title: An acidic phospholipase A(2) (RVVA-PLA(2)-I) purified from Daboia russelli venom exerts its anticoagulant activity by enzymatic hydrolysis of plasma phospholipids and by non-enzymatic inhibition of factor Xa in a phospholipids/Ca(2+) independent manner.
abstract: A homodimeric acidic PLA(2) (RVVA-PLA(2)-I) of 58.0 kDa molecular weight purified from Russell's viper (Daboia russelli) venom demonstrated dose-dependent catalytic, strong anticoagulant and indirect hemolytic activities and inhibited blood coagulation cascade in both enzymatic and non-enzymatic mechanisms. In in vitro condition, RVVA-PLA(2)-I showed preferential hydrolysis of phosphatidylcholine with a K(m) and V(max) values of 0.65 mM and 28.9 μmol min(-1), respectively. Biochemical study and GC-analysis of plasma phospholipids hydrolysis by PLA(2) revealed that anticoagulant activity of RVVA-PLA(2)-I was partly attributed by the enzymatic hydrolysis of pro-coagulant phospholipids PC, followed by PS. The spectrofluorometric and gel-filtration analyses documented binding of RVVA-PLA(2)-I with activated factor X and PC; however, it does not bind with factor Va, prothrombin and thrombin. Therefore, this anticoagulant PLA(2) inhibits the blood coagulation cascade non-enzymatically by binding with coagulation factor Xa, even in the absence of phospholipids and Ca(2+) and thus slows down the blood coagulation by partially inhibiting the prothrombin activation. Chemical modification of essential amino acids present in the active site, neutralization with Azadirachta indica leaves extract (AIPLAI) and heat-inactivation study reinforce the association of catalytic and anticoagulant activity of RVVA-PLA(2)-I and also throw a light on its non-enzymatic mechanism of anticoagulant action.
authors.affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur 784 028, Assam, India.
authors.forename: Debashree
authors.initials: D
authors.lastname: Saikia
authors.forename: Rupamoni
authors.initials: R
authors.lastname: Thakur
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Mukherjee
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000302:isolation & purification* / Q000633:toxicity; D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D065427:Factor Xa Inhibitors; D005453:Fluorescence; D006868:Hydrolysis; D007194:India; D010740:Phospholipases / Q000302:isolation & purification* / Q000633:toxicity; D010743:Phospholipids / Q000097:blood / Q000378:metabolism*; D011516:Prothrombin; D017840:Daboia; D014757:Viper Venoms / Q000201:enzymology*
chemical_list: D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D010743:Phospholipids; D014757:Viper Venoms; D011516:Prothrombin; D010740:Phospholipases; D002118:Calcium