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Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

Florian Beck, Jörg Geiger, Stepan Gambaryan, Fiorella A. Solari, Margherita Dell'Aica, Stefan Loroch, Nadine Mattheij, Igor Mindukshev, Oliver Pötz, Kerstin Jurk, Julia M. Burkhart, Christian Fufezan, Johan W.M. Heemskerk, Ulrich Walter, René P. Zahedi and Albert Sickmann

Key points

  • Temporal profiles of more than 4000 phosphopeptides after stimulating human platelets (a) with ADP and (b) consecutively with ADP and Iloprost.

  • Reciprocal phosphorylation profiles of ADP and Iloprost point to central players of platelet homeostasis.

Abstract

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 that is antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful anti-platelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (i) critical hubs in platelet signaling towards aggregation, and (ii) novel molecular targets for anti-platelet treatment strategies. We thus 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 4,797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal re-organization, 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 (e.g. 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.

  • Submitted May 2, 2016.
  • Accepted November 3, 2016.