Epinephrine restores platelet functions inhibited by ticagrelor: A mechanistic approach

Comparative In Vitro Study of Various α2-Adrenoreceptor Agonist Drugs for Ticagrelor Reversal

“…Kafka et al demonstrated that clonidine was as potent as epinephrine and norepinephrine in binding to the receptor but was only 1/10 as potent in inhibiting PGE1-stimulated cAMP production [14]. Second, we previously showed that restoration of platelet aggregation by epinephrine is due to cAMP pathway inhibition and PI3K pathway activation, but simultaneously requires ADP-induced P2Y 1 -receptor-related signaling and Ca 2+ mobilization [11]. We cannot exclude that dexmedetomidine and clonidine exert only partial activation of the PI3K pathway, thereby limiting subsequent signaling involved in sustained activation, granule secretion, and stable thrombus formation.…”

“…Experiments were performed on washed platelets in order to investigate the direct effects of α 2 -adrenoreceptor agonists on platelets, independently of the plasma proteins, and the anticoagulant used for blood sampling. The washed platelet suspension was prepared as previously described [11]: briefly, blood samples collected in ACD tubes were mixed with wash buffer (citric acid 36 mM, glucose 5 mM, KCl 5 mM, CaCl 2 2 mM, MgCl 2 1 mM, and NaCl 103 mM; pH = 6.5) containing 0.2 µM prostaglandin E1 (PGE1, Sigma-Aldrich, St. Louis, MO, USA) and 0.03 IU/mL apyrase (Agro-Bio, La Ferté-Saint-Aubin, France) to avoid platelet activation during preparation. Platelet-rich plasma was obtained after the first spin (216 g for 11 min at room temperature).…”

“…Ticagrelor was extracted from tablets (Brilique ® 90 mg, AstraZeneca, Mölndal, Sweden) as previously described [11]. Briefly, tablets were dispersed in 10 mM HCl and the water-soluble excipient extracted by centrifugation (5 min, 5000× g, 20 • C).…”

“…Washed platelets were incubated with ticagrelor for 10 min at 37 • C prior to use. A final concentration of 100 nM ticagrelor was selected from a dose-ranging study since it inhibited at least 80% of platelet aggregation [11]. ADP (Roche Molecular Biochemicals, Meylan, France) diluted in suspension buffer was used at either 5 µM final concentration, selected to induce less than 50% platelet aggregation in all samples, in order to capture potential α 2 -adrenoreceptor agonist synergism, or 10 µM final concentration, to maximize potential restoration in the presence of inhibitor.…”

“…Epinephrine acts through stimulation of α 2A -adrenoreceptors coupled with G z -protein on the platelet membrane surface. The α subunit of G z -protein binds to adenylate cyclase and inhibits the synthesis of the second messenger cyclic adenosine monophosphate (cAMP), which plays a central role in platelet inhibition, while the dissociated βγ subunit activates phosphoinositide 3-kinase (PI3K) pathway [10,11]. Therefore, α 2A -adrenoreceptor stimulation participates in platelet activation.…”