title: Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition.
abstract: Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbβ3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Beck
authors.affiliation: Interdisciplinary Bank of Biomaterials and Data, Würzburg, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Geiger
authors.affiliation: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Saint Petersburg, Russia.
authors.forename: Stepan
authors.initials: S
authors.lastname: Gambaryan
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors.forename: Fiorella A
authors.initials: FA
authors.lastname: Solari
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors.forename: Margherita
authors.initials: M
authors.lastname: Dell'Aica
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Loroch
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.
authors.forename: Nadine J
authors.initials: NJ
authors.lastname: Mattheij
authors.affiliation: Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Saint Petersburg, Russia.
authors.forename: Igor
authors.initials: I
authors.lastname: Mindukshev
authors.affiliation: Natural and Medical Sciences Institute at the University of Tübingen, Tübingen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Pötz
authors.affiliation: Center for Thrombosis and Hemostasis, Universitätsklinikum der Johannes Gutenberg-Universität Mainz, Mainz, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Jurk
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Burkhart
authors.affiliation: Institute for Biology and Biotechnology of Plants, University Münster, Münster, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Fufezan
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.
authors.forename: Johan W M
authors.initials: JW
authors.lastname: Heemskerk
authors.affiliation: Center for Thrombosis and Hemostasis, Universitätsklinikum der Johannes Gutenberg-Universität Mainz, Mainz, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Walter
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors.forename: René P
authors.initials: RP
authors.lastname: Zahedi
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.|Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, United Kingdom; and.|Medizinisches Proteom Center, Ruhr Universität Bochum, Bochum, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D040901:Proteomics / Q000379:methods; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D010975:Platelet Aggregation Inhibitors; D000244:Adenosine Diphosphate; D016285:Iloprost