Figure 1.
Purification and determination of molecular masses of PLA2 isoenzymes isolated from N. kaouthia venom.
(a) Fractionation of crude N. kaouthia 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) Chromatogram resulting from anion-exchange fractionation of cation-exchange unbound peak [Nk(H)CEXP1] by using a Hiprep DEAE FF16/10 FPLC column. (c) and (d) MALDI-TOF mass spectrum of Nk-PLA2α [peak Nk(H)AEXP4], and Nk-PLA2β [peak Nk(H)AEXP5], respectively.
Table 1.
A summary of the purification of PLA2 isoenzymes from the venom of N. kaouthia.
Figure 2.
Comparison of anticoagulant activity and plasma phospholipids hydrolytic activity of Nk-PLA2α and Nk-PLA2β.
(a) 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) Effect of PPP/PLA2 pre-incubation time on anticoagulant activity. (c) Effect of PPP/PLA2 pre-incubation on release of free fatty acids from plasma phospholipids. Values are mean ± SD of triplicate determination.
Figure 3.
Inhibition of thrombin-induced platelet aggregation by different doses of Nk-PLA2β.
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.
Figure 4.
Effects of Nk-PLA2 isoenzymes on inhibition of amidolytic activity of FXa.
(a)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) Michaelis-Menten plot to determine the inhibitory constant (Ki) of Nk-PLA2α (50 nM and 100 nM) on amidolytic activity of FXa (0.15 µM) at 37°C, pH 7.4.
Figure 5.
Inhibition of prothrombin activation by FXa pre-treated with NkPLA2 isoenzymes.
(a) 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) 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.
Table 2.
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.
Figure 6.
Inhibitory effect of Nk-PLA2α and Nk-PLA2β on amidolytic activity and fibrinogen clotting property of thrombin.
(a) The thrombin (0.03 NIH U/ml) was pre-incubated with Nk-PLA2α (1.0 µg), or Nk-PLA2β (1.0 µg), or buffer (control) for 30 min at 37°C, pH 7.4 before addition of its chromogenic substrate T1637 (0.2 mM). The values are mean of triplicate determination. (b) Inhibition of fibrinogen clotting activity of thrombin through different doses (0–0.2 µg) of Nk-PLA2α or Nk-PLA2β. Before the addition of fibrinogen (final concentration ∼7.5 µM in 20 mM K-phosphate buffer, pH 7.4), thrombin (0.03 NIH U/ml) was pre-incubated with different doses of PLA2 (dissolved in 20 mM K-phosphate buffer, pH 7.4) for 30 min at 37°C. Values are mean ± SD of triplicate determination. (c) Michaelis-Menten plot to show the Inhibition of amidolytic activity of thrombin (0.03 NIH U/ml) through two different concentrations (50 nM and 100 nM) of Nk-PLA2β. Before the addition of substrate T1637 (0.2 mM), thrombin was pre-incubated with two different doses of the above PLA2 in 20 mM Tris-HCl, pH 7.4 for 30 min at 37°C (d) The effect of pre-incubation of Nk-PLA2β/thrombin on inhibition of fibrinogen clotting activity of thrombin. A fixed amount (0.05 µg) of Nk-PLA2β was pre-incubated with thrombin (0.03 NIH U/ml) for 0–30 min at 37°C and then assayed for fibrinogen clotting activity. The values are mean ± SD of triplicate determinations.
Figure 7.
Spectrofluorometric assay of the interaction of Nk-PLA2 isoenzymes with FXa, thrombin and PC.
(a) FXa incubated with Nk-PLA2α or Nk-PLA2β (at 1∶10 ratio), (b) interaction of thrombin with Nk-PLA2α or Nk-PLA2β (at 1∶10 ratio), (c) 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.
Table 3.
Effect of pBPB and monovalent antivenom on catalytic, anticoagulant and thrombin inhibitory activity of Nk-PLA2α and Nk-PLA2β.