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.
Br J Pharmacol. 2010 Nov;161(5):1150-60. doi: 10.1111/j.1476-5381.2010.00951.x.
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.
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.
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.
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.
噻氯匹定和氯吡格雷在肝脏转化为其活性形式后，通过对 P2Y₁₂ 受体的不可逆抑制发挥抗血小板作用。核苷酸（如 ADP，血小板 P2Y₁ 和 P2Y₁₂ 受体的生理激动剂）的浓度受血管外核苷酸酶的调节，主要是核苷三磷酸二磷酸水解酶（NTPDase）1 和外核苷酸 5′-磷酸二酯酶。在这里，我们评估这些前药对血管外核苷酸酶活性以及这些酶对调节血小板聚集的天然功能的影响。
通过 HPLC 和 P(i)测定法进行核苷酸酶测定，使用人脐静脉内皮细胞（HUVEC）和转染 COS-7 细胞的蛋白提取物作为酶的来源。使用富含血小板的人血浆测定血小板聚集。
每种前药均抑制内皮细胞外核苷酸酶活性，并降低其在体外阻断血小板聚集的能力。在治疗浓度下，噻氯匹定（60 μM）和氯吡格雷（20 μM）抑制 HUVEC 中 ADP 水解约 80%，并使 AMP 水解减少三分之一。因此，这些化合物对重组人 NTPDase1 表现出混合抑制作用，表观 K(i)（K(i,app)）为 10 μM（氯吡格雷）和 14 μM（噻氯匹定）。重组大鼠外切 5′-核苷酸酶，但不是其人类同源物，被噻氯匹定抑制，K(i,app)为 4.5 mM。
这些前药通过体外抑制血管 NTPDase1 促进血小板聚集。应进一步研究是否在体内也会发生这种作用，特别是在开始治疗时，在肝脏产生足够水平的活性代谢物之前。
