Assessment of platelet functional activity in healthy individuals and patients receiving antiplatelet therapy. Possible inconsistencies between aggregation and flow cytometry tests.

Platelet functional activity was assessed in healthy volunteers (HV, n=92), patients with stable angina pectoris (SA, n=42) and acute coronary syndrome (ACS, n=73), treated with acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor, respectively. In all HV and patients we have compared parameters of platelet aggregation (maximum light transmission and velocity, Tmax and Vmax) and parameters, characterizing exposure of platelet activation markers, evaluated by flow cytometry. HV platelets were activated by 10 µM, 1 µM TRAP, and 20 µM, 5 µM, 2.5 µM ADP; patient platelets were activated by 10 µM TRAP and by 20 µM and 5 µM ADP. Strong and significant correlations between the aggregation and flow cytometry parameters (the r correlation coefficient from 0.4 up to >0.6) most frequently were registered in HV platelet during activation by 1 µM TRAP and in SA patients during platelet activation by 20 µM and 5 µM ADP. However, in many other cases these correlations were rather weak (r < 0.3) and sometimes statistically insignificant. In HV the differences in PAC-1 binding parameters between platelets activated by 10 µM TRAP (the strongest agonist) and all ADP concentrations were negligible (≤ 10%), while CD62P binding (at all ADP concentrations) and LTA parameters for (5 µM and 2.5 µM ADP) were significantly lower (by 40-60%). Antiplatelet therapy in patients decreased all parameters as compared to HV, but to varying extents. For 10 µM TRAP the MFI index for PAC-1 binding (40-50% decrease) and for both ADP concentrations the Tmax values (60-85% decrease) appeared to be the most sensitive in comparison with the other parameters that decreased to a lesser extent. The data obtained indicate a possibility of inconsistency between different LTA and flow cytometry parameters in assessing platelet activity and efficacy of antiplatelet drugs.

Flow cytometry for comprehensive assessment of platelet functional activity in response to ADP stimulation.

OBJECTIVES: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment. METHODS: For activation, 2.5 µM CaCl2 , 5 µM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation. RESULTS: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP. CONCLUSIONS: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation.

Thromboelastography 6s for assessment of platelet function during coil embolization of unruptured intracranial aneurysms.

OBJECTIVES: Methods for assessing platelet function in patients with neurovascular disease remain controversial and poorly studied. This study aimed to assess associations between thromboelastography 6s (TEG6s) measurements and postoperative ischemic complications in patients with unruptured intracranial aneurysms (UIAs) treated by coil embolization. METHODS: Eighty-four patients with UIAs taking a combined aspirin and clopidogrel protocol were retrospectively reviewed from January 2021 to May 2022. Blood samples were obtained for TEG6s to assess platelet function on the day of coil embolization. To identify acute ischemic complications, diffusion-weighted imaging (DWI) was performed within 24 h after coil embolization. Multivariate logistic regression analysis was conducted to identify potential risk factors for postoperative positive DWI (DWI (+)) lesions. RESULTS: Forty-three of the 84 patients (51%) with DWI (+) lesions were identified. Compared with patients without DWI (+) lesions, Adenosine diphosphate (ADP)-induced platelet-fibrin clot strength (MAADP) was significantly higher (53.6 mm [Interquartile range (IQR): 48.3-58.3 mm] vs 46.7 mm [IQR: 36.8-52.2 mm]; p=0.001) and ADP inhibition rate (ADP%) was significantly lower (19% [IQR: 11-31%] vs 31% [IQR: 21-44%]; p=0.001) in DWI (+) patients. Multivariate analysis identified MAADP, ADP%, and procedure time as significant independent predictors of subsequent DWI (+) lesions (odds ratios: 1.07, 0.96, and 1.02, respectively). Based on receiver operating characteristic curve analysis, MAADP >50.9 mm and ADP% <28.8% were associated with postoperative DWI (+) lesions in patients undergoing coil embolization for UIAs. CONCLUSIONS: MAADP and ADP% as assessed by TEG6s can offer reliable parameters to predict postoperative ischemic complications after coil embolization of UIAs. Lower MAADP values and higher ADP% may decrease the risk of postoperative ischemic complications.

Assessment of on-treatment platelet reactivity at high and low shear stress and platelet activation status after the addition of dipyridamole to aspirin in the early and late phases after TIA and ischaemic stroke.

BACKGROUND: Data are limited on the ability of dipyridamole to additionally inhibit platelet function/reactivity in ischaemic cerebrovascular disease (CVD) patients on aspirin. AIMS: To assess inhibition of platelet function/reactivity and platelet activation with dipyridamole in CVD. METHODS: This prospective, observational study assessed TIA/ischaemic stroke patients before (baseline; N = 60), at 14 ±7 days (14d, N = 39) and ≥ 90 days (90d, N = 31) after adding dipyridamole to aspirin. Platelet function/reactivity at high shear stress (PFA-100® C-ADP) and low shear stress (VerifyNow® P2Y12 and Multiplate® ADP assays), and platelet activation status (% expression of CD62P, CD63 and leucocyte-platelet complexes on whole blood flow cytometry) were quantified. 'Dipyridamole-high on-treatment platelet reactivity (HTPR)' was defined as failure to inhibit ADP-induced platelet aggregation +/- adhesion compared with the patient's baseline on aspirin monotherapy by more than twice the coefficient-of-variation of the assay after adding dipyridamole to aspirin. RESULTS: Dipyridamole-HTPR was identified in 71.4-75% of patients on PFA-100 C-ADP, 83.9-86.8% of patients on VerifyNow P2Y12, and 81.5-83.3% of patients on Multiplate ADP assays. There were no changes in CD62P/CD63 expression (P ≥ 0.18), or consistent changes in leucocyte-platelet complexes in CVD patients overall at 14d or 90d vs. baseline after commencing dipyridamole. Monocyte-platelet complexes increased in the patient subgroup with dipyridamole-HTPR at 14d and 90d on PFA-100, and at 14d on VerifyNow (P ≤ 0.04), but not in those without dipyridamole-HTPR. DISCUSSION: Additional antiplatelet effects of dipyridamole are detectable under high and low shear stress conditions with user-friendly platelet function/reactivity tests ex vivo. Increasing circulating monocyte-platelet complexes over time are associated with dipyridamole-HTPR.

Peripheral versus central venous blood sampling does not influence the assessment of platelet activation in cirrhosis.

Cirrhotic patients have an increased risk of bleeding and thromboembolic events, with platelets being involved as key players in both situations. The impact of peripheral versus central blood sampling on platelet activation remains unclear. In 33 cirrhotic patients, we thus analyzed platelet function in peripheral (P) and central (C) blood samples. Platelet surface expression of P-selectin, activated glycoprotein (GP) IIb/IIIa, and leukocyte-platelet aggregate formation were measured by flow cytometry in response to different agonists: thrombin receptor-activating peptide-6, adenosine diphosphate, collagen-related peptide (CrP), epinephrine, AYPGKF, Pam3CSK4, and lipopolysaccharide. Unstimulated platelet surface expression of P-selectin (p = .850) and activated GPIIb/IIIa (p = .625) were similar in peripheral and central blood samples. Stimulation with various agonists yielded similar results of platelet surface expression of P-selectin and activated GPIIb/IIIa in peripheral and central samples, except for CrP-inducible expression of activated GPIIb/IIIa (median fluorescence intensity, MFI in P: 7.61 [0.00-24.66] vs. C: 4.12 [0.00-19.04], p < .001). The formation of leukocyte-platelet aggregate was similar in central and peripheral blood samples, both unstimulated and after stimulation with all above-mentioned agonists. In conclusion, peripheral vs. central venous blood sampling does not influence the assessment of platelet activation by flow cytometry in cirrhosis.Abbreviations: ACLD: advanced chronic liver disease; ADP: adenosine diphosphate; ALD: alcoholic liver disease; AYPGKF: PAR-4 agonist AYPGKF; CrP: collagen related protein; EPI: epinephrine; FACS: fluorescence-activated cell sorting; GP: glycoprotein; HVPG: hepatic venous pressure gradient; IQR: interquartile range; LPS: lipopolysaccharide; LSM: liver stiffness measurement; MFI: median fluorescence intensity; NAFLD: nonalcoholic fatty liver disease; PAM: lipopeptide Pam3CSK4; PAR: protease-activated receptor; PBS: phosphate-buffered saline; PH: portal hypertension; TIPS: transjugular intrahepatic portosystemic stent shunt; TLR: toll-like receptor; TRAP-6: thrombin receptor-activator peptide-6; vWF: von Willebrand factor.

Comparison of Aggregometry with Flow Cytometry for the Assessment of Agonists´-Induced Platelet Reactivity in Patients on Dual Antiplatelet Therapy.

Data on the agreement between aggregometry and platelet activation by flow cytometry regarding the measurement of on-treatment platelet reactivity to arachidonic acid (AA) and adenosine diphosphate (ADP) are scarce. We therefore sought to compare three platelet aggregation tests with flow cytometry for the assessment of the response to antiplatelet therapy. Platelet aggregation in response to AA and ADP was determined by light transmission aggregometry (LTA), the VerifyNow assays, and multiple electrode aggregometry (MEA) in 316 patients receiving aspirin and clopidogrel therapy after angioplasty with stent implantation. AA- and ADP-induced P-selectin expression and activated glycoprotein (GP) IIb/IIIa were determined by flow cytometry. LTA, the VerifyNow P2Y12 assay and MEA in response to ADP correlated significantly (all p<0.001), and the best correlation was observed between LTA and the VerifyNow P2Y12 assay (r = 0.63). ADP-induced platelet reactivity by all aggregation tests correlated significantly with ADP-induced P-selectin expression and activated GPIIb/IIIa (all p<0.001). The best correlation was seen between the VerifyNow P2Y12 assay and activated GPIIb/IIIa (r = 0.68). The platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP were significantly higher in patients with high on-treatment residual platelet reactivity (HRPR) to ADP by all test systems (all p<0.001). A rather poor correlation was observed between AA-induced platelet reactivity by LTA and the VerifyNow aspirin assay (r = 0.15, p = 0.007), while both methods did not correlate with MEA. AA-induced platelet reactivity by all aggregation tests correlated significantly, but rather poorly with AA-induced P-selectin expression (all p<0.05), while only AA-induced platelet reactivity by LTA correlated significantly with AA-induced activated GPIIb/IIIa (r = 0.21, p<0.001). The platelet surface expression of P-selectin in response to AA was significantly higher in patients with HRPR by LTA AA and MEA AA (both p<0.02). In contrast, P-selectin expression in response to AA was similar in patients without and with HRPR by the VerifyNow aspirin assay (p = 0.5), and platelet surface activated GPIIb/IIIa in response to AA did not differ significantly between patients without and with HRPR to AA by all test systems (all p>0.1). In conclusion, ADP-induced platelet reactivity by aggregometry translates partly into flow cytometry. In contrast, AA-induced platelet reactivity correlates poorly between different platelet aggregation tests, and between aggregometry and flow cytometry. Overall, both approaches capture different aspects of platelet function and are therefore not interchangeable in the assessment of agonists´-induced platelet reactivity. Clinical outcome data are needed to determine which test systems and settings are associated with different in vivo consequences.

Discrepancies in assessment of adherence to antiplatelet treatment after myocardial infarction.

The poor response to clopidogrel is multifactorial and includes, amongst others, low patient adherence to medication. The aim of this study was to assess the reported patient adherence to treatment with clopidogrel and confront it with adherence assessed by drug availability. We evaluated determinants of adherence and its impact on platelet aggregation and clinical outcome. The study population comprised 184 patients treated with primary percutaneous coronary intervention for acute myocardial infarction. Follow-up visits were scheduled at 3, 6 and 9 months after discharge. Patient adherence to clopidogrel was defined according to self-reported drug intake and verified based on data from the National Health Fund regarding the purchase of prescribed drugs. The patients were judged as adherent when the proportion of drug availability exceeded 80%. According to drug availability, 100 (54.3%) patients were adherent and 84 (45.7%) were nonadherent. The analysis identified the following factors as predictors of low adherence (<80%): adenosine diphosphate-induced platelet aggregation (ADP-PA) during hospitalization ≤45 U, male gender and occurrence of ST-elevation myocardial infarction [(STEMI) vs. non-STEMI (NSTEMI)], while three-vessel disease was predictive of high adherence to medication. Compared with drug availability-based assessment, self-reported drug intake was significantly different: 172 (94.5%) patients reported regular and 10 (5.5%) patients reported irregular intake of clopidogrel. Clinical follow-up suggested that the self-reported nonregular clopidogrel intake may discriminate patients with a high risk of cardiovascular events. We demonstrated a huge discrepancy between the two most widely used methods for the evaluation of adherence to clopidogrel in secondary prevention treatment in patients after STEMI and NSTEMI. ADP-PA during hospitalization ≤45 U, male gender and STEMI (vs. NSTEMI) were independent predictors of nonadherence while three-vessel disease was independently predictive of adherence to treatment with clopidogrel in the investigated population.

Assessment of DNA binding to human Rad51 protein by using quartz crystal microbalance and atomic force microscopy: effects of ADP and BRC4-28 peptide inhibitor.

The interaction of human Rad51 protein (HsRad51) with single-stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au-ArSO3 H. The Au-ArSO3 H layer was activated by using a solution of PCl5 in CH2 Cl2 to give a Au-ArSO2 Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4-28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4-28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA-protein interactions and for the screening of inhibitors.

High on-treatment platelet reactivity in patients with ischemic cerebrovascular disease: assessment of prevalence and stability over time using four platelet function tests.

High on-treatment platelet reactivity (HTPR), referred to as a higher than expected platelet reactivity in patients under antiplatelet therapy, could influence outcome in cerebrovascular disease (CVD), but its prevalence and its stability over time is uncertain. Platelet reactivity was assessed in 18 patients with ischemic stroke/transient ischemic attack (TIA) 7 days (D7) and 90 days (D90) after prescription of clopidogrel, using four methods: light transmission aggregometry with 5 µmol/l ADP (LTA-ADP), vasodilator-stimulated phosphoprotein (VASP), Verify Now P2Y12 and platelet function analyzer (PFA) P2Y. HTPR was defined as LTA-ADP more than 46%; PFA-100-P2Y closure time less than 106 s; VerifyNow P2Y12, PRU greater than 235, VASP, PRI greater than 50%. Patients displayed, both at D7 and D90, a marked inhibition of platelet reactivity towards ADP in all tests as compared with reference levels. Correlations between the results obtained with all the tests at D7 and D90 and between measurements on each day in each test were low-to-moderate. The prevalence of HTPR for all the tests was 40% at D7 and 42% at D90. There was a moderate degree of agreement (k statistic < 0.5) between tests with regard to categorizing patients as HTPR/No-HTPR (D7 and D90). The on-clopidogrel platelet reactivity phenotype, HTPR/No-HTPR, remained stable in 55-72% of patients, depending on the test. A high prevalence of HTPR is found among CVD patients treated with clopidogrel and this platelet reactivity phenotype remains over time. There is poor agreement between the different platelet function tests for categorizing the platelet reactivity phenotype in these patients. The new PFA-100 P2Y equals other platelet function assays for evaluating HTPR in CVD.

Assessment of orally dosed commercial silver nanoparticles on human ex vivo platelet aggregation.

Enhanced in vitro human and ex vivo rat platelet aggregation from direct exposure to silver nanoparticles is previously reported. Given the increasing human use of engineered silver nanoscale products, platelet aggregation prompted by silver nanoparticles may contribute to human cardiovascular events. To understand how direct washed platelet exposure to silver nanoparticles translates to ex vivo platelet aggregation, the authors conducted a placebo-controlled, single-blind, dose-monitored, cross-over study design in 18 healthy human volunteers. After 2 weeks of daily oral silver nanoparticle ingestion, platelet aggregation was evaluated by light transmission aggregometry in response to collagen and ADP agonists, both at baseline and after silver nanoparticle or placebo diluent oral dosing. Final percent aggregation (PA) and the changes in PA were determined using a paired design (i.e., active and placebo solutions). Enhanced ex vivo platelet activation was not detectable at peak serum silver concentrations <10 µg/L. Further studies of colloidal silver nanoparticles on human platelet activities are warranted.

Methodological considerations for the assessment of ADP induced platelet aggregation using the Multiplate® analyser.

Factors affecting the Multiplate® assay's analytical precision have not been well defined. We investigated the effect of methodological factors on the measurement of ADP-induced platelet aggregation using the Multiplate® assay. ADP-induced platelet aggregation was analysed in whole blood using the Multiplate® assay. We tested the reproducibility of measurement, the effect of different anticoagulants (hirudin, citrate and heparin) and the effect of time delay (15, 30, 45, 60, 120 and 180 minutes) between sampling and analysis in patients. The use of a manual calibrated pipette with the Multiplate® analyser was also tested. The mean coefficient of variation (CV) using the manufacturers recommended methods was 10.8 ± 8.7% (n = 30). When compared to hirudin (359.5 ± 309 AU*min) the use of heparin (521.0 ± 316 AU*min, p = 0.0015) increased platelet aggregation, while the use of sodium citrate (245.0 ± 209 AU*min, p = 0.003) decreased the platelet aggregation (n = 20). The addition of CaCl2 to the citrate-anticoagulated blood resulted in platelet aggregation levels similar to hirudin. Platelet aggregation varied with time delay (n = 20). When compared to platelet aggregation at 30 minutes (391.1 ± 283 AU*min), platelet aggregation was reduced at 60 minutes (335.2 ± 251.6 AU*min, p < 0.05), 120 minutes (198.8 ± 122.9 AU*min, p < 0.001) and 180 minutes (160.7 ± 92 AU*min, p < 0.001). The use of a manual calibrated pipette did not significantly reduce the mean CV in the assay (n = 20). Methodological factors such as the anticoagulant used and the time delay should be standardised where possible to reduce variability, and allow thresholds derived from one study to be comparable across multiple studies.

Comparison of venous and arterial blood sampling for the assessment of platelet aggregation with whole blood impedance aggregometry.

Platelet monitoring is presently under evaluation in the clinic as a tool to improve antiplatelet treatment in patients with coronary artery disease (CAD). Measuring platelet function has, however, many inherent problems. It is important not only to evaluate the method used, but also to evaluate and standardize sampling and sample handling. As platelet monitoring is often performed in connection to coronary angiography and percutaneous coronary interventions, arterial sampling may be more convenient. However, in the outpatient follow-up setting venous sampling is, for obvious reasons, more practical and convenient. In the present study we compared platelet aggregation in blood collected from the arterial sheath to blood collected from the antecubital vein using multiple electrode aggregometry in whole blood in 28 patients with CAD. We found that sampling from artery and vein give similar data and that an identical number of patients with insufficient antiplatelet responses ('low responders' to aspirin and clopidogrel, respectively, according to predefined criteria) were detected with respect to adenosine diphosphate induced and arachidonic-acid induced aggregation. Thus both arterial and venous blood samples can be used in the monitoring of platelet function when multiple electrode aggregometry is applied to detect 'low responders'.

Assessment and characterization of purinergic contractions and relaxations in the rat urinary bladder.

The aim of the present study was to assess the purinoceptor functional responses of the urinary bladder by using isolated rat urinary bladder strip preparations. ATP elicited a transient bladder contraction followed by a sustained relaxation and ADP, UDP and UTP generated predominantly potent relaxations (relaxatory potencies: ADP = ATP > UDP = UTP). The ATP contractions were desensitized with the P2X(1/3) purinoceptor agonist/desensitizer alpha,beta-meATP and reduced by the P2 purinoceptor antagonist PPADS but unaffected by the P2 purinoceptor antagonist suramin. Electrical field stimulation (1-60 Hz) evoked frequency-dependent bladder contractions that were decreased by incubation with alpha,beta-meATP but not further decreased by PPADS. Suramin antagonized relaxations generated by UDP but not those by ADP, ATP or UTP. PPADS antagonized and tended to antagonize UTP and UDP relaxations, respectively, but did neither affect ADP nor ATP relaxations. ADP relaxations were insensitive to the P2Y(1) purinoceptor antagonist MRS 2179 and the ATP-sensitive potassium channel antagonist glibenclamide. The ATP relaxations were inhibited by the P1 purinoceptor antagonist 8-p-sulfophenyltheophylline but unaffected by the A2A adenosine receptor antagonist 8-(3-chlorostyryl)caffeine and glibenclamide. Adenosine evoked relaxations that were antagonized by the A2B adenosine receptor antagonist PSB 1115. Thus, in the rat urinary bladder purinergic contractions are elicited predominantly by stimulation of the P2X(1) purinoceptors, while UDP/UTP-sensitive P2Y purinoceptor(s) and P1 purinoceptors of the A2B adenosine receptor subtype are involved in bladder relaxation.

Evaluation of the platelet count drop method for assessment of platelet function in comparison with "gold standard" light transmission aggregometry.

INTRODUCTION: Hyporesponsiveness to antiplatelet agents has been linked to an increased risk of major adverse cardiovascular events. However, light transmission aggregometry (LTA), the gold standard methodology for assessing platelet function, requires expertise and is labour-intensive, which render its use in clinical settings impractical. We assessed whether platelet count drop (PCD), a technique widely available in any haematology laboratory, could replace LTA in testing for inhibition of platelet aggregation induced by antiplatelet agents. MATERIALS AND METHODS: One hundred and sixty-one coronary artery disease patients taking aspirin alone and 91 patients taking a combination of aspirin and clopidogrel were enrolled. Platelet aggregation was measured by LTA and PCD stimulated with 1.6 mM of arachidonic acid (AA) for aspirin and 5 and 20 microM of adenosine diphosphate (ADP) for clopidogrel. RESULTS: Correlation between AA-induced LTA and PCD was nonexistent (r=-0.043, p=0.587), while correlation between ADP-induced LTA and PCD was low (r=0.374, p<0.0001 for ADP 5 microM and r=0.402, p<0001 for ADP 20 microM ). PCD, whether stimulated with AA or ADP, overestimated platelet aggregation as assessed by LTA, by 13-18%. The wide 95% limits of agreement suggest that the assays can disagree significantly in individual patients. CONCLUSIONS: Although the PCD method is widely available in non-specialized laboratories, our results demonstrate that there is poor correlation with the current gold standard, i.e. LTA. Thus, PCD should not be used in replacement of LTA to assess antiplatelet responsiveness.

Platelet concentrates transfusion in cardiac surgery and platelet function assessment by multiple electrode aggregometry.

BACKGROUND: Platelet dysfunction contributes to the pathophysiology of bleeding complications during and after cardiac surgery. In most surgical institutions, no peri-operative point-of-care monitoring of platelet function is used. We evaluated the usefulness of the Multiplate platelet function analyser based on impedance aggregometry for identifying groups of patients at a high risk of transfusion of platelet concentrates (PC). METHODS: Platelet function parameters were determined in 60 patients before and after routine cardiac surgery. Impedance aggregometry measurements were performed on Multiplate using ADP (ADPtest), collagen (COLtest) and thrombin receptor activating peptide (TRAPtest) as platelet activators. The correlations between the aggregometry results and the transfusion of PC were calculated. The results of the aggregation tests were also divided into tertiles and the differences in PC transfusion between the low and the high tertile were assessed. RESULTS: Low aggregometry delimited groups of patients with significantly higher PC transfusion. In the receiver operating characteristic curve, low pre-operative aggregation in the ADPtest identified patients with high total transfusion of PC (area under the curve 0.74, P=0.001), while the ADPtest performed at the end of the operation identified patients with high PC transfusion on the intensive care unit (ICU) (area under the curve 0.76, P=0.002). CONCLUSIONS: Near-patient platelet aggregation may allow the identification of patients with enhanced risk of PC transfusion, both pre-operatively and upon arrival on the ICU.

Assessment of VerifyNow P2Y12 assay accuracy in evaluating clopidogrel-induced platelet inhibition.

The emergence of point-of-care assays enabling bedside testing such as the VerifyNow P2Y12 system might prove useful in clinical settings. The aim of this study was to evaluate the ability of the VerifyNow P2Y12 assay to estimate the inhibition of platelet aggregation provided by clopidogrel in the absence of baseline off-drug aggregation data. Sixty-eight patients with coronary artery disease scheduled to initiate clopidogrel therapy underwent platelet aggregation testing by VerifyNow P2Y12 at baseline and after clopidogrel administration. The inhibition reported by the VerifyNow assay (relative to thrombin receptor activating peptide-induced platelet aggregation, serving as baseline) was compared with that calculated with the actual adenosine diphosphate-induced baseline obtained with the same methodology. The postclopidogrel thrombin receptor activating peptide-induced aggregation showed a great discordance with that induced by adenosine diphosphate before clopidogrel with a bias of 24 units (95% limits of agreement from -142 to 190 units). Moreover, the inhibition reported by the assay overestimated the standard before-and-after testing data by an average of 8% (95% limits of agreement from -49% to 65%), making its use without a true baseline comparator unsatisfactory. The VerifyNow P2Y12 assay fails to accurately quantify platelet inhibition achieved by clopidogrel compared with before-and-after testing. Further studies are required to establish the clinical usefulness of the VerifyNow P2Y12 assay to accurately predict the occurrence of major adverse cardiovascular events in patients with reduced clopidogrel efficacy before it can be implemented in clinical practice. At present, the use of this assay in clinical care cannot be recommended for monitoring clopidogrel therapy.

Assessment of clopidogrel responsiveness: measurements of maximum platelet aggregation, final platelet aggregation and their correlation with vasodilator-stimulated phosphoprotein in resistant patients.

BACKGROUND: Controversy surrounds the optimal platelet aggregation measurement to assess clopidogrel non-responsiveness. The P2Y12 reactivity ratio (PRR) determined by vasodilator-stimulated phosphoprotein phosphorylation levels has been used to indicate the extent of P2Y(12) blockade. OBJECTIVES: We sought to compare the prevalence of non-responsiveness measured by maximum (MA) and 6 min aggregation (FA) and correlate these measurements with PRR in patients with non-responsiveness. METHODS: MA and FA were measured in stented patients (n=100) before and after clopidogrel treatment. The PRR was determined in 22 non-responsive patients. Responsiveness was defined as pre-treatment minus post-treatment aggregation; and non-responsiveness was defined as <10% change in platelet aggregation. RESULTS: Responsiveness was greater as determined by FA, p=0.006 (5 microM ADP) and p=0.003 (20 microM ADP)). There was a strong correlation between MA and FA stimulated by 5 microM (r=0.84, p<0.0001) and 20 microM ADP (r=0.90, p<0.001). The prevalence of non-responsiveness rose with agonist concentration but did not differ significantly between methods: 5 microM ADP [22% (MA) vs. 17% (FA), p=0.186] and 20 microM ADP [33% (MA) vs. 29% (FA), p=0.270]. PRR correlated with both MA (r=0.66, p<0.001) and FA (r=0.74, p<0.001) in non-responsive patients indicating incomplete receptor blockade. CONCLUSION: Clopidogrel responsiveness is higher when measured by FA as compared to MA. However, these measurements are equivalent in determining the prevalence of non-responsiveness: FA and MA are affected to the same degree in patients with non-responsiveness. These findings are relevant to ongoing studies assessing platelet inhibition by P2Y(12) inhibitors and support previous studies that employed MA to assess non-responsiveness.

Monitoring platelet inhibition after clopidogrel with the VerifyNow-P2Y12(R) rapid analyzer: the VERIfy Thrombosis risk ASsessment (VERITAS) study.

INTRODUCTION: Clopidogrel inhibits platelet P2Y12 ADP receptors, while ADP, as an inductor of aggregation, stimulates both P2Y12 and P2Y1 platelet receptors. Despite a clinical loading dose routine with clopidogrel, some patients still experience coronary stent thrombosis suggesting persistent platelet activation. The VerifyNow-P2Y12 is a rapid assay that test platelet activity over 3 min and uses of the combination of ADP and prostaglandin E1 (PGE1) to directly measure the effects of clopidogrel on the P2Y12 receptor. ADP is used to maximally activate the platelets by binding to the P2Y1 and P2Y12 platelet receptors, while PGE1 is used to suppress the ADP-induced P2Y1-mediated increase in intracellular calcium levels. OBJECTIVE: The VERIfy Thrombosis risk ASsessment (VERITAS) was a prospective study designed to measure platelet response to clopidogrel therapy in subjects with multiple risk factors or history of vascular disease using this novel point-of-care assay. METHODS: 166 participants were enrolled in 4 participating sites. Data from 147 participants were analyzed after exclusion of 19 patients due to protocol violations. Platelets were assessed twice at baseline (before clopidogrel) and at 24 h post-loading 450 mg (110 participants) or 7 days after chronic clopidogrel treatment (75 mg/day) (37 patients). All participants received aspirin 81-325 mg for at least 2 days before the study enrollment. Results from the VerifyNow-P2Y12 assay are reported in P2Y12 reaction units (PRU). RESULTS: Clopidogrel therapy resulted in a mean 64.0+/-25.3% PRU reduction. No participant reached PRU inhibition below 10% of baseline. Distribution of PRU values for the VerifyNow-P2Y12 assay shows a separation from baseline to post-clopidogrel assay values with some overlap due to high inter-individual variations in response. CONCLUSIONS: VerifyNow-P2Y12 is a reliable, fast and sensitive device suitable for monitoring of platelet inhibition during clopidogrel therapy.

Assessment of the antiplatelet effects of low to medium dose aspirin in the early and late phases after ischaemic stroke and TIA.

Vascular events commonly recur in stroke patients on aspirin, and may reflect incomplete inhibition of platelet function with aspirin therapy. The platelet function analyser (PFA-100) activates platelets by aspirating a blood sample at a moderately high shear rate through a capillary to a biologically active membrane with a central aperture. The membrane is coated with collagen, and either ADP (C-ADP) or epinephrine (C-EPI). The time taken for activated platelets to adhere, aggregate, and occlude the aperture is called the closure time. Previous studies have shown that aspirin prolongs the C-EPI closure time, without prolongation of the C-ADP closure time, in the majority of control subjects. We hypothesised that the PFA-100 would provide a sensitive assay for the detection of early and convalescent phase cerebrovascular disease (CVD) patients who had incomplete inhibition of platelet function with aspirin. We investigated potential cyclooxygenase-dependent and -independent mechanisms that might influence the responsiveness to aspirin using the PFA-100. Patients were studied during the early (< or = 4 weeks, n=57) and convalescent phases ((< or = 3 months, n=46) after ischaemic stroke or TIA. To investigate potential mechanisms that could contribute to aspirin responsiveness on the PFA-100, we measured von Willebrand factor antigen levels, and carried out platelet aggregometry experiments in platelet-rich plasma in response to sodium arachidonate (1 mM) and ADP (5 microM). Sixty percent of patients in the early phase and 43% of patients in the convalescent phase did not have prolonged C-EPI closure times on 75-300 mg of aspirin daily, and were defined as aspirin non-responders. Median C-ADP closure times were significantly shorter in aspirin non-responders than aspirin-responders in both the early and convalescent phases after symptom onset (P=0.008), suggesting platelet hyper-reactivity to collagen or ADP in the aspirin non-responder subgroup. There was a significant inverse relationship between plasma von Willebrand factor antigen levels and C-EPI closure times in both early and convalescent phase CVD patients (P=0.008). Mean von Willebrand factor antigen levels were significantly higher in aspirin non-responders than aspirin responsive patients in the early (P=0.001), but not convalescent phase (P=0.2) after stroke and TIA. None of the patients studied were defined as being aspirin-resistant using sodium arachidonate- or ADP-induced platelet aggregometry. A large proportion of ischaemic CVD patients have incomplete inhibition of platelet function with low to medium dose aspirin using the PFA-100. The results suggest that cyclooxygenase-independent mechanisms, including elevated von Willebrand factor antigen levels, play an important role in mediating aspirin non-responsiveness on the PFA-100.