title: 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 A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-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 PLA2-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 μmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-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 NnPLA2-I showed a non-enzymatic, 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 NnPLA2-I. The NnPLA2-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 NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-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 NnPLA2-I for treatment and prevention of cardiovascular disorders is therefore suggested.
authors.affiliation: Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Science, Tezpur University, Tezpur 784028, Assam, India.
authors.forename: Sumita
authors.initials: S
authors.lastname: Dutta
authors.affiliation: Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Science, Tezpur University, Tezpur 784028, Assam, India.
authors.forename: Debananda
authors.initials: D
authors.lastname: Gogoi
authors.affiliation: Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Science, Tezpur University, Tezpur 784028, Assam, India. Electronic address: akm@tezu.ernet.in.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Mukherjee
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000187:drug effects; D055162:Biocatalysis; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D004546:Elapid Venoms / Q000201:enzymology; D017815:Elapidae; D015951:Factor Xa / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D006495:Heparin, Low-Molecular-Weight / Q000494:pharmacology*; D006801:Humans; D010713:Phosphatidylcholines / Q000378:metabolism; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D000925:Anticoagulants; D004546:Elapid Venoms; D006495:Heparin, Low-Molecular-Weight; D010713:Phosphatidylcholines; D054467:Phospholipases A2; D013917:Thrombin; D015951:Factor Xa
keywords: Anticoagulant activity; Heparin neutralization; Naja naja; Phospholipase A(2); Platelet deaggregation; Thrombin inhibition