Snake Venom Phospholipases A2And Blood Coagulation : Competition Between Phospholipases And Blood Clotting Factors U And U At The Phospholipid Interface

In a preceeding paper (J. Biol. Chem., 255, 7734-7739, 1980) we have shown that phospholipases A2 from snake venoms have different phospholipid binding affinities according, to their anticoagulant activities : only the anticoagulant one are able to bind with neutral and negative phospholipid bilayers in EDTA and therefore in coagulation test could hydrolyse both classes of lipids. A step forward in the understanding of the mechanism of the inhibitory action of phospholipases on coagulation is to know if competition between clotting factors and phospholipase can occur at the plasma phospholipid interface as a consequence of the strong affinity of anticoagulant phospholipase for lipids. The method used is mainly to look at the intrinsic fluorescence of lipids bound phospholipase when blood clotting factors II and IX are added to the complex. Results show that both factors can expel the phospholipase from the interface when the factor to phospholipase molar ratio is about 1, suggesting a better affinity of the clotting proteins for the interface. When hydrolysis of phospholipids is allowed to occur (ie in presence of calcium ion) factors also have a good affinity for degraded products. By the same technic, it was also possible to dectect direct interactions between phospholipase and factors, which differ for factors II and factor IX. This can also occur in presence of phospholipids. In summary, as it was demonstrated that inactivated phospholipase has lost its anticoagulant activity lipid hydrolysis is a necessary step. However, we show here that competition between clotting factors and phospholipase could also occur if drastic concentrations of phospholipase are used in coagulation test, i.e. molar ratio much higher than 1. It is also proposed that factor to phospholipase binding may be important in the inhibitory mechanism since clotting proteins could be no longer available for the coagulation.