title: A clopidogrel-insensitive inducible pool of P2Y12 receptors contributes to thrombus formation: inhibition by elinogrel, a direct-acting, reversible P2Y12 antagonist.
abstract: It is known that hepatic metabolism limits the antiaggregatory activity of clopidogrel and, as a consequence, its clinical benefits. In this study, we investigated whether other factors exist that could account for clopidogrel's suboptimal antithrombotic activity. Using an in vivo murine FeCl(3) thrombosis model coupled with intravital microscopy, we found that at equivalent, maximal levels of inhibition of ADP-induced platelet aggregation, clopidogrel (50 mg/kg p.o.) failed to reproduce the phenotype associated with P2Y(12) deficiency. However, elinogrel (60 mg/kg p.o.), a direct-acting reversible P2Y(12) antagonist, achieved maximal levels of inhibition in vivo, and its administration (1 mg/kg i.v.) abolished residual thrombosis associated with clopidogrel dosing. Because elinogrel is constantly present in the plasma, whereas the active metabolite of clopidogrel exists for ∼2 h, we evaluated whether an intracellular pool of P2Y(12) exists that would be inaccessible to clopidogrel and contribute to its limited antithrombotic activity. Using saturation [(3)H]2-(methylthio)ADP ([(3)H]2MeSADP) binding studies, we first demonstrated that platelet stimulation with thrombin and convulxin (mouse) and thrombin receptor activating peptide (TRAP) (human) significantly increased surface expression of P2Y(12) relative to that of resting platelets. We next found that clopidogrel dose-dependently inhibited ADP-induced aggregation, signaling (cAMP), and surface P2Y(12) on resting mouse platelets, achieving complete inhibition at the highest dose (50 mg/kg), but failed to block this inducible pool. Thus, an inducible pool of P2Y(12) exists on platelets that can be exposed upon platelet activation by strong agonists. This inducible pool is not blocked completely by clopidogrel, contributes to thrombosis in vivo, and can be blocked by elinogrel.
authors: Portola Pharmaceuticals Inc, 270 East Grand Avenue, South San Francisco, California 94080, USA.
authors: Helena
authors: H
authors: Haberstock-Debic
authors: Patrick
authors: P
authors: Andre
authors: Scott
authors: S
authors: Mills
authors: David R
authors: DR
authors: Phillips
authors: Pamela B
authors: PB
authors: Conley
mesh_terms: D000818:Animals; D000077144:Clopidogrel; D000242:Cyclic AMP / Q000097:blood; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019007:P-Selectin / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D058912:Purinergic P2Y Receptor Agonists / Q000494:pharmacology; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology*; D052999:Quinazolinones / Q000494:pharmacology*; D011869:Radioligand Assay; D058925:Receptors, Purinergic P2Y12 / Q000235:genetics / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology*; D013927:Thrombosis / Q000139:chemically induced / Q000517:prevention & control*; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D005343:Fibrinolytic Agents; D019007:P-Selectin; D010975:Platelet Aggregation Inhibitors; D058912:Purinergic P2Y Receptor Agonists; D058921:Purinergic P2Y Receptor Antagonists; D052999:Quinazolinones; D058925:Receptors, Purinergic P2Y12; D013449:Sulfonamides; C549473:elinogrel; D000077144:Clopidogrel; D000242:Cyclic AMP; D013988:Ticlopidine