title: Modification of platelet proteins by 4-hydroxynonenal: Potential Mechanisms for inhibition of aggregation and metabolism.
abstract: Platelet aggregation is an essential response to tissue injury and is associated with activation of pro-oxidant enzymes, such as cyclooxygenase, and is also a highly energetic process. The two central energetic pathways in the cell, glycolysis and mitochondrial oxidative phosphorylation, are susceptible to damage by reactive lipid species. Interestingly, how platelet metabolism is affected by the oxidative stress associated with aggregation is largely unexplored. To address this issue, we examined the response of human platelets to 4-hydroxynonenal (4-HNE), a reactive lipid species which is generated during thrombus formation and during oxidative stress. Elevated plasma 4-HNE has been associated with renal failure, septic shock and cardiopulmonary bypass surgery. In this study, we found that 4-HNE decreased thrombin stimulated platelet aggregation by approximately 60%. The metabolomics analysis demonstrated that underlying our previous observation of a stimulation of platelet energetics by thrombin glycolysis and TCA (Tricarboxylic acid) metabolites were increased. Next, we assessed the effect of both 4-HNE and alkyne HNE (A-HNE) on bioenergetics and targeted metabolomics, and found a stimulatory effect on glycolysis, associated with inhibition of bioenergetic parameters. In the presence of HNE and thrombin glycolysis was further stimulated but the levels of the TCA metabolites were markedly suppressed. Identification of proteins modified by A-HNE followed by click chemistry and mass spectrometry revealed essential targets in platelet activation including proteins involved in metabolism, adhesion, cytoskeletal reorganization, aggregation, vesicular transport, protein folding, antioxidant proteins, and small GTPases. In summary, the biological effects of 4-HNE can be more effectively explained in platelets by the integrated effects of the modification of an electrophile responsive proteome rather than the isolated effects of candidate proteins.
authors.affiliation: Department of Pathology; UAB Mitochondrial Medicine Laboratory; Center for Free Radical Biology.
authors.forename: Saranya
authors.initials: S
authors.lastname: Ravi
authors.affiliation: Department of Pathology; UAB Mitochondrial Medicine Laboratory; Center for Free Radical Biology.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Johnson
authors.affiliation: Department of Pathology; UAB Mitochondrial Medicine Laboratory; Center for Free Radical Biology.
authors.forename: Balu K
authors.initials: BK
authors.lastname: Chacko
authors.affiliation: Department of Pathology; UAB Mitochondrial Medicine Laboratory; Center for Free Radical Biology.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Kramer
authors.affiliation: Department of Pathology; UAB Mitochondrial Medicine Laboratory; Center for Free Radical Biology.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Sawada
authors.affiliation: Department of Pathology; UAB Mitochondrial Medicine Laboratory; Center for Free Radical Biology.
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Locy
authors.affiliation: The Targeted Metabolomics and Proteomics Laboratory.
authors.forename: Landon S
authors.initials: LS
authors.lastname: Wilson
authors.affiliation: The Targeted Metabolomics and Proteomics Laboratory; Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Barnes
authors.affiliation: Department of Pathology.
authors.forename: Marisa B
authors.initials: MB
authors.lastname: Marques
authors.affiliation: Department of Pathology; UAB Mitochondrial Medicine Laboratory; Center for Free Radical Biology. Electronic address: darley@uab.edu.
authors.forename: Victor M
authors.initials: VM
authors.lastname: Darley-Usmar
mesh_terms: D000447:Aldehydes / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002952:Citric Acid Cycle / Q000187:drug effects; D004734:Energy Metabolism / Q000187:drug effects; D006019:Glycolysis / Q000187:drug effects; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects*; D020543:Proteome / Q000378:metabolism*; D013917:Thrombin / Q000502:physiology
chemical_list: D000447:Aldehydes; D020543:Proteome; D013917:Thrombin; C027576:4-hydroxy-2-nonenal
keywords: 4-hydroxynonenal; Aggregation; Bioenergetics; Click chemistry; Metabolomics; Platelet