Vasodilator-stimulated phosphoprotein (VASP) is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and antiplatelet effect of prasugrel in rats.

Vasodilator-stimulated phosphoprotein (VASP) is a member of actin regulatory proteins implicated in platelet adhesion. In addition, phosphorylation of VASP is utilised for the assessment of platelet reactivity in patients treated with P2Y12 receptor antagonists, a class of antiplatelet agents. However, the role of VASP in platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of P2Y12 receptor antagonists remains unclear. We investigated these effects using heterozygous and homozygous VASP knockout rats generated with a CRISPR/Cas9 system. Baseline characteristics, such as haematology and other biochemical parameters, were comparable among the genotypes. In vitro platelet aggregation stimulated by adenosine diphosphate (ADP) or collagen, P-selectin expression of rat platelets treated with ADP, and in vivo thrombocytopenia induced by collagen were also comparable among the genotypes. In addition, in vivo thrombogenesis in a ferric chloride-induced arterial thrombosis model and bleeding time were also comparable among the genotypes. Furthermore, the in vitro antiplatelet effect of prasugrel, a third-generation P2Y12 receptor antagonist, was unaffected by VASP knockout. Although phosphorylated VASP is still an important surrogate marker specific for P2Y12 antagonists, our findings demonstrate that VASP is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of prasugrel in rats.

Prasugrel, a Platelet P2Y12 Receptor Antagonist, Improves Abnormal Gait in a Novel Murine Model of Thrombotic Hindlimb Ischemia.

BACKGROUND: The efficacy of P2Y12 inhibition for the prevention of cardiovascular events in patients with peripheral arterial disease (PAD) has been established. However, the therapeutic effects on ischemic limb complications are less clear. Accordingly, we aimed to develop a novel murine model of thrombotic hindlimb ischemia to reflect that found in patients with PAD exhibiting ischemic limb symptoms. We further investigated the effects of P2Y12 deficiency and P2Y12 inhibition by prasugrel in this model. METHODS AND RESULTS: Thrombus formation induced by application of ferric chloride to the femoral artery resulted in a significant reduction in blood flow in the injured limb. In gait analysis using the CatWalk system, moderate difficulties in grounding and weight bearing of the ischemic limb, including reduction of maximum contact area and stance phase duration and increasing in swing phase duration in the ischemic limb, were observed in this model. Blood flow reduction and gait abnormalities gradually recovered over 21 days to levels present before arterial injury. Compared to wild-type (WT) mice, significant increases in blood flow and improvement in gait were observed in P2Y12-deficient mice. In addition, daily oral administration of prasugrel (3 mg/kg per day) to WT mice resulted in significant inhibition of blood flow reduction and gait abnormalities to levels found in P2Y12 deficient mice. CONCLUSIONS: Acute femoral artery thrombosis resulted in hindlimb ischemia and moderate gait abnormalities in mice. In addition, the present study suggests a possible role of P2Y12 in the complications with thrombotic limb ischemia.

Prevention of occlusive arterial thrombus formation by a single loading dose of prasugrel suppresses neointimal hyperplasia in mice.

The present study examined the effects of prasugrel in a mouse model of thrombosis-induced neointimal hyperplasia. Following carotid artery injury by application of ferric chloride solution, thrombus formation was assessed on Day 1 and neointimal thickening was assessed on Day 21. Single administrations of prasugrel at 0.3-3mg/kg (p.o.) resulted in a dose-related and sustained inhibition of ADP-induced platelet aggregation through 24h. Single and multiple (1 and 3 weeks) administration of prasugrel (3mg/kg loading and 1mg/kg/day maintenance doses) resulted in a marked inhibition of neointimal thickening in the injured artery. In the dose-response study, a single administration of prasugrel at 0.3-3mg/kg (p.o.) dose-relatedly inhibited thrombus formation and neointimal thickening on Days 1 and 21, respectively. The degree of neointimal hyperplasia in the injured artery correlated significantly with the thrombus indices, time to occlusion and patency rate. To explore possible mechanisms of inhibition of neointimal hyperplasia by prasugrel, mRNA expression levels of inflammatory and fibrosis markers were determined in injured arteries. Prasugrel treatment resulted in reduced MCP-1, ICAM-1 and TGF-ß mRNA levels on Day 2 (24h after the injury) and Day 8 (1 week after the injury) in the target arteries. In conclusion, we found that a single oral loading dose of prasugrel markedly prevented neointimal hyperplasia by inhibiting platelet activation and thrombus formation and was associated with inhibition of the expression of inflammatory and fibrosis markers, including MCP-1, ICAM-1 and TGF-ß, in the injured arteries.

Prasugrel reduces ischaemic infarct volume and ameliorates neurological deficits in a non-human primate model of middle cerebral artery thrombosis.

Several clinical trials have demonstrated the benefits of thienopyridine monotherapy in ischaemic stroke patients. Non-human primate models of ischaemic stroke have been used for various antithrombotic agents; however, to the best of our knowledge, there is no data on the effects of P2Y12 antagonists in models, such as the thrombotic middle cerebral artery occlusion (MCAO) monkey model. Accordingly, it remains unclear what level of inhibition of platelet aggregation (IPA) is required for optimal treatment of ischaemic stroke. In the present study, we investigated the effects of prasugrel, a third-generation thienopyridine antiplatelet drug, on platelet aggregation, thrombus formation and cerebral infarct volume in a non-human primate model. Daily oral administration of prasugrel resulted in significant and stable platelet inhibitory effects on Day 3, with IPA values ranging from 31% to 36% at 0.3mg/kg/day and from 44% to 50% at 1mg/kg/day. These IPA levels encompassed values observed in clinical trials of clopidogrel, and were thus selected for further study. In the thrombotic MCAO model, prasugrel increased MCA patency in a dose-dependent manner and significantly reduced ischaemic infarct volume by approximately 70% at 0.3mg/kg/day and 90% at 1mg/kg/day without increasing haemorrhagic infarction. Prasugrel also significantly reduced neurological deficit scores by 60% at 0.3mg/kg/day and 80% at 1mg/kg/day. In conclusion, prasugrel treatment resulted in effective reduction of ischaemic infarction and an associated improvement in neurological function without increasing haemorrhagic infarction. These data suggest that prasugrel monotherapy would be effective for the prevention of thrombotic stroke.

Characterization of platelet aggregation responses in microminipigs: Comparison with miniature pigs and the influence of dual antiplatelet administration of aspirin plus prasugrel.

We aimed to characterize platelet aggregation responses and the impact of dual antiplatelet therapy in microminipigs. In this in vitro study, both adenosine-5'-diphosphate (ADP, 5-50µM) and collagen (2-20µg/ml) induced concentration-related platelet aggregation in the microminipigs; 20µM ADP and 5 and 12.5µg/ml collagen were selected for further ex vivo studies. Aspirin plus prasugrel were administered orally for 7days (n=4/each group). Ex vivo platelet aggregation was analyzed on Day 1 (1 and 4h after administration), Day 4 (4h), and Day 7 (4h) under three different prasugrel dosing regimens: LD0/MD1 (1mg/kg/day), LD0/MD3 (3mg/kg/day), and LD10/MD1 (10mg/kg loading dose and 1mg/kg/day maintenance dose). Aspirin (10mg/kg/day) was administered to all groups. In the presence of aspirin, prasugrel at 3 and 10mg/kg significantly inhibited ADP-induced platelet aggregation on Day 1. On Days 4 and 7, significant inhibition of platelet aggregation (IPA) was also observed in each group. With 5µg/ml collagen-induced platelet aggregation, all three groups showed significant IPA at 4h on Day 1 or later. In 12.5µg/ml collagen-induced platelet aggregation, all groups showed significant effects on Days 4 and 7; however, the 30%-35% IPA was considerably lower than that (50%-60%) found with 5µg/ml collagen. In Clawn miniature pigs, similar inhibitory patterns were observed for both ADP- and collagen-induced ex vivo platelet aggregation. In conclusion, these results indicated that microminipigs as well as miniature pigs may represent useful experimental animals for thrombosis research.

Impaired responsiveness to the platelet P2Y12 receptor antagonist clopidogrel in patients with type 2 diabetes and coronary artery disease.

BACKGROUND: Several studies have shown that patients with diabetes mellitus (DM) exhibit an impaired response to clopidogrel. This may contribute to their increased risk of recurrent atherothrombotic events, despite the use of dual-antiplatelet therapy. The mechanisms for impaired clopidogrel response in DM patients have not been fully elucidated. OBJECTIVES: The aim of this study was to explore the mechanisms for impaired clopidogrel-mediated platelet inhibition in patients with DM using a comprehensive methodological approach embracing both pharmacokinetic (PK) and pharmacodynamic (PD) assessments as well as ex vivo and in vitro investigations. METHODS: Patients (DM, n = 30; non-DM, n = 30) with stable coronary artery disease taking aspirin 81 mg/day and P2Y12 antagonist naive were enrolled. Blood was collected before and at various times (0.5, 1, 2, 4, 6, and 24 h) after a 600-mg loading dose of clopidogrel. PD assessments included vasodilator-stimulated phosphoprotein, light transmission aggregometry, and VerifyNow P2Y12 ex vivo, before and after dosing and following in vitro incubation with escalating concentrations (1, 3, and 10 µM) of clopidogrel's active metabolite (Clop-AM). Exposure to Clop-AM was also determined. RESULTS: PD assessments consistently showed that during the overall 24-h study time course, residual platelet reactivity was higher in DM patients compared with non-DM patients. In vitro incubation with Clop-AM revealed altered functional status of the P2Y12 signaling pathway in DM platelets as measured by vasodilator-stimulated phosphoprotein, but not with other PD assays. Clop-AM exposure was ∼40% lower in DM patients than in non-DM patients. CONCLUSIONS: The present study suggests that among DM patients, impaired P2Y12 inhibition mediated by clopidogrel is largely attributable to attenuation of clopidogrel's PK profile. This is characterized by lower plasma levels of Clop-AM over the sampling time course in DM patients compared with non-DM patients and only modestly attributed to altered functional status of the P2Y12 signaling pathway.

Platelet activation biomarkers in Berkeley sickle cell mice and the response to prasugrel.

Vaso-occlusive crisis (VOC) is a common complication that occurs in sickle cell disease (SCD) patients. Although underlying mechanisms of VOC remain unclear, platelet activation has been associated with VOC. In the present study, plasma adenine nucleotide measurements using LC-ESI-MS/MS showed that plasma ADP in the Berkeley murine model of SCD was significantly higher (applox. 2.7-fold increase) compared with control mice. Assessment of platelet activation markers using flow cytometry indicated that in SCD mice at steady state (8 weeks old), circulating platelets were partially activated and this tended to increase with age (15 weeks old). The administration of prasugrel, a thienopiridyl P2Y12 antagonist, did not affect the activation state of circulating platelets suggesting P2Y12 independent mechanism of activation. In this murine SCD model, ex vivo addition of ADP or PAR4 TRAP resulted in further platelet activation as assessed by expression of activated GPIIb/IIIa and P-selectin both at 8 and 15 weeks. In 15 weeks old SCD mice, agonist-induced increases in activation markers were enhanced compared to control mice. Oral administration of prasugrel effectively inhibited ex vivo platelet activation consistent with clinical data in patients with SCD. In conclusion, in the Berkeley murine model of SCD, we found evidence of basal and agonist-stimulated platelet activation which could in part be attenuated by prasugrel. These data are consistent with observations made in patients with SCD and suggest possible utility of this murine model and prasugrel therapy in exploring treatment options for patients with SCD.

Comparison of antiplatelet effects of prasugrel and ticagrelor in cynomolgus monkeys by an ELISA-based VASP phosphorylation assay and platelet aggregation.

Prasugrel is the third generation thienopyridine prodrug, and ticagrelor is a non-competitive direct acting P2Y12 antagonist. In phase 3 studies, both agents reduced ischaemic event rates compared to clopidogrel. The present in vitro human and monkey studies showed that ticagrelor's active metabolite (AM) was more potent than ticagrelor and prasugrel's AM on inhibition of ADP-induced platelet aggregation by light transmission aggregometry and ELISA-based vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay. In contrast, on an oral dosage basis (mg/kg), prasugrel showed more potent platelet inhibition compared to ticagrelor on ex vivo aggregation and VASP phosphorylation assays in monkeys. Single oral doses of prasugrel (0.3 and 1 mg/kg) resulted in robust antiplatelet effects, which were sustained up to 24 hours after administration. Ticagrelor (3 and 10 mg/kg, p.o.) also showed significant antiplatelet effects but its effects were diminished at 24 hours after the dosing. Repeat administration of prasugrel (1.8 mg/kg loading dose [LD], 0.3 mg/kg once daily maintenance dose [MD]) showed more rapid antiplatelet effects and longer duration of action throughout the entire day. Twice a day repeat administration of ticagrelor (10 mg/kg bid MD following a single 20 mg/kg LD) also showed significant antiplatelet effects but with more intra-day variability compared to prasugrel. The in vitro and ex vivo studies showed strong correlations between platelet aggregation and VASP phosphorylation for prasugrel, ticagrelor and their AMs. These strong correlations between platelet aggregation and VASP phosphorylation in non-human primates also suggest that ELISA-based human VASP assay can be utilised for non-human primate platelet studies.

In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel.

INTRODUCTION: Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. METHODS: The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. RESULTS: PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. CONCLUSIONS: Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings.

P2Y12 receptor blockade augments glycoprotein IIb-IIIa antagonist inhibition of platelet activation, aggregation, and procoagulant activity.

BACKGROUND: New antiplatelet agents that provide greater, more consistent inhibition of the platelet ADP receptor P2Y12 may be used in combination with glycoprotein (GP) IIb-IIIa antagonists, but their combined effect on platelet function and procoagulant activity is not well studied. Therefore, the objective of this study was to evaluate the independent and complementary effects of P2Y12 and GPIIb-IIIa inhibition on platelet function and procoagulant activity. METHODS AND RESULTS: Healthy donor blood was treated with the active metabolite of prasugrel (R-138727 5 µmol/L), GPIIb-IIIa antagonists (abciximab 3 µg/mL or eptifibatide 0.9 µg/mL), and combinations thereof, exposed to physiologically relevant agonists (collagen and ADP) and then evaluated for markers of platelet activation and procoagulant activity. Significant interactions between R-138727 and GPIIb-IIIa antagonists were observed. R-138727 and the GPIIb-IIIa antagonists had additive inhibitory effects on collagen-stimulated platelet aggregation and on the collagen plus ADP-stimulated level of activated platelet surface GPIIb-IIIa. R-138727 and abciximab each inhibited collagen plus ADP-stimulated platelet phosphatidylserine expression and prothrombin cleavage, and the combination produced greater inhibition than achieved with abciximab alone. In contrast, eptifibatide did not inhibit, but instead enhanced, collagen plus ADP-stimulated prothrombin cleavage. Addition of R-138727 reduced prothrombin cleavage in eptifibatide-treated samples, suggesting a novel mechanism for potential benefit from combined prasugrel and eptifibatide treatment. CONCLUSIONS: The complementary effects of abciximab and R-138727 on platelet activation, aggregation, and procoagulant activity suggest their combined use may, to a greater degree than with either agent alone, reduce thrombus formation in vivo.

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.

Comparison of a new ELISA assay with the flow cytometric assay for platelet vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation in whole blood to assess P2Y(12) inhibition.

Thienopyridines and other agents target the platelet P2Y(12) receptor and inhibit several platelet activities mediated by adenosine diphosphate (ADP). The measurement of vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation, expressed as platelet reactivity index (PRI), mirrors the degree of P2Y(12) receptor inhibition and can detect the well-known variable response to clopidogrel. The commercially available VASP assay uses flow cytometry (FC) and requires that the test be run within 48 hours of blood collection. A new ELISA VASP assay offers the advantages of using more widely available technology and the potential to freeze and store samples before analysis. The objectives of the present study were to compare the performance of the ELISA and FC methods and to describe the relative flexibility of the ELISA-based assay. Human blood samples encompassing a wide range of levels of P2Y(12) blockade achieved in vitro by preincubation with P2Y(12) antagonists or in vivo from patients treated with clopidogrel were included, reflecting the wide spread of values reported in clinical studies. The correlation between the PRI measured by ELISA and FC was highly significant (r=0.95, p<0.001), (n=80). After the initial activation, samples were stable for at least four weeks when frozen (-20°C) prior to analysis by ELISA. Frozen samples from patients treated with clopidogrel appeared stable for up to nine weeks. Based on these results, the ELISA-based assay appears to provide a reliable and more flexible alternative to the FC method to determine P2Y(12) receptor blockade and may enable more extensive utilisation of the VASP assay in clinical studies.

Selective blockade of P2Y12 receptors by prasugrel inhibits myocardial infarction induced by thrombotic coronary artery occlusion in rats.

We evaluated the effects of prasugrel, a third-generation thienopyridyl prodrug, on P2Y12 receptors, adenosine 5'-diphosphate (ADP)-induced platelet aggregation, and myocardial infarction (MI) in rats. Oral administration of prasugrel (0.3-3 mg/kg) resulted in the dose-related inhibition of washed platelet aggregation induced by ADP (1-10 µM). Ex vivo [H]-2-MeS-ADP binding to platelet P2Y12 receptors was also inhibited by prasugrel in a similar dose range. The antiaggregatory effects of prasugrel correlated strongly with P2Y12 blockade with correlation coefficients of 0.85-0.92, suggesting that the antiaggregatory activity of prasugrel largely reflected P2Y12 blockade achieved in vivo. We further examined the effects of the in vivo P2Y12 inhibition by prasugrel (1-10 mg/kg, po) on MI induced by thrombotic coronary artery occlusion in rats. In surviving rats, infarct size at 24 hours after photoirradiation was evaluated. In the vehicle group, necrosis area/total left ventricular area was 37.9% ± 6.8% (mean ± SE, n = 7). At all prasugrel doses tested (n = 7 for each dose), necrosis area/total left ventricular area was significantly smaller than that in the vehicle group: 14.4% ± 4.0% for 1 mg/kg (P < 0.01), 19.8% ± 4.5% for 3 mg/kg (P < 0.05), and 14.8% ± 3.6% for 10 mg/kg (P < 0.01). At the highest administered dose of prasugrel (10 mg/kg), blood pressure and heart rate were unchanged. Arrhythmia was observed in 5 of 7 animals in the vehicle group at 24 hours after irradiation; in contrast, no arrhythmia was found in the group treated with prasugrel (10 mg/kg). Taken together, these results demonstrate that prasugrel is a selective P2Y12 inhibitor in vivo, providing effective inhibition of platelet aggregation and MI in rats.

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.

Regulation of functionally active P2Y12 ADP receptors by thrombin in human smooth muscle cells and the presence of P2Y12 in carotid artery lesions.

OBJECTIVE: The platelet P2Y12 ADP receptor is a well-known target of thienopyridine-type antiplatelet drugs. This study is the first to describe increased transcriptional expression of a functionally active P2Y12 in response to thrombin in human vascular smooth muscle cells (SMC). METHODS AND RESULTS: On exposure to thrombin, P2Y12 mRNA was transiently increased, whereas total protein and cell surface expression of P2Y12 were markedly increased within 6 hours and remained elevated over 24 hours. This effect was mediated by activation of nuclear factor κB. Preincubation with thrombin significantly enhanced the efficacy of the P2Y receptor agonist 2-methylthio-ADP to induce interleukin 6 expression and SMC mitogenesis. Effects induced by 2-methylthio-ADP were prevented by RNA interference-mediated knockdown of P2Y12 and a selective P2Y12-antagonist R-138727, the active metabolite of prasugrel. In addition, positive P2Y12 immunostaining was shown in SMC of human carotid artery plaques and was found to colocalize with tissue factor, the rate-limiting factor of thrombin formation in vivo. CONCLUSIONS: These data suggest that the P2Y12 receptor not only is central to ADP-induced platelet activation but also may mediate platelet-independent responses, specifically under conditions of enhanced thrombin formation, such as local vessel injury and atherosclerotic plaque rupture.

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 P2Y(12) 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. P2Y(12) receptor antagonism was determined using a radioligand binding assay ((33)P 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 micromol/L and cangrelor 1 micromol/L completely inhibited (33)P 2-MeSADP binding, compared to partial inhibition following clopidogrel administration. Platelet activation and granule secretion did not expose an additional pool of P2Y(12) receptors. Prasugrel's active metabolite effectively blocks the P2Y(12) receptor with the highest concentrations tested yielding complete inhibition of P2Y(12)-mediated amplification of several important platelet responses.

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.

The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin.

The novel thienopyridine prodrug prasugrel, a platelet P2Y(12) ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation, there are few data on the direct effects of the prasugrel's active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates, platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent inhibition of these ADP-stimulated thrombo-inflammatory markers. These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC(50) values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y(12)-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y(12). In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y(12)-mediated up-regulation of thrombo-inflammatory markers of platelet activation. This inhibition is enhanced in the presence other blood cells and calcium, but not aspirin.