Light Transmission Aggregation Research Articles

Platelet function testing in hirudin and BAPA anticoagulated blood

Sodium citrate is the most commonly used anticoagulant for platelet function testing. However, the use of citrated blood for platelet function analysis has been criticized due to creation of a non-physiological milieu. Moreover, platelet function measurements performed with citrated blood need to be completed within 4 h after blood collection. Alternatively, hirudin and recently, a dual thrombin/factor Xa inhibitor benzylsulfonyl-D-Arg-Pro-4-amidinobenzylamide (BAPA), can be used to improve the reactivity after prolonged storage of platelets. The present study investigated platelet function tests using hirudin and BAPA anticoagulated blood. Blood was obtained from 30 healthy individuals and 20 patients on aspirin or clopidogrel therapy, and stored for 2, 12, 24 or 48 h. Light transmission aggregometry and impedance platelet aggregometry were performed using adenosine 5-diphosphate (ADP) and arachidonic acid as agonists. The vasodilator stimulated phosphoprotein (VASP) phosphorylation assay was evaluated. Platelet aggregation measurements of healthy individuals and patients showed stable platelet aggregation values induced by arachidonic acid, after 24 h, when hirudin or BAPA anticoagulated blood was used. However, citrated blood resulted in significantly reduced platelet response after 12 h. ADP-induced light transmission aggregation of healthy individuals and patients exhibited unchanged platelet aggregation after 12 h using hirudin or BAPA anticoagulated blood, while significantly reduced platelet response was observed after 12 h when using citrated blood. In contrast, measurement of ADP-induced aggregation by use of impedance aggregometry resulted in reduced stability over 12 h using hirudin or BAPA anticoagulated blood. The VASP assay exhibited no significant changes in results over a storage period of 48 h, independent of the anticoagulants used. Use of hirudin or BAPA anticoagulated blood resulted in improvement of stability of platelet function measurements.

The Platelet Activity After Clopidogrel Termination (PACT) Study

"Rebound" platelet hyperreactivity after discontinuation of clopidogrel has been proposed to lead to increased thrombotic risk, including late stent thrombosis. However the hypothesis that discontinuation of clopidogrel results in platelet hyperreactivity has never been rigorously tested. We therefore performed a randomized, double-blind, placebo-controlled, crossover study: the Platelet Activity after Clopidogrel Termination (PACT) study. Platelet reactivity in 15 healthy subjects was measured at baseline, during clopidogrel 75 mg or placebo daily for 14 days, and on days 1, 4, 8, 11, 15, and 45 after discontinuation of clopidogrel or placebo. Platelet reactivity was assessed by (1) platelet surface activated GPIIb-IIIa and surface P-selectin (by whole blood flow cytometry) in response to ADP 0.5, 1, and 20 μmol/L; thrombin receptor activating peptide (TRAP) 1 and 20 μmol/L; and collagen/epinephrine 5 μg/mL/5 μmol/L, (2) light transmission aggregation with ADP 2.5, 5, and 20 μmol/L; TRAP 2 and 20 μmol/L; and collagen 6 μg/mL, (3) whole blood impedance aggregation with ADP 1.6 and 6.5 μmol/L; TRAP 4 and 32 μmol/L; and collagen 3.2 μg/mL, and (4) plasma soluble CD40 ligand (by ELISA). Immature platelet fraction was measured in the Sysmex XE-2100. At no time point after discontinuation of clopidogrel was platelet reactivity, as determined by any assay end point, or the immature platelet fraction significantly greater than after discontinuation of placebo. This randomized, double-blind, placebo-controlled, crossover study demonstrates that discontinuation of clopidogrel does not result in "rebound" platelet hyperreactivity, as determined by multiple time points, assays, agonists, and agonist concentrations. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00619073.

Adenosine diphosphate–induced platelet-fibrin clot strength: A new thrombelastographic indicator of long-term poststenting ischemic events

Poststenting ischemic events occur despite dual-antiplatelet therapy, suggesting that a "one size fits all" antithrombotic strategy has significant limitations. Ex vivo platelet function measurements may facilitate risk stratification and personalized antiplatelet therapy. We investigated the prognostic utility of the strength of adenosine diphosphate (ADP)-induced (MA(ADP)) and thrombin-induced (MA(THROMBIN)) platelet-fibrin clots measured by thrombelastography and ADP-induced light transmittance aggregation (LTA(ADP)) in 225 serial patients after elective stenting treated with aspirin and clopidogrel. Ischemic and bleeding events were assessed over 3 years. Overall, 59 (26%) first ischemic events occurred. Patients with ischemic events had higher MA(ADP), MA(THROMBIN), and LTA(ADP) (P < .0001 for all comparisons). By receiver operating characteristic curve analysis, MA(ADP) >47 mm had the best predictive value of long-term ischemic events compared with other measurements (P < .0001), with an area under the curve = 0.84 (95% CI 0.78-0.89, P < .0001). The univariate Cox proportional hazards model identified MA(ADP) >47 mm, MA(THROMBIN) >69 mm, and LTA(ADP) >34% as significant independent predictors of first ischemic events at the 3-year time point, with hazard ratios of 10.3 (P < .0001), 3.8 (P < .0001), and 4.8 (P < .0001), respectively. Fifteen bleeding events occurred. Receiver operating characteristic curve and quartile analysis suggests MA(ADP) <or=31 as a predictive value for bleeding. This study is the first demonstration of the prognostic utility of MA(ADP) in predicting long-term event occurrence after stenting. The quantitative assessment of ADP-stimulated platelet-fibrin clot strength measured by thrombelastography can serve as a future tool in investigations of personalized antiplatelet treatment designed to reduce ischemic events and bleeding.

Comparison of methods for monitoring residual platelet reactivity after clopidogrel by point-of-care tests on whole blood in high-risk patients

Cardiovascular events are more frequent in high-risk coronary artery disease (CAD) patients on dual antiplatelet therapy with a residual platelet reactivity (RPR) than in those showing inhibition of ADP-inducible platelet activation. It is known that post-interventional RPR is a clinically important entity confirming it as a risk factor for thrombo-ischaemic events. Multiple electrode platelet aggregometry (MEA) on whole blood has been recently proposed as a rapid tool to evaluate RPR in high-risk CAD patients on clopidogrel therapy. It was the aim of this study to detect RPR in 801 high-risk CAD patients on dual antiplatelet therapy comparing MEA with the VerifyNow P2Y12 assay on whole blood and classical light transmission aggregation (LTA) on platelet-rich plasma. ADP (10 microM) was employed as agonist for MEA and LTA. The prevalence of RPR was 20.6% by MEA, 16.1% by LTA and 30.8% by VerifyNow. MEA showed a significant correlation (rho=0.62, p<0.0001) with VerifyNow and a moderate agreement (k=0.52, p<0.001) with 81.5% of concordant values. A significant correlation was found between MEA and LTA (rho=0.71, p<0.001) with a good agreement (k=0.63, p<0.001) and 88.8% of concordant values. MEA in relation to LTA showed a sensitivity of 80% and a specificity of 91%. MEA might represent a reliable method and valid alternative in comparison with other available platelet function assays. It might help to guide antiplatelet therapy and thus improve clinical outcome of high-risk CAD patients.

Lack of reproducibility of assessment of aspirin responsiveness by optical aggregometry and two platelet function tests

The term aspirin-resistance describes the failure of aspirin to inhibit thromboxane A2 production. Many new tests have become available for potentially measuring aspirin responses but some are non-specific and do not isolate COX-1 activity. We previously demonstrated that agreement between two tests (PFA-100® and VerifyNow®-ASA) and light transmission aggregation (LTA) was no greater than would be expected by chance. In this study we re-tested the same patients using identical methods after 1 year to determine whether poor agreement might have been due to assessment in the acute phase and whether the results of the individual tests are consistent over time. Platelet function by all three tests was re-tested in the 72 patients who were alive and still receiving low dose ASA therapy one year after the first tests were performed. On re-testing the prevalence of ASA non-responsiveness compared with baseline was 10% vs 17% by the VerifyNow®-ASA test, 25% vs 22% by the PFA-100®, and 1% vs 5% by LTA. Agreement between the tests at 1 year remained poor (kappas: 0.02–0.17) and only one patient was identified as a non-responder by all three tests, in keeping with the theoretical differences between the tests. Within test comparisons of baseline vs 1 year showed moderate agreement for the PFA-100® (kappa = 0.44, 95% CI 0.19–0.68, p = 0.0006), a fair agreement for VerifyNow®-ASA (kappa = 0.34, 0.04–0.64, p = 0.12) and poor agreement for LTA (kappa = 0.14, −0.11 −0.39, p = 0.24 for ADP; kappa = 0.09, −0.21–0.39, p = 0.41 for arachidonic acid). Agreement between the three tests in identifying aspirin non-responsiveness remained poor in patients who had been taking aspirin for at least 1 year follow-up. Reproducibility over time was no greater than chance for LTA and only moderate for VerifyNow®-ASA and PFA-100®. Lack of consistency over time in identification of apparently non-responsiveness individuals is likely to substantially undermine any ability of these tests to predict risk of recurrent vascular events.

An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts

Light transmission platelet aggregation tests are important for diagnosing platelet function defects. However, uncertainties exist about the best procedures to determine aggregation reference intervals. We investigated methods for determining reference intervals for light transmission aggregation tests, using the % maximal aggregation values for prospectively collected data on healthy control samples. Reference intervals for samples tested at 250 x 10(9) platelets/l were determined by mean +/- 2 standard deviations and non-parametric analyses. To establish reference intervals for tests on thrombocytopenic subjects, regression analyses were used to estimate 95% confidence limits for % maximal aggregation, according to sample platelet counts, using data for control samples diluted to match the platelet count of undiluted thrombocytopenic patient platelet-rich plasma samples. For samples tested at 250 x 10(9) platelets/l, non-parametric analyses described 95% of data for healthy control samples better than mean +/- 2 standard deviations. For samples tested at lower counts, to match thrombocytopenic samples, the % maximal aggregation was influenced by platelet count and derived limits were wider at very low platelet counts for almost all agonists. With ristocetin, it proved feasible to test samples with very low platelet counts to exclude Bernard-Soulier syndrome and type 2B von Willebrand disease. Non-parametric analyses should be the preferred method to establish light transmission aggregation reference intervals for samples tested at normal platelet counts. The derived limits for thrombocytopenic samples provide guidance for evaluating thrombocytopenic platelet function disorders, including which agonists to test, based on the sample platelet count.

Comparison of Different Methods to Evaluate the Effect of Aspirin on Platelet Function in High-Risk Patients With Ischemic Heart Disease Receiving Dual Antiplatelet Treatment

Patients with coronary artery disease (CAD) receiving aspirin therapy with a residual platelet reactivity (RPR) may be at increased risk of ischemic vascular events. Point-of-care (POC) methods PFA-100 (Dade-Behring, Marburg, Germany) and VerifyNow (Accumetrics, San Diego, CA) assays have been suggested as rapid tools to evaluate RPR. We compared PFA-100 closure times by collagen/epinephrine and VerifyNow Aspirin assays with light transmission aggregation (LTA) induced by 1 mmol/L of arachidonic acid in 484 patients with CAD undergoing percutaneous coronary intervention and receiving dual antiplatelet therapy. RPR was detected in 30.0% of patients by LTA, in 32.4% by PFA-100, and in 14.3% by VerifyNow. Significant correlations were found among 3 methods (all P < .0001). In relation to the presence or absence of RPR by LTA and PFA-100, by LTA and VerifyNow, and by PFA-100 and VerifyNow, samples were significantly concordant (all P < .0001). Assuming LTA as the reference method, PFA-100 and VerifyNow showed sensitivity of 62.1% and 39.3% and specificity of 80.2% and 96.4%, respectively. The cutoff values for POC methods need to be defined for clinical use.

Evaluation of Dose-Related Effects of Aspirin on Platelet Function

The antiplatelet effect of aspirin is attributed to platelet cyclooxygenase-1 inhibition. Controversy exists on the prevalence of platelet resistance to aspirin in patients with coronary artery disease and effects of aspirin dose on inhibition. Our primary aim was to determine the degree of platelet aspirin responsiveness in patients, as measured by commonly used methods, and to study the relation of aspirin dose to platelet inhibition. We prospectively studied the effect of aspirin dosing on platelet function in 125 stable outpatients with coronary artery disease randomized in a double-blind, double-crossover investigation (81, 162, and 325 mg/d for 4 weeks each over a 12-week period). At all doses of aspirin, platelet function was low as indicated by arachidonic acid (AA)-induced light transmittance aggregation, thrombelastography, and VerifyNow. At any 1 dose, resistance to aspirin was 0% to 6% in the overall group when AA was used as the agonist, whereas it was 1% to 27% by other methods [collagen and ADP-induced light transmittance aggregation, platelet function analyzer (PFA-100)]. Platelet response to aspirin as measured by collagen-induced light transmittance aggregation, ADP-induced light transmittance aggregation, PFA-100 (81 mg versus 162 mg, P < or = 0.05), and urinary 11-dehydrothromboxane B2 was dose-related (81 mg versus 325 mg, P = 0.003). No carryover effects were observed. The assessment of aspirin resistance is highly assay-dependent; aspirin is an effective blocker of AA-induced platelet function at all doses, whereas higher estimates of resistance were observed with methods that do not use AA as the stimulus. The observation of dose-dependent effects despite nearly complete inhibition of AA-induced aggregation suggests that aspirin may exert antiplatelet properties through non-cyclooxygenase-1 pathways and deserves further investigation.

Different methodologies for evaluating the effect of clopidogrel on platelet function in high-risk coronary artery disease patients.

Two point-of-care (POC) systems have been recently proposed as rapid tools with which to evaluate residual platelet reactivity (RPR) in coronary artery disease (CAD) patients. We compared Platelet Function Analyzer-100 (PFA-100) closure times (CTs) by collagen/adenosine 5'-diphosphate (ADP) (C/ADP CT) cartridge and the VerifyNow P2Y12 Assay (VerifyNow) with light transmission aggregation (LTA) induced by 2 and 10 micromol L(-1) ADP in 1267 CAD patients on dual antiplatelet therapy who underwent percutaneous coronary intervention. We also performed the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay by cytofluorimetric analysis in a subgroup of 115 patients. Cut-off values for identifying RPR were: > or = 54% and > or = 66% for LTA induced by 2 and 10 micromol L(-1) ADP respectively, and > or = 264 P2Y12 Reaction Units (PRU) for VerifyNow. The cut-off for PFA-100 C/ADP CT was > or = 68 s. RPR was detected in 25.1% of patients by 2 mumol L(-1) ADP-induced LTA (ADP-LTA), in 23.2% by 10 micromol L(-1) ADP-LTA, in 24.4% by PFA-100, and in 24.7% by VerifyNow. PFA-100 results did not parallel those obtained with LTA. VerifyNow showed a significant correlation (rho = 0.62, P < 0.001) and significant agreement (k = 0.34, P < 0.001) with LTA induced by 2 micromol L(-1) ADP. The correlation was similar but the agreement was better between VerifyNow and 10 micromol L(-1) ADP-LTA (rho = 0.64, P < 0.0001; k = 0.43, P < 0.001). Significant relationships were found between VASP platelet reactivity index and both ADP-LTA and VerifyNow. PFA-100 C/ADP CT did not significantly correlate with any of the other assays. Our results show a significant correlation between LTA and VerifyNow but not the PFA-100 C/ADP assay. Clinical validation studies for POC systems are necessary.

Clopidogrel Effect on Platelet REactivity in Patients With Stent Thrombosis: Results of the CREST Study

We investigated whether patients who suffered subacute stent thrombosis (SAT) have higher post-treatment reactivity than those who do not encounter stent thrombosis. High post-treatment platelet reactivity has been reported after coronary stenting after clopidogrel therapy and may be an important factor in the occurrence of SAT. We identified patients with SAT treated at two tertiary care centers over a 1.5-year period. Light transmittance aggregation induced by adenosine diphosphate (ADP) and arachidonic acid, total and activated glycoprotein (GP) IIb/IIIa after stimulation with ADP, and vasodilator-stimulated phosphoprotein phosphorylation levels to measure P2Y12 receptor inhibition were determined (n = 20) and compared with an age-matched group of patients without SAT (n = 100). High post-treatment platelet reactivity was defined as >75th percentile ADP-induced aggregation in the group without SAT. The SAT patients had higher mean platelet reactivity than those without SAT by all measurements (p < 0.05): 49 +/- 4% versus 33 +/- 2% for 5 micromol/l ADP-induced aggregation and 65 +/- 3% versus 51 +/- 2% for 20 micromol/l ADP-induced aggregation (p < 0.001), 69 +/- 5% versus 46 +/- 9% for P2Y12 reactivity ratio (p = 0.03), and 138 +/- 19 mean fluorescence intensity (MFI) versus 42 +/- 4 MFI for stimulated GP IIb/IIIa expression (p < 0.001). Of patients with SAT, 60% had high platelet reactivity. High post-treatment platelet reactivity and incomplete P2Y12 receptor inhibition are risk factors for SAT. Measures to uniformly determine platelet reactivity after coronary stenting and treatment strategies to improve P2Y12 receptor inhibition in patients with high post-treatment platelet reactivity should be further investigated.