The product of oxygenation of arachidonic acid by the prostaglandin H synthases (PGHS), prostaglandin H 2 (PGH 2 ), undergoes rearrangement to the highly reactive ␥-ketoaldehydes, levuglandin (LG) E 2 , and LGD 2 . We have demonstrated previously that LGE 2 reacts with the ⑀-amine of lysine to form both the levuglandinyl-lysine Schiff base and the pyrrole-derived levuglandinyl-lysine lactam adducts. We also have reported that these levuglandinyl-lysine adducts are formed on purified PGHSs following the oxygenation of arachidonic acid. We now present evidence that the levuglandinyl-lysine lactam adduct is formed in human platelets upon activation with exogenous arachidonic acid or thrombin. After proteolytic digestion of the platelet proteins, and isolation of the adducted amino acid residues, this adduct was identified by liquid chromatography-tandem mass spectrometry. We also demonstrate that formation of these adducts is inhibited by indomethacin, a PGHS inhibitor, and is enhanced by an inhibitor of thromboxane synthase. These data establish that levuglandinyllysine adducts are formed via a PGHS-dependent pathway in whole cells, even in the presence of an enzyme that metabolizes PGH 2 . They also demonstrate that a physiological stimulus is sufficient to lead to the lipid modification of proteins through the levuglandin pathway in human platelets. Prostaglandin H synthase (PGHS)1 catalyzes the oxygenation of arachidonic acid to the endoperoxide, prostaglandin H 2 (PGH 2 ). PGH 2 is further metabolized to the prostanoids PGD 2 , PGE 2 , PGF 2a , thromboxane A 2 , and prostacyclin by specific enzymes. Also, PGH 2 in aqueous solutions undergoes non-enzymatic rearrangement to yield PGE 2 and PGD 2 , and 20% of it rearranges to the highly reactive ␥-ketoaldehydes, levuglandins (LG) E 2 and D 2 (1, 2) (Fig. 1). Levuglandins are known to react covalently with primary amines, such as the ⑀-amine of lysine, with proteins and with DNA (3, 4). We have characterized the adducts that are formed by the reaction of lysine with LGE 2 or PGH 2 (2, 5), and knowledge of their structures has provided a basis for analysis of the adducts in protein digests utilizing liquid chromatography-tandem mass spectrometry. Utilizing this analytical approach, we have demonstrated formation of LG-lysine adducts on PGHS-1 and PGHS-2 following the oxygenation of arachidonic acid (6). Formation of covalent adducts also was observed with proteins co-incubated with PGHS and arachidonic acid. These findings formed the basis for a hypothesis that PGHS activity in cells could generate levuglandinyl adducts of proteins.Oxygenation of arachidonic acid by PGHS-1 in platelet microsomes has been shown to produce arachidonic acid-derived adducts of multiple proteins (7). Such labeling also has been reported in whole platelets (8) and is increased when thromboxane A 2 synthase is inhibited. However, the reactive product of arachidonic acid that forms these protein adducts has not yet been characterized. We hypothesized that these adducts of platelet proteins...
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