The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity

Abstract

We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 μM PL) to platelets (2 × 108/ml) was measured after incubations of 1 h at 37°C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 μM); esculetin, a 12-lipoxygenase inhibitor (20 μM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor (100 μM); 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca2+ chelator (20 μM); bromoenol lactone (BEL), a Ca2+- independent phospholipase A2 (iPLA2) inhibitor (100 nM); and 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 μM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A2 (cPLA2) and iPLA2, only cPLA2 is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA2-dependent process. The inhibition of iPLA2 by BEL had no effect on PL transfers.

Keywords

• thrombin
• lipoprotein lipase
• metabolic inhibitors
• Ca2+- independent phospholipase A2
• very low density lipoprotein