Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: Inhibition of anticoagulant activity by low molecular weight heparin

Abstract In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A 2 (NnPLA 2 -I) purified from Indian cobra Naja naja venom was investigated. The NnPLA 2 -I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA 2 -I showed its significant similarity with phospholipase A 2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA 2 -I demonstrated putative conserved domains of the PLA 2 -like superfamily. The Km and Vmax values of NnPLA 2 -I toward hydrolysis of its most preferred substrate—phosphotidylcholine (PC)—were determined to be 0.72 mM and 29.3 μmol min −1  mg −1 , respectively. The anticoagulant activity of NnPLA 2 -I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA 2 -I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA 2 -I, albeit its anticoagulant activity was significantly reduced. The NnPLA 2 -I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC 50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA 2 -I. The NnPLA 2 -I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA 2 -I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA 2 -I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA 2 -I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA 2 -I did not show cytotoxicity against human glioblastoma U87MG cells, bactericidal or hemolytic activity. The future therapeutic application of NnPLA 2 -I for treatment and prevention of cardiovascular disorders is therefore suggested.