Reversal of the anti‐platelet effects of aspirin and clopidogrel

Summary. Background: Guidelines recommend stopping aspirin and clopidogrel 7 to 10 days before surgery to allow time for replacement of permanently inhibited platelets by newly released uninhibited platelets.Objectives: The purpose of the present study was to determine the rate of offset of the anti‐platelet effects of aspirin and clopidogrel after stopping treatment and the proportion of untreated donor platelets that are required to reverse their anti‐platelet effects.Methods: Cohort 1 consisted of 15 healthy subjects who received aspirin 81 mg day^?1 or clopidogrel 75 mg day^?1 for 7 days and underwent serial blood sampling until platelet function testing results normalized. Cohort 2 consisted of 36 healthy subjects who received aspirin 325 mg day^?1, clopidogrel 75 mg day^?1, aspirin 81 mg day^?1 plus clopidogrel 75 mg day^?1 or no treatment for 7 days and underwent a single blood sampling.Results: In cohort 1, arachidonic acid (AA)‐induced light transmission aggregation (LTA) returned to baseline levels in all subjects within 4 days of stopping aspirin, coinciding with the partial recovery of plasma thromboxane B₂ concentrations. ADP‐induced LTA did not return to baseline levels until 10 days after stopping clopidogrel. In cohort 2, AA‐induced LTA in patient treated with aspirin reached control levels after mixing with 30% untreated donor platelets whereas ADP‐induced LTA in patients treated with clopidogrel reached control levels only after the addition of 90% or more donor platelets.Conclusions: Platelet aggregation recovers within 4 days of stopping aspirin but clopidogrel must be stopped for 10 days to achieve a normal aggregatory response.     

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.     

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.     

Sequential ADP-stimulated light transmission and multiple electrode aggregometry in patients taking aspirin and clopidogrel after non ST-elevation myocardial infarction

Abstract

Background. Fast platelet function tests can identify weak clopidogrel responders, but data on variability over time in clopidogrel responsiveness in several clinical settings are lacking. We wanted to explore long-term variability of multiple electrode aggregometry (MEA) measurements and the agreement between MEA and light transmission aggregometry (LTA) in patients with non-ST elevation myocardial infarction (NSTEMI) treated with aspirin and clopidogrel. Methods. Parallel MEA and LTA were performed at baseline and after 6 and 12 weeks in 31 patients treated with percutaneous coronary intervention after NSTEMI. Adenosine diphosphate (ADP) concentrations 2 μM, 6.5 μM and 10 μM were used. Parallel testings in both arterial and venous blood were performed at baseline. MEA and LTA cut-off levels were applied to discriminate aggregation values suggesting presence or absence of high platelet reactivity (HPR). Results. Arterial and venous MEA and LTA aggregation were similar. Within-subject variability in both MEA and LTA aggregation throughout the study was moderate. According to MEA, eight patients had HPR at baseline (MEA aggregation >?47 U). Defining >?47% as the LTA aggregation HPR cut-off level, the same number of patients (eight) had HPR according to LTA. Of the 93 MEA/LTA observations 81 (87.1%) gave the same HPR classification. MEA vs. LTA agreement at baseline was slightly inferior to that obtained after 12 weeks. Conclusions. MEA and LTA aggregation in arterial and venous blood seem similar. Within-subject variability over time was moderate, and the agreement between LTA and MEA was good, and stable in most patients.     

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.     

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.     

Variability in aggregometry response before and after initiation of clopidogrel therapy

Abstract

Background: Evaluation of clopidogrel therapy by in vitro methods has limitations which may be of clinical importance. We wanted to explore the variability in aggregometry response in aspirin sensitive patients before and after initiation of clopidogrel therapy. Methods: ADP 9.37 μM, AA 1.2mM and TRAP 25mM stimulated light transmissions aggregometry (LTA) were performed twice before (Exams 1 and 2; 3 weeks apart)-and within one year after-initiation of clopidogrel therapy (Exam 3) in 79 patients treated with PCI. Repeated ADP aggregometry was also performed in 16 healthy volunteers in order to estimate LTA measurement error. Result:. Inter-individual differences in ADP aggregation e.g. at Exam 1 were substantial (range 17–77%, SD 15.8%). Intra-individual changes between Exams 1 and 2 were significant (?27 to +36%, SD 14.6%, p<0.05). Inter-individual differences at Exam 3 (on clopidogrel treatment) were larger than expected from Exams 1 and 2 (p<0.01). AA aggregation was the same before and during clopidogrel treatment. In controls, inter-individual differences were smaller at ADP 10 than at ADP 5μM. Conclusions. Inter-individual differences in ADP aggregation were significant both before and during clopidogrel therapy, and there were significant intra-individual variations over time. Therefore, prediction of aggregometry response before or during clopidogrel therapy based on single tests may be unreliable. Inter-individual differences in healthy controls are smaller at high concentrations of ADP, and comparisons of aggregometry response should be performed with caution unless ADP concentrations are standardized.     

Residual platelet thromboxane A2 and prothrombotic effects of erythrocytes are important determinants of aspirin resistance in patients with vascular disease

Background: Permanent inactivation of cyclooxygenase‐1 and inhibition of platelet thromboxane A₂ (TxA₂) constitute the main mechanisms underlying the prevention of vascular disease by aspirin. Methods and Results: We studied platelet TxA₂ synthesis and its impact on platelet reactivity and platelet–erythrocyte [platelet‐rich plasma (PRP)–RBC] interactions in 533 aspirin‐treated patients with vascular disease. Seventy aspirin‐free and 16 aspirin‐treated normal subjects were evaluated as controls. Collagen (1 μg mL^?1)‐induced platelet activation (¹⁴C‐5HT release) and recruitment (proaggregatory activity of cell‐free releasates from activated platelets) were assessed in PRP, PRP + RBC, and whole blood (WB). TxA₂ was quantified in releasates from WB. Aspirin inhibited TxA₂ synthesis and platelet function in all patients, but to different degrees. Forty‐two patients (8%) displayed partial (<95%) inhibition of TxA₂ relative to that of aspirin‐free controls. They produced >3.5 ng mL^?1 TxA₂ and had higher platelet reactivity than 491 patients who had undetectable TxA₂ or produced residual TxA₂ (R‐TxA₂; ≤3.5 ng mL^?1). Patients with R‐TxA₂ were distributed into TxA₂ quartiles. Patients in the third and fourth quartiles had significantly elevated ¹⁴C‐5HT release in PRP, which was markedly amplified in PRP + RBC and WB. TxA₂ in the fourth quartile translated into increased platelet aggregation and recruitment. Significant correlations were found between R‐TxA₂ and platelet hyperfunction. Conclusion: Biochemical markers (TxA₂ synthesis, ¹⁴C‐5HT release) and biological assays (platelet aggregation and recruitment) used to monitor the aspirin effect in a large population of patients presenting with vascular disease have evidenced the importance of R‐TxA₂ and the prothrombotic effects of RBC in aspirin resistance.     

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.     

Clopidogrel discontinuation and platelet reactivity following coronary stenting

See also Lordkipanidzé M, Harrison P. Beware of being caught on the rebound. This issue, pp 21–3. Summary. Aims: Antiplatelet therapy with aspirin and clopidogrel is recommended for 1 year after drug‐eluting stent (DES) implantation or myocardial infarction. However, the discontinuation of antiplatelet therapy has become an important issue as recent studies have suggested a clustering of ischemic events within 90 days of clopidogrel withdrawal. The objective of this investigation was to explore the hypothesis that there is a transient ‘rebound’ increase in platelet reactivity within 3 months of clopidogrel discontinuation. Methods and Results: In this prospective study, platelet function was assessed in patients taking aspirin and clopidogrel for at least 1 year following DES implantation. Platelet aggregation was measured using a modification of light transmission aggregometry in response to multiple concentrations of adenosine diphosphate (ADP), epinephrine, arachidonic acid, thrombin receptor activating peptide and collagen. Clopidogrel was stopped and platelet function was reassessed 1 week, 1 month and 3 months later. Thirty‐two patients on dual antiplatelet therapy were recruited. Discontinuation of clopidogrel increased platelet aggregation to all agonists, except arachidonic acid. Platelet aggregation in response to ADP (2.5, 5, 10, and 20 μm ) and epinephrine (5 and 20 μm ) was significantly increased at 1 month compared with 3 months following clopidogrel withdrawal. Thus, a transient period of increased platelet reactivity to both ADP and epinephrine was observed 1 month after clopidogrel discontinuation. Conclusions: This study demonstrates a transient increase in platelet reactivity 1 month after clopidogrel withdrawal. This phenomenon may, in part, explain the known clustering of thrombotic events observed after clopidogrel discontinuation. This observation requires confirmation in larger populations.     

Obituaries

Platelet aggregation by adenosine diphosphate (ADP) is a self-limited and reversible process. An application of this phenomenon is described which allows removal of the platelets from large volumes of fresh platelet rich plasma (PRP) for the preparation of platelet concentrates. The macroscopic platelet clumps resulting from a concentration of 10 μgm ADP per ml of PRP are removed by centrifugation at 50 × g for 10 minutes. Resuspension of these platelets in 20 cc of native plasma results in a platelet concentrate that is 80 to 90 per cent as effective per unit as PRP in its ability to elevate the platelet count in recipients. Such concentrates are superior to concentrates prepared by other methods. The posttransfusion survival of ADP platelets compares favorably with the survival of platelets administered as PRP. There is evidence of minor sequestration but there is no apparent irreversible damage to platelets handled in this manner. Alkaline plasma and increase in plasma ionized calcium enhance the ADP aggregation and improve the efficiency of in vitro separation of platelets from PRP. However, the resulting concentrate is less effective in vivo, because of prolonged and slowly reversible clumping, and failure of these platelets to circulate.     

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’.     

Fibrinogen γ‐chain peptide‐coated,ADP‐encapsulated liposomes rescue thrombocytopenic rabbits from non‐compressible liver hemorrhage

Summary. Background: We developed a fibrinogen γ‐chain (dodecapeptide HHLGGAKQAGDV [H12])‐coated, ADP‐encapsulated liposome (H12‐[ADP]‐liposome) that accumulates at bleeding sites via interaction with activated platelets via glycoprotein IIb–IIIa and augments platelet aggregation by releasing ADP. Objective: To evaluate the efficacy of H12‐(ADP)‐liposomes for treating liver hemorrhage in rabbits with acute thrombocytopenia. Methods: Thrombocytopenia (platelets < 50 000 μL^?1) was induced in rabbits by repeated blood withdrawal (100 mL kg^?1 in total) and isovolemic transfusion of autologous washed red blood cells. H12‐(ADP)‐liposomes with platelet‐poor plasma (PPP), platelet‐rich plasma (PRP), PPP, ADP liposomes with PPP or H12‐(PBS)‐liposomes/PPP, were administered to the thrombocytopenic rabbits, and liver hemorrhage was induced by penetrating liver injury. Results: Administration of H12‐(ADP)‐liposomes and of PRP rescued all thrombocytopenic rabbits from liver hemorrhage as a result of potent hemostasis at the liver bleeding site, although rabbits receiving PPP or ADP liposomes showed 20% survival in the first 24 h. Administration of H12‐(ADP)‐liposomes and of PRP suppressed both bleeding volume and time from the site of liver injury. H12‐(phosphate‐buffered saline)‐liposomes lacking ADP also improved rabbit survival after liver hemorrhage, although their hemostatic effect was weaker. In rabbits with severe thrombocytopenia (25 000 platelets μL^?1), the hemostatic effects of H12‐(ADP)‐liposomes tended to be attenuated as compared with those of PRP treatment. Histologic examination revealed that H12‐(ADP)‐liposomes accumulated at the bleeding site in the liver. Notably, neither macrothombi nor microthrombi were detected in the lung, kidney or liver in rabbits treated with H12‐(ADP)‐liposomes. Conclusions: H12‐(ADP)‐liposomes appear to be a safe and effective therapeutic tool for acute thrombocytopenic trauma patients with massive bleeding.     

In the presence of strong P2Y12 receptor blockade,aspirin provides little additional inhibition of platelet aggregation

Summary. Background: Aspirin and antagonists of platelet ADP P2Y₁₂ receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y₁₂ receptors can inhibit thromboxane A₂ (TXA₂)‐dependent pathways of platelet activation independently of aspirin. Objectives: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y₁₂ receptor blockade. Methods: With the use of platelet‐rich plasma from healthy volunteers, determinations were made in 96‐well plates of platelet aggregation, TXA₂ production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP‐6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1–10 μmol L^?1), aspirin (30 μmol L^?1), PAM + aspirin or vehicle. Results: PAM concentration‐dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 μmol L^?1. In further tests of PAM (3 μmol L^?1), aspirin (30 μmol L^?1) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA₂ and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. Conclusions: P2Y₁₂ receptors are critical to the generation of irreversible aggregation through the TXA₂‐dependent pathway. As a result, strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.     

Clinical implications of clopidogrel non‐response in cardiovascular patients: a systematic review and meta‐analysis

Summary. Background: Previous studies have shown an important risk of cardiovascular events in patients with clopidogrel biological non‐response, and data have shown considerable, unexplored heterogeneity. Objectives: To evaluate the magnitude of cardiovascular risk associated with clopidogrel non‐response and to explore heterogeneity. Methods: This was a systematic review and meta‐analysis of prospective studies of patients treated with clopidogrel for symptomatic atherothrombosis, evaluated by light transmission aggregometry with ADP and monitored prospectively for clinical ischemic events. Results: Fifteen studies were included, totaling 3960 patients, of whom 25% were considered to be clopidogrel non‐responders. The global relative risk (RR) for recurrent ischemic events in clopidogrel non‐responders was 3.5 [95% confidence interval (CI) 2.4–5.2, P < 0.0001]. The results of the different studies were heterogeneous (Cochran P = 0.01 and I² = 52%). The most recent studies yielded lower RRs [global RR = 2.9 (95% CI 2.3–3.8) after 2007, and global RR = 6.6 (95% CI 3.7–11.9) before 2007, P = 0.01]. Heterogeneity was present in the group of studies in which more than 10% of patients took glycoprotein (GP)IIb–IIIa inhibitors [Cochran P = 0.003 and I² = 70%; RR = 3.8 (95% CI 2.9–5.1)] and was absent in the other studies [Cochran P = 0.88 and I² = 0; RR = 2.5 (95% CI 1.7–3.6)]. The RR was significantly higher in studies using higher ADP maximal aggregation cut‐offs (> 65%) for clopidogrel non‐response than in studies using lower cut‐offs [RR = 5.8 (95% CI 3.2–10.3) and RR = 2.9 (95% CI 2.2–3.7), respectively, P = 0.03]. Conclusions: The risk of ischemic events associated with clopidogrel non‐response is now more precisely defined. The risk is heterogeneous across studies, possibly because of an interaction with GPIIb–IIIa inhibitors and the use of different cut‐offs to identify non‐responders.     

Endothelin does not affect aggregation of human platelets

Summary. Endothelin (ET-1) is a recently discovered endothelial-derived peptide with pronounced vasoconstrictor activity. The present study addressed whether ET-1, in analogy with several other vasoactive agents, can induce or modulate aggregation of human platelets in vitro. Venous blood from healthy donors was collected in citrate or heparin and platelet-rich plasma (PRP) was prepared. Portions of the PRP were added to drugs, and platelet aggregation was recorded according to Born & Cross (1963). ET-1 added to the PRP (final concentrations 1–100 nM) did not induce aggregation of platelets, either in citrate- or heparin-containing plasma. Adenosine-diphosphate (0·5-2 μM) or thrombin (0·1-0·4 NIH units ml^-1) induced dose-dependent aggregation of platelets in citrate- or heparin-containing PRP; such aggregation was, however, not affected by ET-1 (1–100 μM) either. We conclude that ET-1, in contrast to other endothelial-derived vasoactive agents, lacks direct effect on platelet aggregation in vitro.     

Dual antiplatelet therapy unmasks distinct platelet reactivity in patients with coronary artery disease

Summary. Background: Platelet‐induced thrombosis is a major risk factor for recurrent ischemic events, although platelet function in patients with cardiovascular disease taking aspirin and clopidogrel is very poorly characterized. The aim of this study was to assess platelet reactivity in patients with cardiovascular disease taking aspirin and clopidogrel. Methods: We developed a rapid assay to measure platelet aggregation in response to arachidonic acid, collagen, adenosine diphosphate (ADP), epinephrine and thrombin receptor activating peptide (TRAP) in 80 healthy volunteers. We then recruited 200 consecutive patients from outpatient clinics and the cardiac catheterization laboratory and tested platelet function. Platelet aggregation induced by epinephrine is a marker of global platelet reactivity. We tested platelet function in 146 patients compliant with antiplatelet therapy. Platelet aggregation to epinephrine was divided into quartiles. The platelet response to the other agonists was analysed based on the response to epinephrine. Results: Platelet reactivity increased significantly across the quartiles in response to epinephrine in healthy volunteers and patients (P < 0.0001). A significant increase in response across quartiles was seen with all agonists in healthy volunteers (P < 0.001). In contrast, a significant increase in response across quartiles was only seen with ADP in patients (P < 0.0001). Hypertension, smoking and diabetes were significantly associated with increasing platelet reactivity to epinephrine (P < 0.05). Conclusion: This study shows that platelet response differs between healthy volunteers and patients on dual antiplatelet therapy. In patients with cardiovascular disease, dual antiplatelet therapy unmasks a distinct type of platelet reactivity in response to epinephrine and ADP but not other agonists.     

Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement

Summary. Background: In patients undergoing percutaneous coronary intervention (PCI), a link between bleeding and excess mortality has been demonstrated. A potential association of platelet response to clopidogrel and bleeding has not been well established yet. Objectives: The aim of the present study was to assess the impact of clopidogrel responsiveness on the risk of bleeding in clopidogrel‐treated patients undergoing PCI. Methods: Patients (n = 2533) undergoing PCI after pretreatment with 600 mg of clopidogrel were enrolled in this study. Blood was obtained directly before PCI. Adenosine‐diphosphate (ADP)‐induced platelet aggregation was assessed on a Multiplate analyzer. The primary endpoint was the incidence of in‐hospital Thrombolysis in Myocardial Infarction (TIMI) major bleeding and the secondary endpoint was in‐hospital TIMI minor bleeding. Receiver‐operator curve (ROC) analysis was used to derive the optimal platelet aggregation value defining enhanced clopidogrel responders for the association of measurements with major bleeding. Results: Thirty‐four (1.3%) major bleeding events and 137 (5.4%) minor bleeding events were observed. The risk of a major bleeding was significantly higher in patients (n = 975) with an enhanced response to clopidogrel as compared with the remaining patients (n = 1558) (2.2 vs. 0.8%, unadjusted odds ratio (OR) 2.6, 95% confidence interval (CI) 1.3–5.2, P = 0.005; adjusted OR 3.5, 95% CI 1.6–7.3, P = 0.001). No significant differences between both groups were observed for the occurrence of minor bleeding events (P = 0.68). Conclusions: Enhanced clopidogrel responsiveness is associated with a higher risk of major bleeding. Whether guidance of antiplatelet treatment based on platelet function testing proves useful for avoiding bleeding events warrants further investigation.     

Normalization of platelet reactivity in clopidogrel-treated subjects

BACKGROUND: Aspirin (ASA) + clopidogrel are commonly used in acute coronary syndrome (ACS), but persistent antiplatelet effects may complicate surgery. METHODS AND RESULTS: To study the possibility of normalizing platelet reactivity after ASA + clopidogrel treatment, 11 healthy subjects received a 325-mg ASA + clopidogrel loading dose (300 or 600 mg dependent on study arm), followed by 81 mg of ASA + 75 mg of clopidogrel daily for 2 days. Platelet reactivity was assessed by light transmittance aggregometry (LTA) [challenged by adenosine diphosphate (ADP), arachidonic acid (AA), collagen, and thrombin receptor activating peptide (TRAP)] and flow cytometry for platelet activation by GPIIb/IIIa receptor exposure pretreatment, 4 and 72 h postload. To normalize platelet reactivity, increasing amounts of pooled platelets from five untreated volunteers [volunteers (V)-platelet-rich plasma (PRP)] were added ex vivo to the subject's PRP (S-PRP). At both 4 and 72 h, 40% and 50% V-PRP were needed to overcome platelet disaggregation in the 300 or 600 mg arms, respectively, after ADP challenge; an additional 10% V-PRP fully normalized aggregation. Recovery of function was linear with each incremental increase of V-PRP. ADP-induced GPIIb/IIIa activation showed the same pattern as LTA (r = 0.74). Forty percent V-PRP was required to normalize platelet function to AA, collagen, and TRAP. CONCLUSION: Our results suggest that the pre-operative transfusion of 10 platelet concentrate units (the equivalent of 40% V-PRP) after a 300-mg clopidogrel loading or 12.5 units (50% V-PRP) after a 600 mg loading may adequately reverse clopidogrel-induced platelet disaggregation to facilitate postoperative hemostasis. An additional 2.5 units fully normalized platelet function. The potential clinical implications of our observations could include shorter hospitalizations and reduced bleeding complications. But these observations should be fully explored in an in vivo clinical setting with clopidogrel-treated patients before and after surgery.     

Earlier recovery of platelet function after discontinuation of treatment with ticagrelor compared with clopidogrel in patients with high antiplatelet responses

Summary. Background: The rate of recovery of platelet function after discontinuation of P2Y₁₂ inhibitors depends on the reversibility of the antiplatelet effect and the extent of the on‐treatment response. P2Y₁₂ inhibition increases the bleeding risk in patients requiring surgery. Objectives: To evaluate recovery of platelet function after discontinuation of ticagrelor vs. clopidogrel in stable coronary artery disease (CAD) patients with high levels of platelet inhibition (HPI) during the ONSET/OFFSET study. Methods: Patients received aspirin 75–100 mg per day and either ticagrelor 90 mg twice‐daily or clopidogrel 75 mg daily for 6 weeks. This subanalysis included patients with HPI after the last dose of maintenance therapy, defined as: inhibition of platelet aggregation (IPA) > 75% 4 h post‐dose (ADP 20 μm , final extent); < 120 P2Y₁₂ reaction units 8 h post‐dose (VerifyNow P2Y₁₂ assay); or platelet reactivity index < 50% 8 h post‐dose (VASP‐P assay). Results: IPA > 75% was observed in 39 out of 47 ticagrelor‐treated and 17 out of 44 clopidogrel‐treated patients. The rate of offset of IPA over 4–72 h was greater with ticagrelor (IPA %/hour slope: ?1.11 vs. ?0.67 for clopidogrel; P < 0.0001). Mean IPA was significantly lower with ticagrelor than clopidogrel between 48 and 168 h post‐dose (P < 0.01). Similar findings were observed with the other assays. The average time for IPA to decline from 30% to 10% was 50.8 h with ticagrelor vs. 110.4 h with clopidogrel. Conclusions: In patients with HPI, recovery of platelet function was more rapid after discontinuation of ticagrelor than clopidogrel leading to significantly greater platelet reactivity by 48 h after the last dose in the ticagrelor group.