Predictive values of post-clopidogrel platelet reactivity assessed by different platelet function tests on ischemic events in East Asian patients treated with PCI

Abstract

An accumulating number of studies are revealing that platelet reactivity above specific cut-off scores leads to exponentially increased rates of post-percutaneous coronary intervention (PCI) ischemic events. To evaluate the optimal predictive values for three different platelet function measurement assays of platelet reactivity on early clinical outcomes in Korean patients undergoing PCI, we enrolled 228 patients receiving clopidogrel prior to PCI. Platelet reactivity was measured by light transmittance aggregometry (LTA), VerifyNow P2Y₁₂ assay, and multiple electrode platelet aggregometry (MEA). The primary endpoint was the 30-day occurrence of ischemic events after PCI. MACE occurred in 36 patients (15.8%), including 35 patients (15.4%) with periprocedural MI and the death of one patient (0.4%). ADP-induced LTA and VerifyNow values (pre- and post-PCI) were significantly higher in patients with the subsequent occurrence of periprocedural MI, but the MEA assay data (PCI and post-PCI) displayed no significant differences (pre-PCI p?=?0.25 and post-PCI p?=?0.33). ROC curve analysis demonstrated HPR values for LTA (pre-PCI, >66% and post-PCI, >53 %, all p?<?0.001), VerifyNow (pre-PCI, >347 PRU and post-PCI >272 PRU, all p?<?0.001) and MEA (pre-PCI, >50 U and post-PCI >39 U, all p?>?0.05). The platelet reactivity measurements by LTA and the VerifyNow assay can discriminate the risk of 30-day ischemic events after PCI. The predictive cut-off values for adverse events are dependent on sampling time.     

Defining predictive values using three different platelet function tests for CYP2C19 phenotype status on maintenance dual antiplatelet therapy after PCI

Abstract

Published data suggests that the presence of CYP2C19*2 or *3 loss of function (LOF) alleles is indicative of increased platelet aggregation and a higher risk of adverse cardiovascular events after clopidogrel administration. We sought to determine cut-off values using three different assays for prediction of the CYP2C19 phenotype in Korean percutaneous coronary intervention (PCI) patients. We enrolled 244 patients with drug-eluting stent implantation who were receiving clopidogrel and aspirin maintenance therapy for one month or more. Platelet reactivity was assessed with light transmittance aggregometry (LTA), multiple electrode aggregometry (MEA) and the VerifyNow P2Y12 assay (VN). The CYP2C19 genotype was analyzed by polymerase chain reaction (PCR) and snapshot method. The frequency of CYP2C19 LOF allele carriers was 58.6%. The cut-off values from LTA, MEA and VerifyNow for the identification of LOF allele carriers were as follows: 10?µM ADP-induced LTA?≥?48 %, VN?>?242 PRU and MEA?≥?37?U. Between the three tests, correlation was higher between LTA vs. VN assays (r?=?0.69) and LTA vs. MEA (r?=?0.56), with moderate agreement (κ?=?0.46 and κ?=?0.46), but between VN assay and MEA, both devices using whole blood showed a lower correlation (r?=?0.42) and agreement (κ?=?0.3). Our results provide guidance regarding cut-off levels for LTA, VerifyNow and MEA assays to detect the CYP2C19 LOF allele in patients during dual antiplatelet maintenance therapy.     

Dipstick proteinuria is an independent predictor of high on treatment platelet reactivity in patients on clopidogrel,but not aspirin,admitted for major adverse cardiovascular events

The effectiveness of aspirin and clopidogrel in patients with chronic kidney disease (CKD) suffering from acute cardiovascular events is unclear. High on treatment platelet reactivity (HTPR) has been associated with worse outcomes. Here, we assessed the association of dipstick proteinuria (DP) and renal function on HTPR and clinical outcomes. Retrospective cohort analysis of 261 consecutive, non-dialysis patients admitted for Major Adverse Cardiovascular Events (MACE) that had VerifyNow P2Y12 and VerifyNow Aspirin assays performed. HTPR was defined as P2Y12 reactivity unit (PRU)?>?208 for clopidogrel and aspirin reaction units (ARU)?>?550 for aspirin. Renal function was classified based on the estimated glomerular filtration rate (eGFR), and dipstick proteinuria was defined as ≥30?mg/dl of albumin detected on a spot analysis. All cause mortality, readmissions, and cardiac catheterizations were reviewed over 520 days. In patients on clopidogrel (n?=?106), DP was associated with HTPR, independent of eGFR, diabetes mellitus, smoking or use of proton pump inhibitor (AOR?=?4.76, p?=?0.03). In patients with acute coronary syndromes, HTPR was associated with more cardiac catheterizations (p?=?0.009) and readmissions (p?=?0.032), but no differences in in-stent thrombosis or re-stenosis were noted in this cohort. In patients on aspirin (n?=?155), no associations were seen between DP and HTPR. However, all cause mortality was significantly higher with HTPR in this group (p?=?0.038). In this cohort, DP is an independent predictor of HTPR in patients on clopidogrel, but not aspirin, admitted to the hospital for MACE.     

Upregulation of CD40/CD40L system in rheumatic mitral stenosis with or without atrial fibrillation

Thrombelastography (TEG) analyses the status of blood coagulation including abnormalities associated with low platelet count. The aim of this study was to investigate the changes in TEG parameters in idiopathic thrombocytopenic purpura (ITP) patients. Thirty nine patients with ITP (platelet count?<?100?×?103 µl^?1) were included in the study. Age-matched 17 patients with thrombocytopenia due to chemotherapy were selected as a control group. Platelet count was positively correlated with maximum clot formation (MCF) in INTEM (r?=?0.716, p?<?0.001) and MCF in EXTEM (r?=?0.679, p?<?0.001); negatively correlated with clot formation time (CFT) in INTEM (r?=??0.755, p?<?0.001) and CFT in EXTEM (r?=??0.585, p?<?0.001) in ITP patients. Platelet count was positively correlated with MCF in INTEM (r?=?0.776, p?<?0.001) and MCF in EXTEM (r?=?0.878, p?<?0.001); negatively correlated with CFT in INTEM (r?=??0.627, p?<?0.001) in control group. Receiver operating characteristic curves to describe the critical platelet count and fibrinogen level that affect MCF revealed 31?×?103?µl^?1 and 375 mg?dl^?1 as cut-off values, respectively. In conclusion, ROTEM determines the contribution of fibrinogen and platelets to clot strength in patients with ITP. MCF appears to be the most important TEG parameter in predicting bleeding in ITP patients that makes TEG superior to other hemostatic tests.     

Inhibition of platelet aggregation by prostaglandin E1 (PGE1) in diabetic patients during therapy with clopidogrel and aspirin

Diabetes mellitus (DM) is associated with increased platelet activation and reduced platelet inhibition by clopidogrel. Prostaglandin E1 (PGE1) stimulates adenyl cyclase activity in platelets and increases cyclic AMP concentrations, which inhibit Ca²⁺release and platelet aggregation induced by P2Y1 receptor activation. PGE1 is included in the VerifyNow P2Y12 assay to suppress P2Y1 induced platelet aggregation. We hypothesized that diabetes mellitus may be associated with altered response to PGE1 in subjects treated with clopidogrel. Subjects with established coronary artery disease who were taking clopidogrel 75?mg daily and aspirin for >14 days were enrolled (n?=?96). Diabetic (n?=?34) were compared with non-diabetic subjects (n?=?62). VerifyNow P2Y12 assay and light transmittance aggregometry (LTA) were performed using ADP as agonist with and without addition of PGE1. Genomic DNA was genotyped for common cytochrome P450 (CYP) 2C19 variants using Taqman assays. Residual on-treatment platelet aggregation induced by 20?µM ADP was not significantly different between subjects with and without DM. Addition of 22?nM and 88?nM PGE1 to 20?µM ADP resulted in a significant reduction of maximal platelet aggregation (MPA). Residual LTA platelet aggregation with PGE1 and VerifyNow P2Y12 platelet reactivity were significantly higher in subjects with DM than those without DM and in carriers of CYP 2C19*2 polymorphism. We conclude that an impaired inhibitory response to PGE1 may contribute to the high platelet reactivity phenotype in subjects with DM treated with clopidogrel. Addition of PGE1 to ADP agonist platelet assays may identify subjects with blunted inhibitory response to prostaglandins and result in a higher proportion of subjects with DM being classified as non-responders.     

Platelet function measured by VerifyNow™ identifies generalized high platelet reactivity in aspirin treated patients

Selected aspirin treated patients may exhibit high platelet reactivity to agonists other than arachidonic acid. This study aimed to determine whether the VerifyNow? identifies generalized high platelet reactivity supported by correlations with other established methods that stimulate platelets with various agonists. Stable outpatients with coronary artery disease (n?=?110) were treated with aspirin in a two 3?×?3 Latin square design (81, 162 and 325?mg/day for 4 weeks each). VerifyNow? (arachidonic acid (AA) cartridge); light transmittance aggregometry; thrombelastography; PFA-100®; flow cytometry; PlateletWorks®; and urinary 11- dehydro thromboxane levels were measured. Multianalyte profiling measured fibrinogen and von Willebrand factor (vWF). Patients with ≥550 ARU by VerifyNow? had increased 5?mM AA-, 5?µM ADP-, and 2?µg/mL collagen-induced platelet aggregation compared to patients with <550 ARU (p?≤?0.001). The highest ADP- and collagen-induced aggregation was present in the upper quartile by VerifyNow? (p?<?0.05). Fibrinogen or vWF levels did not differ between VerifyNow? quartiles. Patients with high platelet reactivity to arachidonic acid identified by VerifyNow? also exhibit increased reactivity to other important platelet agonists that is independent of fibrinogen and vWF levels. These data suggest that VerifyNow? may identify a generalized high platelet reactivity phenotype.     

Reduced antiplatelet effect of aspirin during 24 hours in patients with coronary artery disease and type 2 diabetes

Reduced antiplatelet effect of aspirin has been reported in patients with type 2 diabetes, and recent studies suggest that once-daily aspirin provides insufficient platelet inhibition. We investigated if the effect of aspirin declined during the 24-hour dosing interval in patients with coronary artery disease and type 2 diabetes, and whether this correlated with increased platelet turnover. Furthermore, the intra-individual variation in platelet aggregation was determined during a 28-day period. We included 47 patients with coronary artery disease and type 2 diabetes treated with aspirin 75?mg daily. Blood samples were obtained 1 and 24 hours after aspirin intake, and this was repeated three times with a 2-week interval between each visit. Platelet aggregation was evaluated by impedance aggregometry (Multiplate® Analyzer) using arachidonic acid (1.0?mM) and collagen (3.2?µg/ml) as agonists. Markers of platelet turnover were measured by flow cytometry. Compliance was confirmed by serum thromboxane B₂. Platelet aggregation levels measured 1 and 24 hours after aspirin intake were compared using the mean of 1- and 24-hour measurements at the three study visits. The difference in platelet aggregation was 70?±?97?AU?×?min (p?<?0.0001) when using arachidonic acid as agonist and 33?±?76?AU?×?min (p?=?0.01) when using collagen. Markers of platelet turnover correlated positively, though not significantly, with residual platelet aggregation 24 hours after aspirin intake (p values 0.06 and 0.07). Median intra-individual variation of platelet aggregation was 9–16%. Patients with coronary artery disease and type 2 diabetes had increased platelet aggregation at the end of the 24-hour aspirin dosing interval. Platelet turnover did not correlate significantly with residual platelet aggregation, although a trend was observed. The intra-individual variation of platelet aggregation after aspirin intake was low.     

Platelet factor XIIIa release during platelet aggregation and plasma clot strength measured by thrombelastography in patients with coronary artery disease treated with clopidogrel

Abstract

It has been estimated that up to half of circulating factor XIIIa (FXIIIa) is stored in platelets. The release of FXIIIa from platelets upon stimulation with adenosine diphosphate (ADP) in patients with coronary artery disease treated with dual antiplatelet therapy has not been previously examined. Samples from 96 patients with established coronary artery disease treated with aspirin and clopidogrel were examined. Platelet aggregation was performed by light transmittance aggregometry in platelet-rich plasma (PRP), with platelet-poor plasma (PPP) as reference, and ADP 5?µM as agonist. Kaolin-activated thrombelastography (TEG) was performed in citrate PPP. PRP after aggregation was centrifuged and plasma supernatant (PSN) collected. FXIIIa was measured in PPP and PSN. Platelet aggregation after stimulation with ADP 5?µM resulted in 24% additional FXIIIa release in PSN as compared to PPP (99.3?±?27 vs. 80.3?±?24%, p?<?0.0001). FXIIIa concentration in PSN correlated with maximal plasma clot strength (TEG-G) (r?=?0.48, p?<?0.0001), but not in PPP (r?=?0.15, p?=?0.14). Increasing quartiles of platelet-derived FXIIIa were associated with incrementally higher TEG-G (p?=?0.012). FXIIIa release was similar between clopidogrel responders and non-responders (p?=?0.18). In summary, platelets treated with aspirin and clopidogrel release a significant amount of FXIIIa upon aggregation by ADP. Platelet-derived FXIIIa may contribute to differences in plasma TEG-G, and thus, in part, provide a mechanistic explanation for high clot strength observed as a consequence of platelet activation. Variability in clopidogrel response does not significantly influence FXIIIa release from platelets.     

Impaired responsiveness to clopidogrel and aspirin in patients with recurrent stent thrombosis following percutaneous intervention for peripheral artery disease

Patients with peripheral artery disease (PAD) following peripheral percutaneous transluminal angioplasty (PTA) with stent implantation are prone to stent thrombosis despite treatment with aspirin and clopidogrel. Impaired clopidogrel responsiveness is associated with increased risk of ischemic events in patients following coronary stent implantation. We sought to assess platelet responsiveness to clopidogrel and aspirin in patients with PAD and recurrent stent thrombosis. Platelet aggregation induced by 5 and 20?µmol/l adenosine diphosphate (ADP) and 0.5?mmol/l arachidonic acid (AA), together with platelet reactivity index (PRI) and serum thromboxane B₂ (TXB₂), were determined in 11 patients with PAD and a history of stent thrombosis (mean, 3.1?±?1.14) after PTA and in 15 patients with PAD with no such history, also in 11 controls with coronary artery disease (CAD) and previous stent thrombosis. Platelet aggregation to 5?µmol/l ADP was higher in subjects with PAD and stent thrombosis than in those without stent thrombosis (p?=?0.0003) and CAD subjects (p?=?0.002). Aggregation induced by 20?µmol/l ADP was higher in PAD group with stent thrombosis than in PAD subjects without thrombosis (p?=?0.004). The PAD group with stent thrombosis had higher AA-induced platelet aggregation than CAD controls (p?=?0.007) and serum TXB₂ concentrations higher than PAD group without thrombosis (p?=?0.002) and CAD group (p?=?0.02). Concluding, platelet responsiveness to clopidogrel and aspirin is impaired in patients with PAD and recurrent stent thrombosis following PTA, as compared with similar individuals with CAD, and PAD with no history of stent thrombosis. This indicates that atherosclerosis burden affects platelet function and might contribute to stent thrombosis following percutaneous intervention in peripheral arteries.     

A randomized clinical trial comparing point-of-care platelet function assays and bleeding time in healthy subjects treated with aspirin or clopidogrel

Traditional assays of the coagulation status of patients, bleeding time assessment (BT) and light transmission aggregometry (LTA), are useful in clinical drug development. However, these assays are both labor intensive and expensive. BT results can be operator dependent and by its nature can inhibit subject enrollment in a clinical trial. The preparation of platelet-rich plasma necessary for LTA requires specialized training and laboratory support. Alternatives to these methods are desirable. The goal of this study was identification of a quantitative, easy-to-use, point-of-care device with minimal technical variables that could facilitate assessment of platelet aggregation in clinical drug development. This was a double-blind, placebo-controlled, randomized, three-period cross-over study in healthy volunteers designed to compare the abilities of BT, LTA, and three point-of-care devices, Multiplate?, Platelet Function Analyzer-100?, and VerifyNow? to quantitate the effects on platelet function of 3 days of treatment with aspirin, clopidogrel, or placebo. The effect size (difference in treatment means divided by the pooled standard deviations [SD]) of the three point-of-care devices was greater than or similar to BT and LTA for all treatment comparisons examined. VerifyNow? had the highest effect size comparing ASA to placebo. Multiplate? had the highest effect size comparing clopidogrel to placebo. From this study, we conclude that any one of the three simple-to-use point-of-care devices can reliably assess the treatment effect of ASA and CLP on platelet function in comparison with BT or LTA at the study population level     

Comparison of aspirin and indobufen in healthy volunteers

The aim of this study is to quantify the extent and recovery of platelet inhibition after administration of indobufen and aspirin in healthy volunteers. Indobufen inhibits platelet aggregation by reversibly inhibiting the platelet cyclooxygenase enzyme, thereby suppressing thromboxane synthesis. Twenty healthy volunteers completed the study and received aspirin (200?mg/day for 2 weeks) followed by a 4-week washout period and then indobufen (200?mg twice a day for 2 weeks). The percent (%) inhibition of platelet aggregation (IPA) was assessed using arachidonic acid (0.5?mg/ml) and adenosine diphosphate (5?µM) at 4, 12, 24 and 48 hours after last dose of each drug. IPA assessed using arachidonic acid as the agonist was similar at 4 hours after the last dose of indobufen (81.07?±?9.36%) and aspirin (96.99?±?0.29%, p?=?0.10), but significantly lower at 12 hours (74.04?±?9.55% vs. 97.94?±?0.28%, p?=?0.02), 24 hours (33.39?±?11.13% vs. 97.48?±?0.32%, p?p?p?=?0.002). Indobufen (200?mg twice a day) caused equivalent initial inhibition of platelet aggregation to aspirin (200?mg daily), and the anti-aggregation effect diminished faster than after aspirin.     

A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease.

AIMS: We sought to compare the results obtained from six major platelet function tests in the assessment of the prevalence of aspirin resistance in patients with stable coronary artery disease. METHODS AND RESULTS: 201 patients with stable coronary artery disease receiving daily aspirin therapy (> or =80 mg) were recruited. Platelet aggregation was measured by: (i) light transmission aggregometry (LTA) after stimulation with 1.6 mM of arachidonic acid (AA), (ii) LTA after adenosine diphosphate (ADP) (5, 10, and 20 microM) stimulation, (iii) whole blood aggregometry, (iv) PFA-100, (v) VerifyNow Aspirin; urinary 11-dehydro-thromboxane B(2) concentrations were also measured. Eight patients (4%, 95% CI 0.01-0.07) were deemed resistant to aspirin by LTA and AA. The prevalence of aspirin resistance varied according to the assay used: 10.3-51.7% for LTA using ADP as the agonist, 18.0% for whole blood aggregometry, 59.5% for PFA-100, 6.7% for VerifyNow Aspirin, and finally, 22.9% by measuring urinary 11-dehydro-thromboxane B(2) concentrations. Results from these tests showed poor correlation and agreement between themselves. CONCLUSION: Platelet function tests are not equally effective in measuring aspirin's antiplatelet effect and correlate poorly amongst themselves. The clinical usefulness of the different assays to classify correctly patients as aspirin resistant remains undetermined.     

The mean platelet volume in subjects with impaired fasting glucose

Mean platelet volume (MPV), a determinant of platelet function, is a newly emerging risk factor for atherothrombosis. Impaired fasting glucose (IFG) is probably a frequent glycemic disorder in the general population and is considered as a prediabetic state. The present study was designed to evaluate MPV in subjects with IFG compared with diabetic patients and normoglycemic control subjects. We selected 50 patients with type 2 diabetes mellitus, 50 subjects with IFG, and 50 normoglycemic healthy subjects matched for age, gender, and body mass index. MPV was very significantly higher in diabetic and IFG groups than in control group (p?<?0.00, p?<?0.05, respectively); it was also higher in diabetic group than in IFG group (p?<?0.05). Platelet counts were not different among the study groups (p?>?0.05). Platelet mass was significantly higher in diabetic and IFG groups than in normotensives (p?<?0.00, p?<?0.05, respectively); and it was also higher in diabetic group than in IFG group (p?<?0.05). MPV and platelet mass were positively correlated with fasting glucose and HbA1c in diabetic and IFG groups (p?<?0.05). In conclusion, our data suggests one possible mechanism by which subjects with IFG may be at increased cardiovascular risk.     

Overestimation of platelet aspirin resistance detection by thrombelastograph platelet mapping and validation by conventional aggregometry using arachidonic acid stimulation.

OBJECTIVES: This study sought to determine the prevalence of platelet aspirin resistance using methods that directly indicate the degree of platelet cyclooxygenase inhibition. BACKGROUND: Aspirin resistance in platelets may be overestimated by nonspecific laboratory measurements that do not isolate cyclooxygenase activity. METHODS: Arachidonic acid (AA)-induced light-transmittance platelet aggregation (LTA) and thrombelastography (TEG) platelet mapping were performed on the blood of healthy subjects (n = 6) before and 24 h after receiving 325 mg aspirin, and on 223 patients reporting compliance with long-term daily aspirin treatment (n = 203 undergoing percutaneous intervention [PCI] and n = 20 with a history of stent thrombosis). Aspirin resistance was defined as >20% aggregation by LTA or >50% aggregation by TEG. RESULTS: In healthy subjects, AA-induced aggregation by LTA was 82 +/- 10% before and 2 +/- 1% at 24 h after aspirin (p < 0.001), and aggregation by TEG was 86 +/- 14% before and 5 +/- 7% at 24 h after aspirin (p < 0.001). In compliant patients, AA-induced aggregation by LTA was 3 +/- 2% before PCI and 3 +/- 2% after PCI (p = NS), and aggregation by TEG was 5 +/- 9% before PCI and 6 +/- 14% after PCI (p = NS). Seven PCI patients were noncompliant, and all were aspirin sensitive after in-hospital aspirin treatment. Among 223 patients, only one patient ( approximately 0.4%) was resistant to aspirin treatment. CONCLUSIONS: Platelet aspirin resistance assessed by methods that directly indicate inhibition of cyclooxygenase is rare in compliant patients with coronary artery disease.     

Prevalence and Risk Factors for Aspirin Resistance in Elderly Patients With Type 2 Diabetes

Background

Aspirin resistance in patients with diabetes is recognized. However, the prevalence and related risk factors for aspirin resistance in elderly patients with Type 2 diabetes have not been reported, which is why we undertook this study.

Methods

One hundred and forty elderly patients (age, 73.84 ± 8.02 years) with Type 2 diabetes receiving daily aspirin therapy (≥75 mg) over 1 month were recruited. Platelet aggregation was measured by light transmission aggregometry (LTA) and thrombelastography (TEG) platelet mapping assay. The definitions of aspirin resistance were 20% or greater arachidonic acid-induced and 70% or greater adenosine diphosphate-induced aggregation by LTA. Aspirin semiresponders were defined as meeting one (but not both) of these criteria. Aspirin resistance by TEG was defined as 50% or greater aggregation induced by arachidonic acid.

Results

By LTA, 6 (4.3%) patients with Type 2 diabetes were found to be resistant to aspirin therapy; 44 (31.4%) patients were semiresponders. By TEG, 31 patients (22.1%) were aspirin resistant. Of the 31 patients who were aspirin-resistant by TEG, 3 were aspirin-resistant by LTA. Eight of 44 semiresponders by LTA were aspirin-resistant by TEG. In the multivariate logistic regression analysis, being female (odds ratio: 5.54, 95% confidence interval: 1.17–27.47, p = 0.036) and homocysteine levels (odds ratio: 1.15, 95% confidence interval: 1.00–1.31, p = 0.043) were significant risk factors for aspirin resistance by TEG.

Conclusion

The prevalence of aspirin resistance in elderly patients with Type 2 diabetes was considerably higher in female patients and in patients with higher serum levels of homocysteine.     

Brachial artery endothelial function predicts platelet function in control subjects and in patients with acute myocardial infarction

Platelet activation occurs in an endothelium-dependent flow-mediated dilation (FMD) impairment environment. The aim of this study was to explore the association between platelet reactivity and brachial artery FMD in individuals without established cardiovascular disease (controls) and acute myocardial infarction (AMI) patients. We prospectively assessed brachial artery FMD in 151 consecutive subjects, 104 (69%) controls, and 47 (31%) AMI patients; 115 (76%) men, mean age 53?±?11 years. Following overnight fasting and discontinuation of all medications for?≥?12?h, percent change in brachial artery FMD (%FMD) and endothelium-independent, nitroglycerin-mediated vasodilation (%NTG) were assessed. Platelet aggregation was assessed by conventional aggregometry, and platelet adhesion and aggregation under flow conditions by cone-and-plate(let) technology (Impact-R). Smoking, diabetes, and hypertension were more common in AMI compared to control subjects (p?<?0.01 for all). Furthermore, aspirin, clopidogrel, beta-blockers, angiotensin-converting enzyme inhibitors, and statin administration were more common in AMI compared to controls (p?<?0.01 for all). %FMD but not %NTG was significantly lower in AMI patients compared to controls (10.2?±?4.2% vs. 15.4?±?4.4%; p?<?0.001 and 17.2?±?3.9% vs. 18.0?±?3.7%, p?=?0.803, respectively). %FMD was significantly and inversely associated with all platelet functions tests (p?<?0.001) in all study participants. In a multivariate logistic regression (unadjusted and adjusted for age, gender, smoking status, diabetes mellitus, hypertension, hypercholesterolemia, overweight, family history, and concomitant medications), %FMD remained the best predictor of platelet function, irrespective of group allocation (AMI patients or controls). In conclusion, FMD is inversely correlated to platelet reactivity in both controls and AMI patients.     

Association of Thrombin Generation Potential with Platelet PAR-1 Regulation and P-Selectin Expression in Patients on Dual Antiplatelet Therapy

We studied the association of thrombin generation potential with platelet protease activated receptor (PAR)-1 regulation and platelet activation in 52 stable coronary artery disease patients on continuous therapy with aspirin and clopidogrel (n?=?42) or prasugrel (n?=?10). Compared to controls, peak thrombin generation potential was elevated in only 11 patients (p?>?0.05), while F1.2 was elevated in 26 patients (p?<?0.0001). PAR-1 and thrombin inducible P-selectin expression were significantly elevated in patients compared to controls (p?<?0.001). There were no significant correlations between levels of thrombin generation potential or F1.2 and PAR-1 regulation. However, there was a significant inverse correlation between levels of peak thrombin generation potential and in vitro thrombin-inducible expression of P-selectin (p?=?0.002), suggesting in vivo depletion of platelet alpha granules due to ongoing platelet activation.     

“Short” thrombelastography as a test of platelet reactivity in response to antiplatelet therapy: Validation and reproducibility

Background: A significant proportion of patients receiving dual antiplatelet therapy following percutaneous coronary intervention experience recurrent ischaemic events despite standard doses of treatment. Although clinical studies show significant heterogeneity in antiplatelet therapy responses, routine testing is not undertaken due to (i) lack of a standardized test, and (ii) poor clarity with regards to definition of abnormal responses. Short Thrombelastography (s-TEG) is a validated test that allows rapid measurement of clotting responses to antiplatelet therapy.

Objectives: This study sought to determine the reproducibility of s-TEG and to compare s-TEG with VerifyNow in assessment of responses to antiplatelet therapy.

Methods: (i) intra-individual variability was assessed using s-TEG Area Under the Curve (AUC15) and maximum amplitude (MA) in one volunteer at 20 time-points on no medication and at 10 time-points pre and post 300?mg aspirin treatment (ii) inter-individual variability was determined from a retrospective analysis of data on MA and AUC15 obtained from 56 volunteers on no medication, 25 volunteers pre and post 300?mg aspirin treatment and 28 patients pre and post 600?mg clopidogrel treatment (iii) a comparison between AUC15 arachidonic-acid (AA) channel and VerifyNow aspirin response units (VN ARU) and between AUC15 adenosine diphosphate (ADP) channel and VerifyNow P2Y12 reactivity units (VN PRU) was obtained from retrospective analysis of data at 370 and 296 time-points respectively.

Results: There was minimal intra-and inter-individual variability in MA and AUC15 in the AA, ADP and thrombin channels. There was a good correlation between AA AUC15 and VN ARU (r?=?0.701, p?<?0.001) and between ADP AUC15 and VN PRU (r?=?0.609, p?<?0.001).

Conclusions: s-TEG is a reproducible and reliable near-patient test that correlates well with VerifyNow. Large scale studies are needed to determine its potential role in individually tailored antiplatelet therapy.     

Comparison of factors affecting platelet reactivity in various platelet function tests

Previous studies have reported that various factors affect ADP-induced platelet reactivity during clopidogrel therapy. The aim of this study was to determine whether clinical and laboratory variables for platelet reactivity during dual antiplatelet therapy (DAPT) are dependent on the assay used. We enrolled 904 patients receiving DAPT following coronary intervention. Platelet reactivity was measured using three methods: the VerifyNow P2Y12 assay, multiple electrode aggregometry (MEA) ADP assay, and the light transmittance aggregometry (LTA) ADP assay at 24–48 h following coronary intervention. The VerifyNow results demonstrated a significant inverse correlation with hematocrit value (r = –0.268, p < 0.0001); however, MEA results had no such correlation with hematocrit (r = 0.044, p = 0.188). There was a positive correlation between the MEA results and platelet count (r = 0.255, p < 0.0001). LTA was weakly influenced by hematocrit (r = –0.064, p = 0.057) and platelet count (r = 0.069, p = 0.040). Gender (odds ratio 1.53, 95% CI 1.10–2.14, p = 0.013) and hematocrit (odds ratio 0.91,95% CI 0.88–0.94, p < 0.0001) were the independent variables for HPR by VerifyNow. Smoking (odds ratio 0.38, 95% CI 0.16–0.94, p = 0.036) and platelet count (odds ratio 1.01, 95% CI 1.00–1.01, p < 0.0001) were independent factors for HPR when using the MEA assay, whereas platelet count (odds ratio 1.00, 95% CI 1.00–1.01, p = 0.006) was identified as the only independent variable for HPR when using LTA. The incidence of HPR and the influencing variables involved are dependent on the platelet function test used.     

Downregulation of platelet activation markers during long-term immobilization

Immobilization and sedentary lifestyle are risk factors for venous thromboembolism and cardiovascular disease, yet little is known about platelet function during long-term physical inactivity. Our aim was to investigate platelet activation markers and their coupling to standardized immobilization: platelet-derived growth factor (PDGF-BB) and P-selectin. We studied 15 healthy females participating in the Women International Space simulation for Exploration study. Following a 20-day ambulatory control period, the subjects underwent 60 days of bed rest in head-down tilt position (?6°) 24 hours a day, finalized by 20 days of recovery. The subjects were randomized into two groups during bed rest: a control group (n?=?8) that remained physically inactive and an exercise group (n?=?7) that participated in both supine resistance and aerobic exercise training. Blood samples for the analysis of platelet activation markers were collected at baseline (5 days before bed rest), after 44 days of bed rest and 8 days into the recovery period. Compared to baseline, the levels of P-selectin and PDGF-BB decreased after bed rest (by 55%, p?=?0.01 and 73%, p?<?0.03, respectively) and remained decreased in the recovery period (by 76%, p?<?0.001 and 78%, p?<?0.02, respectively, compared to baseline). Platelet count (baseline value for the exercise group 260?000/µl?±?34?000 and baseline value for the control group 210?000/µl?±?30?000) did not change during the bed rest study (two-way repeated measurements ANOVA, p?=?ns). There were no statistical differences between the physically inactive and the exercise group. During long-term immobilization, a known risk factor for thrombosis, the levels of P-selectin and PDGF-BB decreased. Our findings indicate downregulation of platelet activation during immobilization.