Rat platelet phospholipase A₂

Abstract

We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2-dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2-acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.Platelet phospholipids are rich in arachidonic acid [l -31, the precursor of thromboxanes [4], hydroxylated-polyunsaturated fatty acids [S] and prostacyclin 161, whosc role in stimulating and inhibiting platelet activation is now well established [7 -91. Platelets contain two major possible pathways for arachidonate release. The most direct of these involves the action of phospholipase A2 towards all major platelet glycerophospholipids. The alternative pathway is initiated by phospholipase C action on inositolphospholipids followed by diacylglycerol lipases and monoacylgl ycerol lipases producing free arachidonate (for reviews see [lo, 111). A quantitative evaluation of the contribution of these pathways in human platelets upon stimulation by thrombin indicates that the phospholipase Az route is of major importance [I2 -361. Furthermore, rat platelets secrete an extracellular phospholipase

Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction.

“…However, local conditions that increase substrate accessibility can influence sPLA2 activity, allowing for a better penetration into the interface. Indeed, on a monolayer PC substrate, the surface pressure or packing density of phospholipid molecules modulates mammalian sPLA2 activity (39). Thus, one can conclude from these data that the physicochemical organization of phospholipids is an important determinant of their hydrolysis by sPLA2.…”

“…However, no causal relationship has been directly demonstrated. In addition, extrapolation from experimental acute lung injury induced by the intratracheal injection of Naja naja venom (37) is questionable, since venom sPLA2s exhibit a much higher ability than mammalian sPLA2-II to hydrolyze phospholipids on packed monolayer structures (38,39). Hence, the ability of mammalian sPLA2-II to generate lipid derivatives in acute lung injury has remained unknown.…”

“…The ability of a mammalian sPLA2-II to hydrolyze surfactant PC was unexpected, since in contrast to venom sPLA2s, this enzyme was reported to act poorly on PC substrate and to exhibit a headgroup preference for negatively charged phospholipids such as phosphatidylglycerol (39,42), which is unusually abundant in pulmonary surfactant as compared with other mammalian tissues and fluids (43). However, local conditions that increase substrate accessibility can influence sPLA2 activity, allowing for a better penetration into the interface.…”

The Anticoagulant Effect of the Human Secretory Phospholipase A2 on Blood Plasma and on a Cell‐Free System is Due to a Phospholipid‐Independent Mechanism of Action Involving the Inhibition of Factor Va

“…The low efficiency of hsPLA, grII to hydrolyze platelet phospholipids was not surprising given its poor ability to act on substrates with a high surface pressure (Ransac et al, 1992). Indeed, the estimated pressure at the cellular surface of resting platelets is not compatible with a catalytic action of this enzyme (Mounier et al, 1994).…”

“…Two major characteristics have been considered as predictive of an anticoagulant action of sPLA,: first, the ability of the enzyme to hydrolyze phospholipids at a high surface pressure; and secondly, the basic PI of the protein correlated with the presence of basic amino acids located between residues 55-77 (Kini and Evans, 1987). Based on these two criteria, the sPLA, grII is predicted to exert a moderate anticoagulant effect since it has a basic PI (Kramer et al, 1989) but a low efficiency to hydrolyze condensed phospholipid monolayers (Ransac et al, 1992). Recently, human sPLA, grII (hsPLA, grII) was indeed shown to exert a moderate anticoagulant effect on plasma (Cirino et al, 1993) and to inhibit prothrombinase activity (Inada et al, 1994).…”