Extracellular Ca(2+) modulates ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation.

P>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 A(2) 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 mu mol/l)-evoked platelet aggregation from a transient to a sustained response in both platelet-rich plasma and washed suspensions. Blocking thromboxane A(2) 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 A(2) generation was enhanced at low [Ca2+](o), however this was independent of ectonucleotidase activity(.) P2Y receptor antagonists cangrelor and MRS2179 demonstrated the necessity of P2Y(12) receptors for sustained ADP-evoked aggregation, with a minor role for P2Y(1). 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.