Effects of Platelet Agonists and Priming on the Formation of Platelet Populations

Fig. 1. Effect of agonist dose on histogram changes of platelet activation markers. Washed platelets were stimulated with a suboptimal or optimal dose of CRP-XL, 2-MeSADP, or TRAP6, established per donor, for 15 minutes. After stimulation, the cells were labelled with AF647 anti-P-selectin mAb (secretion marker) and FITC-PAC1 mAb (integrin α IIb β 3 activation marker) and analysed by multicolour flow cytometry. Shown are representative histograms and fluorescence dot plots ( n = 5) per agonist; CRP-XL (0.5 µg/mL; 5 µg/mL), 2-MeSADP (25 nM; 2 µM), or TRAP6 (7.5 µM; 15 µM), including the median fluorescence intensity (MFI). Colour code representing different samples: unstimulated platelets ( red ), platelets stimulated with suboptimal agonist dose ( blue ), and platelet stimulated with optimal agonist dose ( black ). ( A ) Staining with FITC-PAC1 mAb, ( B ) AF647 α-P-selectin mAb staining, ( C ) dual-colour dot plots. ( D ) Representative flow cytometric histograms of PAC1 staining of unstimulated platelets ( red ) and platelets stimulated with suboptimal dose of 0.1 µM 2-MeSADP ( total blue area ). Indicated are platelets positive by 2% marker setting (right of vertical line) as well as…

Fig. 2. Agonist- and dose-dependent formation of platelet populations with differential integrin activation and/or secretion. Washed platelets were stimulated with indicated concentrations of CRP-XL (0.5–5 μg/mL), 2-MeSADP (0.06–2 μM), or TRAP6 (2.5–15 μM), and then labelled and analysed as for Fig. 1 . Fluorescence thresholds in quadrant were set for unstimulated sample (2% positive events) per label. ( A ) Representative dot plot of unstimulated sample with 2% marker setting. ( B ) Platelet populations in response to 7.5 µM TRAP6 were (clockwise): PAC1 − /Psel − (gray), PAC1 + /Psel − (blue), PAC1 + /Psel + (yellow), and PAC1 − /Psel + (red). ( C–E ) Mean fractions of different platelet populations as a function of indicated agonist and dose. Bar colours correspond to colour code in quadrant plot. Mean + SD, n = 4–5, (C) † p < 0.05 compared with 2.5 and 5 µg/mL CRP-XL, § p < 0.05 compared with 1, 2.5, and 5 µg/mL CRP-XL; (D) * p < 0.05 compared with all other conditions; (E) * p < 0.05 compared with all other conditions, § p < 0.05 compared with 7.5, 10, and 15 µM TRAP6, † p < 0.05 compared with 10 and 15 µM, # p < 0.05 compared with 15 µM TRAP6; changes in population frequencies…

Fig. 3. Analysis of agonist-induced platelet populations by dimensionality reduction and clustering algorithms. Washed platelets were maximally stimulated with 5 µg/mL CRP-XL, 2 µM 2-MeSADP, or 15 µM TRAP6 followed by labelling with coloured PAC1, anti-CD62P, anti-CD63, anti-TLT1, and anti-GPIX mAbs for multicolour flow cytometry. ( A ) Representative tSNE plots of unstimulated, CRP-XL-, stable ADP- and TRAP6-stimulated samples. ( B ) Characterisation of the FlowSOM clusters (5 populations). −/+ indicates within ± 1 SD from the mean population MFI,–/+ + indicates within ± 2 SD from the mean population MFI. ( C ) Stacked bar graph of the percentage platelets per cluster/population. Mean + SD, n = 5, # p < 0.05 compared with CRP-XL and TRAP6, ¤ p < 0.05 compared with TRAP6; changes in population frequencies between conditions tested by chi-square multi-comparison test with false discovery rate correction. mAb, monoclonal antibody; MFI, median Fluorescence intensity; SD, standard deviation; tSNE, t-distributed stochastic neighbor embedding.

Fig. 4. Effect of adenosine priming on agonist-induced formation of platelet populations. Washed platelets were primed with adenosine before stimulation with suboptimal dose of CRP-XL, 2-MeSADP, or TRAP6 and stained with coloured PAC1 and anti-P-selectin, or additionally with anti-CD63, anti-TLT1, and anti-GPIX mAbs for flow cytometry. ( A ) Flow cytometric events and corresponding histograms as overlaying two-colour plots: black = unstimulated (with 2% marker setting visible), purple = agonist (1.25 µg/mL CRP-XL, 0.1 µM ADP, and 7.5 µM TRAP6), orange = 1 µM adenosine plus agonist. ( B ) Distribution profile of platelet populations after priming with adenosine and indicated stimulation (2% threshold setting). ( C ) Representative tSNE plots of the vehicle- or 1 µM adenosine-primed conditions. ( D ) Characterisation of the FlowSOM clusters (5 populations). ( E ) Distribution profile of platelet populations after priming with adenosine and indicated stimulation resulting from FlowSOM analysis. Mean + SD, n = 4–5, * p < 0.05 compared with all other conditions, ‡ p < 0.05 compared with agonist only, 0.003, 0.01, 0.03, and 0.1 µM adenosine, # p < 0.05 compared with agonist only, 0.003, 0.01,…

Fig. 5. Effect of succinate priming on agonist-induced formation of platelet populations. Platelets were primed with succinate before stimulation with a suboptimal dose of CRP-XL, 2-MeSADP, or TRAP6 and stained with coloured PAC1 and anti-P-selectin, or additionally with anti-CD63, anti-TLT1, and anti-GPIX mAbs for flow cytometry. ( A ) Flow cytometric events and corresponding histograms as overlaying two-colour plots: black = unstimulated (with 2% marker setting visible), purple = agonist (1.25 µg/mL CRP-XL, 0.1 µM ADP, and 7.5 µM TRAP6), orange = succinate plus agonist. ( B ) Distribution profile of platelet populations after priming with succinate and indicated stimulation (2% threshold setting). ( C ) Representative tSNE plots of the vehicle- or 10 mM succinate-primed conditions. ( D ) Characterisation of the FlowSOM clusters (5 populations). ( E ) Distribution profile of platelet populations after priming with succinate and indicated stimulation resulting from FlowSOM analysis. Mean + SD, n = 4–5, ¤ p < 0.05 compared with agonist only, 0.1, 1, 10, and 100 µM succinate, ‡ p < 0.05 compared with agonist only, 0.1, 1, and 10 µM succinate, † p < 0.05 compared with agonist only, 0.1 and…

Fig. 6. Effect of platelet priming with serum or coagulated plasma on agonist-induced formation of platelet populations. Platelets were primed with serum or coagulated PRP or PPP; the plasmas were triggered with tissue factor (TF) or ellagic acid (EL), as indicated; thrombin was inactivated in all serum and plasma samples. For priming, undiluted serum or plasma was added, after which the platelets were stimulated with a suboptimal dose of CRP-XL, ADP, or TRAP6. The stimulated platelets were stained with fluorescently labelled PAC1 and anti-P-selectin, or additionally with anti-CD63, anti-TLT1, and anti-GPIX mAbs. ( A ) Flow cytometric events and corresponding histograms as overlaying two-colour plots: black = unstimulated, purple = agonist (1.25 µg/mL CRP-XL, 0.1 µM ADP, and 7.5 µM TRAP6), orange = serum plus agonist. Quadrant gate was set for unstimulated sample of 2% positive for each fluorescent label. ( B ) Distribution profile of platelet populations after priming with serum or coagulated PRP or PPP, and stimulation with indicated agonist (2% threshold setting). ( C ) Representative tSNE plots of the vehicle- or serum-primed conditions. ( D ) Characterisation of the FlowSOM clusters…