Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A(2) purified from Indian cobra (Naja naja) venom: Inhibition of anticoagulant activity by low molecular weight heparin

作者：Dutta Sumita; Gogoi Debananda; Mukherjee Ashis K^*

来源：Biochimie, 2015, 110: 93-106.

DOI：10.1016/j.biochi.2014.12.020

• Naja naja
• Phospholipase A(2)
• Anticoagulant activity
• Thrombin inhibition
• Heparin neutralization
• Platelet deaggregation

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 NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA(2)-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 mu mol 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 NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA(2)-I showed a nonenzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 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. NnPLA2-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. NnPLA2-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.

• 出版日期2015-3