Phosphoproteome of Resting Human Platelets

Abstract

Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbalpha is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG.

What Can Proteomics Tell Us About Platelets?

“…115 Owing to the enrichment provided by specific probes, activity-based protein profiling 116-121 B, These studies are mainly (40%) based on 2-dimensional gel electrophoresis (2-DE)/ difference gel electrophoresis (DIGE), 46,52,53,80,82,83,85,87,88,90,91,[93][94][95][96]98,102,106,110,112,113 whereas gelfree methods (23%) are still under-represented. 21,42,89,97,[114][115][116][117][118]121 Combination refers to studies using 2-DE/DIGE in combination with other approaches. COFRADIC indicates combined fractional diagonal chromatography; and MudPIT, multidimensional protein identification technology.…”

“…135,136 To date, only few platelet studies used dedicated phosphopeptide enrichment procedures (Text Box 1) in conjunction with LC-MS to identify phosphopeptides and phosphorylation sites. 42,116,121 The first one published by us in 2008 identified >500 phosphorylation sites in resting human platelets. 116 Another recent study used phosphotyrosine peptide immunoprecipitation in conjunction with quantitative MS to quantify changes in 28 of 214 identified phosphotyrosine sites in platelets stimulated via the glycoprotein VI collagen receptor.…”

“…42,116,121 The first one published by us in 2008 identified >500 phosphorylation sites in resting human platelets. 116 Another recent study used phosphotyrosine peptide immunoprecipitation in conjunction with quantitative MS to quantify changes in 28 of 214 identified phosphotyrosine sites in platelets stimulated via the glycoprotein VI collagen receptor. 121 Among these was Tyr370 in oligophrenin-1, which may play a role in platelet filopodia formation.…”

“…Whereas RGS18 Ser49 is phosphorylated during platelet activation, Ser216 is phosphorylated on platelet inhibition by protein kinase A (PKA) and protein kinase G. 140 These 2 cyclic nucleotide-dependent protein kinases govern inhibitory pathways in platelets which, despite their important role, have been addressed by only few proteomic studies to date. 115,116,141 Up to now, only 15 PKA and 11 protein kinase G substrates have been confirmed. 142 Lately, a new PKA and protein kinase G substrate was identified, namely calcium and diacylglycerol-regulated guanine nucleotide exchange factor I, the main activator for Rap1b in platelets.…”

Mapping N-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling

“…Thus, the N-terminus phosphorylation site can be excluded for all three phosphopeptides, confirming the internal phosphorylation sites near the Nterminal region: pSer 232 for Ras-GTPase-activating protein-binding protein 1, pThr401 for Src substrate cortactin, and pSer 641 for ␣-1 catenin. Among these excluded sites, the site on Ser230 of G3BP1 and the site on Thr399 of SRC8 proteins were assigned to be phosphorylation sites in previous reports [29,30] without confirmation. It is worth noting that the synthetic peptides shown in Figures 2b and 3b without and with dimethyl labeling, respectively, were the same sequence (SSpSPAPADIAQTVQEDLR) derived from Ras-GTPase-activating protein-binding protein 1 as shown in Figure 4a, whereas, unlike those detected from the synthetic peptides, no y 16 or y 17 ions (Figure 4a) could be detected from A431 cells to help in identifying the exact phosphorylation site.…”