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The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

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Abstract

Various radioligands have been used to characterize and quantify the platelet P2Y12 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 P2Y1 and P2Y12. We used the [3H]PSB-0413 selective P2Y12 receptor antagonist radioligand to reevaluate the number of P2Y12 receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [3H]PSB-0413 bound to 425 ± 50 sites/platelet (K D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [3H]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 P2Y12, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y1 ligand MRS2179 and the P2X1 ligand α,β-Met-ATP did not displace [3H]PSB-0413 binding. Patients with severe P2Y12 deficiency displayed virtually no binding of [3H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y12 receptor had normal binding. Studies in mice showed that: (1) [3H]PSB-0413 bound to 634 ± 87 sites/platelet (K D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [3H]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, [3H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y12 receptors, to identify patients with P2Y12 deficiencies or quantify the effect of P2Y12 targeting drugs.

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Abbreviations

AR-C67085MX:

2-Propylthioadenosine-5′-adenylic acid (1,1-chloro-1-phosphonomethyl-1-phosphonyl)anhydride

AR-C69931MX (cangrelor):

N 6-(2-Methylthioethyl)-2-(3,3,3-trifluoropropylthio)adenosine-5′-adenylic acid (1,1-chloro-1-phosphonomethyl-1-phosphonyl)anhydride

[3H]PSB-0413:

[3H]2-Propylthioadenosine-5′-adenylic acid (1,1-chloro-1-phosphonomethyl-1-phosphonyl)anhydride

PSB-0412 (precursor of [3H]PSB-0413):

2-Propargylthioadenosine-5′-adenylic acid (1,1-chloro-1-phosphonomethyl-1-phosphonyl)anhydride

MRS2179:

N 6-Methyl-2′-deoxyadenosine-3′,5′-bisphosphate

MRS2500:

2-Iodo-N 6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate)

pCMBS:

para-Chloromercuribenzene sulfonic acid

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Authors and Affiliations

  1. UMR_S949 INSERM-Université de Strasbourg, EFS-Alsace, 10 rue Spielmann, 67065, Strasbourg, France

    Philippe Ohlmann & Christian Gachet

  2. Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal Medicine, Foundation IRCCS Ca’Granda Ospedale Maggiore Policlinico and Università degli Studi di Milano, Milan, Italy

    Anna Lecchi

  3. PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany

    Ali El-Tayeb & Christa E. Müller

  4. Unità di Medicina 3 Ospedale San Paolo, Dipartimento di Medcina, Chirurgia e Odontoiatra, Università degli Studi di Milano, Milan, Italy

    Marco Cattaneo

Authors
  1. Philippe Ohlmann
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  2. Anna Lecchi
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  3. Ali El-Tayeb
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  4. Christa E. Müller
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  5. Marco Cattaneo
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  6. Christian Gachet
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Correspondence to Christian Gachet.

Additional information

A. El-Tayeb is on leave from the University of Al-Azhar, Assiut, Egypt.

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Ohlmann, P., Lecchi, A., El-Tayeb, A. et al. The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413. Purinergic Signalling 9, 59–66 (2013). https://doi.org/10.1007/s11302-012-9329-0

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  • DOI: https://doi.org/10.1007/s11302-012-9329-0

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