Two Acidic, Anticoagulant PLA(2) 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

BACKGROUND: The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A(2) (PLA(2); 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 PLA(2) isoenzymes, Nk-PLA(2)α (13463.91 Da) and Nk-PLA(2)β (13282.38 Da) purified from the venom of N. kaouthia. PRINCIPAL FINDINGS: By LC-MS/MS analysis, these PLA(2)s showed highest similarity (23.5% sequence coverage) with PLA(2) III isolated from monocled cobra venom. The catalytic activity of Nk-PLA(2)β exceeds that of Nk-PLA(2)α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA(2) isoenzymes. The anticoagulant potency of Nk-PLA(2)α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA(2)β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA(2)s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA(2)α and Nk-PLA(2)β 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-PLA(2) isoenzymes inhibit their “pharmacological target(s)” by uncompetitive mechanism without the requirement of phospholipids/Ca(2+). The anticoagulant potency of Nk-PLA(2)β which is higher than that of Nk-PLA(2)α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA(2) isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA(2)β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation. CONCLUSION/SIGNIFICANCE: In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA(2)α and Nk-PLA(2)β for the treatment and/or prevention of cardiovascular disorders are proposed.