The phospholipase A 1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Abstract

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purifi cation of a phospholipase A 1 (PLA 1 ) from thrombin-activated human platelets using sequential chromatographic steps followed by fl uorophosphonate (FP)-biotin affi nity labeling and proteomics characterization that identifi ed acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA 1 . Addition of this recombinant PLA 1 signifi - cantly increased the production of sn -2-esterifi ed polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl- sn -glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn -1 to the sn -2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1 ) Activated platelets release PLA Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purifi cation of a phospholipase A 1 (PLA 1 ) from thrombin-activated human platelets using sequential chromatographic steps followed by fl uorophosphonate (FP)-biotin affi nity labeling and proteomics characterization that identifi ed acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA 1 . Addition of this recombinant PLA 1 signifi - cantly increased the production of sn -2-esterifi ed polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl- sn -glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn -1 to the sn -2 regioisomer of lyso- PAF. We propose the following LPA production pathway in blood: 1 ) Activated platelets release PLA 1 ; 2 ) PLA 1 generates a pool of sn-2 lysophospholipids; 3 ) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4 ) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.-Bolen, A. L., A. P. Naren, S. Yarlagadda, S. Beranova- Giorgianni, L. Chen, D. Norman, D. L. Baker, M. M. Rowland, M. D. Best, T. Sano, T. Tsukahara, K. Liliom, Y. Igarashi, and G. Tigyi. The phospholipase A 1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation. J. Lipid Res. 2011. 52: 958-970.1 ; 2 ) PLA 1 generates a pool of sn-2 lysophospholipids; 3 ) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4 ) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids. Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc.