title: Extracellular Ca(2+) 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 Ca(2+) [(Ca(2+) )(o) ]. Lowering [Ca(2+) ](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 [Ca(2+) ](o) -dependence of platelet activation. Reducing [Ca(2+) ](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 A(2) production with aspirin had no effect on this [Ca(2+) ](o) -dependence. Prevention of ADP degradation abolished the differences between low and physiological [Ca(2+) ](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 [Ca(2+) ](o) . As reported previously, thromboxane A(2) generation was enhanced at low [Ca(2+) ](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, Ca(2+) -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.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Jones
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Evans
authors.forename: Martyn P
authors.initials: MP
authors.lastname: Mahaut-Smith
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology*; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000008:administration & dosage / Q000494:pharmacology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects*; D058925:Receptors, Purinergic P2Y12 / Q000502:physiology*; D013928:Thromboxane A2 / Q000096:biosynthesis
chemical_list: C548576:P2RY12 protein, human; D058925:Receptors, Purinergic P2Y12; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D000241:Adenosine; D002118:Calcium