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 ϳ2 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) 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.

Modulation and pre-amplification of PAR1 signaling by ADP acting via the P2Y12 receptor during platelet subpopulation formation

“…By using previously determined Δψ* we plotted the analogical dependencies for activation with ADP by the fitting of the P 2 Y 12 receptors number. This number (P 2 Y 12 = 750 number/cell) was in agreement with experimental data [49,50,51]. …”

P2Y12 receptors: structure and function

“…Studies in healthy subjects revealed that the selective P2Y 12 [16]. An inducible pool of P2Y 12 R also exists that can be exposed upon platelet activation by strong agonists [17].…”

Activators, receptors and signal transduction pathways of blood platelets

“…При активации тромбоцита под действием сильного активатора (тромбина или коллагена), количество этих рецепторов на его поверхности увеличивается, т.к. эти рецепторы содержатся в его a-гранулах, секретируемых тромбоцитом при его активации [64,65].…”