Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs
Carolyn J. Foster, … , Sergio A. Lira, Madhu S. Chintala
Carolyn J. Foster, … , Sergio A. Lira, Madhu S. Chintala
Published June 15, 2001
Citation Information: J Clin Invest. 2001;107(12):1591-1598. https://doi.org/10.1172/JCI12242.
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Article

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs

Abstract

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein–coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.

Authors

Carolyn J. Foster, Dina M. Prosser, Jacqueline M. Agans, Ying Zhai, Michelle D. Smith, Jean E. Lachowicz, Fang L. Zhang, Eric Gustafson, Frederick J. Monsma Jr., Maria T. Wiekowski, Susan J. Abbondanzo, Donald N. Cook, Marvin L. Bayne, Sergio A. Lira, Madhu S. Chintala

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In vitro characterization of washed platelets prepared from wild-type an...
In vitro characterization of washed platelets prepared from wild-type and SP1999-null mice. (a) ADP-induced inhibition of adenylyl cyclase. Wild-type platelets, filled bars; SP1999-null platelets, open bars. PGE1-induced cAMP production is inhibited by ADP in a concentration-dependent manner in platelets from wild-type, but not SP1999-null, mice. In wild-type platelets, inhibition of cAMP production by 10 μM ADP is blocked by the selective P2Y12 antagonist, 2-MeS-AMP (or 2-MeS; 100 μM). Epinephrine (EPI) at 1 or 10 μM inhibits cAMP production in platelets from both wild-type and SP1999-null mice. Results are mean plus or minus SD of triplicate determinations. Maximum cAMP production was 250 pmol/108 platelets. (b) Representative tracings of ADP-induced calcium mobilization in platelets from wild-type and SP1999-null mice. Platelets from two mice were pooled for each measurement. Intracellular calcium mobilization was initiated by addition of 50 μM ADP at the time indicated on the graph. (c) [3H]-2MeS-ADP binding to washed platelets. Wild-type platelets, filled bars; SP1999-null platelets, open bars. Platelets from wild-type mice, but not SP1999-null mice, bound [3H]-2MeS-ADP specifically, and the selective P2Y12 antagonist, 2-MeS-AMP (100 μM), but not the selective P2Y1 antagonist, A3P5P (100 μM), displaced this binding. Bars represent the average plus or minus SD of triplicate determinations.