Synthesis of 1-acyl-2-[ 3H]acetyl- [formula omitted]-glycero-3-phosphocholine, a structural analog of platelet activating factor, by vascular endothelial cells

Abstract

Human umbilical vein endothelial cells (HUVECS) were challenged with thrombin in the presence of [ 3H]acetate to stimulate the production of radiolabeled platelet activating factor (PAF, 1- O -alkyl-2-[ 3H]acetyl- sn -glycero-3-phosphocholine, 1- O -alkyl-2-[ 3H]acetyl-GPC). The 3H-product was isolated by thin-layer chromatography, and 1-radyl-2[ 3H],3-diacetylglycerols were prepared by phospholipase C digestion and subsequent acetylation at the sn -3 position. When the 1-radyl-2[ 3H],3-diacetylglycerols were analyzed by zonal thin-layer chromatography, 96–97% of the radiolabeled derivative migrated with 1-acyl-2,3-diacetylglycerol standard. Only minor amounts (3–4%) of 1-alkyl-2[ 3H],3-diacetylglycerol were observed, demonstrating that the predominant acetylated product synthesized by thrombin-stimulated HUVECS was 1-acyl-2-[ 3H]acetyl-GPC. This relative abundance of 1-acyl-2-[ 3H]-acetyl-GPC was not significantly affected by thrombin dose, incubation time, or cell passage, and was also observed in HUVECS challenged with ionophore A23187. In addition, the acetylated product from ionophore A23187- or bradykinin-stimulated bovine aortic endothelial cells contained 90% 1-acyl-2-[ 3H]acetyl-GPC, suggesting that the synthesis of the 1-acyl PAF analog is not unique to HUVECS. These findings demonstrate that PAF is a minor synthetic component of HUVECS and bovine aortic endothelial cells. In light of the integral role which the vascular endothelial cell plays in the regulation of thrombosis, these findings also suggest that the production of 1-acyl-2-acetyl-GPC may be biologically important.