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.

BackgroundThe 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 findingsBy 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.Conclusion/significanceIn 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

A summary of the purification of PLA₂ isoenzymes from the venom of <i>N. kaouthia</i>.

<p>The PLA₂ activity was assayed by egg-yolk phospholipids hydrolysis method using 0.1 M Tris-HCl, pH 8.0 buffer. Data are from a typical experiment.</p>a<p>One unit of PLA₂ activity is defined as decrease of 0.01 absorbance at 740 nm per 10 min at 25°C.</p

Effects of Nk-PLA₂ isoenzymes on inhibition of amidolytic activity of FXa.

<p>(<b>a</b>)The Nk-PLA₂α (1.0 µg) or Nk-PLA₂β (1.0 µg) was pre-incubated with FXa (0.15 µM) against its chromogenic substrate F3301 (0.2 mM) for 60 min at 37°C, pH 7.4 before the amidolytic activity assay. A control (FXa treated with buffer) was also run in parallel. The values are mean of triplicate determinations. (<b>b</b>) Michaelis-Menten plot to determine the inhibitory constant (K_i) of Nk-PLA₂α (50 nM and 100 nM) on amidolytic activity of FXa (0.15 µM) at 37°C, pH 7.4.</p

Kinetics of inhibition of FXa (0.15 µM) and thrombin (0.03 NIH U/ml) by Nk-PLA₂α and Nk-PLA₂β at 37°C, pH 7.4, respectively.

<p>The kinetic parameters (<i>Km</i>, <i>Vmax</i>, and <i>Kcat</i>) values were determined from Michaelis-Menten plot as described in the text. The chromogenic substrates (0.2 mM) used for the amidolytic activity assay of thrombin and FXa were T1637 and F3301, respectively. The values are mean of triplicate determinations and SD was found within 10% of the mean value.</p

Purification and determination of molecular masses of PLA₂ isoenzymes isolated from <i>N. kaouthia</i> venom.

<p>(<b>a</b>) Fractionation of crude <i>N. kaouthia</i> venom (25 mg dry weight) done on a HiPrep CM FF16/10 FPLC cation-exchange column. The fractionation conditions are described in the text. The peak showing major PLA₂ and anticoagulant activity is marked with an arrow. (<b>b</b>) Chromatogram resulting from anion-exchange fractionation of cation-exchange unbound peak [Nk(H)CEXP1] by using a Hiprep DEAE FF16/10 FPLC column. (<b>c</b>) and (<b>d</b>) MALDI-TOF mass spectrum of Nk-PLA₂α [peak Nk(H)AEXP4], and Nk-PLA₂β [peak Nk(H)AEXP5], respectively.</p

Effect of pBPB and monovalent antivenom on catalytic, anticoagulant and thrombin inhibitory activity of Nk-PLA₂α and Nk-PLA₂β.

<p>The activity of enzymes in absence of inhibitor/antivenom was considered as 100% activity and the other values were compared with that. The values are mean ± S. D. of triplicate determinations. ND: not determined; pBPB: p-bromophenacyl bromide. Significance of difference with respect to Nk-PLA₂α;</p>a<p>p<0.01,</p>b<p>P<0.05.</p

Spectrofluorometric assay of the interaction of Nk-PLA₂ isoenzymes with FXa, thrombin and PC.

<p>(<b>a</b>) FXa incubated with Nk-PLA₂α or Nk-PLA₂β (at 1∶10 ratio), (<b>b</b>) interaction of thrombin with Nk-PLA₂α or Nk-PLA₂β (at 1∶10 ratio), (<b>c</b>) PC with Nk-PLA₂α/Nk-PLA₂β (at 10∶1 ratio) in presence of 0.5 mM EDTA (to prevent the phospholipids hydrolysis). The data shown above represent a typical experiment; however, the experiments were repeated three times to assure the reproducibility.</p

Inhibition of thrombin-induced platelet aggregation by different doses of Nk-PLA₂β.

<p>The platelet aggregation was induced by with thrombin (1.0 NIH U/ml) pre-incubated with Nk-PLA₂β (0.5–2.0 µg/ml) or 1× PBS (control) for 30 min at 37°C. The platelet aggregation was monitored in a Chrono-log dual channel aggregometer for 10 min. The data represent a typical experiment; however, the experiment was repeated three times to assure the reproducibility.</p

Inhibition of prothrombin activation by FXa pre-treated with NkPLA₂ isoenzymes.

<p>(<b>a</b>) The FXa (20 nM) was pre-incubated with Nk-PLA₂α (1.0 µg), or Nk-PLA₂β (1.0 µg), or buffer (control) for 60 min at 37°C, pH 7.4. The prothrombin (1.4 µM) activation by FXa (treated or control) was determined by formation of thrombin by using its chromogenic substrate T1637 (0.2 mM). (<b>b</b>) SDS-PAGE (15%) analysis (reduced conditions) of affect of Nk-PLA₂α and Nk-PLA₂β on inhibition of prothrombin activation by FXa. Before addition of prothrombin (10.0 µg), FXa (20 nM) was pre-incubated with Nk-PLA₂α or Nk-PLA₂β for 60 min at 37°C, pH 7.4. Lane 1, prothrombin treated with FXa for 60 min at 37°C (control); lane 2, prothrombin treated with FXa (pre-incubated with 2.0 µg of Nk-PLA₂β) for 60 min at 37°C; lanes 3 and 4, prothrombin treated with FXa (pre-incubated with 1 and 2 µg of Nk-PLA₂α, respectively) for 60 min at 37°C. The experiment was repeated three times to assure the reproducibility.</p

Comparison of anticoagulant activity and plasma phospholipids hydrolytic activity of Nk-PLA₂α and Nk-PLA₂β.

<p>(<b>a</b>) A comparison of dose-dependent anticoagulant activity (Ca-clotting time of citrated PPP) of Nk-PLA₂α, Nk-PLA₂β, heparin and warfarin. Unit is defined as 1s increase in clotting of PPP in presence anticoagulants compared to the clotting time of control PPP. (<b>b</b>) Effect of PPP/PLA₂ pre-incubation time on anticoagulant activity. (<b>c</b>) Effect of PPP/PLA₂ pre-incubation on release of free fatty acids from plasma phospholipids. Values are mean ± SD of triplicate determination.</p