Effect of clopidogrel administration to healthy volunteers on platelet phosphorylation events triggered by ADP.
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 aggregation, 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.


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 pathway stimulation triggered by the P2Y1 receptor. P47 is strongly affected by the A3P5P, and this appears to be highly dependent on P2Y12. However, P20 phosphorylation 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 modulate 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 aggregation, 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.
Contreres
Jean O
JO
INSERM U348, IFR Circulation Lariboisière, Hôpital Lariboisière, Paris, France.
Dupuy
Evelyne
E
Job
Bernard
B
Habib
Aïda
A
Bryckaert
Margke
M
Rosa
Jean P
JP
Simoneau
Guy
G
Herbert
Jean M
JM
Savi
Pierre
P
Levy-Toledano
Sylviane
S
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000293:Adolescent; D000328:Adult; D001596:Benzylamines; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D000077144:Clopidogrel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008297:Male; D008565:Membrane Proteins; D010766:Phosphorylation / Q000187:drug effects; D010880:Piperidines; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors; D018048:Receptors, Purinergic P2 / Q000502:physiology; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013844:Thiazoles; D013873:Thionucleotides / Q000494:pharmacology*; D013912:Threonine / Q000378:metabolism; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D001596:Benzylamines; D008565:Membrane Proteins; C546382:P2RY1 protein, human; C548576:P2RY12 protein, human; D010880:Piperidines; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; C106774:SR 121566; D013844:Thiazoles; D013873:Thionucleotides; D013912:Threonine; C002480:methylthio-ADP; D014443:Tyrosine; D012694:Serine; D000244:Adenosine Diphosphate; D000077144:Clopidogrel; C010840:adenosine 3'-phosphate-5'-phosphate; D013988:Ticlopidine