Abstract
Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.
Highlights
From the ‡Systems Immunity Research Institute and Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom, the §Department of Pharmacology, University of Colorado at Denver, Aurora, Colorado 80045, the ¶Vanderbilt Institute of Chemical Biology, Centre in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232-0146, and the ʈWilliam Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom
We show by using lipidomics that thrombinactivated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine
From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8
Summary
Lipids and lipid standards were purchased from Avanti Polar Lipids (Alabaster, AL) or Cayman Chemical (Ann Arbor, MI). Deuterated standards are as follows: arachidonic acid-d8, 5Z,8Z,11Z,14Z-eicosatetraenoic-5,6,8,6:54 PM 5/12/20169, 11,12,14,15-d8 acid, Ն99% deuterated forms; PGE2-d4, 9-oxo11␣,15S-dihydroxy-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid, Ն99% deuterated forms; and PGD2-d4, 9␣,15S-dihydroxy-11oxo-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid, Ն99% deuterated forms. HPLC grade solvents were from Thermo Fisher Scientific (Hemel Hempstead, Hertfordshire, UK). PAR-1 and PAR-4 agonists were from Tocris Biosciences (Bristol, UK). COX-1 inhibitor (Sc-560) was from Cayman Chemical. Platelet signaling inhibitors (PP2, oleyloxyethylphosphocholine (OOEPC), bromoenol lactone, cytosolic phospholipase A2␣ (cPLA2␣) inhibitor (N-{(2S,4R)-4-(biphenyl-2-yl-methylisobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolisatetraenoic acid; PG, prostaglandin; NMBHA, N-methyl benzohydroxamic acid; ANOVA, analysis of variance; HPETE, hydroperoxyeicosatetraenoic acid; FTMS, Fourier MS; CID, collision-induced dissociation; AA, arachidonate; PLC, phospholipase C; LOX, lipoxygenase
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