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

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.

Principal Findings

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/Ca²⁺. 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.

Conclusion/Significance

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.