Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacyclin

BACKGROUND: Activation of two receptors for adenosine diphosphate (ADP), P2Y(1) and P2Y(12), is necessary for ADP-induced platelet aggregation (PA). It is generally believed that the antithrombotic effects of drugs inhibiting P2Y(12), such as clopidogrel, are uniquely mediated by inhibition of P2Y(12)-dependent PA. However, as P2Y(12) is negatively coupled to adenylyl cyclase (AC), its inhibition may also exert antithrombotic effects through the potentiation of prostacyclin (PGI(2)), which inhibit PA by stimulating AC. OBJECTIVES: To test whether inhibition of P2Y(12) potentiates the antiplatelet effects of PGI(2). METHODS: We measured the effects of PGI(2) (0.01-10 microm) on PA of washed human platelets induced by thrombin (0.5 U mL(-1)) in the presence or absence of ARC69931MX (anti-P2Y(12)) or MRS2500 (anti-P2Y(1)). Results: PGI(2) inhibited PA in the presence of anti-P2Y(12), but not in the presence of anti-P2Y(1) or in the absence of inhibitors. In contrast, dibutyryl-cyclicAMP inhibited PA both in the presence and absence of anti-P2Y(1) or anti-P2Y(12). PGI(2) increased platelet cyclicAMP levels only in the absence of thrombin or in the presence of thrombin plus anti-P2Y(12). CONCLUSIONS: PGI(2) did not inhibit PA induced by thrombin, because its effect on AC was prevented by released ADP interacting with P2Y(12). Anti-P2Y(12) drugs, by rescuing AC activity, potentiate the antiplatelet effect of PGI(2), which may contribute to their antithrombotic effect.     

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation

Summary. Background: P2Y₁₂ plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. Objective: To characterize the receptor biology of the first reversibly binding oral P2Y₁₂ antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. Methods: Ticagrelor displayed apparent non‐competitive or insurmountable antagonism of ADP‐induced aggregation in human washed platelets. This was investigated using competition binding against [³H]ADP, [³³P]2MeS‐ADP and the investigational CPTP compound [¹²⁵I]AZ11931285 at recombinant human P2Y₁₂. Functional receptor inhibition studies were performed using a GTPγS‐binding assay, and further binding studies were performed using membranes prepared from washed human platelets. Results: Radioligand‐binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y₁₂ with K_on and K_off of (1.1 ± 0.2) × 10^?4 nm ^?1 s^?1 and (8.7 ± 1.4) × 10^?4 s^?1, respectively. Ticagrelor does not displace [³H]ADP from the receptor (K_i > 10 μm ) but binds competitively with [³³P]2MeS‐ADP (K_i = 4.3 ± 1.3 nm ) and [¹²⁵I]AZ11931285 (K_i = 0.33 ± 0.04 nm ), and shows apparent non‐competitive inhibition of ADP‐induced signaling but competitive inhibition of 2MeS‐ADP‐induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non‐competitive binding for ADP and ticagrelor. Conclusions: These data indicate that P2Y₁₂ is targeted by ticagrelor via a mechanism that is non‐competitive with ADP, suggesting the existence of an independent receptor‐binding site for CPTPs.     

The reversible P2Y12 antagonist cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to produce irreversible inhibition of platelet function

Summary.  Background:  Agents that act as antagonists at P2Y₁₂ ADP receptors on platelets are in use (clopidogrel), and in development for use (cangrelor and prasugrel), in patients with cardiovascular disease. Cangrelor is a direct-acting reversible antagonist being developed for short-term infusion; clopidogrel and prasugrel are oral prodrugs that provide irreversible inhibition via transient formation of active metabolites. At the cessation of cangrelor infusion, patients are likely to receive clopidogrel or prasugrel as a means of maintaining antiplatelet therapy. Objectives:  To apply an experimental in vitro approach to investigate the possibility that cangrelor influences the ability of the active metabolites of clopidogrel and prasugrel to inhibit ADP-mediated platelet function. Methods:  The effects of cangrelor and the active metabolites of clopidogrel (C-AM) and prasugrel (P-AM) on platelet function were assessed by ADP-induced platelet P-selectin expression in whole blood. The method involved rapid removal of the antagonists by dilution, and measurement of residual platelet inhibition. Results:  Cangrelor, C-AM and P-AM markedly inhibited P-selectin expression. The effect of cangrelor, but not of C-AM and P-AM, was reversible following antagonist removal. Preincubation of blood with cangrelor prior to addition of C-AM or P-AM reduced the ability of metabolites to irreversibly antagonize P2Y₁₂. Irreversible inhibition was maintained when blood was preincubated with metabolites prior to cangrelor. Conclusions:  Cangrelor influences the ability of the active metabolites of clopidogrel or prasugrel to inhibit platelet function irreversibly. Careful consideration should be given to the timing of administration of an oral P2Y₁₂ antagonist following cangrelor infusion.     

Inhibition of localized thrombosis in P2Y1-deficient mice and rodents treated with MRS2179, a P2Y1 receptor antagonist

Summary.  Previous studies in experimental models revealed a role for the P2Y₁ platelet ADP receptor in systemic vascular thromboembolism models. In the present work, we used models of localized arterial and venous thrombosis to assess the role of the P2Y₁ receptor in these processes. Arterial thrombosis was induced in one mesenteric arteriole of a mouse using FeCl₃, while venous thrombosis was studied in a Wessler model adapted to rats. P2Y₁-deficient mice and mice treated with the P2Y₁ antagonist MRS2179 displayed significantly less arterial thrombosis than their respective controls. Combination of P2Y₁ deficiency with P2Y₁₂ inhibition led to a significant additive effect. Venous thrombosis was slightly but significantly inhibited in MRS2179-treated rats. These results demonstrate a role for the P2Y₁ receptor in both arterial and venous thrombosis, further establishing this receptor as a potential target for antithrombotic drugs.     

P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

BACKGROUND: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation. RESULTS: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. CONCLUSIONS: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.     

(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .     

Gi-dependent and -independent mechanisms downstream of the P2Y12 ADP-receptor

Summary.  The P2Y₁₂ ADP receptor is one of the major regulators of platelet activation and the target of antithrombotic thienopyridines (ticlopidine and clopidogrel). It has been recently cloned but the signaling pathways triggered by this receptor are still poorly documented. Here, we show that stimulation of the human P2Y₁₂ receptor stably expressed in Chinese hamster ovary cells activates two major intracellular signaling mechanisms leading either to cell proliferation or to actin cytoskeleton reorganization. Both effects were blocked by the active metabolite of clopidogrel, a specific antagonist of P2Y₁₂. The P2Y₁₂-mediated stimulation of proliferation required the pertussis toxin-sensitive activation of PI3-kinase/Akt upstream of MAP-kinases. A partial contribution of a transactivation mechanism, through the tyrosine kinase receptor platelet-derived growth factor (PDGF)-R-β, was also observed. Conversely, the P2Y₁₂-mediated reorganization of the actin cytoskeleton was Gi-independent, requiring activation of RhoA and Rho-kinase. Our results provide new insights into the molecular basis of P2Y₁₂-mediated intracellular signaling. These data may prove to be useful for a better understanding of the physiological role of P2Y₁₂, particularly in platelets and glial cells which express this important therapeutic target.     

Clinical implications of drug–drug interactions with P2Y12 receptor inhibitors

Polypharmacy in patients undergoing coronary artery stenting or in those presenting with an acute coronary syndrome is common. Nevertheless, the risk of drug–drug interactions in patients treated simultaneously with P2Y₁₂ receptor inhibitors is less well considered in routine clinical practice. Whereas the irreversible P2Y₁₂ receptor inhibitors clopidogrel and prasugrel are prodrugs requiring cytochrome P450 (CYP) enzymes for metabolic activation, such activation is not necessary for the direct‐acting reversible P2Y₁₂ receptor inhibitor ticagrelor. Several drugs frequently used in cardiology have been shown to interact with the metabolism of P2Y₁₂ receptor inhibitors in pharmacodynamic studies. Whereas several drug–drug interactions have been described for clopidogrel and ticagrelor, prasugrel seems to have a low potential for drug–drug interactions. The clinical implications of these interactions have raised concern. In general, concomitant administration of P2Y₁₂ receptor antagonists and strong inhibitors or inducers of CYP3A/CYP2C19 should be performed with caution in patients treated with clopidogrel/ticagrelor. Under most circumstances, clinicians have the option of prescribing alternative drugs with less risk of drug–drug interactions when used concomitantly with P2Y₁₂ receptor inhibitors.     

Decreased platelet inhibition by P2Y12 receptor blockers in anaemia

Background

Anaemic patients undergoing angioplasty and stenting are at an increased risk of ischaemic events, which may be caused by an inadequate response to antiplatelet therapy with adenosine diphosphate (ADP) P2Y₁₂ inhibitors. In the current study, we investigated the associations between anaemia and on‐treatment platelet reactivity in clopidogrel‐treated (group 1, n = 306) and prasugrel‐/ticagrelor‐treated (group 2, n = 109) patients undergoing elective and acute angioplasty with stent implantation, respectively.

Materials and methods

Monocyte‐platelet aggregate (MPA) formation was determined by flow cytometry in both groups. On‐treatment residual platelet reactivity in response to ADP was assessed by light transmission aggregometry (LTA) in both groups, and by the VerifyNow P2Y₁₂ assay and the Impact‐R in group 1. P‐selectin expression was measured by flow cytometry in group 2.

Results

In both groups, anaemia was associated with significantly higher MPA formation in response to ADP (both P ≤ .02). Moreover, by LTA maximal aggregation in response to ADP was significantly higher in patients with anaemia in both groups (both P < .05), and anaemic patients in group 1 had a significantly higher on‐treatment platelet reactivity by the VerifyNow P2Y₁₂ assay and the Impact‐R than those without anaemia (both P < .001). In group 2, significantly higher platelet surface expression of P‐selectin was seen in anaemia after stimulation with ADP (P = .02).

Conclusion

Anaemia is associated with decreased platelet inhibition by ADP P2Y₁₂ receptor antagonists after elective and acute percutaneous interventions with stent implantation. However, due to inconsistencies between different platelet function tests additional data are needed to clarify the role of anaemia for platelet inhibition.     

Critical role of ADP interaction with P2Y12 receptor in the maintenance of αIIbβ3 activation: association with Rap1B activation

OBJECTIVE: Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state. METHODS AND RESULTS: Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. CONCLUSIONS: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.     

Aspirin withdrawal in patients treated with ticagrelor presenting with non‐ST elevation myocardial infarction

Essentials

• Strong P2Y₁₂ blockade may cause platelet inhibition that is only minimally enhanced by aspirin.
• We evaluated aspirin withdrawal on platelet reactivity in ticagrelor treated patients.
• Aspirin withdrawal resulted in increased platelet reactivity to arachidonic acid.
• Aspirin withdrawal caused little difference in adenosine diphosphate‐induced platelet aggregation.

Summary

Background

Recent studies have shown that the thromboxane A₂‐dependent pathway is dependent on the ADP–P2Y₁₂ pathway, and that strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is minimally enhanced by aspirin. Data from the PLATO trial suggested that, among ticagrelor‐treated patients, high‐dose versus low‐dose (< 100 mg day^?1) aspirin is associated with an increased risk fof ischemic events.

Objectives

To evaluate the impact of aspirin withdrawal on platelet reactivity in acute coronary syndrome (ACS) patients treated with a potent P2Y₁₂ blocker.

Patients/Methods

This was a current prospective, randomized, placebo‐controlled, double‐blind, cross‐over study. The study population comprised 22 consecutive ACS patients who underwent percutaneous coronary intervention and were treated with aspirin (100 mg day^?1) and ticagrelor. Thirty days post‐ACS, open‐label aspirin was stopped, and patients were randomized to either blinded aspirin or placebo for 2 weeks, with each patient crossing over to the other arm for an additional 2 weeks. Platelet reactivity to arachidonic acid and ADP determined with light‐transmission aggregometry (LTA) and VerifyNow was evaluated at baseline, and 2 weeks and 4 weeks later.

Results

Aspirin withdrawal resulted in an increase in arachidonic‐acid induced platelet reactivity as determined with both LTA (77.0% ± 11.3% versus 20.8% ± 4.4%) and VerifyNow (607.7 ± 10.6 aspirin reaction units [ARU] versus 408.5 ± 14.4 ARU). Platelet response to ADP, as determined with both LTA and VerifyNow, did not differ with either aspirin or placebo (32.9% ± 2.6% versus 35.8% ± 3.6%, and 33.5 ± 6.4 P2Y₁₂ reaction units (PRU) versus 29.6 ± 5.7 PRU, respectively).

Conclusions

Aspirin withdrawal early post‐ACS results in increased platelet reactivity in response to arachidonic acid, despite concomitant treatment with the potent P2Y₁₂ blocker ticagrelor.

     

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.     

P2Y12基因C34T和G52T位点多态性与氯吡格雷疗效的相关性研究

目的研究血小板膜受体P2Y12基因C34T和G52T位点多态性与氯吡格雷疗效的相关性。方法选取2011年1月1日至2012年1月1日首都医科大学附属北京天坛医院心内科收治的规律服用氯吡格雷的急性冠状动脉综合征患者84例为研究对象。采用单核苷酸多态性(SNP)分型检测方法检测血小板受体基因C34T和G52T,应用血栓弹力仪测定二磷酸腺苷(ADP)途径诱导的血小板抑制率,观察血小板膜受体P2Y12基因C34T和G52T位点多态性与氯吡格雷疗效的相关性。结果氯吡格雷血小板抑制率达标58例(达标组),未达标26例(未达标组);在C34T基因位点,将所有患者按CC、CT、TT分型,分别为53、31、0例;氯吡格雷血小板抑制率达标组与未达标组基因型分布比较差异无统计学意义(P0.05)。在G52T基因位点,将所有患者按GG、GT、TT分型,分别为64、19、1例;氯吡格雷血小板抑制率达标组与未达标组基因型分布比较差异有统计学意义(P0.05)。结论血小板膜受体P2Y12基因G52T位点多态性,可能是影响患者口服氯吡格雷疗效的重要因素之一,治疗前进行基因检测有利于指导用药,降低心血管事件的发生风险。     

Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic mice

Summary.  In order to investigate the role of the platelet P2Y₁ receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [³³P]2MeSADP in the presence or absence of the selective P2Y₁ antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 ± 31 P2Y₁ receptors and TG platelets 276 ± 34, representing an 84% increase in P2Y₁ receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y₁ receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y₁ expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y₁ receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y₁ receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.     

Impaired platelet function in a patient with P2Y12 deficiency caused by a mutation in the translation initiation codon

In this study, we have identified a patient (OSP-1) with a congenital P2Y12 deficiency showing a mild bleeding tendency from her childhood and examined the role of P2Y12 in platelet function. At low concentrations of agonists OSP-1 platelets showed an impaired aggregation to several kinds of stimuli, whereas at high concentrations they showed a specifically impaired platelet aggregation to adenosine diphosphate (ADP). ADP normally induced platelet shape change and failed to inhibit PGE1-stimulated cAMP accumulation in OSP-1 platelets. Molecular genetic analysis revealed that OSP-1 was a homozygous for a mutation in the translation initiation codon (ATG to AGG) in the P2Y12 gene. Heterologous cell expression of wild-type or mutant P2Y12 confirmed that the mutation was responsible for the deficiency in P2Y12. OSP-1 platelets showed a markedly impaired platelet spreading onto immobilized fibrinogen. Real-time observations of thrombogenesis under a high shear rate (2000 s(-1)) revealed that thrombi over collagen were small and loosely packed and most of the aggregates were unable to resist against high shear stress in OSP-1. Our data suggest that secretion of endogenous ADP and subsequent P2Y12-mediated signaling are critical for platelet aggregation, platelet spreading, and as a consequence, for stabilization of thrombus.     

Potentiation of clopidogrel active metabolite formation by rifampicin leads to greater P2Y12 receptor blockade and inhibition of platelet aggregation after clopidogrel

Summary. Background: The thienopyridine P2Y₁₂ receptor antagonist clopidogrel reduces the risk of arterial thrombosis and individual pharmacodynamic responses to clopidogrel are believed to reflect the levels of active metabolite (AM) generated. Rifampicin increases the inhibitory effect of clopidogrel on platelet aggregation (PA). We studied the response to clopidogrel before and during administration of rifampicin in order to study the relationship between individual AM levels and P2Y₁₂ blockade. Methods: Healthy volunteers received a 600‐mg loading dose of clopidogrel followed by 75 mg daily for 7 days and, after a washout period and treatment with rifampicin [300 mg twice a day (b.i.d.)], received the same regimen of clopidogrel. Clopidogrel AM levels were determined over 4 h after the clopidogrel loading dose and unblocked P2Y₁₂ receptor number was assessed using a ³³P‐2MeSADP binding assay. PA was measured by optical aggregometry with ADP and TRAP. Results: Rifampicin enhanced clopidogrel AM production [area‐under‐the‐curve (AUC): clopidogrel 89 ± 22 ng h mL^?1, clopidogrel + rifampicin 335 ± 86 ng h mL^?1, P < 0.0001], and P2Y₁₂ blockade (unblocked receptors: clopidogrel 48 ± 24, clopidogrel + rifampicin 4 ± 2, P < 0.0001) and reduced PA (5 μmol L^?1 ADP: clopidogrel 20 ± 4, clopidogrel + rifampicin 5 ± 2, P < 0.01). Increasing numbers of unblocked receptors were required for an aggregation response with a decreasing concentration of ADP. PA induced by ADP 2 μmol L^?1 was particularly sensitive to low levels of receptor blockade. Conclusion: Potentiation of clopidogrel AM production by rifampicin leads to greater P2Y₁₂ blockade and consequently greater inhibition of PA. PA responses to low concentrations of ADP are more sensitive to P2Y₁₂ blockade.