Resolution of radiolabeled molecular species of phospholipid in human platelets: Effect of thrombin

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Abstract
Resolution of individual molecular species of human platelet 1,2‐diradyl‐sn‐glycero‐3‐phosphocholines and 1,2‐diradyl‐sn‐glycero‐3‐phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [3H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2‐diacyl molecular species. Eighty percent of [3H]‐arachidonic acid incorporated into 1‐acyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine in resting platelets was equally distributed between 1‐palmitoyl‐2‐arachidonoyl and 2‐stearoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine, while 70% of the radiolabel in 1‐acyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphoethanolamine was found in 1‐stearoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1‐acyl‐2‐[3H]arachidonoyl molecular species of 1‐acyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine and 1‐acyl‐2‐arachidonoyl‐sn‐glycero‐3‐ethanolamine. There was also a slight increase in 1‐O‐alkyl‐2‐[3H]arachidonoyl‐sn‐glycero‐3‐phosphocholine and a significant increase in 1‐O‐alk‐1′‐enyl‐2‐[3H]arachidonoyl‐sn‐glycero‐3‐phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl‐containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin‐stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.

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