Boutaud Olivier, Li Junyu, Zagol Irene, Shipp Elizabeth A, Davies Sean S, Roberts L Jackson, Oates John A
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232-6602, USA.
J Biol Chem. 2003 May 9;278(19):16926-8. doi: 10.1074/jbc.M300940200. Epub 2003 Mar 11.
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.
花生四烯酸经前列腺素H合成酶（PGHS）氧化的产物前列腺素H2（PGH2）会重排为高反应性的γ-酮醛，即左型前列腺素（LG）E2和LGD2。我们之前已经证明，LGE2与赖氨酸的ε-氨基反应，形成左型前列腺素赖氨酸席夫碱和吡咯衍生的左型前列腺素赖氨酸内酰胺加合物。我们还报道过，这些左型前列腺素赖氨酸加合物是在花生四烯酸氧化后在纯化的PGHS上形成的。我们现在提供证据表明，在用外源性花生四烯酸或凝血酶激活后，人血小板中会形成左型前列腺素赖氨酸内酰胺加合物。在对血小板蛋白进行蛋白水解消化并分离出加合的氨基酸残基后，通过液相色谱-串联质谱法鉴定了这种加合物。我们还证明，这些加合物的形成受到PGHS抑制剂吲哚美辛的抑制，并被血栓素合酶抑制剂增强。这些数据表明，即使存在代谢PGH2的酶，左型前列腺素赖氨酸加合物也是通过全细胞中依赖PGHS的途径形成的。它们还表明，生理刺激足以通过左型前列腺素途径导致人血小板中蛋白质的脂质修饰。
