Identification of arachidonate P-450 metabolites in human platelet phospholipids

Phospholipase A2 (Naja mocambique) catalyzed release of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) from phospholipids of isolated human platelets. The amount of EETs released by phospholipase A2 measured by gas chromatography/mass spectrometry (GC/MS) was 4.3 +/- 0.9 pmol/10(6) platelets. No EETs were detected when phospholipase A2 was omitted from the incubations. The relative abundance of EET isomers (14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET) from human platelets was 5.4:4.5:3.7:1, respectively, as established by a new method based on particle-beam liquid chromatography/mass spectrometry (LC/MS). Fractionation of platelet phospholipids by normal-phase high-performance liquid chromatography followed by hydrolysis and GC/MS analyses indicated that the amount of EETs was highest in fractions containing phosphatidylinositol and phosphatidylserine (142 and 61 pmol/nmol of phosphorus, respectively) while low in phosphatidylcholine and phosphatidylethanolamine (19 and 11 pmol/nmol of phosphorus, respectively). The majority of EETs associated with phosphatidylcholine was found in fractions containing 1-O-alkylphosphatidylcholine. Human platelet phospholipids also released 20-HETE on phospholipase treatment (9.7 +/- 1.6 fmol/10(5) cells) and at least three other HETEs, one of which was tentatively identified as 16-HETE. Activation of human platelets by thrombin or platelet-activating factor released 5 to 7 fmol EET/10(6) cells. Receptor-mediated hydrolysis of phospholipids containing EETs and 20-HETE may play a role in stimulus-response coupling in platelets.