Specific Pharmacological Profile of A2A Adenosine Receptor Predicts Reduced Fractional Flow Reserve in Patients With Suspected Coronary Artery Disease

Abstract

Background: The rapid and reliable exclusion of myocardial revascularization is a major unmet clinical need in patients with suspected coronary artery disease (CAD) and non-contributive electrocardiography and troponin. Non-invasive tests have high rates of false positives and negatives, and there is no biomarker to assess myocardial ischemia. The presence of spare adenosine A2A receptors (A2AR)-characterized by a high dissociation constant/half maximal effective concentration (KD/EC50) ratio-expressed on peripheral blood mononuclear cells (PBMC) has been associated with ischemia during exercise stress testing in patients with CAD. In this work, we investigated the diagnostic accuracy of spare A2AR versus fractional flow reserve (FFR) in patients with suspected CAD.

Methods and results: Sixty patients with suspected CAD, but non-contributive electrocardiography and troponin, were consecutively enrolled in this prospective study. The binding (KD), functional response (cyclic adenosine monophosphate [cAMP] production; EC50) on PBMC A2AR were compared with FFR results. Patients were divided into 3 groups: 17 (group 1) with normal coronary angiography (n=13) or stenosis <20% (n=4); 21 with CAD and non-significant FFR (group 2); and 22 with CAD and significant FFR (group 3). Median KD/EC50 was 6-fold higher in group 3 (4.20; interquartile range: 2.81-5.00) than group 2 (0.66; interquartile range: 0.47-1.25) and 7-fold higher than group 1 (0.60; interquartile range: 0.30-0.66).

Conclusions: In patients with suspected CAD and non-contributive electrocardiography and troponin, the absence of spare A2AR on PBMC may help to rule out myocardial ischemia.

Clinical trial registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03218007.

Keywords: adenosine receptor; coronary artery disease; diagnosis.

© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Figures

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.