-helices (61.9%) and random coils (38.1%). The partial N-terminal sequence (E1-V2-X3-W4-W5-W6-A7-Q8-L9-S10) of Ruviprase demonstrated significant similarity (80.0%) with an internal sequence of apoptosis-stimulating protein reported from the venom of Ophiophagus hannah and Python bivittatus; albeit Ruviprase did not show sequence similarity with existing thrombin/FXa inhibitors, suggesting its uniqueness. Ruviprase demonstrated a potent in vitro anticoagulant property and inhibited both thrombin and FXa following slow binding kinetics. Ruviprase inhibited thrombin by binding to its active site via an uncompetitive mechanism with a Ki value and dissociation constant (KD) of 0.42 μM and 0.46 μM, respectively. Conversely, Ruviprase demonstrated mixed inhibition (Ki = 0.16 μM) of FXa towards its physiological substrate prothrombin. Furthermore, the biological properties of Ruviprase could not be neutralized by commercial polyvalent or monovalent antivenom. Ruviprase at a dose of 2.0 mg/kg was non-toxic and showed potent in vivo anticoagulant activity after 6 h of intraperitoneal treatment in mice. Because of the potent anticoagulant property as well as non-toxic nature of Ruviprase, the possible application of the peptide as an antithrombotic agent for combating thrombosis-associated ailments appears promising. © 2014 Elsevier Masson SAS. All rights reserved.


Majumdar S.,Tezpur University | Dutta S.,Tezpur University | Das T.,Indian Institute of Science | Chattopadhyay P.,Defense Research Laboratory | Mukherjee A.K.,Tezpur University
International Journal of Biological Macromolecules | Year: 2015

Fibrin(ogen)olytic enzymes offer great promise for the treatment of thrombosis associated disorders. The present study describes the characterization of an extracellular fibrin(ogen)olytic serine protease (named Bacethrombase) purified from the Bacillus cereus strain FF01. The molecular mass of the Bacethrombase was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix assisted laser desorption/ionization-time-of-flight-mass spectroscopy analyses at 39.5. kDa and 38,450.51. Da, respectively. The peptide mass fingerprinting and analyses of the composition of the amino acids revealed the similarity of the Bacethrombase to the bacterial serine proteases. The secondary structure of the Bacethrombase was composed of 14% helix, 6.6% beta-sheet, and 79.4% random coil. Bacethrombase was found to contain 48% sialic acid and it preferentially degraded the Aα-chain of fibrinogen, as well as fibrin. The anticoagulant potency of the Bacethrombase was comparable with that of warfarin and heparin, and was corroborated by its fibrinogenolytic activity rather than the inhibition of thrombin, prothrombin or FXa. Bacethrombase demonstrated antiplatelet activity, and dose-dependently inhibited the ADP-induced platelet aggregation. Bacethrombase (10. mg/kg) did not show toxicity after i.v. administration in Wistar rats; however, it revealed an in vivo anticoagulant effect and significantly inhibited the carrageenan-induced in vivo thrombus formation in rats. © 2015 Elsevier B.V.


Goswami L.,Tezpur University | Raul P.,Defense Research Laboratory | Sahariah B.,Tezpur University | Bhattacharyya P.,Indian Statistical Institute | Bhattacharya S.S.,Tezpur University
Clean - Soil, Air, Water | Year: 2014

Overload of coal ash (CA) generated from several coal fired plants poses acute problems of disposal and contamination of soil and water resources. Since CA generated from tea factories has not yet been characterized from environmental viewpoints, we attempted to critically characterize the agro-ecological compatibility of CA samples (CA1, CA2, CA3, and CA4) collected from four distantly located tea factories. Among these samples, we found CA3 and CA4 to have low pH and high metal content. Accordingly, we restricted our study to predict the solubility pattern of non-metal and metal ions in CAs by adopting a geochemical model (Visual MINTEQ). Various fractions of metals like Fe, Mn, Cu, Zn, Cr, Ni, and Pb were studied to figure out the mobility pattern of metals in the environment. The results clearly indicate that tea industry derived CA substantially differs from thermal power plant CA as regards several physico-chemical properties, for example, sandy texture, low pH and high nutrient content. Risk evaluation of human exposure to trace elements and the metal fractionation study led us to conclude that CA1 and CA2 are highly compatible with agricultural use. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Thakur R.,Tezpur University | Chattopadhyay P.,Defense Research Laboratory | Ghosh S.S.,Indian Institute of Technology Guwahati | Mukherjee A.K.,Tezpur University
Toxicon | Year: 2015

The procoagulant proteases present in Russell's Viper venom (RVV) are responsible for promoting consumption coagulopathy in victims. In this study, a procoagulant metalloprotease (Rusviprotease) possessing prothrombin activating and α-fibrinogenase properties has been purified and characterized from RVV. Rusviprotease is a 26.8 kDa glycoprotein which also exists in other multimeric forms. The peptide mass fingerprinting and secondary structure analyses of Rusviprotease revealed its similarity with snake venom prothrombin activators and metalloproteases. Similar to group A prothrombin activators, Rusviprotease cleaved prothrombin independent of any co-factor requirement generating meizothrombin which is further cleaved to form thrombin. The Km and Vmax values of Rusviprotease towards prothrombin were determined to be 1.73 μM, and 153.5 nM thrombin generated/min/μmoles of Rusviprotease, respectively. The Km and Vmax values of Rusviprotease towards fibrinogen were calculated to be 3.14 μM and 78.7 nmol/min, respectively. Spectrofluorometric study provided the evidence of interaction between Rusviprotease and factor Xa with a Kd value of 6.64 nM. This interaction augmented the prothrombin activating property of the factor Xa-prothrombinase-Rusviprotease complex by 2.5 fold. Intravenous injection of Rusviprotease to BALB/c mice (0.1 mg/kg) resulted in in vivo defibrinogenation rendering the blood incoagulable. In conclusion, Rusviprotease is the first example of a prothrombin activator with fibrinogenolytic property purified from Daboia russelii russelii venom. © 2015 Elsevier Ltd All rights reserved.


Das B.,Tezpur University | Chattopadhyay P.,Defense Research Laboratory | Mandal M.,Tezpur University | Voit B.,Leibniz Institute of Polymer Research | Karak N.,Tezpur University
Macromolecular Bioscience | Year: 2013

Hyperbranched polyurethanes are synthesized using TDI, PCL diol, butanediol, and pentaerythritol (1-5wt%) as the B4 reactant with and without the monoglyceride of sunflower oil. The biodegradation, physico-mechanical, and thermal properties are found to be tailored by varying the percentage weight of the branching unit. An MTT/hemolytic assay and subcutaneous implantation in Wistar rats followed by cytokine/ALP assay and histopathology studies confirm a better biocompatibility of HBPU with MG than without MG. HBPU supports the proliferation of dermatocytes with no toxic effect in major organs, in addition the in vitro degraded products are non-toxic. Cell adherence and proliferation endorse the bio-based HBPU as a prospective scaffold material in the niche of tissue engineering. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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