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

Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin Alejandro Zimman, Bjoern Titz, Evangelia Komisopoulou, Sudipta Biswas, Thomas G. Graeber, and Eugene A. Podrez 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 now employed mass spectrometry-based tyrosine, serine, and threonine global 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 a new resource to study 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 PLCy2 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 novel insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. Methods Human platelets isolated by gel filtration (2.7 x 108 / mL) were incubated in Tyrode's buffer with 50 uM KODA-PC or PLPC (as control) for 30 min at 37 degrees C. Platelets were then centrifuged at 3,700 g (10 min, 35 degrees C) and lysed in 8 M urea, 50 mM Tris-HCl (pH 7.4), 1 mM Na3VO4, and 1 mM NaF with sonication. Phosphoproteome changes induced by the agonists were assessed based on trypsin digestion of the protein lysate, phosphopeptide enrichment, mass spectrometry analysis, chromatography alignment, and label-free quantitation as described in the Supplemental Materials and Methods of the publication. The same protocol was applied when studying platelet activation by thrombin, where gel-filtered platelets were incubated in Tyrode's buffer with 0.05 U/mL thrombin or in buffer alone (Resting) for 3 min at 37 degrees C.