Enhanced antiplatelet effect of clopidogrel in patients whose platelets are least inhibited by aspirin: a randomized crossover trial

Summary. Objective: We aimed to determine whether adding clopidogrel to aspirin in patients at high risk of future cardiovascular events would suppress laboratory measures of the antiplatelet effects of aspirin; and have greater platelet inhibitory effects in patients with the least inhibition of platelets by aspirin. Methods: We performed a randomized, double‐blind, placebo‐controlled, crossover trial, comparing clopidogrel 75 mg day^?1 versus placebo, in 36 aspirin‐treated patients with symptomatic objectively confirmed peripheral arterial disease. Results: The addition of clopidogrel to aspirin did not suppress platelet aggregation induced by arachidonic acid, urinary 11 dehydro thromboxane B₂ concentrations, or soluble markers of platelet activation markers (P‐selectin, CD40‐ligand) and inflammation (high sensitivity serum C‐reactive protein, interleukin‐6). Clopidogrel significantly inhibited platelet aggregation induced by ADP (reduction 26.2%; 95% CI: 21.3–31.1%, P < 0.0001) and collagen (reduction 6.2%; 95% CI: 3.2–9.3%, P = 0.0003). The greatest inhibition of collagen‐induced platelet aggregation by clopidogrel was seen in patients with the least inhibition of arachidonic acid induced aggregation by aspirin [lower tertile of arachidonic acid‐induced platelet aggregation: 2.8% (95% CI: ?0.8 to 6.3%) reduction in mean collagen‐induced aggregation by clopidogrel; middle tertile: 4.0% (95% CI: 0.4–7.6%); upper tertile 12.6% (95% CI: 4.5–20.8%); P‐value for interaction 0.01]. Conclusions: The greatest platelet inhibitory effect of clopidogrel occurs in patients with the least inhibition of arachidonic acid‐induced platelet aggregation by aspirin. This raises the possibility that the clinical benefits of adding clopidogrel to aspirin may be greatest in patients whose platelets are least inhibited by aspirin. Confirmation in clinical outcome studies may allow these patients to be targeted with antiplatelet drugs that inhibit the ADP receptor, thereby overcoming the problem of laboratory aspirin resistance.     

Which platelet function test is suitable to monitor clopidogrel responsiveness? A pharmacokinetic analysis on the active metabolite of clopidogrel

Summary. Background: Multiple platelet function tests claim to be P2Y12‐pathway specific and capable of capturing the biological activity of clopidogrel. Objectives: The aim of the present study was to determine which platelet function test provides the best reflection of the in vivo plasma levels of the active metabolite of clopidogrel (AMC). Patients/methods: Clopidogrel‐naive patients scheduled for elective percutaneous coronary intervention (PCI) received a 600 mg loading dose of clopidogrel and 100 mg of aspirin. For pharmacokinetic analysis, blood was drawn at 0, 20, 40, 60, 90, 120, 180, 240 and 360 min after clopidogrel loading and peak plasma concentrations (C_max) of the AMC were quantified with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Platelet function testing was performed at baseline and 360 min after the clopidogrel loading. Results: The VASP‐assay, the VerifyNow P2Y12‐assay and 20 μmol L^?1 adenosine diphosphate (ADP)‐induced light transmittance aggregometry (LTA) showed strong correlations with C_max of the AMC (VASP: R² = 0.56, P < 0.001; VerifyNow platelet reactivity units (PRU): R² = 0.48, P < 0.001; VerifyNow %inhibition: R² = 0.59, P < 0.001; 20 μmol L^?1 ADP‐induced LTA: R² = 0.47, P < 0.001). Agreement with C_max of the AMC was less evident for 5 μmol L^?1 ADP‐induced LTA or whole blood aggregometry (WBA), whereas the IMPACT‐R ADP test did not show any correlation with plasmalevels of the AMC. Conclusion: The flow cytometric VASP‐assay, the VerifyNow P2Y12 assay and, although to a lesser extent, 20 μmol L^?1 ADP‐induced LTA correlate best with the maximal plasma level of the AMC, suggesting these may be the preferred platelet function tests for monitoring the responsiveness to clopidogrel.     

Thromboxane Antagonism with terutroban in Peripheral Arterial Disease: the TAIPAD study

Summary. Background: Terutroban is a selective prostaglandin endoperoxide (TP) receptor antagonist with antithrombotic, antivasoconstrictive and antiatherosclerotic properties and is currently in development for long‐term cardiovascular secondary prevention. Objectives: TAIPAD is an international, double‐blind, randomized controlled study comparing the effects of five dosages of oral terutroban vs. aspirin and placebo on platelet aggregation in peripheral arterial disease (PAD) patients. Patients/methods: After 10 day’s placebo run‐in, included patients (n = 435; ankle‐brachial pressure index, 0.7 ± 0.1) were randomly allocated to aspirin 75 mg day^?1, terutroban 1, 2.5, 5, 10 or 30 mg day^?1 or placebo. On day 5, the placebo group was reallocated to one of the terutroban groups for the rest of the study (day 83). Ex vivo platelet aggregation induced by the thromboxane analog U46619 (7 μm ) was measured 24 h after dosing, as well as platelet aggregation induced by arachidonic acid (AA), collagen and ADP. Results: Terutroban dose‐dependently inhibited U46619‐induced platelet aggregation at days 5 and 83. At day 5, the inhibition was significant vs. placebo for all terutroban dosages (P < 0.001). Terutroban (5, 10 and 30 mg day^?1) was at least as effective as aspirin in inhibiting platelet aggregation induced by arachidonic acid (AA), collagen and adenosine diphosphate (ADP). Terutroban was well tolerated, with a safety profile similar to aspirin. Conclusions: In PAD patients, terutroban dose‐dependently inhibited platelet aggregation 24 h after dosing, and was at least as effective as aspirin at 5, 10 and 30 mg day^?1. Terutroban was well tolerated.     

Clopidogrel 150 vs. 75 mg day−1 in patients undergoing percutaneous coronary intervention: a meta‐analysis

Summary. Background: Whether an increase in the daily oral maintenance dose of clopidogrel may improve clinical outcomes in patients undergoing percutaneous coronary intervention (PCI) is still debated. Objectives: This meta‐analysis aimed to estimate the relative effect of a 150‐ vs. 75‐mg daily maintenance dosage of clopidogrel on clinical and laboratory end‐points in patients undergoing PCI. Methods: We searched electronic and printed sources (up to 14 December 2010) for both randomized control trials and observational studies satisfying the predefined inclusion criteria. Results: We retrieved 12 reports of studies including a total of 23 814 patients. Clopidogrel, 150 mg day^?1, was associated with significant reductions in major adverse cardiac and/or cerebrovascular events (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.48–0.94), myocardial infarction (OR, 0.72; 95% CI, 0.60–0.86), target vessel revascularization (OR, 0.27; 95% CI, 0.12–0.62) and stent thrombosis (OR, 0.64; 95% CI, 0.53–0.77) and decreased adenosine diphosphate‐induced maximal platelet aggregation. However, as compared with 75 mg day^?1, the 150‐mg daily maintenance dosage significantly increased the risk of minor bleeding (OR, 1.21; 95% CI, 1.08–1.36). Conclusion: As compared with the currently recommended 75‐mg day^?1 maintenance dosage of clopidogrel, the 150‐mg day^?1 dosage can reduce major adverse cardiac and/or cerebrovascular events but may increase the risk of minor bleeding.     

The influence of aspirin dose and glycemic control on platelet inhibition in patients with type 2 diabetes mellitus

Summary. Background: Low‐dose aspirin seems to offer no benefit in the primary prevention of cardiovascular disease in type 2 diabetes mellitus (DM2). The anti‐platelet effect may be diminished by poor glycemic control or inadequate dosing of aspirin. Objectives: To study the effects of both glycemic control and increasing aspirin dose on platelet response to aspirin in DM2 patients and matched controls. Patients/methods: Platelet effects of increasing doses of aspirin (30, 100 and 300 mg daily) were prospectively assessed in 94 DM2 patients and 25 matched controls by measuring thromboxane levels in urine (11‐dhTxB2) and platelet aggregation using VerifyNow^® and light transmission aggregometry (LTA). DM2 patients were stratified for glycemic control (hemoglobin‐A1c [HbA1c] ≤ 53, 53–69, ≥ 69 mmol mol^?1). Results: At baseline, median 11‐dhTxB2 excretion was higher in the poorly controlled patients (77 ng mmol^?1), and the moderately controlled (84 ng mmol^?1) compared with the well‐controlled patients (64 ng mmol^?1) and controls (53 ng mmol^?1), P < 0.01. Next, 30 mg of aspirin reduced 11‐dhTxB2 excretion to 31, 29 and 24 ng mmol^?1 in the poorly, moderately and well‐controlled patients, respectively, and to 19 ng mmol^?1 in controls, P < 0.001. VerifyNow^® and LTA were also incompletely suppressed in DM2 patients using 30 mg of aspirin, but 100 mg resulted in similar platelet suppression in all groups, with no additional effect of 300 mg. Conclusions: DM2 patients with inadequate glycemic control (HbA1c > 53 mmol mol^?1) have higher baseline platelet activity and incomplete suppression of platelet activity with 30 mg of aspirin. However, 100 mg of aspirin leads to optimal inhibition irrespective of glycemic control, and 300 mg does not further improve platelet suppression.     

Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary

Summary. Background: Light transmittance aggregometry (LTA) is considered to be the ‘gold standard’ of platelet function testing. As LTA has been poorly standardized, we analyzed the results of LTA in healthy subjects and patients with antiplatelet therapy using different concentrations of agonists and performing tests in non‐adjusted and platelet count‐adjusted platelet‐rich plasma (PRP). Methods: LTA was performed in 20 healthy subjects and in patients treated with aspirin (n = 30) or clopidogrel (n = 30) monotherapy, as well as in patients on combination therapy (n = 20), using arachidonic acid (ARA 0.25 and 0.5 mg mL⁻¹) and adenosine diphosphate (ADP 2 and 5 μm ) as agonists and performing platelet function tests in non‐adjusted and platelet count (250 nL⁻¹ ± 10%)‐adjusted PRP. Results: The overall platelet aggregation response is decreased after adjusting the PRP for platelet count compared with measurements in unadjusted PRP. The variability of aggregation results is high in adjusted PRP in the subgroup of healthy subjects, ranging from 9.2–95.3% (5th–95th percentile) relative to 77.6–95.5% in non‐adjusted PRP when determining maximum aggregation to ARA 0.5 mg mL⁻¹. Late aggregation using ADP 2 μm ranges from 3.8–89.9% in adjusted PRP compared with 42.9–92.5% in non‐adjusted PRP. Maximum aggregation using ARA 0.5 mg mL⁻¹ in non‐adjusted PRP differentiates between aspirin‐treated patients and healthy controls well, whereas late aggregation using ADP 2 μm in non‐adjusted PRP offers the best discrimination between clopidogrel‐treated patients and healthy controls. Conclusion: Adjustment of PRP for platelet count does not provide any advantage and therefore the time‐consuming process of platelet count adjustment is not necessary.     

Effect of the selective serotonin reuptake inhibitor paroxetine on platelet function is modified by a SLC6A4 serotonin transporter polymorphism

Summary. Background: Selective serotonin reuptake inhibitors (SSRIs) have been associated with an increased bleeding tendency. Objectives: To prospectively quantify the dose‐response effects of paroxetine and the influence of the serotonin transporter gene (SLC6A4) promoter polymorphism (5‐HTTLPR) on platelet function. Methods: Nineteen drug‐free psychiatric outpatients (44.5 ± 10.8 years) were tested before and after 6 weeks of paroxetine treatment (20 mg day^?1). Based on clinical symptoms, paroxetine dosages were increased (40–50 mg day^?1) for 6 more weeks in 11 patients. Parameters related to platelet function were assessed by bleeding time, platelet function analyzer (PFA), platelet serotonin, platelet factor 4 (PF4), β‐thromboglobulin (β‐TG), and aggregation tests. Results: Paroxetine 20 mg day^?1 increased mean bleeding time by 1.2 min (95% confidence interval (95% CI) ?0.2–2.7) and reduced median platelet serotonin level (463 ng 10^?9 platelets; inter quartile range (IQR) 361–666), and platelet ß‐TG concentration (3.1 IU 10^?6 platelets; IQR 0.3–6.0). Other platelet parameters did not change significantly. Serial platelet aggregation tests did not become abnormal. Paroxetine dose‐escalation did not further influence platelet function. However, 5‐HTTLPR polymorphisms modified these effects: in L_A/L_A‐carriers, bleeding times did not change (?0.2 min; 95% CI ?0.6 to 0.9), while bleeding times significantly increased in <2L_A‐allele carriers (2.3 min; 95% CI 0.5 to 4.07; P = 0.032). Platelet serotonin decreases were larger in patients without L_A‐alleles (868 ng 10^?9 platelets; IQR 585 to 1213) than in ≥1 L_A‐allele carriers (457 ng 10^?9 platelets; IQR 392 to 598; P = 0.035). PFA closure time and PF4 increased significantly in patients without L_A‐alleles. Conclusions: Paroxetine 20 mg day^?1 does not increase overall bleeding time, but impairs platelet function by decreasing the levels of platelet serotonin and platelet ß‐TG. These paroxetine effects appear to be mediated by 5‐HTTLPR, with most pronounced effects in patients without L_A‐alleles.     

Antiplatelet effects of prasugrel vs. double clopidogrel in patients on hemodialysis and with high on‐treatment platelet reactivity

Summary. Background: High on‐treatment platelet reactivity (HTPR) is frequent in patients on hemodialysis (HD) receiving clopidrogel. Objectives: The primary aim of this study was to determine the antiplatelet effects of prasugrel vs. high‐dose clopidogrel in patients on HD with HTPR. Patients/Methods: We performed a prospective, single‐center, single‐blind, investigator‐initiated, randomized, crossover study to compare platelet inhibition by prasugrel 10 mg day^?1 with that by high‐dose 150 mg day^?1 clopidogrel in 21 patients on chronic HD with HTPR. Platelet function was assessed with the VerifyNow assay, and genotyping was performed for CYP2C19*2 carriage. Results: The primary endpoint of platelet reactivity (PR, measured in P2Y12 reaction units [PRU]) was lower in patients receiving prasugrel (least squares [LS] estimate 156.6, 95% confidence interval [CI] 132.2–181.1) than in those receiving high‐dose clopidogrel (LS 279.9, 95% CI 255.4–304.3), P < 0.001). The LS mean differences between the two treatments were ? 113.4 PRU (95% CI ? 152.9 to ? 73.8, P < 0.001) and ? 163.8 PRU (95% CI ? 218.1 to ? 109.2, P < 0.001) in non‐carriers and carriers of at least one CYP2C19*2 allele, respectively. HTPR rates were lower for prasugrel than clopidogrel, in all patients (19% vs. 85.7%, P < 0.001) and in non‐carriers (25.7% vs. 80%, P = 0.003). All carriers continued to show HTPR while receiving high‐dose clopidogrel, but none showed it while receiving prasugrel. Conclusions: In HD patients exhibiting HTPR following standard clopidogrel treatment, prasugrel 10 mg day^–1 is significantly more efficient than doubling the clopidogrel dosage in achieving adequate platelet inhibition. Neither effect seems to be influenced by carriage of the loss‐of‐function CYP2C19*2 allele.     

Antithrombotic effects of S 18886, a novel orally active thromboxane A2 receptor antagonist

Summary. Platelet activation and thrombus formation play a critical role in the onset of acute coronary syndromes. Thromboxane A₂ (TxA₂) is among the different chemical modulators released by activated platelets. TxA₂ is considered one of the most powerful agonists for platelet activation. In addition, TxA₂ exerts a vasoconstrictor effect by serving as an agonist of the thromboxane receptor (TP) on the vascular smooth muscle cell membranes. The putative effect of TxA₂ on thrombosis is demonstrated by the clinical effectiveness of acetylsalicylic acid (ASA) in the prevention of acute coronary syndromes. Among the clinically used antiplatelet agents, clopidogrel has shown to be slightly more effective than ASA in the prevention of atherothrombotic events in patients with peripheral arterial disease, and is one of the most widely used after aspirin. The aims of the study were to study the antithrombotic effects of escalating doses of the TP‐receptor antagonist, S 18886 and to compare its effects with those achieved by the administration of ASA (5 mg kg^?1 day^?1), and clopidogrel (3 mg kg^?1 day^?1). The study was undertaken at high and low shear rate conditions using the Badimon perfusion chamber in a porcine model. Antithrombotic effects were assessed as changes on platelet and fibrin(ogen) deposition. The doses of 30 and 100 µg kg^?1 day^?1 were selected based on a previous platelet aggregation study. S 18886 shows a dose‐dependent antithrombotic response. The dose of S‐100 develops similar antithrombotic effects to those of clopidogrel and superior to those of aspirin. The antithrombotic effects were statistically significant at both studied shear rate conditions. Therefore, the orally active TP‐receptor antagonist, S 18886, appears to be a new and effective agent to prevent atherothrombotic complications.     

Protective effects of triflusal on secondary thrombus growth and vascular cyclooxygenase-2

Summary. Background: Carotid residual mural thrombus predisposes to recurrent thrombosis and/or distal embolization (i.e. cerebrovascular ischemia). Objectives: Our aims were (i) to analyze and compare the efficacy of aspirin, triflusal, and its main metabolite 2‐hydroxy‐4‐trifluorometylbenzoic acid (HTB) on secondary thrombus growth; and (ii) evaluate to what extent the three Cox‐1 inhibitors influenced vascular Cox‐1/Cox‐2 expression and endothelial prostacyclin synthesis. Methods: In a rabbit model of ex vivo thrombosis, a fresh mural thrombus was formed on damaged vessels at flow conditions typical of mild and severe carotid stenoses. The effects of Cox‐1 inhibitors administered both intravenously (i.v.) (aspirin 5 mg kg^?1, triflusal 10 mg kg^?1, and HTB 10 mg kg^?1) and orally (p.o.) (8 days; aspirin 30 mg kg^?1 day^?1, and triflusal 40 mg kg^?1 day^?1) on secondary thrombus growth were assessed by In‐¹¹¹deposited platelets and compared with a placebo control. Arterial Cox‐1/Cox‐2 expression after 8‐day treatment was evaluated at mRNA and protein levels. Additionally, a drug‐related dose‐dependent in vitro assay was performed for endothelial PGI₂ release measurement (Cox‐2 activity). Results: All Cox inhibitors similarly and significantly (P < 0.05) reduced secondary thrombus formation after i.v. and p.o. administration versus placebo control. Treatments exerted no effect on vascular Cox‐1 mRNA whereas Cox‐2 mRNA was moderately reduced by aspirin and triflusal (placebo 100% ± 9%, aspirin 70% ± 2% and triflusal 70% ± 2%; P < 0.05). Cox‐2 protein levels were slightly higher in the triflusal versus aspirin group (placebo 100% ± 6%, aspirin 35% ± 10% and triflusal 61% ± 9%; P < 0.005 versus placebo). Interestingly, in vitro, HTB solely maintained endothelial PGI₂ synthesis levels similar to the control. Conclusions: At a similar level of efficacy in inhibiting secondary thrombosis, triflusal seems to better preserve Cox‐2 expression than aspirin and its metabolite HTB was able to protect endothelial prostacyclin production.     

Potentiation of clopidogrel active metabolite formation by rifampicin leads to greater P2Y12 receptor blockade and inhibition of platelet aggregation after clopidogrel

Summary. Background: The thienopyridine P2Y₁₂ receptor antagonist clopidogrel reduces the risk of arterial thrombosis and individual pharmacodynamic responses to clopidogrel are believed to reflect the levels of active metabolite (AM) generated. Rifampicin increases the inhibitory effect of clopidogrel on platelet aggregation (PA). We studied the response to clopidogrel before and during administration of rifampicin in order to study the relationship between individual AM levels and P2Y₁₂ blockade. Methods: Healthy volunteers received a 600‐mg loading dose of clopidogrel followed by 75 mg daily for 7 days and, after a washout period and treatment with rifampicin [300 mg twice a day (b.i.d.)], received the same regimen of clopidogrel. Clopidogrel AM levels were determined over 4 h after the clopidogrel loading dose and unblocked P2Y₁₂ receptor number was assessed using a ³³P‐2MeSADP binding assay. PA was measured by optical aggregometry with ADP and TRAP. Results: Rifampicin enhanced clopidogrel AM production [area‐under‐the‐curve (AUC): clopidogrel 89 ± 22 ng h mL^?1, clopidogrel + rifampicin 335 ± 86 ng h mL^?1, P < 0.0001], and P2Y₁₂ blockade (unblocked receptors: clopidogrel 48 ± 24, clopidogrel + rifampicin 4 ± 2, P < 0.0001) and reduced PA (5 μmol L^?1 ADP: clopidogrel 20 ± 4, clopidogrel + rifampicin 5 ± 2, P < 0.01). Increasing numbers of unblocked receptors were required for an aggregation response with a decreasing concentration of ADP. PA induced by ADP 2 μmol L^?1 was particularly sensitive to low levels of receptor blockade. Conclusion: Potentiation of clopidogrel AM production by rifampicin leads to greater P2Y₁₂ blockade and consequently greater inhibition of PA. PA responses to low concentrations of ADP are more sensitive to P2Y₁₂ blockade.     

Randomized,double‐blind comparison of effects of abiciximab bolus only vs. on‐label regimen on ex vivo inhibition of platelet aggregation in responders to clopidogrel undergoing coronary stenting

Summary. Background: On top of aspirin, an abciximab bolus‐only regimen results in a 30% drop in platelet inhibition at 6 h as compared with the on‐label regimen. The concomitant administration of high loading dose clopidogrel, by bridging with abciximab bolus, may sustain suppression of platelet activity over time. Objectives: To investigate the non‐inferiority of abciximab bolus‐only and concomitant high loading dose clopidogrel vs. abciximab bolus + infusion with respect to the inhibition of platelet aggregation (IPA) as determined by light transmission aggregometry. Patients/Methods: Seventy‐three patients with non‐ST segment elevation acute coronary syndromes underwent double‐blind randomization to abciximab bolus followed by a 12‐h placebo infusion and concomitant 600‐mg clopidogrel vs. abciximab bolus + a 12‐h infusion and 300 mg of clopidogrel. IPA was determined by light transmission aggregometry throughout 24 h. Clopidogrel poor responsiveness was defined as ≥ 50% 5 μmol L^?1 ADP‐induced maximum platelet aggregation. Results: In clopidogrel responders (n = 68), IPA after 20 μmol L^?1 ADP at 4 h was 89% ± 13% in the bolus‐only arm vs. 92% ± 14% in the bolus + infusion arm (P = 0.011 for non‐inferiority). IPA after 5 or 20 μmol L^?1 ADP and 5 or 15 μmol L^?1 TRAP and the proportion of patients showing ≥ 80% IPA did not differ at any time point, irrespective of clopidogrel responsiveness status. Thirty‐day outcomes were similar, whereas hemoglobin (0.91 ± 0.8 vs. 0.5 ± 0.7 g dL^?1; P = 0.01) and platelet count mean drop (41.7 ± 57 vs. 18.6 ± 34 10⁹ L^?1; P = 0.042) were significantly reduced in the bolus‐only arm. Conclusions: Withholding abciximab post‐bolus infusion in patients receiving high loading dose clopidogrel does not impair platelet inhibition throughout 24 h, and has the potential to improve the safety profile of the drug at reduced costs.     

Aspirin and the in vitro linear relationship between thromboxane A2‐mediated platelet aggregation and platelet production of thromboxane A2

Summary. Background: Currently, ‘aspirin resistance’, the anti‐platelet effects of non‐steroid anti‐inflammatory drugs (NSAIDs) and NSAID‐aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A₂ formation is non‐linear and TXA₂ generation must be inhibited by at least 95% to inhibit TXA₂‐dependent aggregation. This relationship, however, has never been rigorously tested. Objectives: To characterize, in vitro and ex vivo, the concentration‐dependent relationships between TXA₂ generation and platelet activity. Method: Platelet aggregation, thrombi adhesion and TXA₂ production in response to arachidonic acid (0.03–1 mmol L^?1), collagen (0.1–30 μg mL^?1), epinephrine (0.001–100 μmol L^?1), ADP, TRAP‐6 amide and U46619 (all 0.1‐30 μmol L^?1), in the presence of aspirin or vehicle, were determined in 96‐well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. Results: Platelet aggregation, adhesion and TXA₂ production induced by either arachidonic acid or collagen were inhibited in concentration‐dependent manners by aspirin, with logIC₅₀ values that did not differ. A linear relationship existed between aggregation and TXA₂ production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R² 0.92; n = 224). The same relationships were seen in combinations of aspirin‐treated and naïve platelets, and in blood from individuals taking an anti‐thrombotic dose of aspirin. Conculsions: These studies demonstrate a linear relationship between inhibition of platelet TXA₂ generation and TXA₂‐mediated aggregation. This finding is important for our understanding of the anti‐platelet effects of aspirin and NSAIDs, NSAID–aspirin interactions and ‘aspirin resistance’.     

Clinical predictors of dual aspirin and clopidogrel poor responsiveness in stable cardiovascular patients from the ADRIE study

Summary. Background: Poor response to both aspirin and clopidogrel (dual poor responsiveness [DPR]) is a major risk factor for recurrent ischemic events. Objectives: The aim of this study was to identify factors associated with DPR, defined with specific tests, and derive a predictive clinical score. Methods: We studied 771 consecutive stable cardiovascular patients treated with aspirin (n = 223), clopidogrel (n = 111), or both drugs (n = 437). Aspirin responsiveness was evaluated by serum thromboxane (Tx)B₂ assay, and clopidogrel responsiveness by calculating the platelet reactivity index (PRI) on the basis of the phosphorylation status of the vasodilator phosphoprotein. The analysis was focused on patients treated with both drugs, and on independent predictors of DPR. Results: Among patients on dual therapy, there was no relevant correlation between TxB₂ levels and PRI values (r = 0.11). Sixty‐seven patients (15.4%) had DPR. Diabetes [odds ratio (OR) 1.89, 95% confidence interval (CI) 1.06–3.39], high body weight (> 86 kg vs. < 77 kg, OR 4.74, 95% CI 2.49–9.73), low aspirin dose (75–81 mg vs. ≥ 160 mg, OR 0.12, 95% CI 0.09–0.93) and high C‐reactive protein (CRP) level (> 1.6 mg L^?1 vs. < 0.6 mg L^?1, OR 3.66, 95% CI 1.74–8.72) were independently associated with DPR, via increased TxB₂ levels, increased PRI, or both. These associations with TxB₂ and PRI were reproduced across the whole population. With use of a factor‐weighed score (c‐index = 0.74), the predicted prevalence of DPR was 57% in the highest strata of the score as compared with < 4% for the lowest strata. Conclusions: Diabetes, body weight, the aspirin dose and CRP levels are readily available independent predictors of DPR, and some are potential targets for reducing its prevalence.     

Aspirin withdrawal in patients treated with ticagrelor presenting with non‐ST elevation myocardial infarction

Essentials

• Strong P2Y₁₂ blockade may cause platelet inhibition that is only minimally enhanced by aspirin.
• We evaluated aspirin withdrawal on platelet reactivity in ticagrelor treated patients.
• Aspirin withdrawal resulted in increased platelet reactivity to arachidonic acid.
• Aspirin withdrawal caused little difference in adenosine diphosphate‐induced platelet aggregation.

Summary

Background

Recent studies have shown that the thromboxane A₂‐dependent pathway is dependent on the ADP–P2Y₁₂ pathway, and that strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is minimally enhanced by aspirin. Data from the PLATO trial suggested that, among ticagrelor‐treated patients, high‐dose versus low‐dose (< 100 mg day^?1) aspirin is associated with an increased risk fof ischemic events.

Objectives

To evaluate the impact of aspirin withdrawal on platelet reactivity in acute coronary syndrome (ACS) patients treated with a potent P2Y₁₂ blocker.

Patients/Methods

This was a current prospective, randomized, placebo‐controlled, double‐blind, cross‐over study. The study population comprised 22 consecutive ACS patients who underwent percutaneous coronary intervention and were treated with aspirin (100 mg day^?1) and ticagrelor. Thirty days post‐ACS, open‐label aspirin was stopped, and patients were randomized to either blinded aspirin or placebo for 2 weeks, with each patient crossing over to the other arm for an additional 2 weeks. Platelet reactivity to arachidonic acid and ADP determined with light‐transmission aggregometry (LTA) and VerifyNow was evaluated at baseline, and 2 weeks and 4 weeks later.

Results

Aspirin withdrawal resulted in an increase in arachidonic‐acid induced platelet reactivity as determined with both LTA (77.0% ± 11.3% versus 20.8% ± 4.4%) and VerifyNow (607.7 ± 10.6 aspirin reaction units [ARU] versus 408.5 ± 14.4 ARU). Platelet response to ADP, as determined with both LTA and VerifyNow, did not differ with either aspirin or placebo (32.9% ± 2.6% versus 35.8% ± 3.6%, and 33.5 ± 6.4 P2Y₁₂ reaction units (PRU) versus 29.6 ± 5.7 PRU, respectively).

Conclusions

Aspirin withdrawal early post‐ACS results in increased platelet reactivity in response to arachidonic acid, despite concomitant treatment with the potent P2Y₁₂ blocker ticagrelor.

     

Arterial antithrombotic and bleeding time effects of apixaban,a direct factor Xa inhibitor,in combination with antiplatelet therapy in rabbits

Summary. Background: Optimal treatment of arterial thrombosis may include a combination of antiplatelet and anticoagulant drugs. We evaluated apixaban, a direct and highly selective factor Xa inhibitor, in combination with clinically relevant doses of aspirin and/or clopidogrel for prevention of arterial thrombosis in rabbits. Methods: Studies were conducted in rabbit models of electrically induced carotid artery thrombosis and cuticle bleeding time (BT). Apixaban 0.04 and 0.3 mg kg^?1 h^?1 or aspirin 1 mg kg^?1 h^?1 was infused intravenous (i.v.) continuously from 1 h before artery injury or cuticle bleed until the end of the experiment. Clopidogrel at 3 mg kg^?1 was dosed orally once daily for three days, with the last dose given 2 h before injury. Results: Control thrombus weight and BT averaged 8.6 ± 0.9 mg and 181 ± 12 s, respectively (n = 6 per group). Effective doses of apixaban that reduced thrombus weight by 20 and 50% (ED₂₀ and ED₅₀) were 0.04 and 0.3 mg kg^?1 h^?1 i.v., respectively. Addition of aspirin to apixaban ED₂₀ and ED₅₀ significantly reduced the thrombus weight from 7.4 ± 0.5 to 5.3 ± 0.3 and 3.6 ± 0.3 mg, respectively, with no significant increases in BT (190 ± 7 s vs.181 ± 9 and 225 ± 11 s, respectively). Addition of aspirin and apixaban (ED₂₀ dose) to clopidogrel produced a further significant reduction in thrombus weight from 5.3 ± 0.3 to 0.7 ± 0.1 mg. This combination of clopidogrel and aspirin with apixaban (ED₂₀ dose) produced a significant but moderate BT increase of 2.1 times control. Conclusions: The combination of apixaban and aspirin or apixaban, aspirin and clopidogrel can reduce formation of occlusive arterial thrombosis without excessive increases in BT in rabbits.     

High on‐aspirin platelet reactivity as measured with aggregation‐based,cyclooxygenase‐1 inhibition sensitive platelet function tests is associated with the occurrence of atherothrombotic events

Summary. Background: High on‐aspirin platelet reactivity (HAPR) is associated with atherothrombotic events following percutaneous coronary intervention (PCI). The aim of the present study was to identify the platelet function test sensitive for platelet cyclooxygenase‐1 inhibition that best predicts atherothrombotic events. Methods and results: Nine hundred and fifty‐one consecutive patients on dual antiplatelet therapy undergoing elective PCI were enrolled. On‐aspirin platelet reactivity was measured in parallel by arachidonic acid (AA)‐induced light transmittance aggregometry (AA‐induced LTA), the VerifyNow® Aspirin Assay (VerifyNow® Aspirin Assay), the arachidonic acid prestimulated IMPACT‐R (IMPACT‐R AA) and the PFA‐100 collagen/epinephrine cartridge (PFA COL/EPI). Cut‐offs for HAPR were established by receiver‐operator characteristic curve analysis. At 1‐year follow‐up, the composite of all‐cause death, non‐fatal acute myocardial infarction, stent thrombosis and ischemic stroke occurred more frequently in patients with HAPR when assessed by LTA [10.1% vs. 6.0%, P = 0.020 (n = 925)] and VerifyNow^® [13.3% vs. 5.9%, P = 0.015 (n = 422)]. The VerifyNow^® ASA assay (AUC = 0.78) and, to a lesser extent, AA‐induced LTA (AUC = 0.73) added significantly to a model consisting of clinical and procedural risk factors in predicting atherothrombotic events. In contrast, the IMPACT‐R (n = 791) and the PFA Collagen/Epinephrine (n = 719) were unable to discriminate between patients with and without primary endpoint at 1‐year follow‐up. None of the platelet function tests was able to identify patients at risk for bleeding. Conclusions: AA‐induced LTA and the VerifyNow^® ASA test were able to identify aspirin‐treated patients undergoing PCI with stenting at risk for atherothrombotic events. The VerifyNow^® Aspirin Assay had the highest predictive accuracy. None of the tests was able to identify patients at higher risk of bleeding.     

Effect of platelet turnover on whole blood platelet aggregation in patients with coronary artery disease

Summary. Background: Previous studies have demonstrated considerable variation in the antiplatelet effect of aspirin. Objectives: To investigate the impact of platelet turnover on the antiplatelet effect of aspirin in patients with stable coronary artery disease (CAD) and to identify determinants of platelet turnover. Methods: Platelet turnover was evaluated by measurements of immature platelets and thrombopoietin in 177 stable CAD patients on aspirin monotherapy, including 85 type 2 diabetics and 92 non‐diabetics. Whole blood platelet aggregation was determined using the VerifyNow^® Aspirin test and multiple electrode aggregometry (MEA, Multiplate^®) induced by arachidonic acid (AA) (1.0 mm ), adenosine diphosphate (ADP) (10 μm ) and collagen (1.0 μg mL^?1). Results: Immature platelet levels significantly correlated with MEA (r = 0.31–0.36, P‐values < 0.0001) and the platelet activation marker sP‐selectin (r = 0.19, P = 0.014). Contrary to the VerifyNow^® test, MEA significantly correlated with variations in platelet count (r = 0.45–0.68, P‐values < 0.0001). Among patients with residual platelet reactivity according to AA, there were significantly more diabetics (61% vs. 41%, P = 0.027) and higher levels of sP‐selectin (77.7 ± 29 vs. 70.2 ± 25 ng mL^?1, P = 0.070) and serum thromboxane B₂ (0.81 [0.46; 1.70] vs. 0.56 [0.31; 1.12] ng mL^?1, P = 0.034). In a multivariate regression analysis, immature platelet levels were determined by thrombopoietin levels (P < 0.001), smoking (P = 0.020) and type 2 diabetes (P = 0.042). Conclusions: The antiplatelet effect of aspirin was reduced in CAD patients with an increased platelet turnover. Once‐daily dosing of aspirin might not suffice to adequately inhibit platelet aggregation in patients with an increased platelet turnover.     

Involvement of circulating platelets on the hyperalgesic response evoked by carrageenan and Bothrops jararaca snake venom

Background: The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. Objective: To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. Methods: Rats were (i) rendered thrombocytopenic by administration of polyclonal anti‐rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. Results: Platelet counts were markedly diminished in rats administered with either ARPI (± 88%) or busulfan (± 96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre‐treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre‐incubation with alkaline phosphatase. Conclusions: Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.     

Influence of high‐density lipoprotein and paraoxonase‐1 on platelet reactivity in patients with acute coronary syndromes receiving clopidogrel therapy

Summary. Background: The paraoxonase activity of the enzyme paraoxonase‐1 (PON‐1) associated with high‐density lipoprotein (HDL) may significantly influence clopidogrel’s antiplatelet and clinical efficacy as a result of its involvement in the clopidogrel biotransformation to the pharmacologically active thiol metabolite. We evaluated the possible relationships of HDL levels as well as PON‐1 activities and the Q192R genotype with clopidogrel’s antiplatelet efficacy in acute coronary syndrome (ACS) patients. Methods and results: The platelet aggregation, P‐selectin expression and platelet/leukocyte conjugates as well as the clopidogrel response variability (evaluated by the VASP phosphorylation test and expressed as platelet reactivity index, PRI) were assessed in 74 ACS patients undergoing percutaneous coronary intervention (PCI) in relation to the PON‐1 Q192R genotype and to serum HDL‐cholesterol levels, and PON‐1 (paraoxonase and arylesterase) activities. Patients were loaded with 600 mg of clopidogrel followed by 75 mg per day. HDL‐cholesterol levels and PON‐1 activities at baseline (before clopidogrel loading) were not altered at 5‐ and 30‐day post‐clopidogrel loading, whereas baseline platelet activation parameters were significantly attenuated. At 5 days, 17 patients were clopidogrel non‐responders (PRI: 64.2 ± 11.1%). HDL‐cholesterol was inversely associated with platelet activation parameters independently on platelet response variability to clopidogrel whereas a negative association between platelet activation parameters and paraoxonase activity was observed in patients adequately responding to clopidogrel but not in clopidogrel non‐responders. Similarly, the platelet activation markers were significantly higher in PON‐1 Q192Q genotype carriers compared with those having one or two R alleles only in patients adequately responding to clopidogrel. Conclusions: PON‐1 is an important determinant of clopidogrel antiplatelet efficacy only in patients adequately responding to clopidogrel. These findings may be clinically important in ACS patients receiving clopidogrel therapy, especially the first days after the episode.