Effect of clopidogrel administration to healthy volunteers on platelet phosphorylation events triggered by ADP Jean O. Contreres, 1 Evelyne Dupuy, 1 Bernard Job, 2 Aı¨da Habib, 1 Mar € gke Bryckaert, 1 Jean P. Rosa, 1 Guy Simoneau, 3 Jean M. Herbert, 4 Pierre Savi 4 and Sylviane Levy-Toledano 1 1 INSERM U.348, IFR Circulation Lariboisie `re, 2 SANOFI-SYNTHELABO, Chilly Mazarin, 3 Unite ´ de Recherche The ´rapeutique-Me ´decine, Ho ˆpital Lariboisie `re, Paris, and 4 SANOFI-SYNTHELABO Recherches, Toulouse, France Received 20 February 2002; accepted for publication 12 September 2002 Summary. The action of clopidogrel on platelet receptors was analysed using platelets obtained from 11 healthy volunteers given 75 mg of clopidogrel daily for 8 d. Samples of blood were taken before treatment and after 8 d of medication. Determination of 2-methylthioadenosine diphosphate trisodium (2MesADP)-induced platelet aggre- gation, serine/threonine and tyrosine phosphorylations were performed in the absence or presence of the P2Y1-receptor-specific antagonist: adenosine 3¢-phosphate 5¢-phosphate (A3P5P) or the strong inhibitor of GPIIb/IIIa activation: SR121566. Major conclusions: 1) Serine and threonine phosphorylations of the myosin light chain (P20) and pleckstrin (P47) do not behave similarly, although they are both recognized as the result of phospholipase C path- way stimulation triggered by the P2Y1 receptor. P47 is strongly affected by the A3P5P, and this appears to be highly dependent on P2Y12. However, P20 phosphoryla- tion occurs in the presence of A3P5P, suggesting that the P2Y12 receptor signal contributes to P20 phosphorylation mediated by a calcium-independent pathway. The results suggest that P2Y1 and P2Y12 receptors interact to modu- late the phosphorylation of P20 and P47. 2) The inside-out signalling dependent on both P2Y12 and P2Y1 is necessary for GPIIb/IIIa activation. 3) Clopidogrel and SR121566 inhibited the increase in tyrosine phosphorylation induced by 2MesADP and concomitantly inhibited platelet aggre- gation, indicating that most of the phosphorylations are GPIIb/IIIa dependent. However, neither clopidogrel nor SR121566 inhibited the first wave of 80 kDa substrate (cortactin) which is involved in the reorganization of the cytoskeleton necessary for shape change and which appeared to be essentially P2Y1 dependent. Keywords: platelets, ADP receptors, serine/threonine phosphorylation, tyrosine phosphorylation, clopidogrel. Adenosine 5¢-diphosphate (ADP) induces human blood platelets to aggregate and change shape, and it has been suggested that these two responses are mediated by more than one subtype of ADP receptor. A number of authors (Daniel et al, 1998; Hechler et al, 1998; Jin et al, 1998a; Savi et al, 1998; Jantzen et al, 1999) have proposed a three- receptor model for human platelets. One receptor is coupled to the inhibition of adenylylcyclase via the heterotrimeric G protein, Gi, and is designated P2Y12. A second receptor is coupled to the heterotrimeric protein Gq and is designated P2Y1. Activation of the P2Y1 receptor results in the activation of phospholipase Cb, production of diacylglycerol and mobilization of cytosolic Ca 2+ in response to inositol phosphate formation, leading to the activation of two kinases: protein kinase C (PKC) and myosin light chain kinase (MLCK) allowing the phosphorylation of pleckstrin (P47) and myosin light chain (P20) respectively. A third receptor is coupled to rapid calcium influx and is designated as the intrinsic calcium channel P2X1 (MacKenzie et al, 1996). Using selective agonists and antagonists of the ADP receptors, it has been demonstrated that ADP-induced shape change is mediated by the P2Y1 receptor (Daniel et al, 1998; Jin & Kunapuli, 1998), and that co-activation of the P2Y12 and P2Y1 receptors is essential for ADP-induced platelet aggregation (Daniel et al, 1998; Jin et al, 1998; Jantzen et al, 1999). Whereas the function of the P2X1 receptor remains to be elucidated, it has been reported recently that the transient elevation in calcium (within 10 min of exposure to the agonist) resulting from the receptor stimulation would be expected to sensitize inositol-1,4,5-trisphosphate (Ins Correspondence: Dr Sylviane Levy-Toledano, INSERM U.348, Ho ˆpital Lariboisie `re, 8 rue Guy Patin, 75475, Paris cedex 10, France. E-mail: sylviane.levy-toledano@lrb.inserm.fr British Journal of Haematology, 2003, 120, 633–642 Ó 2003 Blackwell Publishing Ltd 633