The platelet P2Y(12) receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [(3)H]PSB-0413.
Various radioligands have been used to characterize and quantify the platelet P2Y(12) receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y(1) and P2Y(12). We used the [(3)H]PSB-0413 selective P2Y(12) receptor antagonist radioligand to reevaluate the number of P2Y(12) receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [(3)H]PSB-0413 bound to 425 ± 50 sites/platelet (K                 (D) = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K                 (D) = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y(12), with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y(1) ligand MRS2179 and the P2X(1) ligand α,β-Met-ATP did not displace [(3)H]PSB-0413 binding. Patients with severe P2Y(12) deficiency displayed virtually no binding of [(3)H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y(12) receptor had normal binding. Studies in mice showed that: (1) [(3)H]PSB-0413 bound to 634 ± 87 sites/platelet (K                 (D) = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K                 (D) = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [(3)H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y(12) receptors, to identify patients with P2Y(12) deficiencies or quantify the effect of P2Y(12) targeting drugs.
UMR_S949 INSERM-Université de Strasbourg, EFS-Alsace, 10 rue Spielmann, 67065, Strasbourg, France.
Philippe
P
Ohlmann
Anna
A
Lecchi
Ali
A
El-Tayeb
Christa E
CE
Müller
Marco
M
Cattaneo
Christian
C
Gachet
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000097:blood / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects; D000077144:Clopidogrel; D003627:Data Interpretation, Statistical; D006801:Humans; D051379:Mice; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D058920:Purinergic P2X Receptor Antagonists; D011869:Radioligand Assay; D019275:Radiopharmaceuticals; D058925:Receptors, Purinergic P2Y12 / Q000097:blood* / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013872:Thionucleosides / Q000097:blood / Q000378:metabolism; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000494:pharmacology
C117445:AR C67085MX; C548576:P2RY12 protein, human; C511278:PSB-0413; D010975:Platelet Aggregation Inhibitors; D058920:Purinergic P2X Receptor Antagonists; D019275:Radiopharmaceuticals; D058925:Receptors, Purinergic P2Y12; D013439:Sulfhydryl Reagents; D013872:Thionucleosides; D000249:Adenosine Monophosphate; D000077144:Clopidogrel; D013988:Ticlopidine