Levuglandinyl adducts of proteins are formed via a prostaglandin H2 synthase-dependent pathway after platelet activation.

The product of oxygenation of arachidonic acid by the prostaglandin H synthases (PGHS), prostaglandin H(2) (PGH(2)), undergoes rearrangement to the highly reactive gamma-ketoaldehydes, levuglandin (LG) E(2), and LGD(2). We have demonstrated previously that LGE(2) reacts with the epsilon-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 levuglandinyl-lysine 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 are formed from LGE 2 and LGD 2 .
This report provides evidence that levuglandinyl-lysine adducts of proteins are formed in cells as a consequence of the oxygenation of arachidonic acid by a PGHS.

EXPERIMENTAL PROCEDURES
Materials-Dazoxiben was a generous gift from Pfizer Ltd. (Sandwich, UK). Methanol was ordered from Burdick and Jackson (Muskegon, MI). Arachidonic acid, sodium citrate, citric acid, indomethacin, and butylated hydroxytoluene were purchased from Sigma. Sepharose 2B is from Amersham Biosciences (Uppsala, Sweden). Oasis TM Sep-Pak cartridges were obtained from Waters Corp. (Milford, MA), and dimethyl formamide and trisphenylphosphine were from Aldrich. Pronase and aminopeptidase M from porcine kidney were from Calbiochem. Thrombin was obtained from Pharmingen.
Preparation of Washed Human Platelets-Human blood was obtained following a protocol approved by the Institutional Review Board of Vanderbilt University. Washed human platelets were isolated following the protocol described previously (9). The blood was drawn with a syringe containing 5 ml of 3.8% sodium citrate (final volume: 50 ml), then centrifuged in plastic tubes at 300 ϫ g for 10 min at room temperature (23°C). The supernatant (platelet-rich plasma) was acidified to pH 6.4 with 0.15 M citric acid (10) and then centrifuged at 1,000 ϫ g for 10 min at room temperature. The pellet was resuspended with 5 ml of washing buffer (24.4 mM sodium phosphate, pH 6.5, 0.113 M NaCl, 5.5 mM glucose). After 15 min at room temperature, the platelets were purified on a Sepharose 2B column equilibrated with washing buffer. The eluted platelets were counted with a Coulter counter and diluted with resuspension buffer (8.3 mM sodium phosphate, pH 7.5, 0.109 M NaCl, 5.5 mM glucose) for a final count of 600,000 platelets/l. Analysis of LG-Lysine Lactam Adduct in Human Platelets-After incubation, platelets were pelleted at 2,000 ϫ g for 10 min at room temperature. After centrifugation, the LG-lysine lactam adduct was isolated from proteins and analyzed by LC MS/MS as described previously (5, 11). In short, 10 ml of cold ethanol (containing 50 mg/liter of * This work was supported in part by National Institutes of Health Grants GM 15431, CA 68485, and GM 42056. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: butylated hydroxytoluene and 500 mg/liter of trisphenylphosphine) were added to the cell pellets, and the proteins were precipitated by centrifugation at 2,000 ϫ g for 10 min at 4°C. Proteins were then reprecipitated in 10 ml of cold solution of methanol/chloroform 1:2 (v:v) and washed with 10 ml of methanol (each containing butylated hydroxytoluene and trisphenylphosphine). Then, partial digestion of proteins to single amino acid was performed using 3 mg of Pronase and 3 l of aminopeptidase M (0.15 unit) per sample. After purification the LG-lysine lactam adduct was analyzed by LC MS/MS as described previously (5,6). The 13 C-labeled internal standard was prepared by reaction of synthetic LGE 2 (12) and [ 13 C]lysine; the LG-lysine lactam standard was then purified as described previously (11).

RESULTS
We examined the formation of LG-lysine lactam adducts on platelet proteins following oxygenation of exogenous arachidonic acid. Following proteolytic digestion of platelet proteins, the products were analyzed by LC MS/MS. Selected reaction monitoring was used to analyze the LG-lysine lactam fragment ions derived from the levuglandinyl moiety (m/z 332.1) and the lysyl moiety (m/z 84.1) of the adduct (5) (Fig. 2). From platelets incubated with 20 M arachidonic acid, both of these fragment ions are detected (Fig. 3). The simultaneous elution of the two fragment ions concurrently with the [ 13 C]LG-lysine lactam standard provides consistent evidence that identifies the LGlysine lactam adduct. Preincubation of the cells with indomethacin for 30 min markedly reduces the formation of the LG-lysine lactam from 169 to 20 pg of lactam/1 ϫ 10 9 platelets. These findings demonstrate that levuglandins are generated in human platelets in a PGHS-dependent fashion and form covalent adducts with proteins.
Because of the theoretical possibility that activation of platelets with exogenous arachidonic acid might generate an amount of PGH 2 that saturates its catalytic disposition by the thromboxane synthase, we also examined the formation of LGlysine lactam adducts after activation of platelets with the physiological agonist, thrombin. As depicted in Fig. 4, LGlysine adducts are formed following thrombin. Inhibition of formation of the adducts by indomethacin confirms that they are derived from oxygenation of arachidonic acid by the PGHS.
Further evidence that the adducts are derived from PGH 2 was obtained by examining the effect of dazoxiben, an inhibitor of thromboxane synthase. As shown in Fig. 5, inhibition of thromboxane synthase by dazoxiben led to an increase in the levels of LG-lysine lactam adduct (357 pg of lactam/1 ϫ 10 9 platelets) by 2.1-fold compared with the control experiment in which no inhibitor was present.

DISCUSSION
This report provides the first evidence that levuglandinyl adducts of proteins can be formed in a cell as a consequence of oxygenation of arachidonic acid by a PGHS.
The immediate product of the PGHS, PGH 2 , is the substrate for synthases that catalyze its conversion to specific prostanoids. PGH 2 also undergoes non-enzymatic rearrangement to several products, and the highly reactive ketoaldehydes, LGE 2 and LGD 2 , account for ϳ20% of these products. Even though levuglandinyl adducts of proteins have been demonstrated after exposure of the proteins to PGH 2 in vitro (2,6), the competing enzymatic biotransformation of PGH 2 in cells has provided a reason to question whether non-enzymatic rearrangement of PGH 2 to LGE 2 could occur in intact cells. To address this question, we chose to examine the platelet, in which there is robust biotransformation of PGH 2 via the thromboxane synthase. Attention also has been focused on the platelet by the findings of Lecomte et al. (8) of uncharacterized, PGH 2 -derived adducts of at least 10 platelet proteins; one of these adducted proteins was PGHS-1, which we have shown to be adducted by levuglandin as a consequence of arachidonic acid oxygenation in vitro (6). Our finding that formation of the LG-lysine adduct of platelet proteins is inhibited by indomethacin indicates that its formation is a consequence of the oxygenation of arachidonic acid by the PGHS. The increased levels of LG-lysine adducts after treatment of the platelets with dazoxiben supports their origin from PGH 2 . The evidence that a levuglandinyl adduct of platelet proteins is derived from PGH 2 provides support for the levuglandin pathway of arachidonic acid metabolism; it, of course, does not exclude the formation of adducts derived from other products of arachidonic acid. It may be concluded from these findings that LGE 2 is formed in platelets and adducts platelet proteins even in the presence of an enzyme that uses PGH 2 as a substrate.
The known properties of levuglandins provide a context for considering any possible functional consequences of the formation of such adducts of proteins. Clearly, adduct formation will add a lipophilic moiety to the protein. Considerable information on other lipid adducts of proteins indicates that they may alter processes such as localization, degradation, or function (13). As an example specific to the levuglandins, formation of levuglandinyl adducts on proteins significantly reduces their clearance by the 20 S proteasome (14). Also, the initial species of the levuglandinyl adducts are known to be highly reactive. This can lead to cross-linking between proteins and DNA (4) and between proteins and small molecules that contain amine groups (6). In addition, levuglandinyl adducts can produce in-termolecular cross-linking between proteins (3). For example, we have found that PGH 2 accelerates the formation of amyloid ␤ oligomers in vitro (15). Because of the irreversible character of the adducts and cross-linked proteins, they have the potential to accumulate in cells over time.
In conclusion, cyclooxygenase-dependent formation of levuglandinyl adducts of proteins occurs during platelet activation. This finding provides a basis for investigations that address the possible function of these reactive lipid adducts of proteins in platelets, as well as in other cells in which there is abundant or protracted production of PGH 2 .