Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin

PLoS One. 2014 Jan 6;9(1):e84488. doi: 10.1371/journal.pone.0084488. eCollection 2014.

Abstract

Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Platelets / drug effects*
  • Blood Platelets / metabolism*
  • CD36 Antigens / metabolism
  • Cluster Analysis
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Phospholipase C gamma / metabolism
  • Phospholipids / pharmacology*
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Platelet Activation / drug effects*
  • Platelet Activation / physiology*
  • Protein Interaction Maps
  • Protein-Tyrosine Kinases / metabolism
  • Proteome*
  • Proteomics
  • Signal Transduction / drug effects
  • Syk Kinase
  • Thrombin / pharmacology*
  • Thrombosis / metabolism
  • src-Family Kinases / metabolism

Substances

  • CD36 Antigens
  • Intracellular Signaling Peptides and Proteins
  • Phospholipids
  • Phosphoproteins
  • Proteome
  • Protein-Tyrosine Kinases
  • SYK protein, human
  • Syk Kinase
  • src-Family Kinases
  • Phospholipase C gamma
  • Thrombin