Figure 3.
Quantitation of GPVI signaling with feedback from purinergic and thromboxane receptors. (A) Platelets were prepared from n = 5 donors. To analyze phosphopeptides downstream of GPVI with feedback from purinergic and thromboxane receptors, platelets from each donor were incubated Integrilin in the absence of apyrase and indomethacin (condition 2). Principal component analysis of quantified phosphopeptides from pTyr (top) and TiO2 enrichment (below) reveal distinct groupings following CRP-XL stimulation. (B) Scatter plot of measured mean phosphopeptide reporter ion intensities for control vs +CRP-XL samples. (C) Volcano plots of phosphopeptide reporter ion intensity ratios (+CRP-XL/ control) vs P values, colored by FDR find 185 pTyr-enriched phosphopeptides and 1358 TiO2 phosphopeptides significantly increase (FDR < 0.01) in +CRP-XL samples. (D) Phosphopeptides from conditions 1 and 2 represented 893 and 4571 potential phosphorylation events, respectively (2132 regulated phosphorylation sites specific to condition 1; 2550 specific to condition 2; 894 shared). Phosphorylation events occurred on 952 and 1026 proteins, respectively. (E) Summary of significant (FDR < 1×10−15) gene ontology term (Biological Process) enrichment and number (No.) of high confidence (FDR < 0.01) candidate proteins conditions 1 and 2. (F) Summary of No. of proteins modified in conditions 1 and 2 significantly associated with Reactome pathways (supplemental Table 2). (G) Western blot analysis and quantitation of site-specific protein phosphorylation from resting (yellow) and +CRP-XL stimulated platelets (n = 3) in the presence of apyrase and indomethacin (green) or Integrilin alone (blue). Blot pixel intensities quantified with Image J (a.u.). Significance of blot intensities for +CRP-XL conditions were determined by ratio paired Student t test (*P < .1). Note: Interactive and searchable versions of the phosphoproteomics data and graphs above are available at: https://kcvi.shinyapps.io/STARTapp_515_pTyr/ and https://kcvi.shinyapps.io/STARTapp_515_TiO2/.

Quantitation of GPVI signaling with feedback from purinergic and thromboxane receptors. (A) Platelets were prepared from n = 5 donors. To analyze phosphopeptides downstream of GPVI with feedback from purinergic and thromboxane receptors, platelets from each donor were incubated Integrilin in the absence of apyrase and indomethacin (condition 2). Principal component analysis of quantified phosphopeptides from pTyr (top) and TiO2 enrichment (below) reveal distinct groupings following CRP-XL stimulation. (B) Scatter plot of measured mean phosphopeptide reporter ion intensities for control vs +CRP-XL samples. (C) Volcano plots of phosphopeptide reporter ion intensity ratios (+CRP-XL/ control) vs P values, colored by FDR find 185 pTyr-enriched phosphopeptides and 1358 TiO2 phosphopeptides significantly increase (FDR < 0.01) in +CRP-XL samples. (D) Phosphopeptides from conditions 1 and 2 represented 893 and 4571 potential phosphorylation events, respectively (2132 regulated phosphorylation sites specific to condition 1; 2550 specific to condition 2; 894 shared). Phosphorylation events occurred on 952 and 1026 proteins, respectively. (E) Summary of significant (FDR < 1×10−15) gene ontology term (Biological Process) enrichment and number (No.) of high confidence (FDR < 0.01) candidate proteins conditions 1 and 2. (F) Summary of No. of proteins modified in conditions 1 and 2 significantly associated with Reactome pathways (supplemental Table 2). (G) Western blot analysis and quantitation of site-specific protein phosphorylation from resting (yellow) and +CRP-XL stimulated platelets (n = 3) in the presence of apyrase and indomethacin (green) or Integrilin alone (blue). Blot pixel intensities quantified with Image J (a.u.). Significance of blot intensities for +CRP-XL conditions were determined by ratio paired Student t test (*P < .1). Note: Interactive and searchable versions of the phosphoproteomics data and graphs above are available at: https://kcvi.shinyapps.io/STARTapp_515_pTyr/ and https://kcvi.shinyapps.io/STARTapp_515_TiO2/.

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