From the Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tennessee 37232-6602
Received for publication, January 28, 2003, and in revised form, March 7, 2003
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ABSTRACT |
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The product of oxygenation of arachidonic acid by
the prostaglandin H synthases (PGHS), prostaglandin
H2 (PGH2), undergoes rearrangement to the
highly reactive Prostaglandin H synthase
(PGHS)1 catalyzes the
oxygenation of arachidonic acid to the endoperoxide, prostaglandin
H2 (PGH2). PGH2 is further
metabolized to the prostanoids PGD2, PGE2,
PGF2a, thromboxane A2, and prostacyclin by
specific enzymes. Also, PGH2 in aqueous solutions undergoes
non-enzymatic rearrangement to yield PGE2 and
PGD2, and 20% of it rearranges to the highly reactive -ketoaldehydes, levuglandin (LG) E2, and
LGD2. We have demonstrated previously that LGE2
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 levuglandinyl-lysine adducts are formed via a
PGHS-dependent pathway in whole cells, even in the presence
of an enzyme that metabolizes PGH2. They also demonstrate
that a physiological stimulus is sufficient to lead to the lipid
modification of proteins through the levuglandin pathway in human platelets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ketoaldehydes, levuglandins (LG) E2 and D2
(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 LGE2
or PGH2 (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.
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Fig. 1.
Structures of levuglandins
E2 and D2.
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 A2 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 LGE2 and LGD2.
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.
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EXPERIMENTAL PROCEDURES |
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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). OasisTM 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 × 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 × 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/µl.
Formation of LG-Lysine Adducts in Human Platelets-- Washed platelets were then preincubated with indomethacin (final concentration of 100 µM), dazoxiben (final concentration of 10 µM), or vehicle for 30 min at room temperature. At this time, the platelets were activated by adding arachidonic acid (final concentration of 20 µM) or thrombin (final concentration of 1 Unit/ml) and incubated at room temperature for 15 min.
Analysis of LG-Lysine Lactam Adduct in Human
Platelets--
After incubation, platelets were pelleted at 2,000 × 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 butylated hydroxytoluene and 500 mg/liter of trisphenylphosphine) were added to the cell pellets, and the proteins were precipitated by centrifugation at 2,000 × 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 µl 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 13C-labeled internal standard was
prepared by reaction of synthetic LGE2 (12) and
[13C]lysine; the LG-lysine lactam standard was then
purified as described previously (11).
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RESULTS |
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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 µM
arachidonic acid, both of these fragment ions are detected (Fig.
3). The simultaneous elution of the two
fragment ions concurrently with the [13C]LG-lysine lactam
standard provides consistent evidence that identifies the LG-lysine
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 × 109 platelets. These findings
demonstrate that levuglandins are generated in human platelets in a
PGHS-dependent fashion and form covalent adducts with
proteins.
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Because of the theoretical possibility that activation of platelets
with exogenous arachidonic acid might generate an amount of
PGH2 that saturates its catalytic disposition by the
thromboxane synthase, we also examined the formation of LG-lysine
lactam adducts after activation of platelets with the physiological
agonist, thrombin. As depicted in Fig. 4,
LG-lysine 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.
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Further evidence that the adducts are derived from PGH2 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 × 109 platelets) by 2.1-fold compared with the control
experiment in which no inhibitor was present.
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DISCUSSION |
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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, PGH2, is the substrate for synthases that catalyze its conversion to specific prostanoids. PGH2 also undergoes non-enzymatic rearrangement to several products, and the highly reactive ketoaldehydes, LGE2 and LGD2, account for ~20% of these products. Even though levuglandinyl adducts of proteins have been demonstrated after exposure of the proteins to PGH2 in vitro (2, 6), the competing enzymatic biotransformation of PGH2 in cells has provided a reason to question whether non-enzymatic rearrangement of PGH2 to LGE2 could occur in intact cells. To address this question, we chose to examine the platelet, in which there is robust biotransformation of PGH2 via the thromboxane synthase. Attention also has been focused on the platelet by the findings of Lecomte et al. (8) of uncharacterized, PGH2-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 PGH2. The evidence that a levuglandinyl adduct of platelet proteins is derived from PGH2 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 LGE2 is formed in platelets and adducts platelet proteins even in the presence of an enzyme that uses PGH2 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
intermolecular cross-linking between proteins (3). For example, we have
found that PGH2 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 PGH2.
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ACKNOWLEDGEMENT |
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We thank Dr. Sheila Timmons for her valuable advice.
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FOOTNOTES |
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* 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. The 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: Dept. of Pharmacology,
Vanderbilt University, Nashville, TN 37232-6602. Tel.: 615-343-7398;
Fax: 615-322-4707; E-mail: olivier.boutaud@vanderbilt.edu.
§ Thomas F. Frist, Sr. Professor of Medicine.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M300940200
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ABBREVIATIONS |
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The abbreviations used are: PGHS, prostaglandin H2 synthase; PGH2, prostaglandin H2; LC, liquid chromatography; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; LG, levuglandin; HPLC, high performance liquid chromatography.
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REFERENCES |
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