Inhibition of vascular ectonucleotidase activities by the pro-drugs ticlopidine and clopidogrel favours platelet aggregation.
OBJECTIVE
After conversion to their active forms by the liver, ticlopidine and clopidogrel exert antiplatelet effects through irreversible inhibition of the P2Y₁₂ receptor. Concentrations of nucleotides such as ADP, the physiological agonist at platelet P2Y₁ and P2Y₁₂ receptors, are regulated by vascular ectonucleotidases, mainly nucleoside triphosphate diphosphohydrolase (NTPDase)1 and ecto-5'-nucleotidase. Here we evaluate the effect of these pro-drugs on vascular ectonucleotidase activity and on the natural function of these enzymes in regulating platelet aggregation.


METHODS
Nucleotidase assays were performed by HPLC and by P(i) determination, using human umbilical vein endothelial cells (HUVEC) and protein extracts from transfected COS-7 cells as sources of enzymes. Platelet aggregation was assayed using human platelet-rich plasma.


RESULTS
Each pro-drug inhibited endothelial ectonucleotidase activities and decreased their ability to block platelet aggregation in vitro. At their therapeutic concentrations, ticlopidine (60 µM) and clopidogrel (20 µM) inhibited ADP hydrolysis by HUVEC by about 80%, and AMP hydrolysis by one-third. Accordingly, these compounds showed a mixed-type inhibition of recombinant human NTPDase1 with an apparent K(i) (K(i,app) ) of 10 µM (clopidogrel) and 14 µM (ticlopidine). Recombinant rat ecto-5'-nucleotidase, but not its human orthologue, was inhibited by ticlopidine with a K(i,app) of 4.5 mM.


CONCLUSIONS
These pro-drugs facilitated platelet aggregation via the inhibition of vascular NTPDase1 in vitro. Further studies should be performed to assess whether this effect also occurs in vivo, especially at the beginning of treatment, before sufficient levels of active metabolites are produced by the liver.
Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec (Pavillon CHUL) and Départament de Microbiologie Infectiologie, Faculté de Médecine, Université Laval, 2705 Boulevard Laurier, Québec, QC, Canada.
Joanna
J
Lecka
Manjit Singh
MS
Rana
Jean
J
Sévigny
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD; D001081:Apyrase / Q000037:antagonists & inhibitors; D019556:COS Cells; D002522:Chlorocebus aethiops; D000077144:Clopidogrel; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D011355:Prodrugs; D051381:Rats; D013045:Species Specificity; D013988:Ticlopidine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D015703:Antigens, CD; D010975:Platelet Aggregation Inhibitors; D011355:Prodrugs; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000077144:Clopidogrel; D015720:5'-Nucleotidase; D001081:Apyrase; C067869:CD39 antigen; D013988:Ticlopidine