Evidence for the existence of two different ADP-binding sites on rat platelets

doi:10.1016/0049-3848(94)90186-4

Thrombosis Research

Volume 76, Issue 2, 15 October 1994, Pages 157-169

https://doi.org/10.1016/0049-3848(94)90186-4 Get rights and content

Abstract

[³H]-2-Methylthio-ADP ( [³H]-2-MeS-ADP ), a stable analogue of ADP bound to one type of specific binding sites on rat platelets ( K_D = 0.77 ± 0.07 nM, Bmax = 160 ± 11 fmol/10⁸ cells ). 2-MeS-ADP and ADP antagonized [³H]-2-MeS-ADP binding, showing respective Ki values of 1.4 ± 0.1 nM and 486 ± 78 nM. Clopidogrel, a potent and specific inhibitor of ADP-induced platelet aggregation partially inhibited ( ∼70% inhibition ) the binding of [³H]-2-MeS-ADP at the same time it abrogated 2-MeS-ADP-and ADP-induced adenylyl cyclase inhibition and aggregation. A population of clopidogrel-resistant [³H]-2-MeS-ADP binding sites was detected on platelets from treated animals. These receptor sites ( K_D = 0.9 ± 0.2 nM, Bmax = 47 ± 5 fmol/10⁸ platelets ) which showed high affinity for both ADP and 2-MeS-ADP (Ki values in the nanomolar range) might be involved in the ADP-induced shape change, a clopidogrel-resistant ADP-induced event. Using clopidogrel which acts via a direct and irreversible inhibition of ADP binding to its adenylyl cyclase-coupled receptor sites on platelets, we were able to discriminate between two types of ADP receptor sites.

The former which was clopidogrel-sensitive represented about 70% of the total [³H]-2-MeS-ADP receptors and was responsible for ADP-induced platelet aggregation and adenylyl cyclase inhibition. The latter which was not affected by clopidogrel might be involved in ADP-induced shape-change.

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ADP and ATP play a crucial role in hemostasis and thrombosis and their receptors are potential targets for antithrombotic drugs. The ATP-gated channel P2X₁ and the two G protein-coupled P2Y₁ and P2Y₁₂ ADP receptors selectively contribute to platelet aggregation. Due to its central role in the formation and stabilization of a thrombus, the P2Y₁₂ receptor is a well established target of antithrombotic drugs like clopidogrel which has proved efficacious in many clinical trials and experimental models of thrombosis. Competitive P2Y₁₂ antagonists have also been shown to be effective in experimental thrombosis as well as in several clinical trials. Studies in P2Y₁ and P2X₁ knock-out mice and experimental thrombosis models using selective P2Y₁ and P2X₁ antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs. Since both P2X₁ and P2Y₁ receptor inhibition result in milder prolongation of the bleeding time as compared to P2Y₁₂ inhibition, the idea is put forward that combination of P2 receptor antagonists could improve efficacy with diminished hemorrhagic risk. However, further studies are required to validate such a point of view.
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P2Y12 is a G‐protein‐coupled seven transmembrane receptor expressed exclusively in the brain, in megakaryocytic cells and in blood platelets [1]. In platelets, it is now clear that P2Y12 is the low‐affinity ADP receptor which was pharmacologically characterized 10 years ago [2]. It binds 2‐MeS‐ADP at nanomolar range and ADP at submicromolar range when expressed in Chinese hamster ovary (CHO) cells (CHO‐P2Y12 cells) and this binding is antagonized by the active metabolite of clopidogrel, a well‐known antiADP antiaggregating drug [3].
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The binding characteristics of ³³P-2MeS-ADP, a stable analogue of ADP, were determined on CHO cells transfected with the human P2Y₁₂ receptor, a novel purinergic receptor. These transfected CHO cells displayed a strong affinity for ³³P-2MeS-ADP, the binding characteristics of which corresponded in all points to those observed on platelets. In particular, this receptor recognised purines with the following order of potency: 2MeS-ADP = 2MeS-ATP > ADP = ATPγS = ATP ⪢ UTP, a binding profile which is similar to that obtained in platelets. The binding of ³³P-2MeS-ADP was antagonised by pCMPS but not by MRS2179 and FSBA, antagonists of P2Y₁ and aggregin, respectively. Moreover, the binding of ³³P-2MeS-ADP to these cells was strongly and irreversibly inhibited by the active metabolite of clopidogrel with a potency which was consistent with that observed for this compound on platelets. Like in platelets, 2MeS-ADP induced adenylyl cyclase down-regulation in these P2Y₁₂ transfected CHO cells, an effect which was absent in the corresponding non-transfected cells. As already shown in platelets, the active metabolite of clopidogrel antagonised 2MeS-ADP-induced inhibition of adenylyl cyclase on transfected cells. Our results confirm that P2Y₁₂ is the previously called “platelet P2t_AC” receptor and show that this receptor is antagonised by the active metabolite of clopidogrel.
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PaperEvidence for the existence of two different ADP-binding sites on rat platelets

Abstract

Blood

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Clin. Chim. Acta

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Blood

Blood

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

ADP receptor : a review, Nouv

Rev. Fr. Hématol.

ADP plays a key role in thrombogenesis in rats

Thrombos. Haemostas.

ADP receptor antagonists and converting enzyme systems reduce platelet deposition onto collagen

Thrombos. Haemostas.

Neuromodulatory roles of purine nucleosides and nucleotides: their receptors ands ligands

Neurotransmissions

2-methylthioadenosine[β32P] diphosphate an agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets

J. Clin. Invest.

Aggregin: a platelet ADP receptor that mediates activation

Faseb J.

Binding of adenosine diphosphate to intact human platelets

J. Physiol.

Identification of a receptor for ADP on blood platelets by photoaffinity labelling

Biochem. J.

Binding of 14C-ADP by thrombasthenic platelet membranes

Haemostasis.

Paper
Evidence for the existence of two different ADP-binding sites on rat platelets

2-methylthioadenosine[β³²P] diphosphate an agonist and radioligand for the receptor that inhibits the accumulation of cyclic AMP in intact blood platelets

Binding of ¹⁴C-ADP by thrombasthenic platelet membranes