Intrinsic platelet reactivity before P2Y12 blockade contributes to residual platelet reactivity despite high-level P2Y12 blockade by prasugrel or high-dose clopidogrel. Results from PRINCIPLE-TIMI 44

It was the objective of this study to determine whether the intrinsic platelet response to adenosine diphosphate (ADP) before thienopyridine exposure contributes to residual platelet reactivity to ADP despite high level P2Y12 blockade by prasugrel (60 mg loading dose [LD]), 10 mg daily maintenance dose [MD]) or high-dose clopidogrel (600 mg LD, 150 mg daily MD). High residual platelet function during clopidogrel therapy is associated with poor clinical outcomes. It remains unknown whether the relationship between platelet reactivity prior to treatment with clopidogrel (300 mg LD, 75 mg daily MD) and residual on-treatment platelet reactivity is maintained after more potent P2Y12 inhibition. PRINCIPLE-TIMI 44 was a randomised, double-blind, two-phase crossover study of prasugrel compared with high-dose clopidogrel in 201 patients undergoing cardiac catheterisation for planned percutaneous coronary intervention. ADP-stimulated platelet-monocyte aggregates, platelet surface P-selectin and platelet aggregation were measured pre-treatment, during LD (6 h and 18-24 h) and MD (15 d). Correlations of pre-treatment to on-treatment values were determined by Spearman rank order. Prasugrel resulted in greater platelet inhibition than high-dose clopidogrel for each measure. However, for both drugs, pre-treatment reactivity to ADP predicted 6 h, 18-24 h and 15 day reactivity to ADP (correlations 0.24-0.62 for platelet-monocyte aggregates and P-selectin). In conclusion, a patient's intrinsic platelet response to ADP before exposure to thienopyridines contributes to residual platelet reactivity to ADP despite high level P2Y12 blockade with high-dose clopidogrel or even higher level P2Y12 blockade with prasugrel. Patients who are hyper-responsive to ADP pre-treatment are more likely to be hyper-responsive to ADP on-treatment, which may be relevant to therapeutic strategies.     

A comparison of the antiplatelet effects of prasugrel and high-dose clopidogrel as assessed by VASP-phosphorylation and light transmission aggregometry

Platelet inhibition as measured by vasodilator-stimulated phosphoprotein (VASP) and light transmission aggregometry (LTA) have shown concordance following dosing of clopidogrel. No reports have directly compared the VASP assay and LTA at the levels of P2Y(12) blockade after loading doses (LDs) of prasugrel or high dose clopidogrel (600 and 900 mg). The aim was to compare the VASP assay and LTA during the loading dose phase of a comparative study of prasugrel and clopidogrel. Prasugrel 60 mg LD/10 mg maintenance dose (MD) and clopidogrel 300 mg/75 mg and 600 mg/75 mg LD/MD regimens were compared in a 3-way crossover study in 41 healthy, aspirin-free subjects. Each LD was followed by seven daily MDs and a 14-day washout period. P2Y(12) receptor blockade was estimated using the VASP assay, expressed as platelet reactivity index (VASP-PRI). Platelet aggregation was assessed by light transmission aggregometry (20 and 5 microM ADP). Twenty-four hours after prasgurel 60 mg or clopidogrel 300 mg and 600 mg, respectively, VASP-PRI decreased from approximately 80% to 8.9%, 54.7%, and 39.0%, and maximal platelet aggregation (MPA) decreased from approximately 79% to 10.8%, 42.7%, and 31.2%, with an overall VASP:MPA correlation of 0.88 (p < 0.01). VASP assay responses after the clopidogrel LDs showed a wider range of values (300 mg: 0-93%; 600 mg: 0-80%) than prasugrel (0-13%); MPA responses followed a similar trend. Pearson's correlation suggested a strong agreement between VASP and LTA (20 microM ADP) for MPA (r = 0.86, p < 0.0001). VASP and LTA demonstrated concordance across the response range of P2Y(12) receptor blockade following thienopyridine LDs.     

The use of the VerifyNow P2Y12 point-of-care device to monitor platelet function across a range of P2Y12 inhibition levels following prasugrel and clopidogrel administration

Variability in response to antiplatelet agents has prompted the development of point-of-care (POC) technology. In this study, we compared the VerifyNow P2Y12 (VN-P2Y12) POC device with light transmission aggregometry (LTA) in subjects switched directly from clopidogrel to prasugrel. Healthy subjects on aspirin were administered a clopidogrel 600 mg loading dose (LD) followed by a 75 mg/d maintenance dose (MD) for 10 days. Subjects were then switched to a prasugrel 60 mg LD and then 10 mg/d MD for 10 days (n = 16), or to a prasugrel 10 mg/d MD for 11 days (n = 19). Platelet function was measured by LTA and VN-P2Y12 at baseline and after dosing. Clopidogrel 600 mg LD/75 mg MD treatment led to a reduction in P2Y(12) reaction units (PRU) from baseline. A switch from clopidogrel MD to prasugrel 60 mg LD/10 mg MD produced an immediate decrease in PRU, while a switch to prasugrel 10 mg MD resulted in a more gradual decline. Consistent with the reduction in PRU, device-reported percent inhibition increased during both clopidogrel and prasugrel regimens. Inhibition of platelet aggregation as measured by LTA showed a very similar pattern to that found with VN-P2Y12 measurement, irrespective of treatment regimens. The dynamic range of VN-P2Y12 appeared to be narrower than that of LTA. With two different thienopyridines, the VN-P2Y12 device, within a somewhat more limited range, reflected the overall magnitude of change in aggregation response determined by LTA. The determination of the clinical utility of such POC devices will require their use in clinical outcome studies.     

Pharmacodynamic assessment of a novel P2Y12 receptor antagonist in Japanese patients with coronary artery disease undergoing elective percutaneous coronary intervention

Introduction

Numerous reports have shown that prasugrel shows a rapid and consistent antiplatelet effect among European and US patients. Previous studies suggest that prasugrel might be expected to achieve an adequate antiplatelet effect in healthy Asian subjects, even at lower doses than those assessed in the TRITON-TIMI 38 study. In this study, the antiplatelet effect of prasugrel was evaluated in Japanese coronary artery disease (CAD) patients undergoing percutaneous coronary intervention (PCI).

Methods and results

Eighty-four patients were randomized into four treatment groups: prasugrel 10/2.5 mg (loading dose [LD]/maintenance dose [MD]), 15/3.75 mg or 20/5 mg, and clopidogrel 300/75 mg. The LD of each regimen was administered the day before PCI, followed by 28-day MD on aspirin background therapy (81-100 mg). Antiplatelet effects were evaluated by light transmission aggregometry and VASP assay.The mean inhibition of platelet aggregation (IPA) induced by 20 μM of adenosine diphosphate at 4 hours after LD was higher among the prasugrel 10/2.5 mg, 15/3.75 mg and 20/5 mg groups compared with the clopidogrel group (12.3%, 20.9%, 29.8% vs. 8.4%, respectively). The proportion of subjects with an IPA of < 10% on Day 28 was lower among the prasugrel 15/3.75 mg, and 20/5 mg groups than in the clopidogrel group (0%, 6.3% vs. 15.8%, respectively). No “major” or “clinically relevant non-major” bleeding was observed.

Conclusions

Prasugrel 15 mg LD/3.75 mg MD or higher doses was well tolerated and achieved a more rapid, higher and consistent antiplatelet effect than clopidogrel in Japanese CAD patients undergoing PCI.     

Delayed thrombin-induced platelet-fibrin clot generation by clopidogrel: a new dose-related effect demonstrated by thrombelastography in patients undergoing coronary artery stenting

BACKGROUND: We have previously demonstrated that clopidogrel reduces platelet activation and aggregation in patients undergoing stenting. However, the effect of the clopidogrel loading dose on the rate of thrombin-induced platelet-fibrin clot formation is unknown in this patient population. METHODS AND RESULTS: Using thrombelastography (TEG) we measured the time to platelet-fibrin clot formation (R), a marker of the speed of thrombin generation, in 120 patients undergoing elective coronary artery stenting treated with standard and high loading doses of clopidogrel. Platelet reactivity to adenosine diphosphate (ADP) by light transmittance aggregometry (LTA) was determined simultaneously. Measurements were made immediately before and at 24 h after clopidogrel treatment. Clopidogrel produced a prolongation in R (4.4+/-1.4 min pre vs. 5.4+/-1.7 min post, p<0.001) that directly correlated with the change in platelet aggregation (r=0.65, p<0.0001). Prolongation in R was greatest in patients treated with a high loading dose (p=0.004). CONCLUSIONS: Delayed thrombin-induced platelet-fibrin clot formation as measured by TEG is a newly reported dose-related effect of clopidogrel that may contribute to the overall antithrombotic properties of the drug in patients undergoing stenting. This effect was more marked in patients loaded with 600 mg, lending further mechanistic support for this dose of clopidogrel as a more effective antithrombotic regimen than the standard 300 mg dose. Measurement of R may serve as a new indicator of clopidogrel responsiveness.     

Functional impact of high clopidogrel maintenance dosing in patients undergoing elective percutaneous coronary interventions. Results of a randomized study

The currently recommended maintenance dose of clopidogrel is often associated with inadequate platelet inhibition, suggesting the need for a higher dose. The aim of this pilot study was to assess the functional impact of a high (150 mg/day) maintenance dose of clopidogrel in patients undergoing elective percutaneous coronary intervention (PCI). This is a prospective, randomized, platelet function study which was performed in elective PCI patients assigned to treatment with either a 75 mg (n = 20) or 150 mg (n = 20) daily maintenance dose of clopidogrel for 30 days; afterwards, all patients resumed standard dosing. Platelet aggregation was performed using light transmittance aggregometry following 20 microM and 5 microM adenosine diphosphate (ADP) stimuli 30 days after randomization and 30 days after resuming standard dosing. Patients treated with 150 mg/day clopidogrel had lower 20 microM ADP-induced platelet aggregation compared to patients on 75 mg/day (52.1 +/- 9% vs. 64.0 +/- 8%; p < 0.001; primary endpoint). The dose-dependent effect was confirmed by the absolute and relative increase in platelet aggregation after resuming standard dosing (p < 0.001). No changes were observed in patients randomized to standard dosing. Parallel findings were observed following 5 microM ADP stimuli for all assessments. A broad variability in clopidogrel-induced antiplatelet effects was observed irrespective of dosing. In conclusion, a 150 mg/day maintenance dose regimen of clopidogrel is associated with reduced platelet reactivity and enhanced platelet inhibition compared to that achieved with the currently recommended 75 mg/day in patients undergoing elective PCI.     

A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation

Introduction

Prasugrel, a P2Y₁₂ adenosine diphosphate (ADP) receptor antagonist effectively inhibits ADP-mediated platelet activation and aggregation, and may be useful in reducing vaso-occlusive crises in sickle cell disease (SCD). In this study, we assess the effect of prasugrel on biomarkers of platelet activation and coagulation in patients with SCD.

Materials and Methods

Twelve adult patients with SCD and 13 healthy subjects were examined before and after 12 ± 2 days of 5.0 or 7.5 mg/day oral prasugrel. Assessed cellular biomarkers included monocyte- and neutrophil-platelet aggregates, activated glycoprotein IIb-IIIa (GPIIbIIIa), P-selectin, CD40 ligand (CD40L), tissue factor (TF) expression on circulating platelets and on monocyte-platelet aggregates, and platelet-erythrocyte aggregates. Soluble biomarkers included CD40L, prothrombin fragment 1.2 (F1.2), thromboxane B₂ (TXB₂), P-selectin, and TF.

Results

Patients with SCD had increased platelet baseline activation compared to healthy subjects, as measured by percentages of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. Likewise, baseline levels of soluble F1.2 and TXB₂ were elevated in patients with SCD compared to healthy subjects. After 12 days of prasugrel, patients with SCD had a significant reduction in platelet-monocyte aggregates that was not observed in healthy subjects. Following prasugrel administration, those with SCD maintained higher levels of monocyte-platelet aggregates and soluble F1.2, but had lower levels of platelet-erythrocyte aggregates and soluble TF compared to healthy subjects.

Conclusions

These results provide evidence for chronic platelet activation in the SCD steady state, activation that was in part attenuated by prasugrel, thereby suggesting that ADP may mediate platelet activation in SCD.     

Flow cytometric analysis of circulating platelet-monocyte aggregates in whole blood: methodological considerations

Platelet-monocyte aggregates are increasingly being used to quantify platelet activation. The variables that influence platelet-monocyte aggregates have not been well defined. We sought to determine the effect of blood collection, handling and processing techniques on detected levels of platelet-monocyte aggregates using a flow cytometric assay. Whole blood was labelled with anti-CD14-PE and anti-CD42a-FITC. Thereafter, samples were fixed and red cells lysed. Analysis was performed with the flow cytometer initially triggering on light scatter and then on FL-2 to identify CD14-PE positive monocytes. Platelet-monocyte aggregates were defined as monocytes positive for CD42a. The effect of collection, handling and processing techniques on this assay were assessed. Anticoagulation with heparin (20.1 +/- 2.0%), PPACK (16.8 +/- 1.9%), sodium citrate (12.3 +/- 1.6%) and EDTA (9.5 +/- 1.0%) resulted in markedly different levels of platelet-monocyte aggregation (P < 0.0001). Platelet-monocyte aggregation was higher in samples obtained from intravenous cannulae compared to those obtained by venepuncture (20.9 +/- 3.9% vs.13.8 +/- 2.4%, P = 0.03). For every 10 minutes of delay prior to processing platelet-monocyte aggregates increased by 2.8% (P = 0.0001) in PPACK anticoagulated blood and 1.7% (P = 0.01) in citrate anticoagulated blood. Erythrocyte lysis together with fixation does not affect platelet-monocyte aggregation. Platelet-monocyte aggregates remained stable over 24 hours when fixed and stored at 4 degrees C. Multiple handling and processing factors may affect platelet-monocyte aggregation. We recommend the measurement of platelet-monocyte aggregates on samples collected by direct venepuncture, using a direct thrombin inhibitor as the anticoagulant and minimising the time delay before sample fixation.     

Reduced thrombus cohesion in an ex vivo human model of arterial thrombosis induced by clopidogrel treatment: kinetics of the effect and influence of single and double loading-dose regimens

OBJECTIVE: To compare the effects of clopidogrel on ex vivo thrombogenesis with those on ADP-dependent platelet aggregation, and to compare single and double loading-dose regimens. METHODS AND RESULTS: Step 1: Volunteers (n=12) received clopidogrel 75 mg/day for 8 days. ADP-induced platelet aggregation was measured in platelet-rich plasma (PRP). Thrombogenesis was measured in an ex vivo model. Clopidogrel produced rapid platelet inhibition, increasing up to day 5. Maximal intensity of platelet aggregation correlated with density of platelet thrombus, surface of collagen covered by platelets and thrombus cross-sectional surface (p<0.001). Step 2: On day 1, volunteers (n=60) randomly received clopidogrel 75 mg, a single 300-mg loading dose or two 300-mg loading doses separated by a 12-h interval. On day 2, all volunteers received clopidogrel 75 mg. Both loading dose regimens enhanced platelet inhibition at all time points (p<0.03 vs. clopidogrel 75 mg). After 3 h, the antiplatelet effect of a loading dose was substantial, and the mean decrease in dense thrombus surface was greater in the loading-dose groups than in the 75 mg group (p=0.041 for the single loading dose). Ex vivo, there were no significant differences between loading-dose groups. CONCLUSIONS: Clopidogrel reduces arterial thrombus cohesion by an effect that correlates with inhibition of ADP-induced platelet aggregation. A single 300-mg loading dose provides a rapid onset of such an antithrombotic effect, which was more significant at 24 h with the double loading dose.     

Biological efficacy of a 600 mg loading dose of clopidogrel in ST-elevation myocardial infarction

Optimal platelet reactivity (PR) inhibition is critical to prevent thrombotic events in primary percutaneous coronary intervention (PCI). We aimed to determine the relationship between high on-treatment platelet reactivity (HTPR) and ST-elevation myocardial infarction (STEMI) following a 600 mg loading dose (LD) of clopidogrel. We performed a prospective monocentre study enrolling patients on clopidogrel undergoing PCI. The VASP index was used to assess PR inhibition after clopidogrel LD. HTPR was defined according to the consensus as a VASP index ≥50%. The present study included 833 patients undergoing PCI. Most patients had PCI for an acute coronary syndrome (58.7%). The mean VASP index was 50 ± 23% with a large inter-individual variability (range: 1-94%). Patients with a VASP index ≥50% were significantly older (p= 0.03), with a higher body mass index (BMI) (p<0.001), more often diabetic (p=0.03), taking omeprazole (p=0.03), admitted for an acute coronary syndrome (ACS) and with a high fibrinogen level compared to good responders (VASP <50%). In multivariate analysis BMI, omeprazole use, ACS and high fibrinogen level (p<0.001) remained significantly associated with HTPR. Of importance, in this analysis STEMI was independently associated with HTPR when compared with the other forms of ACS (NSTEMI and unstable angina) with an odd ratio of 2.14 (95% CI: 1.3 -3.5; p=0.003). In conclusion, STEMI is associated with high on-treatment platelet reactivity following 600 mg of clopidogrel. The present results suggest that 600 mg of clopidogrel may not be able to achieve an optimal PR inhibition in STEMI patients undergoing PCI and more potent drugs may be preferred.     

Platelet Inhibition by Abciximab Bolus-Only Administration and Oral ADP Receptor Antagonist Loading in Acute Coronary Syndrome Patients: The Blocking and Bridging Strategy

Introduction

Current guidelines still recommend the bolus and infusion administration of glycoprotein IIbIIIa inhibitors in patients with high-risk acute coronary syndrome undergoing percutaneous coronary intervention. We sought to evaluate the extent of platelet inhibition by a blocking and bridging strategy with intracoronary abciximab bolus-only administration and oral loading of adenosine diphosphate receptor antagonists.

Patients and methods

Fifty-six consecutive high-risk acute coronary syndrome patients with bolus-only abciximab administration (0.25 mg/kg i.c.) and loading with 600 mg clopidogrel (55%) or 60 mg prasugrel (45%) were included in this study. Platelet aggregation induced by thrombin receptor-activating peptide and adenosine diphosphate was measured by multiple electrode aggregometry up to 7 days.

Results

Thrombin receptor-activating peptide induced platelet aggregation was significantly suppressed for a minimum of 48 h (45 ± 17 U) and returned to a normal range (> 84 U) after 6 days (90 ± 26 U; p < 0.001). Co-medication with prasugrel significantly reduced adenosine diphosphate-induced (p = 0.002) and thrombin receptor-activating peptide-induced (p = 0.02) platelet aggregation compared with clopidogrel throughout the observation period. No stent thrombosis or repeat myocardial infarction occurred at 30-day follow-up.

Conclusions

Immediate blocking of platelet aggregation in high-risk acute coronary syndrome patients by intracoronary abciximab bolus-only administration and bridging to prolonged inhibition via oral blockade of ADP receptors effectively inhibited overall platelet reactivity for at least 48 h, questioning the value of continuous abciximab infusion. Co-medication with prasugrel vs. clopidogrel synergistically augmented platelet inhibition.     

Transitioning patients from cangrelor to clopidogrel: pharmacodynamic evidence of a competitive effect

BACKGROUND: Cangrelor is a direct, parenteral, and reversible inhibitor of the platelet P2Y12 receptor currently undergoing Phase III testing. As many individuals treated acutely with cangrelor will often be treated long-term with a thienopyridine, it is important to determine the effects of concurrent cangrelor and clopidogrel administration. METHODS AND RESULTS: Ten healthy volunteers received a 600 mg oral loading dose of clopidogrel and then underwent serial platelet function monitoring for 6 h. Two weeks later these same individuals received a 600 mg clopidogrel loading dose simultaneously with a cangrelor IV bolus (30 microg/kg) and a 2-hour infusion (4 microg/kg/min). A separate group of ten volunteers received a 600 mg clopidogrel loading dose after administration of a cangrelor bolus and a 1-hour infusion. The effects on ADP-induced platelet activation and aggregation were evaluated by flow cytometry, whole-blood electrical impedance, and light-transmittance aggregometry. Cangrelor and clopidogrel alone achieved the expected levels of platelet inhibition. However, the sustained platelet inhibition anticipated for clopidogrel treatment did not occur when cangrelor was initiated simultaneously. No such effect was found when clopidogrel was started upon completion of the cangrelor infusion. CONCLUSION: To achieve sustained platelet P2Y12 inhibition in patients treated with cangrelor, clopidogrel administration should be started when the cangrelor infusion is terminated.     

Inter-patient variability and impact of proton pump inhibitors on platelet reactivity after prasugrel

Although there is considerable variability of platelet reactivity among patients treated with clopidogrel, little is known about inter-individual differences and possible role of proton pump inhibitors (PPIs) after prasugrel. We defined the extent of inter-patient variability, and evaluated the impact of PPI interaction in prasugrel-treated patients with acute coronary syndrome (ACS). Between January 2010 and May 2011, 104 prospective, high-risk patients with ACS were recruited into this multicentre, prospective, observational study. Twelve to 24 hours after receiving 60 mg loading dose of prasugrel, light transmission aggregometry (LTA) and whole blood impedance aggregometry (Multiplate) were used to assess platelet activity. Platelet function measurements were repeated during maintenance phase on reduced (5 mg) or on conventional (10 mg) doses of prasugrel. High platelet reactivity (HPR) was defined according to the consensus document of the Working Group on High On-Treatment Platelet Reactivity (LTA:>46%; Multiplate:>47U). Compared to maintenance doses, 60 mg loading dose of prasugrel provided significantly greater platelet reactivity inhibition (p<0.05). There were no significant differences between the conventional and reduced maintenance doses. Notably, a remarkable inter-patient variability was present in platelet reactivity after all doses of prasugrel, and the prevalence of HPR was significantly higher during the maintenance doses (p<0.05). Although median platelet reactivity values were consistently higher when prasugrel was used in combination with PPIs, these differences were not significant (p≥0.17). Despite potent platelet inhibition, inter-patient variability is present after all tested doses of prasugrel. The 60 mg loading dose is superior to conventional and reduced maintenance doses in terms of platelet reactivity inhibition and regarding the prevention of HPR.     

Platelet function profiles in the elderly: results of a pharmacodynamic study in patients on clopidogrel therapy and effects of switching to prasugrel 5 mg in patients with high platelet reactivity

Studies specifically designed to assess clopidogrel response in the elderly as well as treatment alternatives to improve platelet inhibition in this high-risk population are lacking. This study aimed to define pharmacodynamic (PD) profiles, including high platelet reactivity (HPR) rates, among elderly patients on maintenance clopidogrel therapy and to assess the PD effects of prasugrel 5 mg/day in elderly with HPR. This was a prospective observational PD study enrolling consecutive ≥ 75-year-old patients on maintenance clopidogrel therapy (75 mg/day) who were tested for clopidogrel response by the VerifyNow P2Y12 assay and light transmittance aggregometry (LTA). HPR rates were estimated using multiple definitions. HPR patients identified by the VerifyNow P2Y12 assay [P2Y12 reaction unit (PRU) ≥ 230] were switched to prasugrel 5 mg/day, and platelet function testing was performed after 15 days of treatment. PD testing was completed in 100 patients. The HPR prevalence varied between 25% and 32%, depending on the definition used. A PRU ≥ 230 was observed in 25 patients; of these, 20 switched to prasugrel 5 mg/day. This resulted in significant reduction in PRU mean values (279.8 ± 45.1 vs. 171.7 ± 65.2, p=0.0002) with an absolute between-treatment difference of 108.1 (95% confidence intervals 75.2-140.9). Accordingly, switching to prasugrel 5 mg/day overcame HPR in most (80%) patients. Consistently, all LTA measures were significantly lower after prasugrel compared with clopidogrel. In conclusion, a considerable proportion of elderly patients exhibit HPR while on standard clopidogrel therapy. Switching to 5 mg/day prasugrel in elderly patients with HPR is associated with enhanced platelet inhibition and overcomes HPR in the majority of these patients.     

Impact of cytochrome P450 3A4-metabolized statins on the antiplatelet effect of a 600-mg loading dose clopidogrel and on clinical outcome in patients undergoing elective coronary stent placement

Early studies suggested interactions between statins and clopidogrel. Based on the outcome and platelet data, there is now huge evidence of no interactions between statins and 75 to 300 mg clopidogrel; however, data with 600-mg loading are lacking. In a pre-specified analysis of the EXCELSIOR cohort, we investigated the interaction between statins, especially cytochrome P4503A4-metabolized atorvastatin and simvastatin, and the antiplatelet effects of a 600-mg loading dose of clopidogrel. We analyzed 1,395 patients scheduled for coronary angiography (CA). Patients received clopidogrel 600 mg at least two hours before CA and 75 mg daily thereafter in case of percutaneous coronary intervention (PCI). Statin medication on admission was continued unaltered until discharge. Platelet function was assessed by optical aggregometry and flow cytometry of adenosine diphosphate (ADP)-stimulated surface expression of CD62P, CD63 and PAC-1 before clopidogrel and immediately before CA. Residual platelet aggregation (RPA) after addition of ADP 5 muM was similar irrespective of statin treatment at baseline (p = 0.968). RPA at CA was 46.2 +/- 16.8% in patients without statin (n = 682), 45.5 +/- 17.0% in patients with atorvastatin (n = 255), 45.8 +/- 16.3% with simvastatin (n = 335), 47.3 +/- 14.9% with fluvastatin (n = 42) and 45.9 +/- 16.2% with pravastatin (n = 81; p = 0.962). Consistent results were obtained by flow cytometry. In patients with PCI (n = 553), the one-year incidence of death, myocardial infarction and target lesion reintervention did not differ between cohorts stratified according to statin co-medication (p = 0.645). Thus, peri-interventional atorvastatin and simvastatin had no effect on the antiplatelet activity of a loading dose of clopidogrel 600 mg and did not affect clinical outcome after PCI.     

Effect of clopidogrel treatment on stress-induced platelet activation and myocardial ischemia in aspirin-treated patients with stable coronary artery disease

Stress may counteract responses to antiplatelet drug treatment. We investigated if adding clopidogrel to aspirin treatment could attenutate stress-induced platelet activation and myocardial ischemia in patients with coronary artery disease (CAD). Thirty-one male patients with documented CAD-treated with aspirin (75-160 mg daily) were randomized to co-treatment with clopidogrel (n = 16) or placebo (n = 15). A symptom-limited exercise test and 48-hour (h) Holter monitoring were performed before and after two weeks of double-blind treatment. Platelet function was assessed by flow cytometry and impedance aggregometry in whole blood. Exercise-induced and ambulatory ischemia was assessed from electrocardiographic (ECG) recordings. Clopidogrel treatment inhibited ADP-induced platelet P-selectin expression by 64% (22-87%), and attenuated the P-selectin response to thrombin (p < 0.001), and platelet aggregation induced by low-dose collagen (p < 0.01). Exercise ( approximately 110W) increased heart rate similarly, and caused approximately 1.8 mm ST-segment depression both before and after treatment. Exercise caused platelet activation, i.e. increased circulating activated single platelets and platelet-platelet aggregates, enhanced the in-vitro responsiveness to ADP or thrombin stimulation, and increased platelet-leukocyte aggregation. Clopidogrel inhibited ADP-induced platelet activation to a similar relative degree at rest and during exercise, but did not attenuate the platelet activating effect of exercise. Addition of clopidogrel to aspirin treatment did not attenuate either ambulatory or exercise-induced ischemia. In conclusion, adding clopidogrel to aspirin treatment inhibited platelet activation by both ADP, thrombin and collagen in vitro, but did not influence the prothrombotic responses to exercise. Intensified antiplatelet treatment did not reduce ECG signs of either exercise-induced or ambulatory myocardial ischemia.     

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.     

Protease activated receptor-1 (PAR-1) mediated platelet aggregation is dependent on clopidogrel response

Introduction

Clopidogrel inhibits ADP mediated platelet aggregation through inhibition of the P2Y12 receptor by its active metabolite. Thrombin induces platelet aggregation by binding to protease activated receptor-1 (PAR-1), and inhibition of PAR-1 has been evaluated in patients treated with clopidogrel to reduce ischemic events after acute coronary syndromes. Residual PAR-1 mediated platelet aggregation may be dependent on extent of clopidogrel response.

Material and Methods

Platelet aggregation was measured in 55 patients undergoing elective PCI at 16-24 hours after 600 mg clopidogrel loading dose by light transmittance aggregometry using ADP 20 μM and thrombin receptor agonist peptide (TRAP) at 15 μM and 25 μM as agonists. Genomic DNA was genotyped for common CYP2C19 variants.

Results

Increasing quartiles of 20 μM ADP induced platelet aggregation after clopidogrel loading were associated with increasing levels of TRAP mediated platelet aggregation. Patients in the highest quartile (clopidogrel non-responders) of post treatment ADP aggregation had significantly higher TRAP mediated aggregation than the patients in the lowest quartile (clopidogrel responders) [TRAP 15 μM: 79.6 ± 5% vs. 69.5 ± 8%, p < 0.001].

Conclusions

Non-responders to clopidogrel show increased residual platelet aggregation induced by TRAP, whereas clopidogrel responders exhibit attenuated response to TRAP. Addition of PAR-1 antiplatelet drugs may be most effective in patients with reduced clopidogrel response and high residual TRAP mediated platelet aggregation.     

The influence of body size on the pharmacodynamic and pharmacokinetic response to clopidogrel and prasugrel: A retrospective analysis of the FEATHER study

Introduction

Patients treated with clopidogrel who have higher body size exhibit greater platelet reactivity than patients with lower body size. In a retrospective analysis of the FEATHER trial, we examined the relationship between platelet response to thienopyridines clopidogrel 75 mg (Clop-75), prasugrel 5 mg (Pras-5), and prasugrel 10 mg (Pras-10) using 3 body size indices: body weight (BW), body mass index (BMI), and body surface area (BSA). Relationships were assessed as continuous variables and as 4 incremental body size groups.

Materials and Methods

Aspirin-treated patients with stable coronary artery disease (N = 72) and a BW range of 45-134 kg received Clop-75, Pras-5, and Pras-10 in a 3-period, blinded, cross-over study. Platelet assays included maximum platelet aggregation (MPA) to 20 μM ADP by light transmission aggregometry, VerifyNow-P2Y12 reaction units (PRU), and vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation platelet reactivity index (PRI). Exposure to active metabolites (AMs) was also assessed.

Results

Body size was a determinant of AM exposure and residual platelet reactivity regardless of type and dose of thienopyridine. BW and BSA demonstrated marginally stronger correlations with platelet reactivity; VASP-PRI demonstrated a stronger correlation with the body size than the other tests. Correlation coefficients ranged from a high of 0.64 (BW vs. PRI on Pras-5) to a low of 0.34 (BMI vs. MPA on Pras-10), but all were statistically significant (p < 0.01).

Conclusions

Using a comprehensive selection of body size indices, AM exposures, platelet function tests, and thienopyridine doses, we demonstrated a consistent inverse relationship between body size and response to clopidogrel and prasugrel.     

Increased expression of platelet P-selectin and formation of platelet-leukocyte aggregates in blood from patients treated with unfractionated heparin plus eptifibatide compared with bivalirudin

INTRODUCTION: Platelet-leukocyte aggregates have been implicated in atherogenesis. This study was designed to determine the influence in vivo of a direct thrombin inhibitor, bivalirudin, compared with unfractionated heparin (UFH) plus the GP IIb-IIIa inhibitor eptifibatide (E) on platelet reactivity, the formation of platelet-leukocyte aggregates, and leukocyte activation. MATERIALS AND METHODS: Blood was taken before and after percutaneous coronary intervention (PCI) from 60 patients randomized to UFH+E (n=26) or bivalirudin (n=34). Platelet function and the formation in vivo of platelet-monocyte aggregates (PMA) and platelet-neutrophil aggregates (PNA) were assessed with the use of flow cytometry. Myeloperoxidase (MPO) elaborated during leukocyte activation was measured by ELISA. RESULTS: Compared with those treated with bivalirudin, patients treated with UFH+E exhibited a 45% decrease in the capacity of platelets to bind fibrinogen (p=0.006) but a 2-fold increase in platelet surface expression of P-selectin (p=0.04) in samples taken from the coronary ostium before PCI. Platelet-leukocyte aggregation in vivo was greater (PMA=2-fold, p=0.04; PNA=3-fold, p=0.006) with UFH+E as was the concentration in blood of MPO (1.5-fold, p=0.007). CONCLUSIONS: Increased platelet surface expression of P-selectin, augmented platelet-leukocyte aggregation in vivo, and consequent activation of leukocytes was seen before PCI in blood from patients treated with UFH+E compared with bivalirudin. Benefits associated with decreased platelet aggregation when PCI is performed with UFH plus GP IIb-IIIa inhibition may be partially offset by increased platelet-leukocyte aggregation.