title: Two acidic, anticoagulant PLA2 isoenzymes purified from the venom of monocled cobra Naja kaouthia exhibit different potency to inhibit thrombin and factor Xa via phospholipids independent, non-enzymatic mechanism.
abstract: BACKGROUND
  The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia.
  
  
  RESULTS
  By LC-MS/MS analysis, these PLA2s showed highest similarity (23.5% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their "pharmacological target(s)" by uncompetitive mechanism without the requirement of phospholipids/Ca(2+). The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.
  
  
  CONCLUSIONS
  In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2α and Nk-PLA2β for the treatment and/or prevention of cardiovascular disorders are proposed.
authors.affiliation: Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Science, Tezpur University, Tezpur, Assam, India.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Mukherjee
authors.affiliation: Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Science, Tezpur University, Tezpur, Assam, India.
authors.forename: Bhargab
authors.initials: B
authors.lastname: Kalita
authors.affiliation: Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Science, Tezpur University, Tezpur, Assam, India.
authors.forename: Rupamoni
authors.initials: R
authors.lastname: Thakur
mesh_terms: D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D004546:Elapid Venoms / Q000201:enzymology*; D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D007700:Kinetics; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D000925:Anticoagulants; D004546:Elapid Venoms; D065427:Factor Xa Inhibitors; D007527:Isoenzymes; D010743:Phospholipids; D054467:Phospholipases A2; D013917:Thrombin; D015951:Factor Xa