Levuglandinyl Adducts of Proteins Are Formed via a Prostaglandin H2 Synthase-dependent Pathway after Platelet Activation

Abstract

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...

Pyridoxamine Analogues Scavenge Lipid-Derived γ-Ketoaldehydes and Protect against H2O2-Mediated Cytotoxicity

“…We have previously demonstrated that levuglandin adducts form in stimulated platelets after activation with arachidonic acid, and that levuglandin adducts levels can be further increased by pretreatment of platelets with dazoxiben, a thromboxane synthase inhibitor (10). We therefore preincubated platelets with dazoxiben and 100 μM or 1 mM PM analog and measured the amount of lysyl-γKA-lactam adduct formed.…”

“…After incubation, platelets were pelleted at 2,000 × g for 10 min at 4 °C. After centrifugation, the lysyl-levuglandin-lactam adduct was isolated from a proteolytic digest of the pelleted proteins and analyzed by high-performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) as described previously (1,10,44). Levels of prostaglandin E 2 (PGE 2 ) in the platelet supernatant were analyzed by GC/MS as previously described (2).…”

“…Levels of γKA adducts significantly increase in pathological conditions including atherosclerosis, end-stage renal disease, and Alzheimer's Disease (5,6). Increased γKA adduct formation has also been characterized in experimental models of oxidative injury and inflammation, including carbon tetrachloride treated rats (7), hyperoxia treated mice (8), septic mice (9), and ex vivo activation of platelets (10). Levels of γKA adducted proteins are expected to be elevated in a wide variety of conditions previously linked to oxidative injury and inflammation (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”

Modification of platelet proteins by malondialdehyde: prevention by dicarbonyl scavengers

“…Samples ( ‫ف‬ 3 mg of protein) were digested with proteases as previously described for lysyl-lactam adducts ( 7 ). In summary, the samples were subsequently heated at 95°C and allowed to cool at room temperature before step digestion with pronase (1 mg) at 37°C for 24 h. Pronase was inactivated by heating samples at 95°C for 10 min and then cooled to room temperature.…”

“…An IS of dilysyl-MDA cross-link was prepared as described above by reaction of TMP in the presence of [ 13 peripheral vein using a 21-gauge needle, and washed platelets were prepared as previously described ( 7 ). The platelets were counted with a Coulter counter and diluted with resuspension buffer to a fi nal concentration of 300,000 platelets/ l and aliquoted in volumes corresponding to 500 µl.…”