An acidic phospholipase A2 (RVVA-PLA2-I) purified from Daboia russelli venom exerts its anticoagulant activity by enzymatic hydrolysis of plasma phospholipids and by non-enzymatic inhibition of factor Xa in a phospholipids/Ca2+ independent manner

Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: Inhibition of anticoagulant activity by low molecular weight heparin

“…Phospholipase A 2 activity was assayed by the turbidometric method of Joubert and Tallijard [22] slightly modified as described previously [6,10]. Briefly, one egg yolk was suspended in 250 ml of 0.9% (w/v) NaCl containing 0.02% (w/v) sodium azide.…”

“…The PLA 2 activity was assayed by microtitration of liberated fatty acids (FA) with 0.01 N NaOH, and the amount of FA liberated was determined from a standard curve of palmitic acid [10]. The LineweavereBurk plot was used to determine kinetic parameters (K m and V max ) of purified PLA 2 using different concentrations (1.0e300 mM) of substrate (PC), and then the values of 1/V were plotted as a function of 1/S.…”

“…To determine the correlation between the kinetics of plasma phospholipids hydrolysis and that of the anticoagulant activity, a fixed amount (100 nM) of purified PLA 2 was incubated with 300 ml of plasma from 3 to 20 min at 37 C. Then, 40 ml of 250 mM CaCl 2 was added and the clotting time was recorded [10]. In another set of experiments, the PLA 2 was incubated with plasma under identical experimental conditions and the liberated fatty acids were titrated with 0.01 N NaOH.…”

“…In another set of experiments, the PLA 2 was incubated with plasma under identical experimental conditions and the liberated fatty acids were titrated with 0.01 N NaOH. The amount of FA liberated was determined from a standard curve of palmitic acid [10]. The anticoagulant potency of N. naja acidic PLA 2 was compared with the commercial drugs heparin and warfarin.…”

“…To measure the inhibitory effect of PLA 2 on amidolytic activity of factor Xa, the procedures described by Saikia et al [10] were followed. Briefly, 500 nmol of purified enzyme was pre-incubated with 0.1 mg of FXa isolated from human plasma (Calbiochem) for 60 min at 37 C. Then, 1.0 mg of human prothrombin (Calbiochem) and chromogenic substrate (0.2 mM final concentration) for thrombin (T1637) were added to the reaction mixture.…”