Vasodilator-stimulated Phosphoprotein Platelet Reactivity Index Research Articles

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

A study of microRNA-223 in evaluating platelet reactivity in patients with acute ischemic stroke.

To investigate the relationship between plasma microRNA-223 expression and platelet reactivity in patients with acute ischemic stroke (AIS) and to evaluate its predictive value in clopidogrel resistance or high on-treatment platelet reactivity (HTPR). A total of 120 patients with acute ischemic stroke were screened in this study, and 60 patients were included in the acute ischemic stroke group according to the inclusion criteria and platelet reactivity after clopidogrel treatment. control group was 60 non-ischemic stroke patients hospitalized. The levels of phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and adenosine diphosphate-induced platelet aggregation (ADP-PAg) in platelets were detected by flow cytometry. The expression level of plasma microRNA-223 was detected before and after clopidogrel treatment using quantitative real-time polymerase chain reaction (PcR). The AIS group was then divided into the clopidogrel non-HTPR and the clopidogrel HTPR groups based on the relative inhibition rate. We found that: 1) the VASP platelet reactivity index (PRI) was positively correlated with ADP-PAg; 2) before administration, the plasma microRNA-223 expression level and VASP-PRI were higher in the AIS group than in the control group; 3) after administration, the expression level of microRNA-223 was negatively correlated with VASP-PRI; 4) before and after treatment, the plasma microRNA-223 expression level in the clopidogrel HTPR group was lower than in the non-AIS patients; 5) before treatment, there was an interaction between the expression level of microRNA-223 in the plasma and the cYP2c19 loss-of-function (LOF) allele. The study showed that decreased plasma microRNA-223 expression levels in AIS patients indicate an increased risk of clopidogrel HTPR. carrying cYP2c19 LOF alleles may result in the microRNA-223 expression being more distinct. The combined detection of plasma microRNA-223 and cYP2c19 gene polymorphisms may be effective in predicting the occurrence of clopidogrel HTPR in patients with AIS.

Comparison of platelet reactivity between prasugrel and ticagrelor in patients with acute coronary syndrome: a meta-analysis

BackgroundThis meta-analysis aimed to compare the effects of prasugrel and ticagrelor on high (HTPR) and low on-treatment platelet reactivity (LTPR) in patients with acute coronary syndrome (ACS).MethodsEligible studies were retrieved from PubMed, Embase, and the Cochrane Library. HTPR and LTPR were evaluated on the basis of the vasodilator-stimulated phosphoprotein platelet reactivity index (VASP-PRI) and P2Y12 reaction units (PRUs). HTPR and LTPR were analyzed using risk ratios (RRs) and their 95% confidence intervals (CIs). Weighted mean difference (WMD) and 95% CI were used to calculate the pooled effect size of platelet reactivity (PR).ResultsFourteen eligible studies were obtained, which included 2629 patients treated with ticagrelor (n = 1340) and prasugrel (n = 1289). The pooled results showed that the prasugrel-treated patients had higher platelet reactivity than the ticagrelor-treated patients (PRU: WMD = − 32.26; 95% CI: − 56.48 to − 8.76; P < 0.01; VASP-PRI: WMD = − 9.61; 95% CI: − 14.63 to − 4.60; P = 0.002). No significant difference in HTPR based on PRU was identified between the ticagrelor and prasugrel groups (P = 0.71), whereas a lower HTPR based on VASP-PRI was found in the ticagrelor-treated patients than in the prasugrel-treated patients (RR = 0.30; 95% CI: 0.12–0.75; P = 0.010). In addition, the results showed a lower LTPR was observed in the prasugrel group than in the ticagrelor group (RR = 1.40; 95% CI: 1.08–1.81; P = 0.01).ConclusionsPrasugrel might enable higher platelet reactivity than ticagrelor. Ticagrelor could lead to a decrease in HTPR and increase in LTPR. However, this result was only obtained in pooled observational studies. Several uncertainties such as the nondeterminancy of the effectiveness of ticagrelor estimated using VASP-PRI or the definition of HTPR (a high or modifiable risk factor) might have affected our results.

2350Evaluation of the pharmacodynamics of a ticagrelor reversal agent PB2452

Abstract Background Ticagrelor is a P2Y12 antagonist used in combination with aspirin to reduce the risk of recurrent thrombotic cardiovascular events in patients with acute coronary syndrome. Ticagrelor is associated with a risk for spontaneous major bleeding or bleeding associated with invasive procedures, particularly cardiac surgery. A rapid-acting reversal agent for ticagrelor would be advantageous. The pharmacodynamic effects of ticagrelor and a ticagrelor reversal agent are best evaluated using a panel of platelet function tests that have different sensitivities and methodologies and using a variety of agonists at different concentrations. Purpose In a first-in-human randomized, double-blind, placebo-controlled, healthy volunteer Phase 1 study, intravenous (IV) PB2452, a monoclonal antibody fragment that binds ticagrelor with high affinity, was evaluated as a ticagrelor reversal agent. Methods Platelet function was assessed using light transmission aggregometry (LTA) and 5 and 20 μM adenosine diphosphate (ADP), 1.6 mM arachidonic acid (AA), and 15 μM thrombin receptor agonist peptide (TRAP-6) as agonists. The VerifyNow P2Y12 cartridges, which assess whole blood platelet function, were evaluated as a point-of-care test. A modified vasodilator-stimulated phosphoprotein (VASP) ELISA was used to assess the extent of P2Y12 signaling. These assessments were performed 48 h prior to ticagrelor administration and at multiple time points up to 48 h after administration of PB2452 or placebo. Results 64 subjects were randomized; 48 received PB2452 and 16 received placebo. After ticagrelor administration, LTA response to 20 μM ADP was inhibited by 87% compared to the pre-ticagrelor values. Platelet function as measured by the VerifyNow P2Y12 cartridge was completely inhibited (&lt;10 platelet reactivity units [PRU]). The VASP platelet reactivity index (PRI) was inhibited by 82%. Ticagrelor reduced TRAP-6 induced LTA by 30% reflecting the effect of ADP on platelet aggregate stability. PB2452 administered as a 10 min IV bolus followed by 16 h infusion, significantly restored platelet function to &gt;80% and &gt;90% of baseline as measured by LTA using ADP and TRAP-6, respectively, to &gt;180 PRU using VerifyNow, and to &gt;90% PRI as measured by VASP. The VASP assay enabled batch analyses in a central laboratory, eliminating the need for special equipment on-site and reducing operator variability. When platelet function was assessed by VASP, PB2452 administration produced rapid ticagrelor reversal within 5 min consistent with restoration of P2Y12 signaling. Onset of reversal by all measurements occurred within 15 min of PB2452 administration and was sustained for 20–24 h. Conclusions PB2452 is a specific reversal agent for ticagrelor and produces a rapid and sustained reversal of ticagrelor inhibition of platelets. Utilizing multiple platelet function assays provided a broader understanding of the PB2452 pharmacodynamics in this first-in-human Phase 1 study. Acknowledgement/Funding PhaseBio Pharmaceuticals

Impact of Antithrombotic Regimen and Platelet Inhibition Extent on Leaflet Thrombosis Detected by Cardiac MDCT after Transcatheter Aortic Valve Replacement.

The impact of antithrombotic regimen and platelet inhibition extent on subclinical leaflet thrombosis (SLT) detected by cardiac multidetector computed tomography (MDCT) after transcatheter aortic valve replacement (TAVR) is not well established. Hypoattenuation affecting motion (HAM) has been proposed as a surrogate marker of SLT, and is characterized by hypoattenuated leaflet thickening (HALT) and concomitant reduction in leaflet motion (RELM). We sought to investigate (i) the prevalence of HAM and HALT after TAVR detected by MDCT, (ii) the predictors of SLT, (iii) the impact of oral anticoagulant (OAC) and platelet inhibition extent assessed by platelet reactivity index vasodilator stimulated phosphoprotein (PRI-VASP) and closure time adenosine diphosphate (CT-ADP) on SLT. Of 187 consecutive patients who underwent TAVR from 1 August 2017 to 31 March 2018, 90 of them had cardiac CT at relevant follow-up. Clinical, biological, echocardiographic, procedural characteristics and treatments were collected before, at discharge, and 1 year after TAVR. P2Y12 platelet inhibition extent and primary haemostasis disorders were investigated using platelet PRI-VASP and CT-ADP point-of-care assays. Eighty-five post-TAVR CTs out of 90 were ranked for clarity and assessed with sufficient diagnostic quality. HAM was evidenced in 13 patients (15.3%) and HALT in 30 patients (35%). Procedural characteristics, including aortic valve calcium score, annulus size, or procedural heparin regimens, were equivalent between groups. Likewise, no impact of P2Y12 inhibition (PRI-VASP) nor primary haemostasis disorders (CT-ADP) on SLT could be evidenced. No impact of SLT on valve deterioration evaluated by transthoracic echocardiography (TTE) and clinical events could be established at 12 months follow-up. By multivariate analysis, lack of oral anticoagulant therapy at discharge (HR 12.130 CI 95% (1.394–150.582); p = 0.028) and higher haemoglobin levels were evidenced as the sole independent predictors of SLT. In four patients with HAM, MDCT follow-up was obtained after initiation of OAC therapy and showed a complete regression of HAM. SLT was evidenced in a sizeable proportion of patients treated by TAVR and was mainly determined by the lack of oral anticoagulant therapy. Conversely, no impact of platelet inhibition extent on SLT could be evidenced.

Benefit of Switching Dual Antiplatelet Therapy After Acute Coronary Syndrome According to On-Treatment Platelet Reactivity: The TOPIC-VASP Pre-Specified Analysis of the TOPIC Randomized Study

ObjectivesThis study sought to evaluate the impact of initial platelet reactivity on the benefit of switched strategy. BackgroundTOPIC (Timing Of Platelet Inhibition after acute Coronary Syndrome) study suggested that switched dual antiplatelet therapy (DAPT) could improve net clinical benefit after acute coronary syndrome by preventing bleeding. MethodsAcute coronary syndrome patients, 1 month after coronary stenting and event free, were randomly assigned to aspirin and clopidogrel (switched DAPT) or continuation of drug regimen (unchanged DAPT). All patients underwent platelet function testing at this time and were classified as low on-treatment platelet reactivity (LTPR) (platelet reactivity index vasodilator-stimulated phosphoprotein ≤20%) or non-LTPR (platelet reactivity index vasodilator-stimulated phosphoprotein >20%). The primary endpoint aimed to evaluate the impact of platelet reactivity on clinical outcomes and benefit of switched DAPT strategy. ResultsA total of 645 patients were included, 305 (47%) of whom were classified as LTPR. LTPR patients were less often diabetic (p = 0.01), had lower body mass index (p < 0.01), and were more often on ticagrelor (p < 0.01). Patients defined as LTPR and randomized to unchanged DAPT were at the highest risk of primary endpoint occurrence (31%; p < 0.01). Conversely, in the switched arm, LTPR patients had no significant difference in primary outcome incidence compared with non-LTPR patients (hazard ratio [HR]: 0.78; 95% confidence interval [CI]: 0.40 to 1.49; p = 0.45). The switched strategy was associated with important reduction in primary endpoint incidence in LTPR patients (HR: 0.29; 95% CI: 0.17 to 0.51; p < 0.01) and only numerically lower incidence in non-LTPR patients (HR: 0.79; 95% CI: 0.46 to 1.35; p = 0.39). ConclusionsSwitched DAPT was superior regardless of initial platelet reactivity but the benefit was greater in LTPR patients. Indeed, the switched strategy was highly effective in this group, which had impaired prognosis with unchanged DAPT but similar prognosis after switching.

Comparative Long-Term Effect of Three Anti-P2Y12 Drugs after Percutaneous Angioplasty: An Observational Study Based on Electronic Drug Adherence Monitoring.

Aims: Dual platelet inhibition using anti-P2Y12 drugs and aspirin is the standard of care in patients after percutaneous coronary interventions (PCI). Prasugrel and ticagrelor have been shown to be more potent than clopidogrel with less high on-treatment platelet reactivity. Whether differences in long-term adherence to these drugs can partly explain different antiplatelet efficacy has not been studied so far. The objective was to compare the long-term P2Y12 receptor inhibition and drug adherence to different anti-P2Y12 drugs, and to assess the impact of adherence on the pharmacodynamic effect.Methods: Monocentric, prospective, observational study. Stable outpatients treated with clopidogrel 75 mg once daily, prasugrel 10 mg once daily or ticagrelor 90 mg twice daily after PCI with stent implantation were included. Drug adherence was recorded during 6 months using electronic monitoring. Platelet responsiveness was assessed with the vasodilator-stimulated phosphoprotein platelet reactivity index (VASP-PRI) at inclusion, 3 and 6 months.Results: 120 patients had VASP-PRI and adherence data available. At 6-months, mean VASP-PRI (±SD) was 17.7 ± 11.0% with ticagrelor, 29.2 ± 15.5% with prasugrel and 47.2 ± 17.6% with clopidogrel (ANOVA, P < 0.0001).Median [IQR] taking adherence was 96 [82–100]% with ticagrelor, 100 [97–101]% with prasugrel and 100 [99–101]% with clopidogrel (p = 0.0001). Median [IQR] correct dosing was 88 [73–95]% with ticagrelor, 97 [92.5–98]% with prasugrel and 98 [96–99]% with clopidogrel (p = 0.0001).Anti-P2Y12 drug (p ≤ 0.001) and diabetes (p = 0.014) emerged as predictors of poor antiplatelet response after adjusting for age, BMI, sex, and CYP2C19∗2 carriers status.Conclusion: Drug adherence to anti-P2Y12 drugs assessed with electronic monitoring was very high. However, anti-P2Y12 drugs showed significant differences in antiplatelet activity, with newer anti-P2Y12 drugs ticagrelor and prasugrel exerting a stronger P2Y12 receptor inhibition.These data suggest that pharmacokinetic-pharmacodynamic differences between oral anti-P2Y12 drugs are more important than adherence in determining antiplatelet efficacy when adherence to prescription is high.The study was registered (Current Controlled Trials ISRCTN85949729).

Effect of long-term adherence to clopidogrel on the VASP-PRI after elective coronary stent implantation: a randomized controlled study.

The biological response to clopidogrel is highly variable and a poor responsiveness is associated with major adverse cardiac events. Adherence to therapy is a major cause of poor responsiveness but its impact on long-term platelet inhibition is unknown. The objective of the present study was to evaluate the effect of different programmes monitoring adherence to clopidogrel on platelet reactivity. The study took the form of a monocentric, parallel group, randomized controlled trial. Adults treated with clopidogrel 75mg after elective coronary stenting were randomized into one of three groups: (i) a standard of care group; (ii) a standard of care+adherence electronic monitoring group, in which drug intake was recorded but kept blinded until the study end; or (iii) an integrated care group, with regular feedback on recorded adherence. Clopidogrel response was assessed with the vasodilator-stimulated phosphoprotein-platelet reactivity index (VASP-PRI) at randomization, 3months and 6months. A total of 123 adults were enrolled and randomized. Baseline VASP-PRI was highly variable, with a mean of 48±18.8%. No difference between groups in VASP-PRI was found at 6months (P = 0.761), despite better adherence to clopidogrel in the integrated care group. However, adherence (P = 0.035) and baseline VASP-PRI (P = 0.015) were associated with VASP-PRI at 3months and 6months. The association between adherence and VASP-PRI was lost in patients with baseline VASP-PRI > 50%. Diabetes, CYP2C19*2 carrier status and body mass index were significant predictors of VASP-PRI. The platelet response to clopidogrel during chronic therapy remained highly variable, despite high adherence. Different adherence monitoring programmes did not affect VASP-PRI at 6months. Poor adherence is associated with lower VASP-PRI only in initial good responders to clopidogrel.

Is platelet inhibition correlated with time from last intake on P2Y12 blockers after an acute coronary syndrome? A pilot study

Delay from the last intake of drug could be an important and unexplored variable in the biological response to antiplatelet agents after acute coronary syndrome (ACS) discharge. The objective was to define the impact of the delay from P2Y12 blocker intake on the platelet inhibition level. We compared ticagrelor-, prasugrel-, and clopidogrel-treated patients.All consecutive patients, who had been addressed between 2013 and 2014 for ACS, treated with aspirin and a P2Y12 blocker as maintenance dose, were eligible. One month after discharge, blood sample and a questionnaire were proposed to the patient by a nurse blinded to the protocol. On this questionnaire, three questions about name of the drug, regularity of the intakes, and hour of the last intake were collected. The response to antiplatelet therapy was assessed using platelet reactivity index vasodilator-stimulated phosphoprotein (PRI VASP) and % of adenosine-5’-diphosphate-induced aggregation (%ADP).The primary objective of this study was to evaluate the correlation between platelet inhibition and delay from drug intake. We enrolled 474 ACS treated with clopidogrel 75 mg in 182 cases (38% patients), prasugrel in 190 cases (40%), or ticagrelor in 102 patients (22%). We observed a significant correlation between delay from intake and PRI VASP and %ADP for ticagrelor (r = 0.25, p = 0.01; r = 0.21, p = 0.03; respectively). On clopidogrel (r = 0.09, p = 0.24; r = 0.02, p = 0.80; respectively) and prasugrel (r = 0.02, p = 0.82; r = 0.11, p = 0.12 respectively), no correlation exists.In conclusion, ticagrelor, unlike thienopyridines, is associated with a significant correlation between delay from the last intake and platelet inhibition.

Expression of miRNA-26a in platelets is associated with clopidogrel resistance following coronary stenting.

The present study aimed to evaluate the association between platelet microRNA (miRNA)-26a expression and clopidogrel resistance in patients who underwent coronary stenting. Between September 2013 and August 2014, 43 patients with coronary heart disease underwent percutaneous coronary intervention at Heibei General Hospital (Shijiazhuang, China). In the same period, 20 healthy volunteers without any history of cardiovascular disease were enrolled in the present study as the control group. Flow cytometry was used to measure the phosphorylation levels of vasodilator-stimulated phosphoprotein (VASP), and to calculate the platelet reactivity index (PRI). Low response to clopidogrel was defined as PRI ≥50% on day 7 following clopidogrel administration. Western blotting was used to measure protein expression of VASP and reverse transcription-quantitative polymerase chain reaction analysis was performed to determine the expression levels of mRNA and miRNAs. Bioinformatics tools were employed to predict that miR-26a, miR-199 and miR-23a may target VASP mRNA. The results of the present study demonstrated that the activity of platelets in patients with low or high clopidogrel response was increased, as compared with healthy subjects. No differences in platelet VASP protein expression levels were detected between patients with high clopidogrel response and healthy subjects; whereas VASP protein expression was elevated in patients with low clopidogrel response. Furthermore VASP gene transcription was maintained at low levels in healthy subjects and patients with high clopidogrel response, whereas patients with low clopidogrel response exhibited increased VASP mRNA expression levels. Platelet expression of miRNA-26a, but not miRNA-199 or miRNA-23a, was associated with high platelet reactivity. Serum miRNA-26a, miRNA-199 and miRNA-23a were not demonstrated to be involved in clopidogrel resistance. Therefore, the present study demonstrated that platelet miRNA-26a has an important role in clopidogrel resistance. Combined miRNA and VASP PRI tests may aid the early diagnosis and prediction of clopidogrel resistance.

Platelet effect of prasugrel and ticagrelor in patients with ST-segment elevation myocardial infarction

Recent studies have suggested that ticagrelor 90mg twice daily provides stronger platelet inhibition than prasugrel 10mg once daily in acute coronary syndrome patients undergoing percutaneous coronary intervention. To compare the effects of ticagrelor 90 mg twice daily and prasugrel 10mg once daily on platelet reactivity in patients with ST-segment elevation myocardial infarction (STEMI), using: the VerifyNow(®) P2Y12 (VN-P2Y12) assay, expressed in P2Y12 reaction units (PRU); measurement of the vasodilator-stimulated phosphoprotein platelet reactivity index (VASP-PRI; %); and light transmission aggregometry (LTA), expressed as residual platelet aggregation (RPA; %). Platelet reactivity was evaluated prospectively using the three assays 30 days after primary PCI in 118 patients with STEMI on a maintenance dose of prasugrel 10mg once daily (n=60) or ticagrelor 90mg twice daily (n=58). On-treatment platelet reactivity, assessed by the VN-P2Y12 assay, was lower for ticagrelor compared with prasugrel (20.91 ± 4.59 PRU vs. 43.50±6.98 PRU; P=0.008) but was not significantly different when using the more specific VASP-PRI assay (13.05 ± 1.61% vs. 17.44 ± 1.97%; P=0.09) or RPA assessed by LTA (10.49 ± 1.44% vs. 7.20 ± 1.27%; P=0.09). The difference in platelet reactivity between ticagrelor and prasugrel varies according to the platelet function test in patients with STEMI. The differences observed may be related more to the tests than to the drugs used.

Aspirin ‘Resistance': Impact on No-Reflow, Platelet and Inflammatory Biomarkers in Diabetics after ST-Segment Elevation Myocardial Infarction

Background: The no-reflow (NR) phenomenon exists despite percutaneous coronary intervention (PCI), and is especially prevalent in diabetics. The causes(s) of NR are not fully elucidated, but may be associated with impaired residual platelet and inflammatory reactivity during dual-antiplatelet therapy. Objective: To assess the relationship between dual-antiplatelet therapy, NR and conventional biomarkers suggestive of platelet and inflammatory response in diabetics following ST-segment elevation myocardial infarction (STEMI) treated with PCI. Methods: Sixty diabetics with (n = 27) and without NR (n = 33) were prospectively enrolled. All patients were treated with clopidogrel and aspirin. Platelet and inflammatory biomarkers were assessed serially in the peripheral blood and right atrium before and after PCI and then at 24 h, 7 days and 30 days. Results: Arachidonic acid (AA)-induced platelet aggregation and the serum thromboxane B₂ level before and after PCI (in the peripheral and right atrium blood) were significantly higher in the NR patients than in those with no NR. AA-induced aggregation >100 (AUC*min) before PCI predicted NR in diabetic patients with 96.2% sensitivity and 38.5% specificity (AUC 0.66; 95% CI 0.52-0.71; p = 0.029). There were no other correlations between NR and platelet reactivity (collagen, adenosine diphosphate, thrombin receptor agonist peptide-induced aggregation, vasodilator-stimulated phosphoprotein platelet reactivity index, soluble P-selectin, soluble CD40 ligand, platelet-derived growth factor AB and the level of platelet-monocyte aggregates) or between NR and inflammatory indices (i.e. high-sensitivity C-reactive protein, interleukin 6 and interleukin 10). Conclusion: An inadequate response to aspirin, but not to clopidogrel, may be associated with the occurrence of the NR phenomenon in diabetics with STEMI who have been treated with primary PCI.

Impact of Red Blood Cell Transfusion on Platelet Aggregation and Inflammatory Response in Anemic Coronary and Noncoronary Patients: The TRANSFUSION-2 Study (Impact of Transfusion of Red Blood Cell on Platelet Activation and Aggregation Studied With Flow Cytometry Use and Light Transmission Aggregometry)

This study sought to determine whether red blood cell (RBC) transfusion increases in vivo platelet aggregation and inflammation in coronary and noncoronary patients. RBC transfusion increases in vitro platelet activation and aggregation in healthy volunteers, providing a possible explanation for the increase in recurrent ischemic events and mortality reported after RBC transfusion in patients with acute coronary syndromes (ACS). Platelet reactivity was measured before and after RBC transfusion in 61 patients (33 with ACS patients and 28 without ACS). Relative changes between baseline and post-transfusion measurements of maximal and residual platelet aggregation were considered with different agonists as well as changes in vasodilator-stimulated phosphoprotein platelet reactivity index and P-selectin expression. Inflammatory and thrombotic biomarkers were also measured before and after transfusion. After RBC transfusion, platelet reactivity was increased when measured using adenosine diphosphate-induced light transmission aggregometry (11.6% relative increase in maximal platelet aggregation, p = 0.004; 10.8% increase in residual platelet aggregation, p = 0.005) and vasodilator-stimulated phosphoprotein platelet reactivity index (20.7% relative increase, p = 0.002), and there was a nonsignificant trend toward an increase in P-selectin expression. Similar results were found with the nonspecific agonist thrombin receptor-activated peptide (relative increases of 11.7% for maximal platelet aggregation, p = 0.04, and 12.7% for residual platelet aggregation, p = 0.02) but not with collagen or arachidonic acid agonists. There were no significant differences in inflammatory and thrombotic biomarkers before and after transfusion. After RBC transfusion, there is an increase in platelet reactivity, especially with tests measuring the adenosine diphosphate-P2Y12 receptor pathway, without significant variations in inflammatory or thrombotic biomarkers. This in vivo effect may account for the excess of ischemic events observed in the context of patients with ACS treated using percutaneous coronary intervention and P2Y12 inhibitors.

Impact of Obesity and the Metabolic Syndrome on Response to Clopidogrel or Prasugrel and Bleeding Risk in Patients Treated After Coronary Stenting

This study aimed to analyze the impact of body mass index (BMI) and the metabolic syndrome (MS) on responses to clopidogrel or prasugrel and bleeding risk after acute coronary syndrome. The study included 1,542 consecutive patients who underwent coronary stenting (287 clopidogrel 75 mg, 868 clopidogrel 150 mg, and 387 prasugrel 10 mg). Platelet reactivity was assessed 1 month after discharge using platelet reactivity index vasodilator stimulated phosphoprotein (PRI VASP). Three hundred thirty-six patients (21.8%) were obese (BMI ≥30), and we observed higher platelet reactivity associated with higher BMI across thienopyridine regimens. Incidence of high on-treatment platelet reactivity (PRI VASP >50%) was higher in obese than nonobese patients (p <0.05 for all regimens). Conversely, incidence of low on-treatment platelet reactivity with prasugrel therapy (PRI VASP <20%) was lower in obese than nonobese patients: 13% (12 of 93) versus 33% (97 of 294); odds ratio 0.30, 95% confidence interval 0.16 to 0.58; p <0.001. Accordingly, incidence of Bleeding Academic Research Consortium bleeding complications was higher in nonobese than in obese patients: 10% (119 of 1,206) versus 6% (20 of 336); odds ratio 1.7, 95% confidence interval 1.1 to 2.8; p = 0.03. This impaired response was only observed in obese patients with the MS, and obese with the MS had significantly higher platelet reactivity than other obese patients with all regimens (p <0.01). Obese patients without the MS had no significant difference in platelet reactivity compared with nonobese patients. In conclusion, the present study confirmed that BMI has a strong impact on response to clopidogrel and prasugrel with higher incidence of high on-treatment platelet reactivity, lower incidence of low on-treatment platelet reactivity, and lower bleeding complication in obese patients. However, among obese patients, the presence of the MS strongly affects response to antiplatelet agents, indicating that the metabolic status might be a better predictor of platelet inhibition than BMI.

Ticagrelor or prasugrel in STEMI patients: a pharmacodynamic evaluation

Background: A recent study has suggested that ticagrelor 90 mg twice daily (bid) provides stronger platelet inhibition than prasugrel 10mg once daily (od) when measured with the VerifyNow P2Y12 assay (VN-P2Y12), in ACS patients. Aim: To compare the effect of ticagrelor 90mg bid and prasugrel 10mg od on platelet reactivity in STEMI patients using different platelet function tests including the VN-P2Y12 Methods: Platelet reactivity was prospectively evaluated using VN-P2Y12 platelet reaction unit (PRU), light transmission aggregometry (LTA) residual platelet agregation (RPA), and Vasodilator-Stimulated-Phosphoprotein (VASP) platelet reactivity index (PRI) 30 days after an acute STEMI. We studied 92 STEMI patients who received either prasugrel 10mg od (n=54) or ticagrelor 90 mg bid (n=38) after primary coronary stenting. Results: On-treatment platelet reactivity evaluated with the VN-P2Y12 assay (PRU) was lower with ticagrelor compared to prasugrel: 20±4.6 vs 55±8.3 (p=0.001). But these results were not confirmed with the other platelet function tests. The more specific test, VASP PRI (%) was 13.6±2.4 on ticagrelor vs. 18.0±2.1 on prasugrel (p=0.18); the more global test, LTA showed a mean RPA (%) of 12.9±2.0 vs. 9.0±1.7 for ticagrelor and prasugrel, respectively (p=0.15) (Figure). High on-treatment platelet reactivity rates were 2.6% with VASP (defined as PRI>50%) and 0% with VN-P2Y12 (PRU>235) and LTA (% RPA > 46.2%) on ticagrelor, while it was 0% with VASP, 1.8% with VN-P2Y12 and LTA on prasugrel. ![Figure][1] Pharmacodynamic Evaluation Conclusions: VASP and LTA measurements do not confirm the pharmacological superiority of ticagrelor over prasugrel when measured with VN-P2Y12 in STEMI Patients. These results suggest that VN-P2Y12 assay could overestimate the platelet inhibitory effect of ticagrelor. [1]: pending:yes

Clinical Implications of Very Low On-Treatment Platelet Reactivity in Patients Treated With Thienopyridine: The POBA Study (Predictor of Bleedings With Antiplatelet Drugs)

This study was designed to define the hyperresponse to thienopyridine (very low on-treatment platelet reactivity [VLTPR]) as the most predictive threshold value of platelet reactivity index vasodilator-stimulated phosphoprotein (PRI VASP) for the prediction of non-access site-related bleeding events. We also aimed to identify predictors of bleeding and VLTPR in patients treated with thienopyridines. New P2Y12 blockers and platelet monitoring has been proposed to optimize platelet inhibition after acute coronary syndromes and improve ischemic outcomes. However, bleeding complications remain the Achilles' heel of antiplatelet therapy, and platelet monitoring could be useful to evaluate this risk. A total of 1,542 consecutive patients undergoing coronary stenting for ACS were included in the present study (287 taking clopidogrel 75 mg, 868 taking clopidogrel 150 mg, and 387 taking prasugrel 10 mg). During 6-month follow-up, 9% of patients (n= 139) experienced nonaccess site-related Bleeding Academic Research Consortium bleeding complications. These patients were more often women and nondiabetic and had lower PRI VASP values than others (p < 0.001). Receiver-operating characteristic curve analysis (0.71, p < 0.01) identified a threshold value for VLTPR of PRI VASP ≤10% to predict bleeding events with a sensitivity of 17% and a specificity of 97%. Although prasugrel was the main predictor of VLTPR in the whole population (odds ratio: 10.2, 95% confidence interval: 3.0 to -34.2; p < 0.001), VLTPR was the strongest and independent predictor of bleeding (odds ratio: 4.7, 95% confidence interval: 2.7 to 8.3; p < 0.001). The present study identified VLTPR (PRI VASP ≤10%) as the strongest predictor of bleeding complications in patients treated with thienopyridines. This marker could be useful for tailored therapy and bleeding prevention.

Clinical and Laboratory Significance of Defective P2Y12 Pathway Function in Patients with Myeloproliferative Neoplasms: A Pilot Study

Background: Patients with myeloproliferative neoplasms (MPN) have an increased risk for thrombosis and bleeding and show a defect in adenosine diphosphate (ADP)-induced platelet aggregation. This risk of thrombosis is further increased in MPN patients bearing the JAK2V617F mutation. Two ADP receptors, P2Y₁ and P2Y₁₂, are present on platelets. Although the pattern of defective ADP-induced platelet aggregation in MPN suggests an abnormality in the P2Y₁₂ pathway, no previous studies have specifically evaluated P2Y₁₂ function in MPN or the relationship between P2Y₁₂ function and the JAK2V617F mutation. Methods: Forty-one MPN patients were enrolled, including 24 with essential thrombocythemia (ET), 16 with polycythemia vera (PV) and 1 with primary myelofibrosis. Platelet P2Y₁₂ function in MPN was evaluated by flow-cytometric measurement of the phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Clinical data were collected by review of medical records. JAK2V617F mutation was detected by allele-specific polymerase chain reaction. JAK2V617F allele burden was measured by the pyrosequencing method. Results: In patients with MPN, platelet P2Y₁₂ function determined by VASP platelet reactivity index (PRI) was inversely correlated with platelet and white blood cell (WBC) counts. In subgroup analysis, PRI was inversely correlated with platelet and WBC counts in PV. PRI was also inversely correlated with platelet counts in ET, but the correlation of PRI and WBC counts did not reach statistical significance. Eight of the 41 patients had a history of thrombosis and only 2 had a bleeding history. Neither thrombosis nor bleeding patients were found to have significantly different PRIs. JAK2V617F mutation data were available in 35 cases. PRI was not different between JAK2V617F mutation and wild-type patients but PRI had a trend towards an inverse correlation with JAK2V617F allele burden for patients with mutations. Conclusions: The present study provides the first explicit demonstration of a defect in the P2Y₁₂ pathway in platelets of patients with MPN. Furthermore, platelet P2Y₁₂ function, assayed by VASP, is inversely correlated with platelet and WBC counts in patients with MPN. Platelet P2Y₁₂ function also appears to be inversely correlated with JAK2V617F allele burden. This compromised P2Y₁₂ function may be a novel mechanism for the bleeding tendency associated with extreme thrombocytosis in MPN.

Effect of CYP2C19*2 and *17 Genetic Variants on Platelet Response to Clopidogrel and Prasugrel Maintenance Dose and Relation to Bleeding Complications

The present study was performed to compare the influence of cytochrome P459 2C19 (CYP2C19) *2 and *17 genetic variants on the platelet response to clopidogrel and prasugrel maintenance therapy and to assess the relation between platelet reactivity and bleeding complications. A total of 730 patients were included (517 patients treated with clopidogrel 150 mg/day and 213 discharged with prasugrel 10 mg). Platelet reactivity was assessed at 1 month with the platelet reactivity index vasodilator-stimulated phosphoprotein (PRI VASP). High on-treatment platelet reactivity was defined as PRI VASP >50% and low on-treatment platelet reactivity (LTPR) as PRI VASP <20%. The patients were classified according to their genotypes as poor metabolizers (*2/non *17), intermediate metabolizers (*2/*17 or non *2/non *17) and ultrametabolizers (non *2/*17). At 1 month, the prasugrel response was significantly better than the clopidogrel response in all groups of patients, with a lower incidence of high on-treatment platelet reactivity but a greater incidence of LTPR, regardless of the genetic variants. The genetic distribution had a significant effect on the mean PRI VASP values, the incidence of high on-treatment platelet reactivity, and LTPR with both clopidogrel and prasugrel (p <0.05 for all). LTPR identified a group of patients at a greater risk of bleeding (odds ratio 4.8, 95% confidence interval 2.7 to 8.3; p <0.0001). In conclusion, the present study showed that both clopidogrel and prasugrel have genetic modulation by CYP2C19 *2 and *17 alleles and that prasugrel provides greater platelet inhibition, regardless of the genotypes. In addition, LTPR was associated with a greater risk of bleeding.

CYP2C19*2 and *17 Alleles Have a Significant Impact on Platelet Response and Bleeding Risk in Patients Treated With Prasugrel After Acute Coronary Syndrome

The present study was designed to assess the effect of genetic variants on chronic biological response to prasugrel and bleeding complications. CYP2C19*2 loss-of-function allele and CYP2C19*17 gain-of-function allele have been linked with response to clopidogrel, but preliminary data did not show any significant influence of these alleles on prasugrel effect. A total of 213 patients undergoing successful coronary stenting for acute coronary syndrome and discharged with prasugrel 10 mg daily were included. Prasugrel response was assessed at 1 month with the platelet reactivity index (PRI) vasodilator-stimulated phosphoprotein (VASP) and high on-treatment platelet reactivity (HTPR) defined as PRI VASP > 50% and hyper-response as PRI VASP <75th percentile (PRI VASP < 17%). CYP2C19*2 and CYP2C19*17 genotyping were performed. Carriers of loss-of-function *2 allele had significantly higher PRI VASP than noncarriers (33 ± 15% vs. 27 ± 14%, p = 0.03) and higher rate of HTPR (16% vs. 4%, p = 0.01). Conversely, carriers of *17 gain-of-function allele had significantly lower PRI VASP than noncarriers (25 ± 13% vs. 31 ± 15%, p = 0.03, p = 0.03), lower rate of HTPR (1% vs. 10%, p = 0.02), higher rate of hyper-response (34% vs. 21%, p = 0.02), and higher rate of bleeding complications than noncarriers: 23% versus 11%, (odds ratio [95% confidence interval]: 2.5 [1.2 to 5.4]; p = 0.02). No significant influence of genotypes on platelet reactivity assessed by adenosine diphosphate-induced platelet aggregation was observed. The present study shows a significant influence of CYP2C19*2 and *17 alleles on response to chronic treatment by prasugrel 10 mg daily and occurrence of bleeding complications.

Relation between clopidogrel active metabolite levels and different platelet aggregation methods in patients receiving clopidogrel and aspirin

Clopidogrel is a prodrug that undergoes bioconversion via cytochrome P450 system to form an active metabolite (AM) that binds to the platelet ADP receptor. The antiplatelet effect of clopidogrel is commonly assessed by measuring the aggregatory response to 5μM ADP by light transmission aggregation (LTA) or multiple electrode aggregometry (MEA) or by the vasodilator-stimulated phosphoprotein platelet reactivity index (VASP-PRI). To determine which of these three tests of platelet ADP receptor pathway inhibition most closely correlates with clopidogrel AM levels. We analyzed blood samples from 82 patients with coronary artery disease who were randomized to receive double-dose or standard dose clopidogrel for 2weeks. We measured peak clopidogrel AM levels, platelet aggregation in response to ADP and VASP-PRI on days 1, and repeated all the measures on days 7 and 14. Linear regression analysis was used to examine the correlation between clopidogrel AM and LTA, MEA and VASP-PRI. Bland-Altman plots were used to explore the agreement between tests of the antiplatelet effects of clopidogrel. Clopidogrel AM on day 1 correlated most closely with VASP-PRI (r=-0.5767) and demonstrated weaker correlations with LTA (r=-0.4656) and MEA (r=-0.3384) (all p<0.01). Intra-class correlation (ICC) between VASP-PRI and LTA was 0.6446; VASP-PRI and MEA was 0.4720; and LTA and MEA was 0.4693. Similar results were obtained on days 7 and 14. Commonly used pharmacodynamic measures of clopidogrel response are only moderately correlated with clopidogrel AM levels and may not be suitable to measure the adequacy of clopidogrel therapy.