Specific Pharmacological Profile of A 2A Adenosine Receptor Predicts Reduced Fractional Flow Reserve in Patients With Suspected Coronary Artery Disease

Figure 1. Study flow chart. CAD indicates coronary artery disease; FFR, fractional flow reserve; GRACE, Global Registry of Acute Coronary Events; ICA, invasive coronary angiography.

Figure 2. A2 AR expression evaluated on peripheral blood mononuclear cells in 60 patients with suspected coronary artery disease. Group 1: normal angiography (n=17); group 2: abnormal angiography but non‐significant FFR (ie, 0.8–1.0) (n=21); group 3: abnormal angiography with significant FFR (ie, ≤0.8) (n=22). Expression was evaluated by Western blot; a representative blot from each group of patients is shown. The staining intensity was measured using densitometry analysis and data (medians [thick lines], IQRs [thin lines]) are expressed in AU (ie, ratio of pixels generated by the A2 AR band to pixels generated by the background signal). A2 AR indicates adenosine A2A receptors; AU, arbitrary units; FFR, fractional flow reserve; IQR, interquartile range. A representative Western blot with the molecular weight markers (left) and the specific A2 AR band (45 kDa) of a patient from each group is shown. The presence of a nonspecific band (NSB) demonstrates that equal amounts of protein were loaded in each lane.

Figure 3. Peripheral blood mononuclear cells A2 AR characterization in patients with suspected coronary artery disease. Group 1: normal angiography (n=17); group 2: abnormal angiography but non‐significant FFR (ie, 0.8–1.0) (n=21); group 3: abnormal angiography with significant FFR (ie, ≤0.8). KD (A) and EC 50 (B) were interpolated from the dose response curves obtained using increasing concentrations of Adonis (see Methods); KD/EC 50 ratio (C). Data are expressed as median (thick lines) and IQRs (thin lines). A2 AR indicates adenosine A2A receptors; EC 50, half maximal effective concentration; FFR, fractional flow reserve; IQR, interquartile range; KD, dissociation constant.

Figure 4. Identification of spare A2 AR in patients with suspected coronary artery disease. The dose–response curves resulting from Adonis binding to A2A adenosine receptors and the resulting cAMP production for each group of patients: (A) patients with normal angiography (n=17); (B) patients with stenosis but non‐significant FFR (ie, 0.8–1.0) (n=21); (C) patients with stenosis and significant FFR (≤0.8) (n=22). White discs: KD (densitometry analysis of the 25 kDa band corresponding to the light [L‐chain], see a representative Western blot at the top of the figure); black discs: EC 50 Results are mean±SD. cAMP indicates cyclic adenosine monophosphate; EC 50, half maximal effective concentration; FFR, fractional flow reserve; KD, dissociation constant; SD, standard deviation.

Figure 5. Receiver operating characteristic curves. For (A) A2 AR expression data and (B) KD/EC 50 ratio data evaluated on peripheral blood mononuclear cells of patients with suspected coronary artery disease (n=60). A2 AR indicates adenosine A2A receptors; EC 50, half maximal effective concentration; KD, dissociation constant.

Figure 6. Mechanism of action of A2 AR on coronary vascular wall. Adonis binds to a linear epitope on the second extracellular loop of A2 AR. Upon stimulation by Adonis, the third intracellular loop of A2 AR couples with the stimulatory subunit of G‐proteins (Gs) and triggers adenylate cyclase (AC) activity. Production of cAMP activates protein kinase‐mediated signaling pathways and acts on Ca++/K+ channels of vascular smooth muscle leading eventually to coronary dilation. PKA indicates protein kinase A.