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.
Boutaud
Olivier
O
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232-6602, USA. olivier.boutaud@vanderbilt.edu
Li
Junyu
J
Zagol
Irene
I
Shipp
Elizabeth A
EA
Davies
Sean S
SS
Roberts
L Jackson
LJ
Oates
John A
JA
D016718:Arachidonic Acid / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism; D015539:Platelet Activation; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011457:Prostaglandins D / Q000378:metabolism; D011458:Prostaglandins E / Q000378:metabolism; D011462:Prostaglandins G / Q000378:metabolism; D011463:Prostaglandins H / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*
D010446:Peptide Fragments; D011457:Prostaglandins D; D011458:Prostaglandins E; D011462:Prostaglandins G; D011463:Prostaglandins H; D011506:Proteins; D016718:Arachidonic Acid; D011451:Prostaglandin-Endoperoxide Synthases