Stimulation of platelet P2Y 1 receptors by different endogenous nucleotides leads to functional selectivity via biased signalling

Figures

• 

  FIGURE 1. ADP and Ap3A, but not NAD, ADP‐ribose or Up4A, induce platelet aggregation and fibrinogen binding. PRP isolated from healthy human donors was stimulated with increasing concentrations of endogenous P2Y1 receptor ligands, and aggregation measured by light transmission aggregometry, with traces showing effect over 16 min, and statistical analysis at 5 min (a, c, e, g, i). In other experiments, fibrinogen‐488 (2 μg·mL−1) was added to gel filtered platelets (5 × 107 mL−1), and platelets were stimulated with endogenous P2Y1 receptor ligands for 30 min before fixation and analysis on a Beckman Coulter FC500 to quantify fibrinogen binding (b, d, f, h, j). (a, b) ADP. (c, d) Ap3A. (e, f) NAD+. (g, h) ADP‐ribose. (i, j) Up4A. Data: Mean ± SEM. n = 6 per group (aggregation), and n = 4 per group (fibrinogen binding). One‐way ANOVA with Dunnett's multiple comparisons undertaken on aggregation data only. *P < 0.05 versus negative control left hand column).

• 

  FIGURE 2. Characterisation of endogenous P2Y1 receptor ligands on in vitro platelet chemotaxis, P‐selectin expression, and in vivo lung accumulation. Washed platelets (5 × 107 mL−1) in RPMI medium were stimulated with increasing concentrations of endogenous ligands for the P2Y1 receptor. Platelets were then added to the top chamber of a 96‐well Transwell plate, with 0/30 nM fMLP in the bottom chamber. After 90 min at 37°C, platelets in the bottom chamber were quantified and normalised to negative controls to give the chemotactic index (CI). (a) ADP. (b) Ap3a. (c) NAD+. (d) ADP‐ribose. (e) Up4A. Separately, platelet P‐selectin expression after incubation with ADP, NAD+, ADP‐ribose, or Up4A was measured by flow cytometry with concentrations (10 nM or 100 nM, as indicated) that induced chemotaxis (f), or 10 μM (g). In other experiments, BALB/c mice were administered ADP, NAD+, ADP‐ribose, or Up4A intranasally, and bronchoalveolar lavage fluid collected 24 h later for platelet enumeration (h). Data: Mean ± SEM. n = 8 per group (a–e), 6 per group (f), 11 per group (g), and 10 per group (h). Analysed by one‐way ANOVA with Dunnett's multiple comparisons. *P < 0.05 versus in the presence of no…

• 

  FIGURE 3. Platelet chemotaxis towards fMLP was induced by ADP, NAD+, ADP‐ribose and Up4A via P2Y1 receptor. Washed platelets (5 × 107 mL−1) in RPMI medium were incubated with specific antagonists for either P2Y1 (MRS2500) or P2Y12 (AR‐C66096) for 10 min at room temperature. Platelets were then stimulated with increasing concentrations of reported P2Y1 receptor ligands and added to the top chamber of a 96‐well Transwell plate, with 0/30 nM fMLP in the bottom chamber. After 90 min at 37°C, platelets in the bottom chamber were quantified and normalised to negative controls to give the chemotactic index (CI). (a, b) ADP‐induced platelet chemotaxis following P2Y1 or P2Y12 receptor inhibition, respectively. (c, d) NAD. (e, f) ADP‐ribose. (g, h) Up4A. Data: Mean ± SEM. n = 8 per group. One‐way ANOVA with Tukey's multiple comparisons. **P < 0.05 versus in the presence of no nucleotide (column D). # P < 0.05 versus positive control (column 5 from left).

• 

  FIGURE 4. Rac1 and RhoA (ROCK) stimulation are necessary for P2Y1 ligand induced platelet chemotaxis. Washed platelets (5 × 107 mL−1) were incubated with specific inhibitors of either PLC (U73122), Rac1 (NSC23766), or ROCK (GSK429286) for 10 min at room temperature. Platelets were then stimulated with increasing concentrations of endogenous P2Y1 receptor ligands and added to the top chamber of a 96‐well Transwell plate, with 0/30 nM fMLP in the bottom chamber. After 90 min at 37°C, platelets in the bottom chamber were quantified and normalised to negative controls to give the chemotactic index (CI). (a–c) ADP‐induced platelet chemotaxis following inhibition with PLC, Rac1, or ROCK, respectively. (d–f) NAD. (g–i) ADP‐ribose. (j–l) Up4A. Data: Mean ± SEM. n = 6 per group. One‐way ANOVA with Tukey's multiple comparisons. *P < 0.05 versus negative control (column A). # P < 0.05 versus positive control (2nd column).

• 

  FIGURE 5. Molecular docking analysis of endogenous P2Y1 receptor ligands reveal unique amino acid interactions for non‐cognate ligands compared with ADP. 2D interaction diagrams showing how endogenous ligands bind to amino acids within the P2Y1 receptor (a), and comparisons (b), identifying common versus unique contacts. 3D interaction diagram showing the relative position of the endogenous ligands within the P2Y1 binding pocket (c).