Incomplete inhibition of platelet function as assessed by the platelet function analyzer (PFA-100) identifies a subset of cardiovascular patients with high residual platelet response while on aspirin

Sixty-six patients with a history of ischemic events (myocardial infarction, unstable angina, or stroke) on chronic aspirin therapy were studied by different platelet function tests: 37 patients had suffered a recurrent event while on aspirin and 29 were without recurrences. Based on results from light transmission aggregometry (LTA) induced by arachidonic acid (AA) and serum TxB(2) both COX-1-dependent methods, only one patient could be identified as aspirin "resistant". However, when methods only partially-dependent on platelet COX-1 activity were considered, the prevalence of aspirin non-responders ranged, according to the different tests, from 0 to 52%. No difference was observed between patients with recurrences and those without. Among patients with recurrent events, those with an incomplete inhibition of platelet function, as assessed by the PFA-100, had significantly higher residual serum TxB(2) (2.4?±?2.4?ng/mL vs 0.4?±?0.1?ng/mL, p?=?0.03), residual LTA-AA (9.2?±?10.6% vs 2.0?±?1.6%, p?=?0.008), LTA-Coll (49.3?±?14.6% vs 10.2?±?8.3%, p?=?0.007) and LTA-ADP (50.9?±?16.2% vs 34.3?±?11.0%, p?=?0.04). In conclusion, laboratory tests solely exploring the AA-mediated pathway of platelet function, while being the most appropriate to detect the effect of aspirin on its pharmacologic target (platelet COX-1), may fail to reveal the functional interactions between minimal residual TxA(2) and additional stimuli or primers potentially leading to aspirin-insensitive platelet aggregation. High residual platelet response in platelet function tests only partially dependent on COX-1 may reveal a condition of persistent platelet reactivity in a subset of aspirin-treated patients characterizing them as a subgroup at higher vascular risk.     

Aspirin response: Differences in serum thromboxane B2 levels between clinical studies

Serum thromboxane B2 (TxB2) is a specific marker of platelet inhibition by aspirin. Yet, TxB2 levels differ by up to 10-fold between some aspirin-treated patient cohorts. This study aimed to identify factors responsible for differences in serum TxB2 between cohorts in the ADRIE study (n?=?657) and the BOSTON study (n?=?678) of aspirin-treated cardiovascular patients originally tested with different ELISA assays. TxB2 levels were assessed in representative subgroups of the two cohorts (34 samples in BOSTON and 39 in ADRIE) by both ELISAs, as well as liquid chromatography and tandem mass spectroscopy (MS). A multivariate analysis was performed on the whole cohort database to identify determinants of the difference of TxB2 levels between cohorts. There was no systematic bias between the original ELISA TxB2 values and the MS values and the median difference was small, 0.12?ng/ml, thus not explaining the difference between median TxB2 levels in the two study populations (7 and 0.6?ng/ml in the ADRIE and BOSTON studies, respectively). In the combined dataset of the ADRIE and BOSTON cohorts (n?=?1342), body mass index, age, gender, aspirin dose, time from aspirin intake to blood draw, NSAID intake, platelet count and C-reactive protein were significantly associated with TxB2 levels. After adjustment for patient characteristics, the difference between cohorts did not decrease. Unexplained differences in serum TxB2 levels in different populations of aspirin-treated cardiovascular patients suggest that further studies are needed to confirm the role of serum TxB2 level as a prognostic factor or rather as a marker of therapeutic observance.     

How to test the effect of aspirin and clopidogrel in patients on dual antiplatelet therapy?

Dual antiplatelet therapy with clopidogrel and aspirin is frequently used for the prevention of recurrent ischemic events. Various laboratory methods are used to detect the effect of these drugs administered in monotherapy, however their value in dual therapy has not been explored. Here, we determined which methods used for testing the effect of clopidogrel or aspirin are influenced by the other antiplatelet agent. One arm of the study included 53 ischemic stroke patients being on clopidogrel monotherapy showing effective inhibition of the P2Y₁₂ ADP receptor. Laboratory tests routinely used for the detection of aspirin resistance (arachidonic acid (AA)-induced platelet aggregation/secretion, AA-induced thromboxane B₂ (TXB₂) production in platelet-rich plasma and VerifyNow Aspirin assay) were carried out on samples obtained from these patients. The other arm of the study involved 52 patients with coronary artery disease being on aspirin monotherapy. Methods used for testing the effect of clopidogrel (ADP-induced platelet aggregation and secretion, flow cytometric analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation and a newly developed P2Y₁₂-specific platelet aggregation (ADP[PGE₁] test)) were performed on samples obtained from these patients. Clopidogrel monotherapy significantly inhibited AA-induced platelet aggregation and secretion, moreover, AA-induced TXB₂ production was also significantly decreased. VASP phosphorylation and AA-induced platelet aggregation showed fair correlation in patients taking clopidogrel only. Clopidogrel did not inhibit the VerifyNow Aspirin test significantly. Aspirin monotherapy influenced ADP-induced platelet aggregation and secretion, but did not have an effect on VASP phosphorylation and on the ADP[PGE₁] platelet aggregation test.     

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.     

Effects of tipranavir, darunavir, and ritonavir on platelet function, coagulation, and fibrinolysis in healthy volunteers

The use of HIV protease inhibitors (PIs) as part of antiretroviral therapy in the treatment of HIV-1 infection may be associated with an increased risk of bleeding. This prospective, randomized, open-label trial in healthy volunteers compared the effects of tipranavir/ritonavir (TPV/r), darunavir/ ritonavir (DRV/r), and ritonavir (RTV) alone on platelet aggregation after a single dose and at steady-state concentrations. Subjects were selected on the basis of normal platelet aggregation and arachidonic acid (AA)-induced platelet aggregation inhibition after administration of a single 325-mg dose of aspirin. All 3 PI therapies were administered twice daily for 10 days. In some but not all subjects, TPV/r inhibited AA-induced platelet aggregation and prolonged PFA-100? closure time with collagen-epinephrine cartridge, which was of lesser magnitude and consistency compared with aspirin, but greater when compared to DRV/r and RTV. At least 2 subjects in each treatment arm showed complete inhibition of AA-induced platelet aggregation on treatment, and the magnitude of change in all platelet-function tests did not correlate with PI plasma concentrations. Effects of TPV/r on platelet aggregation were reversed 24 hours after the last TPV/r dose. None of the PI treatments tested were associated with increases in bleeding time, decreases in plasma coagulation factors, or increase in fibrinolysis. There was large inter-patient variability in antiplatelet effect for all PI treatments, ranging from no effect to complete inhibition of AA-induced platelet aggregation.     

Aspirin sensitivity of platelet aggregation in diabetes mellitus

Although aspirin is cardioprotective in high-risk populations, many with diabetes mellitus (DM) are unresponsive to these benefits. We questioned whether cardiovascular unresponsiveness might be demonstrated by lack of aspirin sensitivity to in vitro platelet functions especially in subjects with poorly controlled diabetes. Six women and 4 men (48+/-8 years [mean+/-S.D.]), selected for poor control (glycohemoglobin 11.9+/-2.2%) and 10 sex-age (+/-5 years) matched controls received 81 mg aspirin daily. There was a 2-week washout from aspirin and related drugs. After the aspirin dose on day-7, blood for platelet aggregation assays, and 24-h urine for 2,3 dinor thromboxane B2 (TxB2) and 2,3 dinor 6-keto (PGF1alpha) were obtained. Aspirin sensitivity was defined as inhibition (i.e., lower than expected) platelet aggregation after exposure to an agonist. Those with diabetes and controls were sensitive to aspirin inhibition of platelet aggregation induced by 1.6 mM arachidonic acid (9.5+/-3.9% versus 9.1+/-3.1%, normal range 40-100%) and by 0.83 microg/mL collagen (17.4+/-13.9% versus 13.2+/-9.3%, normal range 60-93%), respectively. Aspirin sensitivity to 2 microM ADP was present in five with diabetes and five controls. Urinary prostaglandin metabolites were suppressed below reference ranges, without differences between those with DM or controls for TxB2 (350+/-149 pg/mg versus 348+/-93 pg/mg creatinine) and PGF1alpha (255+/-104 pg/mg versus 222+/-88 pg/mg creatinine). In conclusion, in poorly controlled diabetes, there was no differential lack of aspirin sensitivity to platelet aggregation, or lack of aspirin suppression of urinary TxB2 or PGF1alpha, compared with controls on aspirin. Despite suppression of urinary prostaglandin metabolites, aspirin resistance was most apparent to ADP-mediated platelet aggregation. It is not known what level of inhibition of in vitro tests is necessary for the cardioprotective benefits of aspirin in diabetes mellitus. Thus, the lack of aspirin protection in diabetes may be due to undefined aspects of platelet function.     

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.     

Aspirin resistance: does it exist?

Aspirin irreversibly inhibits platelet cyclooxygenase-1 (COX-1). Aspirin sensitivity can be measured easily by its inhibition of arachidonic acid (AA) -induced platelet aggregation. Aspirin resistance has to be defined by its inability to inhibit COX-1. By using this definition, aspirin resistance very likely does not exist. A specific rapid laboratory test using either AA-induced platelet aggregation or AA-induced malondialdehyde production in platelet-rich plasma is needed to test aspirin sensitivity. The reports on so-called aspirin resistance are usually due to noncompliance of aspirin intake or consumption of inadequate doses of aspirin. In addition, data generated from using nonspecific platelet function tests have added confusion to this observed phenomenon of aspirin resistance.     

A study of aspirin and clopidogrel in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is characterised by in situ thrombosis and increased thromboxane (Tx) A2 synthesis; however, there are no studies of antiplatelet therapy in IPAH. The aim of the current study was to determine the biochemical effects of aspirin (ASA) and clopidogrel on platelet function and eicosanoid metabolism in patients with IPAH. A randomised, double-blind, placebo-controlled crossover study of ASA 81 mg once daily and clopidogrel 75 mg once daily was performed. Plasma P-selectin levels and aggregometry were measured after exposure to adenosine diphosphate, arachidonic acid and collagen. Serum levels of TxB2 and urinary metabolites of TxA2 and prostaglandin I2 (Tx-M and PGI-M, respectively) were assessed. A total of 19 IPAH patients were enrolled, of whom nine were being treated with continuous intravenous epoprostenol. ASA and clopidogrel significantly reduced platelet aggregation to arachidonic acid and adenosine diphosphate, respectively. ASA significantly decreased serum TxB2, urinary Tx-M levels and the Tx-M/PGI-M ratio, whereas clopidogrel had no effect on eicosanoid levels. Neither drug significantly lowered plasma P-selectin levels. Epoprostenol use did not affect the results. In conclusion, aspirin and clopidogrel inhibited platelet aggregation, and aspirin reduced thromboxane metabolite production without affecting prostaglandin I2 metabolite synthesis. Further clinical trials of aspirin in patients with idiopathic pulmonary arterial hypertension should be performed.     

Bimodal response to aspirin loading in acute ST-elevation myocardial infarction

Patients with stable coronary disease who exhibit platelet hypo-responsiveness to aspirin (ASA) have worse outcomes. Little data exist regarding platelet response to ASA in ST-elevation myocardial infarction (STEMI) patients. Our objective was to assess acute platelet response to ASA loading in STEMI patients undergoing primary percutaneous coronary intervention (PCI). The study comprised 102 consecutive patients with STEMI. All patients received a loading dose of 300?mg chewable ASA upon admission. Platelet reactivity was assessed immediately prior to primary PCI, at a median of 95(63?139) minutes after ASA loading. A bimodal response to arachidonic acid (AA) stimulation was observed, such that two distinct populations could be discerned: “good responders” had a mean AA-induced platelet aggregation of 36?±?11% vs. 79?±?9% for “poor responders.” Despite equivalent demographic, clinical, and angiographic characteristics, good responders were significantly more likely to demonstrate early ST-segment resolution ≥70% after primary PCI (80% vs. 48%, p?=?0.001), suggestive of better myocardial reperfusion. Early inhibition of AA-induced platelet aggregation post-ASA loading in the setting of STEMI is associated with better tissue reperfusion; however, a sizeable proportion of patients do not achieve significant inhibition of AA-induced platelet aggregation in response to ASA loading at the time of primary PCI.     

Inhibition of platelet thromboxane generation by suloctidil in man

Oral administration of 100 or 200 mg suloctidil to healthy volunteers resulted in serum thromboxane B2 (TxB2) inhibition. This reached a maximum level between 90 min and 4 h after drug ingestion. TxB2 levels returned to 75% of basal values 6 h after 100 mg and more than 8 h after 200 mg, reaching control values at 24 h. Different experiments were performed to define the metabolic step at which suloctidil acts to inhibit serum TxB2 generation. Suloctidil prevented TxB2 synthesis also when platelet-rich plasma was stimulated by exogenous arachidonic acid or whole blood was clotted in the presence of exogenous arachidonic acid. This rules out the possibility that it inhibits phospholipases. No rediversion of prostaglandin synthesis after suloctidil occurred concomitantly with TxB2 inhibition, suggesting that the drug is not a selective thromboxane synthase inhibitor. In contrast, a significant reduction of serum PGE2 formation was found, suggesting a mechanism of action of suloctidil involving inhibition of cyclo-oxygenase. This was supported by the finding that PGI2 production by rat smooth muscle cells stimulated with arachidonic acid was significantly prevented by suloctidil in vitro. Suloctidil, however, did not prevent aspirin-induced inhibition of serum TxB2 generation. Blockade of platelet cyclo-oxygenase activity by suloctidil is therefore exerted at a level different from that of aspirin. In conclusion, suloctidil is a relatively weak nonselective cyclo-oxygenase inhibitor whose effect on platelet TxA2 production hardly accounts for its reported inhibitory effect on platelet aggregation.     

Inconsistency of different methods for assessing ex vivo platelet function: relevance for the detection of aspirin resistance

Background

Assays to evaluate platelet function are often interchangeably used to assess “resistance” to aspirin. We compared different platelet function assays in patients treated or untreated with aspirin.

Design and Methods

Platelet function was evaluated in 162 subjects, 85 of whom were not being treated with any antiplatelet drug and 77 of whom were receiving chronic therapy with low-dose aspirin. Platelet Function Analyzer collagen/ADP- and collagen/epinephrine closure times, as well as light transmittance aggregometry in response to ADP, collagen and arachidonic acid (this last in 47 aspirin-treated patients) were determined. In 43 aspirin-treated patients, serum thromboxane B₂ levels were also measured.

Results

In untreated patients, collagen/ADP- and collagen/epinephrine-closure times were correlated with each other (r=0.5, P=0.0001), but did not correlate with ADP- or collagen-induced aggregation. In patients treated with aspirin, collagen/ADP-closure time values were not different from those in untreated patients, while the collagen/epinephrine-closure time was prolonged. ADP-induced aggregation was unaffected by aspirin, while collagen-induced aggregation was reduced. Arachidonic acid-induced aggregation was almost completely suppressed (% maximum light transmittance aggregometry=5±13%). There was, however, no correlation between the various platelet function tests. Serum thromboxane B₂, an index of platelet cyclooxygenase-1 activity, was almost completely suppressed (down to 8±17 ng/mL) in treated patients, and was not correlated with arachidonic acid-, ADP- and collagen-induced aggregation or with collagen/ADP-closure time, but was inversely correlated with collagen/epinephrine-closure time.

Conclusions

There is a high heterogeneity of results of tests evaluating inhibition of platelet function by aspirin, and the results of functional tests do not match biochemical measurement of cyclooxygenase-1 activity. Extreme caution should, therefore, be used in defining “resistance” to aspirin on the basis of the results of these tests.     

Inhibition of human platelet aggregation and thromboxane-B2 production by melatonin: evidence for a diurnal variation

The effects of melatonin on platelet aggregation and thromboxane-B2 (TxB2) production induced by 1-4 x 10(-6) M adenosine diphosphate (ADP) or 0.6 x 10(-3) M arachidonic acid (AA) were assessed in platelet-rich plasma (PRP). Micromolar concentrations of melatonin inhibited in a dose-dependent way ADP-induced platelet aggregation with individual inhibitions 40% or more at 10(-6)-10(-5) M. A significant depression of AA-induced platelet aggregation was observed only at 10(-5)-10(-4) M melatonin. Morning (0830 h)-evening (1800 h) studies of ADP-induced platelet aggregation in seven normal men showed a higher sensitivity at 1800 h when analyzed as a global inhibitory effect of melatonin (P less than 0.01). Moreover, only during the evening hours did melatonin induce reversible aggregation, an index of inhibition of the platelet secretory process elicited by ADP exposure. No diurnal variability in melatonin inhibition of AA-induced aggregation was detected. TxB2 production elicited by AA in the evening was inhibited significantly in a concentration-related manner by a 2-min preincubation with 10(-9)-10(-5) M melatonin, while during the morning hours the inhibition was significant only at 10(-6) M or higher melatonin concentrations. In the case of ADP, the inhibition of TxB2 release attained significance at 10(-5)-M (0830 h) or 10(-6)-M concentrations (1800 h). In the presence of either stimulatory agent, melatonin depression of TxB2 generation was about 2-fold greater at 1800 h than at 0830 h. The diurnal changes in melatonin effect on TxB2 production were also observed in thrombin-stimulated washed platelets. The present data indicate the existence of circadian variations in platelet responsiveness to melatonin in humans.     

Platelet aggregation and thromboxane A2 production after adrenergic stimulation in young healthy humans

The effects of adrenergic stimulation on platelet aggregation (platelet aggregation ratio; PAR), beta-thromboglobulin (beta-TG) release and plasma thromboxane B2 (TxB2) levels were investigated in 25 healthy young volunteers. Adrenergic stimulation induced by cold application was checked by evaluating the changes in the calculated vascular resistance in the forearm. A prompt increase in platelet aggregates and plasma beta-TG and TxB2 concentrations was observed after adrenergic stimulation. PAR changed from resting values of 0.97 +/- 0.05 to 0.75 +/- 0.08 (p less than 0.001) at the end of cold application. At the same time, beta-TG plasma concentration increased from 32.09 +/- 19.64 to 135.48 +/- 37.97 ng/ml (p less than 0.001) and TxB2 plasma levels changed from 0.49 +/- 0.24 to 0.99 +/- 0.39 pmol/ml (p less than 0.001). TxB2 levels, but not PAR and beta-TG concentration came back to the resting values at the end of the observation period (10 min). Aspirin, as the lysine acetylsalicylate equivalent to 5 mg/kg i.v. of acetylsalicylic acid, although able to completely inhibit platelet cyclooxygenase failed to inhibit the plasma TxB2 increase induced by adrenergic stimulation. This strongly suggests that the increase in plasma TxB2 following adrenergic stimulation is of extraplatelet origin. Also beta-TG and PAR changes were not affected by aspirin administration.     

In vitro and ex vivo effects of indobufen on human platelet aggregation, the release reaction and thromboxane B2 production

We have done a comprehensive study in normal volunteers of the in vitro and ex vivo effects of the antiplatelet agent indobufen on platelet aggregation, the release reaction and thromboxane B2 (TxB2) production as induced by different concentrations of aggregating agents. At low concentrations (10 microM), indobufen completely inhibited secondary platelet aggregation, the release reaction and TxB2 production stimulated by ADP, epinephrine and low concentrations of platelet-activating factor (PAF acether). Higher concentrations of indobufen (100 microM) completely inhibited TxB2 production, platelet aggregation and ATP release induced by arachidonic acid (1 mM) or collagen (2 micrograms/ml). The inhibitory effect was partially overcome by higher concentrations of arachidonic acid (2 mM). Data obtained ex vivo 2 h after the oral administration of 200 mg indobufen to 8 normal volunteers were in keeping with those of the in vitro study. We conclude that indobufen inhibits platelet aggregation and the release reaction by inhibiting the platelet arachidonate pathway.     

Thrombopoietin and platelet aggregation in patients with stable coronary artery disease

Thrombopoietin (TPO) may facilitate platelet activation and aggregation. However, data on the impact of TPO on platelet aggregation in patients with stable coronary artery disease (CAD) are scarce. We aimed to investigate associations between TPO and platelet aggregation and activation in patients with stable coronary artery disease (CAD). We studied 900 stable CAD patients. Serum TPO was assessed by ELISA. Platelet aggregation was evaluated using the Multiplate Analyzer (agonists: arachidonic acid [AA] and collagen) and the VerifyNow Aspirin Assay. Platelet activation was evaluated by soluble (s)P-selectin. Cyclooxygenase-1 inhibition was evaluated by serum thromboxane B₂ (TXB₂). We found that TPO correlated weakly with platelet aggregation evaluated by Multiplate using AA (r = ?0.09, p = 0.01) and collagen as agonists (r = ?0.03, p = 0.43) and by VerifyNow (r = 0.07, p = 0.03). We found no correlation between TPO and sP-selectin (r = ?0.01, p = 0.70). Independent predictors of AA-induced platelet aggregation by Multiplate included high levels of sP-selectin and serum TXB₂, high platelet count, increasing age and body mass index, female sex, and active smoking. Independent predictors of TPO included low AA-induced platelet aggregation by Multiplate, high levels of hs-CRP, active smoking, and high platelet aggregation evaluated by VerifyNow. In conclusion, TPO levels did not correlate with platelet activation and only weak associations were found between TPO and platelet aggregation, suggesting that TPO did not substantially facilitate platelet aggregation in stable CAD patients.     

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.     

Platelet response to low-dose enteric-coated aspirin in patients with stable cardiovascular disease.

OBJECTIVES: We investigated whether use of low-dose enteric-coated (EC) aspirin for secondary prevention of cardiovascular events has sufficient bioavailability to achieve complete platelet cyclooxygenase (COX) inhibition in all individuals. BACKGROUND: Aspirin reduces cardiovascular morbidity and mortality in patients with pre-existing vascular disease; however, there is variability in the way individuals respond. Persistent normal platelet function despite therapy, referred to as "aspirin resistance," is associated with an increased risk of major cardiovascular events. METHODS: We studied 131 stable cardiovascular patients between March and September 2002 who were taking 75 mg EC aspirin. Serum thromboxane (TX) B2 levels were assayed as a measure of COX activity. Mean arachidonic acid (AA)-induced platelet aggregation > or =20% was deemed evidence of persistent platelet activity and an incomplete aspirin response. RESULTS: Patients of median age 63 years (61% men) were enrolled. Forty-four percent of patients had elevated serum TX B2 levels (>2.2 ng/ml). Arachidonic acid-induced platelet aggregation occurred more frequently in these patients (21% vs. 3%; p = 0.004). In all cases addition of exogenous aspirin during the assay abolished platelet aggregation. Patient weight and age were significant independent predictors of an incomplete response to EC aspirin (p = 0.025 and p < 0.001, respectively). These patients were also more likely to have a history of myocardial infarction (MI) (p = 0.038). CONCLUSIONS: Many patients who are prescribed low-dose EC aspirin for secondary prevention of cardiovascular events have persistent uninhibited platelet COX activity. Younger and heavier patients and those with a previous MI are most likely to have an inadequate response to treatment.     

Variation in thromboxane B2 concentrations in serum and plasma in patients taking regular aspirin before and after clopidogrel therapy

Dual antiplatelet therapy with aspirin and a P2Y₁₂ antagonist is widely prescribed for the prevention of thrombotic events in patients with an acute coronary syndrome or undergoing percutaneous coronary intervention (PCI). It is recognised that there is inter-individual variation in the antiplatelet effects of both drugs. Recent data also suggest that P2Y₁₂ antagonists can affect the response to aspirin. A direct indicator of the effect of aspirin on platelets is their ability to generate thromboxane, which if measured as the difference between the level of thromboxane B₂ in serum and plasma ([TxB2]_S-P) avoids the confounding effect of endogenous TxB2 production from other cells. We therefore analysed [TxB2]_S-P as a measure of aspirin response in a group of 123 patients undergoing elective PCI before and after the introduction of clopidogrel. In a subgroup of 40 patients taking aspirin alone, we compared [TxB2]_S-P and VerifyNow Aspirin for the assessment of aspirin response. There was a wide variation in plasma and serum TxB2 concentrations both before and after clopidogrel therapy but only 3.5% of patients had residual serum concentration of TxB2 > 10?ng/ml. There was a strong correlation between the pre and post clopidogrel levels of TxB2 (r?≥?0.78; p?=?0.001) and no significant difference in [TxB2]_S-P. There was no correlation between the magnitude of response to clopidogrel response and the generation of thromboxane B2. Correlation between [TxB2]_S-P and VerifyNow Aspirin was poor. We conclude that the use of a P2Y₁₂ antagonist does not influence the effect of aspirin on the ability of platelets to generate thromboxane. Therefore, measurement of TxB2 levels in serum, after subtracting the contribution from plasma, provides a measure of the response to aspirin in patients taking dual antiplatelet therapy.     

Very-low-dose twice-daily aspirin maintains platelet inhibition and improves haemostasis during dual-antiplatelet therapy for acute coronary syndrome

Higher aspirin doses may be inferior in ticagrelor-treated acute coronary syndrome (ACS) patients and reducing bleeding risk whilst maintaining antithrombotic benefits could improve outcomes. We characterized the pharmacodynamics of a novel dual-antiplatelet-therapy regimen consisting of very-low-dose twice-daily (BD) aspirin with standard-dose ticagrelor. A total of 20 ticagrelor-treated ACS patients entered a randomized crossover to take aspirin 20 mg BD (12-hourly) during one 14-day period and 75 mg once-daily (OD) in the other. After 14 days of treatment, serum thromboxane (TX)B₂ and light-transmittance aggregometry were assessed pre- and 2 h post-morning-dose, bleeding time was measured post-dose, and TXA₂ and prostacyclin stable metabolites were measured in urine collected 2 h post-morning-dose. Data are expressed as mean ± SD. After 14 days treatment, serum TXB₂ levels were significantly greater 2 h post-dosing with aspirin 20 mg BD vs. 75 mg OD (3.0 ± 3.6 ng/mL vs. 0.8 ± 1.9 ng/mL; p = 0.018) whereas pre-dosing levels were not significantly different (3.5 ± 4.1 ng/mL vs. 2.5 ± 3.1 ng/mL, p = 0.23). 1-mmol/L arachidonic acid-induced platelet aggregation was similarly inhibited by both regimens pre-dose (8.5 ± 14.3% vs. 5.1 ± 3.6%, p = 0.24) and post-dose (8.7 ± 14.2% vs. 6.6 ± 5.3%; p = 0.41). Post-dose bleeding time was shorter with 20 mg BD (680 ± 306 s vs. 834 ± 386 s, p = 0.02). Urinary prostacyclin and TX metabolite excretion were not significantly different. In conclusion, compared to aspirin 75 mg OD, aspirin 20 mg BD provided consistent inhibition of platelet TXA₂ release and aggregation, and improved post-dose hemostasis, in ticagrelor-treated ACS patients. Further studies are warranted to assess whether this regimen improves the balance of clinical efficacy and safety.