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

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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.