Table 2.
Iloprost- and riociguat- stimulated phosphorylation of selected phosphosites of ENSA, ARPP19, PP2A-B56δ (PPP2R5D), and VASP in human platelets.
| Gene | Uniprot | Protein Name | Copy Number/Platelet | P-site (1st) (#of Averaged Peptides) | Iloprost (cA) | Riociguat (cG) | ||
|---|---|---|---|---|---|---|---|---|
| Av. Ratio | p-Value Fraction | Av. Ratio | p-Value Fraction | |||||
| ENSA | O43768 | ENSA/ARPP19e | 7800 | S109 (3) | 4.81 | 100% | 3.75 | 100% |
| ARPP19 | P56211 | ARPP19 | 2500 | S104 (1) | 3.53 | 100% | 2.18 | 100% |
| PPP2R5D | Q14738 | PP2A B-subunit B’-δ (B56δ) | 1300 | S573 (1) | 4.52 | 100% | 2.13 | 100% |
| VASP | P50552 | VASP | 44600 | S157* (1) | 2.03 | 100% | 1.67 | 100% |
| VASP | P50552 | VASP | 44600 | S239 (4) | 3.92 | 100% | 4.52 | 100% |
Washed human platelets from three healthy donors (three biological replicates) were incubated with buffer (control), iloprost (5 nM, 2 min at 37 °C) or riociguat (10 µM, 5 min at 37 °C). After incubation, samples were stopped by the addition of 4× lysis buffer, shock frozen in liquid nitrogen and analysed by quantitative phosphoproteomics as described [40]. The fold increase of phosphorylation of various phosphosites compared to control is shown as average ratio of all quantified peptides bearing the corresponding site. As a measure of reliability, the ‘p-value fraction’ (for each site: ∑ peptides p < 0.05/∑ site-bearing peptides) and # of averaged peptides is presented. The change of various phosphosites in response to the test substance compared to control (increase or decrease) is shown as average ratio (av. ratio). The p-value fraction of all sites shown here is 100%, which represents an excellent reliability of these phosphosite measurements. * All trypsin digests except VASP S157 (subtilisin digest).