Evidence for the release of arachidonic acid through the selective action of phospholipase A2 in thrombin-stimulated human platelets

The release of arachidonic acid from thrombin-stimulated platelets can be attributed to the action of phospholipase A2 on membrane phospholipid. Previously, analysis of individual subclasses of phospholipid demonstrated that 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphocholine and to a lesser degree 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphoethanolamine were the main source of [3H]arachidonic acid in thrombin-stimulated cells. In the present work, 1,2-diacyl phospholipid subclasses were analyzed as 1,2-diacylglycerobenzoates by high-pressure liquid chromatography in order to analyze arachidonate release as mass changes in individual molecular species of phospholipid. Following thrombin stimulation (5 U/ml, 5 min, 37 degrees C) all arachidonoyl-containing molecular species of 1,2-diacyl-sn-glycerophosphocholine decreased in mass and [3H]arachidonate content by almost 50%, while those of 1,2-diacyl-sn-glycerophosphoethanolamine decreased by 20%. The mass change was substantial and indicated that these phospholipids are a major source of arachidonate in stimulated cells. No variation was seen in the other non-arachidonate-containing molecular species of either subclass. Thus, deacylation of membrane 1,2-diacylglycerophosphocholine and 1,2-diacylglycerophosphoethanolamine by phospholipase A2 is selective for those molecular species of phospholipid containing arachidonic acid, suggesting that a certain proportion of arachidonoyl-containing molecular species of phospholipid are compartmentalized with the platelet membrane proximal to the site of action of this enzyme. These studies demonstrate that the human platelet is a cell poised and specialized to release rapidly substantial amounts of arachidonic acid upon stimulation.