Extracellular Ca²⁺ modulates ADP‐evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation

Abstract

ADP is considered a weak platelet agonist due to the limited aggregation responses it induces in vitro at physiological concentrations of extracellular Ca2+ [(Ca2+)o]. Lowering [Ca2+]o paradoxically enhances ADP-evoked aggregation, an effect that has been attributed to enhanced thromboxane A2 production. This study examined the role of ectonucleotidases in the [Ca2+]o-dependence of platelet activation. Reducing [Ca2+]o from millimolar to micromolar levels converted ADP (10 μmol/l)-evoked platelet aggregation from a transient to a sustained response in both platelet-rich plasma and washed suspensions. Blocking thromboxane A2 production with aspirin had no effect on this [Ca2+]o-dependence. Prevention of ADP degradation abolished the differences between low and physiological [Ca2+]o resulting in a robust and sustained aggregation in both conditions. Measurements of extracellular ADP revealed reduced degradation in both plasma and apyrase-containing saline at micromolar compared to millimolar [Ca2+]o. As reported previously, thromboxane A2 generation was enhanced at low [Ca2+]o, however this was independent of ectonucleotidase activity. P2Y receptor antagonists cangrelor and MRS2179 demonstrated the necessity of P2Y12 receptors for sustained ADP-evoked aggregation, with a minor role for P2Y1. In conclusion, Ca2+-dependent ectonucleotidase activity is a major factor determining the extent of platelet aggregation to ADP and must be controlled for in studies of P2Y receptor activation.

P2 receptors and platelet function

“…Addition of ADP to washed platelets results in shape change, reversible aggregation at physiological concentrations of calcium (2 mM) and finally desensitization [10,11]. Transduction of the ADP signal involves a transient rise in free cytoplasmic calcium, due to mobilization of internal stores, secondary store-mediated influx and a concomitant inhibition of adenylyl cyclase activity [12].…”

Pharmacokinetic and Pharmacodynamic Effects of Elinogrel

“…Elinogrel is the only selective, competitive, and reversible P2Y 12 inhibitor available in both IV and oral formulations. 8 It is a direct-acting P2Y 12 receptor inhibitor that does not require metabolic activation and thus has low potential for drug-drug interactions or genetic modulation.…”

“…8 It is a direct-acting P2Y 12 receptor inhibitor that does not require metabolic activation and thus has low potential for drug-drug interactions or genetic modulation. 8,9 Recently, the dose-ranging Intravenous and Oral Administration of Elinogrel to Evaluate Tolerability and Efficacy in Nonurgent PCI Patients (INNOVATE-PCI) phase 2b trial conducted in patients undergoing nonurgent PCI showed that elinogrel was not associated with increased major or minor bleeding, defined by Thrombolysis In Myocardial Infarction criteria, although there was an increase in bleeding requiring medical attention, primarily at the PCI access site.…”

“…This artificially low calcium concentration results in inhibition of endogenous ecto-ADPase activity in plasma, which normally degrades ADP in the cuvette that is released during the aggregation reaction. 12 Inhibition of ecto-ADPase activity results in artificially high ADP concentrations in TSC versus other anticoagulants in which physiological calcium concentrations are maintained (ie, C921-78). Because elinogrel is a competitive and reversible P2Y 12 antagonist, and because it is thus sensitive to ADP concentration in the cuvette, use of an anticoagulant such as TSC will underestimate the potency of elinogrel, relative to clopidogrel, the potency of which is minimally affected by ADP concentrations, attributable to its irreversible mechanism of action.…”

“…effects (eg, prasugrel and ticagrelor) and have been associated with a greater reduction in atherothrombotic recurrences, albeit at the expense of an increase in spontaneous bleeding, compared with clopidogrel, which is the most broadly utilized P2Y 12 inhibitor. 6,7 However, all these agents are administered orally and are not available for intravenous (IV) use, a route of administration that remains desirable in the acute coronary syndrome/PCI setting because of its immediate and more predictable pharmacokinetic (PK) and pharmacodynamic (PD) effects.…”