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.................................................................Identication of the platelet ADP receptor targeted by antithrombotic drugs
Zero K+
ADP ADP ADP
a
b
c
Kir3.1 + Kir3.4
+ Rat clone
+ Human clone
+ 500 pool
Gunther Hollopeter*, Hans-Michael Jantzen, Diana Vincent, Georgia Li, Laura England*, Vanitha Ramakrishnan, Ruey-Bing Yang, Paquita Nurden, Alan Nurden, David Julius* & Pamela B. Conley
* Department of Cellular and Molecular Pharmacology and Program in Neuroscience, University of California, San Francisco, San Francisco, California 94143, USA COR Therapeutics, Inc., South San Francisco, California 94080, USA UMR 5533 CNRS, Hopital Cardiologique, 33605 Pessac, France..............................................................................................................................................
Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIbIIIa and subsequent binding of brinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators1. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes2,3. The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation4,5. The second ADP receptor required for aggregation (variously called P2YADP, P2YAC, P2Ycyc or P2TAC) is
coupled to the inhibition of adenylyl cyclase through Gi. The
molecular identity of the Gi-linked receptor is still elusive, even though it is the target of efcacious antithrombotic agents, such as ticlopidine and clopidogrel68 and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and
provide evidence that a patient with a bleeding disorder10 has a
defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.
To identify the Gi-linked platelet ADP receptor, we engineered Xenopus oocytes to allow the detection of Gi-linked responses through a sensitive electrophysiological assay. This strategy is based on the fact that several Gi-coupled receptors, such as the M2 muscarinic receptor, release Gbg subunits from heterotrimeric G proteins, thereby activating inwardly rectifying K+ channels (Kir3.13.4)11. A complementary DNA library from rat platelets was screened in oocytes expressing Kir3.1 and 3.4, and three positive pools that responded to 10 mM ADP...