The active metabolite of prasugrel effectively blocks the platelet P2Y12 receptor and inhibits procoagulant and pro-inflammatory platelet responses

The aim of these studies was to investigate the extent of platelet P2Y₁₂ receptor inhibition by the thienopyridine active metabolite of prasugrel, R-138727. Blood was taken from healthy volunteers and pre-incubated with R-138727 or cangrelor (AR-C66931MX). Platelet aggregation was assessed in platelet rich plasma (PRP) and whole blood (WB). Vasodilator stimulated phosphoprotein (VASP) phosphorylation, platelet procoagulant activity (annexin V binding and microparticle formation) and calcium mobilisation were measured by flow cytometry. Platelet-leukocyte co-aggregate formation and sCD40L release, both pro-inflammatory responses of platelets, were measured by flow cytometry and ELISA, respectively. P2Y₁₂ receptor antagonism was determined using a radioligand binding assay (³³P 2-MeSADP) in resting and stimulated platelets and the effects of clopidogrel administration were also assessed. R-138727 yielded concentration-dependent inhibition of platelet aggregation, VASP phosphorylation inhibition, procoagulant activity and pro-inflammatory responses. In the presence of R-138727 or cangrelor there was increased calcium reuptake following agonist stimulation. R-138727 30 µmol/L and cangrelor 1 µmol/L completely inhibited ³³P 2-MeSADP binding, compared to partial inhibition following clopidogrel administration. Platelet activation and granule secretion did not expose an additional pool of P2Y₁₂ receptors. Prasugrel's active metabolite effectively blocks the P2Y₁₂ receptor with the highest concentrations tested yielding complete inhibition of P2Y₁₂-mediated amplification of several important platelet responses.     

Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel.

Clopidogrel is an effective new antiplatelet agent useful for the treatment of ischemic cerebrovascular, cardiac, and peripheral arterial disease. However, the mechanism of clopidogrel action is not well understood, although it is known to inhibit ADP-evoked platelet aggregation. In the current study, the effect of clopidogrel on recently identified human platelet ADP receptors and their signaling pathways was investigated by using platelets from clopidogrel-treated subjects, 6 healthy volunteers (2 females and 4 males) who received 75 mg of clopidogrel daily for 7 days. Blood was taken and various platelet receptor signaling pathways were analyzed before treatment, after 7 days of medication, and 4 weeks after treatment had ceased. Platelet tests included the analysis of aggregation, rapid calcium influx, calcium mobilization from intracellular stores, adenylyl cyclase, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The data indicate that clopidogrel does not affect those platelet ADP receptors coupled to cation influx (P2X1 ADP receptors) or calcium mobilization (P2Y1 ADP receptors). In contrast, clopidogrel treatment specifically impairs the ADP receptor coupled to G(i)/adenylyl cyclase (P2Y(AC) ADP receptors). Clopidogrel abolishes the inhibitory P2Y(AC) receptor-mediated ADP effects on prostaglandin E(1)-stimulated, cAMP-dependent phosphorylation of VASP without affecting epinephrine, thrombin, and thromboxane signaling. VASP phosphorylation is known to be closely correlated with the inhibition of platelet and fibrinogen receptor (glycoprotein IIb/IIIa) activation. Therefore, inhibition of the platelet P2Y(AC) ADP receptor and its intracellular signaling, including decreased VASP phosphorylation, is suggested as a molecular mechanism of clopidogrel action.     

Glycoprotein IIb/IIIa and P2Y12 receptor antagonists yield additive inhibition of platelet aggregation, granule secretion, soluble CD40L release and procoagulant responses

Glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists, including abciximab and tirofiban, are administered concurrently with clopidogrel, a P2Y12 antagonist, and aspirin in some patients undergoing percutaneous coronary intervention. We studied the effects of, and interactions between, abciximab, tirofiban, aspirin and the P2Y12 antagonist cangrelor on platelet aggregation, alpha and dense granule secretion and procoagulant responses in vitro. Blood was obtained from healthy volunteers. Platelet aggregation, dense granule secretion, alpha granule secretion (PAI-1 and soluble CD40 ligand levels) and procoagulant responses (annexin-V and microparticle formation) were assessed using collagen and thrombin receptor activating peptide (TRAP) as agonists. All the antagonists used singularly inhibited collagen-induced responses. Combinations of abciximab or tirofiban with aspirin and/or cangrelor gave additive inhibition with the greatest effect seen when abciximab or tirofiban was combined with both aspirin and cangrelor. Cangrelor inhibited TRAP-induced responses and, again, there was additive inhibition of these parameters when abciximab or tirofiban were combined with cangrelor. The GPIIb/IIIa receptor plays an important role in amplification of platelet activation such that there are important interactions between GPIIb/IIIa antagonists and inhibitors of both P2Y12 receptor activation and, to a lesser extent, thromboxane A2 generation. These interactions are likely to have important influences on the safety and efficacy of combination anti-platelet therapies.     

Modulation of platelet and leucocyte function by a Chinese herbal formulation as compared with conventional antiplatelet agents

Platelet and leucocyte activity are important in the acute development of thrombosis and in the pathogenesis of ischaemic vascular disease. Dan Shen Di Wan (DS, Cardiotonic Pill or Dantonic(R) Pill) is one of the most commonly used Chinese herbal formulations for treating patients with atherosclerotic disease in China and several Asian countries. We studied the effect of DS on platelet and leucocyte function and compared the effects with conventional antiplatelet agents, cangrelor (ADP P2Y(12) receptor antagonist) and aspirin (acetyl salicylic acid, ASA). Measurements were made by platelet aggregation (%) and activation (CD62P %), platelet-monocyte conjugate formation (P/M, CD42a median fluorescence, mf), platelet-neutrophil conjugate formation (P/N, mf), and leucocyte activation (CD11b median fluorescence on monocytes and neutrophils, mf) in response to 3.3 micromol/L adenosine diphosphate (ADP), 1.0 micromol/L platelet activating factor (PAF), 5.0 micromol/L adrenaline and 0.5 microg/mL collagen. We also evaluated the effect of its main component, water soluble extract of salvia miltiorrhiza (SME) on intracellular calcium mobilization in platelets triggered by 10 micromol/L ADP, 10 micromol/L PAF, 2 microg/mL collagen and 15 micromol/L thrombin receptor activating peptide (TRAP). Overall DS showed inhibition of platelet aggregation, platelet activation, platelet-leucocyte conjugate formation and leucocyte activation in response to all the agonists apart from adrenaline (all p < 0.01). DS showed inhibition of platelet aggregation and leucocyte activation equivalent to cangrelor 100 nmol/L and ASA 100 micromol/L. SME dose-dependently inhibited intracellular calcium mobilization in platelets following stimulation with all the platelet agonists with maximum effective at 0.36 mg/mL (all p < 0.01). When used at 0.18 mg/mL its inhibitory effect was equivalent to cangrelor and ASA. We conclude that DS is a potential inhibitor of both platelet and leucocyte activation.     

Evaluation of the INNOVANCE PFA P2Y test cartridge: sensitivity to P2Y(12) blockade and influence of anticoagulant

Monitoring of platelet ADP receptor P2Y(12) inhibition may be performed by a variety of platelet function assays. Given the lack of sensitivity of the existing PFA-100? cartridge formulations to detect P2Y(12) inhibition, a new cartridge for the PFA-100 (INNOVANCE? PFA P2Y) has recently been developed. The performance of the new PFA-100 test cartridge was compared with standard collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges, light transmission aggregometry, vasodilator-stimulated phosphoprotein, the VerifyNow? P2Y(12) assay and multiple electrode aggregometry. In this study, 20 normal blood samples anticoagulated with either citrate or hirudin were spiked with two different clinically relevant concentrations (1 and 10?μM final concentration) of the prasugrel active metabolite (R-138727, Lilly/Daiichi Sankyo) for 30?min at 37°C. Comparison of the platelet function tests demonstrated that all tests (except CADP and CEPI) were substantially inhibited by 10?μM R-138727. Intermediate results were typically obtained with 1?μM R-138727 in citrated blood. However, both MEA ADP and ADPHS tests were highly sensitive to 1?μM R-138727 in hirudin anticoagulated blood. Further comparison of citrate or hirudin blood samples (N?=?5) revealed that all platelet tests (except CEPI) became more sensitive to 1?μM R-138727 in hirudinized blood. The INNOVANCE PFA P2Y cartridge proved to be sensitive to P2Y(12) inhibition and was comparable to other currently available platelet function tests. The sensitivity of all platelet function tests for detecting in?vitro inhibition of P2Y(12) is markedly different depending on the anticoagulant used.     

Pharmacodynamic effects of cangrelor and clopidogrel: the platelet function substudy from the cangrelor versus standard therapy to achieve optimal management of platelet inhibition (CHAMPION) trials

Cangrelor is an intravenous antagonist of the P2Y(12) receptor characterized by rapid, potent, predictable, and reversible platelet inhibition. However, cangrelor was not superior to clopidogrel in reducing the incidence of ischemic events in the cangrelor versus standard therapy to achieve optimal management of platelet inhibition (CHAMPION) trials. A prospectively designed platelet function substudy was performed in a selected cohort of patients to provide insight into the pharmacodynamic effects of cangrelor, particularly in regard to whether cangrelor therapy may interfere with the inhibitory effects of clopidogrel. This pre-defined substudy was conducted in a subset of patients from the CHAMPION-PCI trial (n = 230) comparing cangrelor with 600 mg of clopidogrel administered before percutaneous coronary intervention (PCI) and from the CHAMPION-PLATFORM trial (n = 4) comparing cangrelor at the time of PCI and 600 mg clopidogrel given after the PCI. Pharmacodynamic measures included P2Y12 reaction units (PRU) assessed by VerifyNow P2Y12 testing (primary endpoint marker), platelet aggregation by light transmittance aggregometry following 5 and 20 μmol/L adenosine diphosphate stimuli, and markers of platelet activation determined by flow cytometry. The primary endpoint was the percentage of patients who achieved <20 % change in PRU between baseline and >10 h after PCI. The main trial was stopped early limiting enrollment in the platelet substudy. A total of 167 patients had valid pharmacodynamic assessments for the primary endpoint. The percent of individuals achieving <20 % change in PRU between baseline and >10 h after PCI was higher with cangrelor + clopidogrel (32/84, 38.1 %) compared with placebo + clopidogrel (21/83, 25.3 %), but this was not statistically significant (difference:12.79 %, 95 % CI: -1.18 %, 26.77 %;p = 0.076). All pharmacodynamic markers as well as the prevalence of patients with high on-treatment platelet reactivity were significantly lower in patients treated with cangrelor. A rapid platelet inhibitory effect was achieved during cangrelor infusion and a rapid offset of action after treatment discontinuation. This CHAMPION platelet function substudy represents the largest pharmacodynamic experience with cangrelor, demonstrating its potent P2Y(12) receptor inhibitory effects, and rapid onset/offset of action. Although there was no significant pharmacodynamic interaction when transitioning to clopidogrel therapy, further studies are warranted given that enrollment in this study was limited due to premature interruption of the main trial.     

Modification of the VerifyNow® P2Y12 test BASE channel to accommodate high levels of P2Y(12) antagonism

The VerifyNow? P2Y12 (VN-P2Y12) test reports thienopyridine-mediated platelet inhibition relative to a "BASE" channel, potentially eliminating the need for predrug patient assessment, by activating platelets through a P2Y(12)-independent pathway. The original formulation of the BASE channel used a protease activated receptor-1 (PAR-1) peptide as agonist. However, more potent P2Y(12) antagonism required more complete activation of platelet thrombin receptors for the BASE measurement in order to negate any contribution of the P2Y(12) receptor. Accordingly, the current BASE channel formulation consists of both PAR-1 and protease activated receptor-4 (PAR-4) activating peptides to facilitate a higher degree of platelet activation. The aim of this study was to compare the performance of PAR-1 versus PAR-1/PAR-4 activating peptides as the BASE channel formulation using prasugrel's active metabolite, R-138727, in?vitro to achieve high-grade P2Y(12) inhibition. Blood samples from 20 healthy donors were spiked in?vitro with R-138727 at concentrations that include plasma levels achieved following prasugrel administration and were incubated for 30 minutes at 37°C. All samples were run in triplicate using both the PAR-1 and the PAR-1/PAR-4 BASE formulation in the VN-P2Y12 test device. The data confirmed the sensitivity of the original BASE formulation to high-grade P2Y(12) inhibition as reflected in the concentration-dependent decrease in values. Incorporation of PAR-4 activating peptide eliminated the effect of P2Y(12) blockade at all concentrations of R-138727. Thus, the use of PAR-1/PAR-4 in the BASE channel of the VN-P2Y12 cartridge addresses the impact of high grade P2Y(12) blockade and may allow more accurate reporting of "% inhibition" in patients treated with more effective P2Y(12) antagonists.     

Comparison of platelet P2Y(12) ADP receptor-mediated pathway inhibition in triple versus dual antiplatelet therapy as assessed by VASP-phosphorylation in Japanese patients undergoing coronary stenting

Despite wide interindividual variability in response to clopidogrel, platelet P2Y(12) ADP receptor inhibition in Japanese patients has not been fully studied using specific methodology. This study compared platelet P2Y(12) ADP receptor inhibition during treatment with clopidogrel versus clopidogrel plus cilostazol in patients undergoing coronary stenting. Forty-two patients in whom platelet function was measured within 2 months after coronary stenting were enrolled. All patients were treated with aspirin 100 or 200 mg/day, and were divided into a dual therapy group (aspirin plus clopidogrel 75 mg/day; n = 34) and a triple therapy group (aspirin plus clopidogrel 75 mg/day plus cilostazol 200 mg/day; n = 8). Vasodilator-stimulated phosphoprotein (VASP) phosphorylation analysis and 5 and 20 μmol/L-induced maximal platelet aggregation were assessed. No differences were found in baseline characteristics except for a higher incidence of diabetes mellitus (DM) in the triple therapy group. Although there were no differences in platelet aggregation between the 2 groups, VASP index was significantly lower in the triple therapy group than in the dual therapy group (23.1 ± 15.3% versus 51.2 ± 19.9%; P = 0.001). The rate of low responsiveness to clopidogrel, defined by VASP index > 50%, was lower in the triple therapy group than in the dual therapy group (12.5% versus 55.9%; P = 0.047). Similarly, in DM patients the triple therapy group had a lower VASP index compared with the dual therapy group (23.1 ± 15.3% versus 47.0 ± 23.5%; P = 0.015).Clopidogrel plus cilostazol is more effective in inhibiting the platelet P2Y(12) ADP receptor pathway than clopidogrel alone. This may be useful for reducing clopidogrel resistance in Japanese patients.     

Prostacyclin receptor stimulation facilitates detection of human platelet P2Y12 receptor inhibition by the PFA-100® system

The rationale for monitoring platelet inhibition by thienopyridines for the identification of patients at risk for future recurrent arterial thrombosis or ischemic events is intensively discussed, as well as which monitoring systems are appropriate, robust and reliable. Flow cytometric measurement of phosphorylated VASP (vasodilator-stimulated phosphoprotein), expressed as platelet reactivity index (PRI), is presently “the gold standard method” for evaluating P2Y₁₂ receptor inhibition. The PFA-100® system, a commercially available and clinically widely used platelet test system, is based on a different principle, not that of VASP phosphorylation. The aim of the present study was to compare the two methods and evaluate whether the conventional PFA-100® collagen/ADP cartridge could be pharmacologically improved to enable its routine clinical use for detection of platelet P2Y₁₂ receptor inhibition. The effects of increasing concentrations of the competitive P2Y₁₂ receptor antagonist cangrelor (AR-C69931MX) and the time-dependent effects of a single oral loading dose of clopidogrel (600 mg) were analysed with human whole blood. P2Y₁₂ receptor inhibition was measured by the VASP/PRI assay and the PFA-100® collagen/ADP cartridge system, with and without preincubation with the prostacyclin analog iloprost (Ilomedin®). In vitro addition of iloprost (0.5 nM) enabled PFA-100® collagen/ADP cartridge system detection of P2Y₁₂ receptor inhibition in whole blood by cangrelor in vitro or by clopidogrel treatment of volunteers. The addition of a prostacyclin analog facilitates PFA-100® collagen/ADP system detection of P2Y₁₂ receptor inhibition, achieving a sensitivity similar to that of the VASP/PRI reference method. Future studies should now evaluate whether this modified PFA-100® system, like the VASP assay, is a reliable test system for monitoring P2Y₁₂ receptor inhibition under clinical conditions.     

Role of G protein-gated inwardly rectifying potassium channels in P2Y12 receptor-mediated platelet functional responses

The role of the G(i)-coupled platelet P2Y(12) receptor in platelet function has been well established. However, the functional effector or effectors contributing directly to alphaIIbbeta3 activation in human platelets has not been delineated. As the P2Y(12) receptor has been shown to activate G protein-gated, inwardly rectifying potassium (GIRK) channels, we investigated whether GIRK channels mediate any of the functional responses of the platelet P2Y(12) receptor. Western blot analysis revealed that platelets express GIRK1, GIRK2, and GIRK4. In aspirin-treated and washed human platelets, 2 structurally distinct GIRK inhibitors, SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) and U50488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate), inhibited adenosine diphosphate (ADP)-, 2-methylthioADP (2-MeSADP)-, U46619-, and low-dose thrombin-mediated platelet aggregation. However, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin, AYPGKF, or convulxin. Furthermore, the GIRK channel inhibitors reversed SFLLRN-induced platelet aggregation, inhibited the P2Y(12)-mediated potentiation of dense granule secretion and Akt phosphorylation, and did not affect the agonist-induced G(q)-mediated platelet shape change and intracellular calcium mobilization. Unlike AR-C 69931MX, a P2Y(12) receptor-selective antagonist, the GIRK channel blockers did not affect the ADP-induced adenlylyl cyclase inhibition, indicating that they do not directly antagonize the P2Y(12) receptor. We conclude that GIRK channels are important functional effectors of the P2Y(12) receptor in human platelets.     

P2Y12, a new platelet ADP receptor, target of clopidogrel

Clopidogrel is a potent antithrombotic drug that inhibits ADP-induced platelet aggregation. The results of large clinical trials have demonstrated an overall benefit of clopidogrel over aspirin in the prevention of vascular ischemic events (myocardial infarction, stroke, vascular death) in patients with a history of symptomatic atherosclerotic disease. The antiaggregating effect of clopidogrel is attributed to an irreversible inhibition of ADP binding to a purinergic receptor present at the platelet surface. Clopidogrel is not active in vitro and can be considered a precursor of an active metabolite formed in the liver. The chemical structure of this active metabolite and its biological activity have been described recently. Several purinergic receptors have been described on platelets; P2X (1), a calcium channel, and P2Y1 a Gq-coupled seven-transmembrane domain receptor, have been found not to be antagonized by clopidogrel. Another Gi (2)-coupled receptor (named P2Y12) has been recently cloned and stably expressed in CHO cells. These cells displayed a strong affinity for (33)P-2MeS-ADP, a stable analogue of ADP, the binding characteristics of which corresponded in all points to those observed on platelets. The binding of (33)P-2MeS-ADP to these cells was strongly inhibited by the active metabolite of clopidogrel with a potency that was consistent with that observed for this compound on platelets. In these transfected CHO cells, as in platelets, ADP and 2MeS-ADP induced adenylyl cyclase downregulation, an effect that was inhibited by the active metabolite of clopidogrel. These results demonstrate that this receptor corresponds to the previously called "P2t" platelet receptor and show that the active metabolite of clopidogrel binds in a covalent manner to this receptor, thus explaining how it blocks the aggregating effect of ADP on platelets.     

Evaluation of the INNOVANCE PFA P2Y test cartridge: Sensitivity to P2Y12 blockade and influence of anticoagulant

Monitoring of platelet ADP receptor P2Y₁₂ inhibition may be performed by a variety of platelet function assays. Given the lack of sensitivity of the existing PFA-100® cartridge formulations to detect P2Y₁₂ inhibition, a new cartridge for the PFA-100 (INNOVANCE® PFA P2Y) has recently been developed. The performance of the new PFA-100 test cartridge was compared with standard collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges, light transmission aggregometry, vasodilator-stimulated phosphoprotein, the VerifyNow® P2Y₁₂ assay and multiple electrode aggregometry. In this study, 20 normal blood samples anticoagulated with either citrate or hirudin were spiked with two different clinically relevant concentrations (1 and 10?µM final concentration) of the prasugrel active metabolite (R-138727, Lilly/Daiichi Sankyo) for 30?min at 37°C. Comparison of the platelet function tests demonstrated that all tests (except CADP and CEPI) were substantially inhibited by 10?µM R-138727. Intermediate results were typically obtained with 1?µM R-138727 in citrated blood. However, both MEA ADP and ADPHS tests were highly sensitive to 1?µM R-138727 in hirudin anticoagulated blood. Further comparison of citrate or hirudin blood samples (N?=?5) revealed that all platelet tests (except CEPI) became more sensitive to 1?µM R-138727 in hirudinized blood. The INNOVANCE PFA P2Y cartridge proved to be sensitive to P2Y₁₂ inhibition and was comparable to other currently available platelet function tests. The sensitivity of all platelet function tests for detecting in vitro inhibition of P2Y₁₂ is markedly different depending on the anticoagulant used.     

Underlying mechanism and specific prevention of hemolysis-induced platelet activation

Thromboembolic complications significantly impair the outcome of hemolytic disorders. We hypothesized that red cell adenosine diphosphate (ADP) release results in significant platelet activation in hemolysis and that this prothrombotic state can be prevented by inhibition of the ADP P2Y₁₂ receptor. In the current study, we therefore sought to investigate the mechanism and inhibition of hemolysis-induced platelet activation. The expression of activated integrin αIIbß3 was determined by flow cytometry, and platelet aggregation was assessed by multiple electrode platelet aggregometry. We demonstrate platelet activation and increased platelet aggregation by adding hemolytic blood (lysates) to whole blood, similarly to that achieved by the platelet agonist ADP. Enhanced platelet activation and reactivity in the presence of hemolytic blood were significantly abolished by apyrase, which catalyzes ADP degradation, and inhibited by blockade of the platelet ADP P2Y₁₂ receptor with cangrelor. Platelets from patients treated with the ADP P2Y₁₂ receptor antagonist clopidogrel showed a reduced response to lysates compared to platelets from healthy controls without antiplatelet treatment. Further, in vitro blood group ABO incompatibility induced hemolysis and led to increased platelet activation. Finally, “spontaneous” platelet aggregation seen in patients with cold agglutinin disease was completely abolished by cangrelor. In conclusion, hemolysis is associated with increased platelet activation and aggregation due to red cell derived ADP, which can be prevented by ADP receptor blockade.     

Modification of the VerifyNow® P2Y12 test BASE channel to accommodate high levels of P2Y12 antagonism

The VerifyNow® P2Y12 (VN-P2Y12) test reports thienopyridine-mediated platelet inhibition relative to a “BASE” channel, potentially eliminating the need for predrug patient assessment, by activating platelets through a P2Y₁₂-independent pathway. The original formulation of the BASE channel used a protease activated receptor-1 (PAR-1) peptide as agonist. However, more potent P2Y₁₂ antagonism required more complete activation of platelet thrombin receptors for the BASE measurement in order to negate any contribution of the P2Y₁₂ receptor. Accordingly, the current BASE channel formulation consists of both PAR-1 and protease activated receptor-4 (PAR-4) activating peptides to facilitate a higher degree of platelet activation. The aim of this study was to compare the performance of PAR-1 versus PAR-1/PAR-4 activating peptides as the BASE channel formulation using prasugrel's active metabolite, R-138727, in vitro to achieve high-grade P2Y₁₂ inhibition. Blood samples from 20 healthy donors were spiked in vitro with R-138727 at concentrations that include plasma levels achieved following prasugrel administration and were incubated for 30 minutes at 37°C. All samples were run in triplicate using both the PAR-1 and the PAR-1/PAR-4 BASE formulation in the VN-P2Y12 test device. The data confirmed the sensitivity of the original BASE formulation to high-grade P2Y₁₂ inhibition as reflected in the concentration-dependent decrease in values. Incorporation of PAR-4 activating peptide eliminated the effect of P2Y₁₂ blockade at all concentrations of R-138727. Thus, the use of PAR-1/PAR-4 in the BASE channel of the VN-P2Y12 cartridge addresses the impact of high grade P2Y₁₂ blockade and may allow more accurate reporting of “% inhibition” in patients treated with more effective P2Y₁₂ antagonists.     

ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Galphaq

Platelets from Galphaq knockout mice are unable to aggregate in response to physiological agonists like adenosine 5'-diphosphate (ADP), thromboxane A(2), thrombin, or collagen, although shape change still occurs in response to all of these agonists except ADP. ADP-induced platelet aggregation results from simultaneous activation of the purinergic P2Y(1) receptor coupled to calcium mobilization and shape change and of a distinct P2 receptor, P2cyc, coupled through Gi to adenylyl cyclase inhibition, which is responsible for completion and amplification of the response. P2cyc could be the molecular target of the antithrombotic drug clopidogrel and the adenosine triphosphate (ATP) analogs AR-C69931MX, AR-C67085, and AR-C66096. The aim of the present study was to determine whether externally added ADP could still act through the Gi pathway in Galphaq-deficient mouse platelets and thereby amplify the residual responses to agonists such as thrombin or collagen. It was found that (1) ADP and adrenaline still inhibited cyclic AMP accumulation in Galphaq-deficient platelets; (2) both agonists restored collagen- but not thrombin-induced aggregation in these platelets; (3) the effects of ADP were selectively inhibited in vitro by the ATP analog AR-C69931MX and ex vivo by clopidogrel and hence were apparently mediated by the P2cyc receptor; and (4) high concentrations of ADP (100 micromol/L) induced aggregation without shape change in Galphaq-deficient platelets through activation of P2cyc. Since adrenaline was not able to induce platelet aggregation even at high concentrations, we conclude that the effects of ADP mediated by P2cyc are not restricted to the inhibition of adenylyl cyclase through Gi(2).     

Prasugrel achieves greater and faster P2Y12receptor-mediated platelet inhibition than clopidogrel due to more efficient generation of its active metabolite in aspirin-treated patients with coronary artery disease.

AIMS: P2Y(12) receptor antagonism and platelet inhibition by prasugrel vs. clopidogrel were investigated in patients with stable coronary artery disease. METHODS AND RESULTS: One hundred and ten aspirin treated subjects were randomized to double-blind treatment with clopidogrel (n = 55) 600 mg loading dose (LD) and 75 mg maintenance dose (MD) or prasugrel (n = 55) 60 mg LD and 10 mg MD for 28 days. Concentrations of prasugrel and clopidogrel active metabolites were determined. Platelet aggregation to 20 microM adenosine diphosphate, measured by light transmission aggregometry, was reported as maximal platelet aggregation (MPA). P2Y(12) function was assessed by the vasodilator-stimulated phosphoprotein assay and reported as platelet reactivity index (PRI). The same pharmacodynamic measurements were performed after ex vivo addition of clopidogrel's active metabolite. At 2 h post-LD, mean MPA was 31 vs. 55%, and mean PRI 8.3 vs. 55.9% for prasugrel and clopidogrel, respectively (P < 0.001). During MD on day 14 and 28, mean MPA was 42 vs. 54% and mean PRI was 25 vs. 51%, respectively (P < 0.001). Peak level of the active metabolite and P2Y(12) inhibition occurred earlier and was greater with prasugrel (P < 0.001). Mean area under the time-concentration curve (AUC; microM.h) of the respective active metabolite was higher with prasugrel vs. clopidogrel post-LD (1.11 vs. 0.24) and post-MD (0.16 vs. 0.062). Ex vivo addition of clopidogrel's active metabolite further reduced PRI in all patients whose platelets were not already maximally inhibited. CONCLUSION: In aspirin-treated subjects with coronary artery disease, prasugrel 60/10 mg provides faster onset and greater inhibition of P2Y(12) receptor-mediated platelet aggregation than clopidogrel 600/75 mg, because of greater and more efficient generation of the active metabolite.     

PAR-1 genotype influences platelet aggregation and procoagulant responses in patients with coronary artery disease prior to and during clopidogrel therapy

Genetic variations of the protease-activated receptor-1 (PAR-1) have been associated with platelet receptor density and linked to thrombin receptor-activating peptide (TRAP)-induced phenotypes of platelet aggregation and P-selectin expression. We investigated whether the PAR-1 intervening sequence-14 A>T dimorphism influences platelet procoagulant activity. We also determined whether the P2Y12 antagonist clopidogrel could offset any observed functional polymorphism of the PAR-1 receptor by inhibiting P2Y12-mediated amplification of TRAP-induced responses. We studied 54 patients listed for elective percutaneous coronary intervention assessing TRAP-induced platelet aggregation and markers of procoagulant activity. Platelet responses were measured at baseline, 4 h post clopidogrel 300 mg, and 10 and 28 days following clopidogrel 75 mg daily. Each patient was genotyped for the PAR-1 intervening sequence-14 A/T dimorphism. Increased platelet aggregation and procoagulant responses were observed with PAR-1 A allele homozygotes. Clopidogrel significantly inhibited these platelet responses regardless of PAR-1 genotype, but did not offset the hyper-reactivity associated with the A/A homozygotes. We conclude that a common sequence variation within the PAR-1 gene influences TRAP-induced platelet procoagulant activity as well as aggregation. Higher platelet reactivity associated with PAR-1 IVSn-14 A allele homozygotes persists despite clopidogrel therapy. These individuals may be at higher risk of thromboembolic events and may require additional anti-platelet medication.     

The P2 receptors in platelet function

After vessel wall injury, platelets adhere to the exposed subendothelium, are activated, and release mediators such as thromboxane A (2) (TXA (2)) and nucleotides stored at very high concentration in the so-called dense granules. Among other soluble agents, released nucleotides act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled adenosine diphosphate (ADP) receptors, namely the P2Y (1) and P2Y (12) receptor subtypes; the P2X (1) receptor ligand-gated cation channel is activated by adenosine triphosphate (ATP). The P2Y (1) receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, whereas the P2Y (12) receptor is responsible for completion of the aggregation to ADP. This receptor, the molecular target of the antithrombotic drug clopidogrel, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen, or immune complexes. The P2X (1) receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all of the sequential events involved in platelet function and hemostasis. As such, they represent potential targets for antithrombotic drugs.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y₁ receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y₁ receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y₁ antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM ) totally inhibited the [Ca²⁺]_i rise induced by ADP (0.1 μM ) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM ) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM ). A3P5P (1 m M ) reduced the number of [³³P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [³³P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M ). In summary, these results demonstrate that the platelet P2Y₁ receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.