Stimulation of Ca2+-activated human platelet phospholipase A2 by diacylglycerol

We examined the effect of diacylglycerol on Ca2+-dependent phospholipase A2 from human platelets. Phospholipase A2 was solubilized and partially purified to a stable form in the presence of n-octyl beta-D-glucopyranoside (octyl glucoside), and its enzymic activity was determined with sonicated 2.5 microM-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (arachidonoyl-PC) as substrate. Phospholipase A2 activity was increased when diacylglycerol was incorporated into the substrate arachidonoyl-PC. Stimulation was maximal in the presence of greater than or equal to 29 mol% (1 microM) diacylglycerol, and was greater than 4-fold for both 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonoylglycerol. 1-Stearoyl-2-arachidonoylglycerol at concentrations of 2-5 mol% increased phospholipase A2 activity 1.3-1.8-fold. Exogenously added 1-oleoyl-2-acetylglycerol also enhanced phospholipase A2 activity, producing a maximal stimulation of 1.6-fold at a concentration of 25 microM. Comparative studies conducted with pancreatic, bee-venom and snake-venom phospholipase A2 showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but stimulation was less than observed for platelet phospholipase A2. Our results suggest that diacylglycerol, known to be generated in stimulated platelets, may enhance Ca2+-activated phospholipase A2.