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Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by enhanced platelet production and thrombotic complications. The inhibition of platelet cyclooxygenase (COX) activity by the standard once-daily aspirin is mostly incomplete due to accelerated thrombopoiesis. The phase II Aspirin Regimens in EsSential thrombocythemia (ARES) trial has recently compared the efficacy of once- vs. twice- or three-times daily low-dose aspirin in inhibiting platelet thromboxane (TX) A 2 production, as reflected by serum (s) TXB 2 measurements. The present substudy characterized the determinants of the highly variable response to the standard aspirin 100 mg once-daily regimen in fully compliant patients with ET and the effects of the experimental dosing regimens on response variability. By multivariable analysis, the platelet count (directly) and cytoreductive treatment (inversely) were significantly associated with sTXB 2 values in 218 patients with ET. However, the platelet count positively correlated with sTXB 2 in patients not being treated with cytoreductive drugs (ρ = 0.51, P < 0.01, n = 84), but not in patients on cytoreduction. Patients in the lowest sTXB 2 quartile were older, more often on cytoreductive drugs, had lower platelet count and Janus-Associated Kinase2 (JAK2)-V617F allele frequency as compared with patients in the upper sTXB 2 quartiles. After 2 weeks of a twice- or 3-times daily aspirin regimen, the association between the platelet count and sTXB 2 became similar in cytoreduced and non-cytoreduced patients. In conclusion, the platelet count appears the strongest determinant of TXA 2 inhibition by once-daily low-dose aspirin in ET, with different patterns depending of cytoreductive treatment. More frequent aspirin dosing restores adequate platelet inhibition and reduces interindividual variability, independently of cytoreduction., (© 2021 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase-1 (COX-1) inhibition. We performed a multicenter double-blind trial to investigate the efficacy of 3 aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2-dependent vascular thromboresistance. Patients on chronic once-daily low-dose aspirin (n = 245) were randomized (1:1:1) to receive 100 mg of aspirin 1, 2, or 3 times daily for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate end points of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated. Evaluable patients assigned to the twice-daily and thrice-daily regimens showed substantially reduced interindividual variability and lower median (interquartile range) values for sTXB2 (ng/mL) compared with the once-daily arm: 4 (2.1-6.7; n = 79), 2.5 (1.4-5.65, n = 79), and 19.3 (9.7-40; n = 85), respectively. Urinary PGIM was comparable in the 3 arms. Urinary TXM was reduced by 35% in both experimental arms. Patients in the thrice-daily arm reported a higher abdominal discomfort score. In conclusion, the currently recommended aspirin regimen of 75 to 100 once daily for cardiovascular prophylaxis appears to be largely inadequate in reducing platelet activation in the vast majority of patients with ET. The antiplatelet response to low-dose aspirin can be markedly improved by shortening the dosing interval to 12 hours, with no improvement with further reductions (EudraCT 2016-002885-30)., (© 2020 by The American Society of Hematology.)

Background: The prevalence and degree of obesity is rising worldwide, increases cardiovascular risk, modifies body composition and organ function, and potentially affects the pharmacokinetics and/or pharmacodynamics of drugs., Objectives: To investigate the pharmacodynamics of once-daily low-dose aspirin in healthy obese subjects, and to assess whether body weight (BW) and body mass index (BMI) affect the pharmacology of aspirin., Patients/methods: Otherwise healthy, obese (BMI > 30 kg/m 2 ) subjects were studied before and after 3-4 weeks of 100-mg once-daily aspirin intake. Aspirin pharmacodynamics were assessed according to serum thromboxane (TX) B 2 levels measured at 4 hours, 24 hours (i.e., posologic interval) and 48 hours after the last witnessed intake; age-matched and sex-matched non-obese controls were included. A previously calibrated pharmacokinetic/pharmacodynamic in silico model of aspirin was used to fit serum TXB 2 data from obese subjects. At baseline, the major urinary TXA 2 and prostacyclin metabolites, urinary isoprostane and plasma inflammatory biomarkers were measured., Results: In 16 obese subjects (aged 47 ± 11 years; BMI of 39.4 ± 5.1 kg/m 2 ), residual serum TXB 2 values between 4 and 48 hours after aspirin intake were increased 3- to 5-fold as compared with controls. At 24 hours, the residual serum TXB 2 level was log-linearly associated with body size over a wide range of BMI and BW values, without any apparent threshold. The in silico model predicted that reduced aspirin bioavailability would be inversely related to body size and rescued by 200 mg of aspirin once daily or 85 mg twice daily. Baseline urinary TXA 2 metabolite, isoprostane and plasma C-reactive protein levels were significantly increased in obese subjects., Conclusions: Obesity is associated with impaired aspirin responsiveness, largely because of body size. Impaired inhibition of platelet activation by conventional low-dose aspirin may affect antithrombotic efficacy., (© 2019 International Society on Thrombosis and Haemostasis.)

Due to its ability to irreversibly inactivate platelet cyclooxygenase, low-dose aspirin (ASA) is the most widely used antithrombotic agent. Although, studies in specific types of patients with cardiovascular disease (CVD) have shown an incomplete inhibition of platelet's cyclooxygenase, which may increase the variability in drug response. Some aspects of ASA pharmacokinetics (PK) still need further investigation. In this study, we aimed to characterise the contribution of esterase enzymes to ASA hydrolysis in the peripheral blood and to assess their activity in 36 healthy subjects (Ctrl) and 77 CVD patients. To this aim, an in vitro assay testing esterase activity in parallel to a liquid chromatography-tandem mass spectrometry method simultaneously detecting ASA and its main metabolites salicylic (SA) and gentisic acids, have been developed. Michaelis-Menten constant (Km) calculation, ASA esterase isoforms characterisation, and ASA PK study were then achieved. The calculated Km identified plasma esterases as the enzymes with the higher affinity for the substrate compared to the RBC ones. Both rivastigmine and 4-bis-nitrophenyl phosphate inhibited plasma esterase activities, suggesting that acetylcholinesterase and carboxylesterase largely contribute to ASA hydrolysis. The feasibility of the method here developed has been explored in Ctrl and CVD patients. The effect of ASA treatment on enzyme activity was further evaluated on an age, sex and BMI matched subgroup of patients and Ctrl (n = 10 for each subgroup, on and off ASA). No overall variations were evidenced in both CVD and Ctrl groups, even when the effect of ASA treatment was tested. This result suggests the absence of any influence of disease state, drug treatments, and comorbidities on plasma esterase and the inability of ASA intake to induce esterase function. In conclusion, the method here developed allows a better characterisation of ASA esterase activity and could be helpful to define the PK-related determinants of ASA responsiveness in order to personalise regimen in specific pathological conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Once-daily (od), low-dose aspirin (75-100 mg) is recommended to reduce the thrombotic risk of patients with essential thrombocytemia (ET). This practice is based on data extrapolated from other high-risk patients and an aspirin trial in polycythemia vera, with the assumption of similar aspirin pharmacodynamics in the two settings. However, the pharmacodynamics of low-dose aspirin is impaired in ET, reflecting accelerated renewal of platelet cyclooxygenase (COX)-1. ARES is a parallel-arm, placebo-controlled, randomized, dose-finding, phase II trial enrolling 300 ET patients to address two main questions. First, whether twice or three times 100 mg aspirin daily dosing is superior to the standard od regimen in inhibiting platelet thromboxane (TX)A 2 production, without inhibiting vascular prostacyclin biosynthesis. Second, whether long-term persistence of superior biochemical efficacy can be safely maintained with multiple vs. single dosing aspirin regimen. Considering that the primary study end point is serum TXB 2 , a surrogate biomarker of clinical efficacy, a preliminary exercise of reproducibility and validation of this biomarker across all the 11 participating centers was implemented. The results of this preliminary phase demonstrate the importance of controlling reproducibility of biomarkers in multicenter trials and the feasibility of using serum TXB 2 as a reliable end point for dose-finding studies of novel aspirin regimens.

The serum TXB 2 (sTXB 2 ) assay reflects the pharmacodynamics of platelet inhibition by low-dose aspirin. However, different studies reported variable sTXB 2 values. sTXB 2 assay requires whole blood incubation at 37 °C as a condition for optimal thrombin generation, arachidonic acid release and its metabolism by platelet cyclooxygenase-1 to form TXA 2 . Access to 37 °C incubation may be variably delayed, and different methods to quantitate sTXB 2 may contribute to variable results between different Centers. We investigated whether delaying 37 °C incubation and/or analytical issues affect sTXB 2 concentrations, biasing the assessment of aspirin responsiveness. Sixty-eight samples from 54 volunteers, on- and off-aspirin, were incubated at 37 °C immediately after sampling (reference sample) or after 5, 10, 15, 20, 30 or 60 minutes at room temperature (RT); 8 samples remained at RT 60 minutes, without subsequent incubation; 314 sera were measured by enzyme immunoassay (EIA) and liquid chromatography-tandem mass-spectrometry (LC/MS-MS) methods. sTXB 2 concentrations decreased exponentially as a function of the delay before 37 °C incubation, ranging from 94 ± 11 % at 5 minutes to 23 ± 22 % of the reference sample after 60 minutes at RT. There was high agreement between EIA and LC/MS-MS. Moreover, we simulated the influence of a 15- or 30-minute delayed incubation on 300 sTXB2 measurements from previously-studied, aspirin-treated patients. Delayed incubation reduced the percentage of aspirin 'non-responders' by 22 % to 52 %, depending on the response threshold. In conclusion, a variable delay in the 37 °C incubation of blood samples may affect the assessment of platelet cyclooxygenase-1 inhibition by aspirin and confound the characterization of the determinants of aspirin responsiveness.

Recent trials suggest that Aspirin (ASA) reduces the incidence of venous thromboembolism in human. However, the molecular mechanisms underlying this effect are still unclear. In this study we assessed the effects of ASA in venous thrombosis mouse model induced by inferior vena cava (IVC) ligation and we investigated the mechanisms responsible for this effect. ASA (3mg/kg daily for 2 days) treatment decreased the thrombus size, the amounts of tissue factor activity in plasma microvesicles (TF-MP) and the levels of 2,3-dinor Thromboxane B2 (TXB-M) in urine compared to control mice. Interestingly, the thrombus size positively correlated with both TF-MP activity and TXB-M. In addition, positive correlation was observed between TF-MP activity and TXB-M. A reduced number of neutrophils and monocytes, and of TF-positive cells accompanied to a lower amount of fibrin and neutrophil extracellular traps (NETs) were also found in thrombi of ASA-treated mice. Similar results were obtained when mice were treated 24h before IVC ligation with SQ29548 (1mg/kg), a selective thromboxane receptor antagonist. In addition, transfusion of platelets in SQ29548 treated-mice excluded the likelihood of a redundant role of platelet-TP receptor in this context. Finally, incubation of macrophages and neutrophils with SQ29548 prevented TF activity and/or NETs formation induced by supernatant of activated platelets or by IBOP, a selective thromboxane analogue. In conclusion, ASA, suppressing TXA2, prevents macrophages and neutrophils activation and markedly reduces thrombus size with a mechanism most likely dependent of the inhibition of TF activity and NETs formation. These results provide a new link between platelet-produced thromboxane and the occurrence of venous thrombosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Platelet activation is persistently enhanced, and its inhibition by low-dose aspirin is impaired in type 2 diabetes mellitus. We investigated in vivo thromboxane (TX) and prostacyclin (PGI2) biosynthesis and their determinants, as well as aspirin responsiveness, in young adult subjects with type 1 diabetes mellitus (T1DM) without overt cardiovascular disease and stable glycemic control. The biosynthesis of TXA2 was persistently increased in subjects with T1DM versus matched healthy subjects, with females showing higher urinary TX metabolite (TXM) excretion than male subjects with T1DM. Microalbuminuria and urinary 8-iso-PGF2α, an index of in vivo oxidative stress, independently predicted TXM excretion in T1DM. No homeostatic increase in PGI2 biosynthesis was detected. Platelet COX-1 suppression by low-dose aspirin and the kinetics of its recovery after drug withdrawal were similar in patients and control subjects and were unaffected by glucose variability. We conclude that patients with T1DM and stable glycemic control display enhanced platelet activation correlating with female sex and microvascular and oxidative damages. Moreover, aspirin responsiveness is unimpaired in T1DM, suggesting that the metabolic disturbance per se is unrelated to altered pharmacodynamics. The efficacy and safety of low-dose aspirin in T1DM warrant further clinical investigation., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Low‐dose aspirin is currently recommended for patients with polycythemia vera (PV), a myeloproliferative neoplasm with increased risk of arterial and venous thromboses. Based on aspirin pharmacodynamics in essential thrombocythemia, a twice‐daily regimen is recommended for patients with PV deemed at particularly high thrombotic risk. We investigated the effects of low‐dose aspirin on platelet cyclooxygenase activity and in vivo platelet activation in 49 patients with PV, as assessed by serum thromboxane (TX) B2 and urinary TXA2/TXB2 metabolite (TXM) measurements, respectively. A previously described pharmacokinetic‐pharmacodynamic in silico model was used to simulate the degree of platelet TXA2 inhibition by once‐daily (q.d.) and twice‐daily (b.i.d.) aspirin, and to predict the effect of missing an aspirin dose during q.d. and b.i.d. regimens. Serum TXB2 averaged 8.2 (1.6–54.7) ng/ml and significantly correlated with the platelet count (γ = 0.39) and urinary TXM (γ = 0.52) in multivariable analysis. One‐third of aspirin‐treated patients with PV displayed less‐than‐maximal platelet TXB2 inhibition, and were characterized by significantly higher platelet counts and platelet‐count corrected serum TXB2 than those with adequate inhibition. Eight patients with PV were sampled again after 12 ± 4 months, and had reproducible serum TXB2 and urinary TXM values. The in silico model predicted complete inhibition of platelet‐derived TXB2 by b.i.d. aspirin, a prediction verified in a patient with PV with the highest TXB2 value while on aspirin q.d. and treated short‐term with a b.i.d. regimen. In conclusion, one in three patients with PV on low‐dose aspirin display less‐than‐maximal inhibition of platelet TXA2 production. Serum TXB2 measurement can be a valuable option to guide precision dosing of antiplatelet therapy in patients with PV. [ABSTRACT FROM AUTHOR]

Given to its ability to irreversibly acetylate the platelet cyclooxygenase-1 enzyme, acetylsalicylic acid (ASA) is successfully employed for the prevention of cardiovascular disease. Recently, an antitumoral effect of ASA in colorectal cancer has been increasingly documented. However, the molecular and metabolic mechanisms by which ASA exerts such effect is largely unknown. Using a new, untargeted liquid chromatography–mass spectrometry approach, we have analyzed urine samples from seven healthy participants that each ingested 100 mg of ASA once daily for 1 week. Of the 2007 features detected, 25 metabolites differing after ASA ingestion (nominal p < 0.05 and variable importance in projection (VIP) score > 1) were identified, and pathway analysis revealed low levels of glutamine and of metabolites involved in histidine and purine metabolisms. Likewise, consistent with an altered fatty acid β-oxidation process, a decrease in several short- and medium-chain acyl-carnitines was observed. An abnormal β-oxidation and a lower than normal glutamine availability suggests reduced synthesis of acetyl-Co-A, as they are events linked to one another and experimentally related to ASA antiproliferative effects. While giving an example of how untargeted metabolomics allows us to explore new clinical applications of drugs, the present data provide a direction to be pursued to test the therapeutic effects of ASA—e.g., the antitumoral effect—beyond cardiovascular protection. [ABSTRACT FROM AUTHOR]

• Persistent greater amount of adrenaline was found in TTS during long-term follow-up. • TTS show endothelial perturbation with low citrulline and high thrombomodulin levels. • TTS display residual TXA 2 production even though low dose ASA treatment. • Increased TXA 2 production is paralleled by enhanced platelet aggregation. Takotsubo (TTS) syndrome is an acute cardiac condition characterized by transient and reversible left ventricle dysfunction that mainly affects postmenopausal women. Catecholamine burst is the most accredited mechanism underpinning TTS onset and leading to endothelial dysfunction and platelet activation. Even if the use of low dose acetylsalycilic acid (ASA) in this clinical setting is based on both clinical presentation and unfavorable long-term prognosis, its efficacy has been recently challenged. This study was designed to assess endothelial function, residual thromboxane formation and platelet aggregation in TTS women on low-dose ASA treatment at long-term follow-up. Twenty-eight females with previously diagnosis of TTS syndrome were enrolled. Data were compared to those obtained from 23 coronary artery disease (CAD) women with a history of acute myocardial infarction, and 26 control subjects with no TTS or clinically evident CAD. Psychological and clinical profile were assessed in all study groups at the enrollment. Main metabolites involved in L-arginine/nitric oxide pathway, urinary prostacyclin, serum and urine thromboxane metabolites were measured by LC MS/MS methods. Thrombomodulin levels were quantified using an ELISA kit, and platelet aggregation, carried out on platelet rich-plasma, was induced by ADP or by epinephrine (EPI), norepinephrine (NORE) and TRAP-6, alone or in association with ADP and evaluated by Born's method. In TTS women an endothelial derangement, characterized by reduced citrulline production and increased thrombomodulin concentration, with no perturbation in prostacyclin levels, was evidenced. In addition, despite ASA treatment, TTS displayed a higher residual thromboxane formation, in parallel with an enhanced platelet response to compared to CAD. Our study highlighted the presence of endothelial perturbation in TTS patients even at long-term from the index event. The residual thromboxane production and platelet aggregation still leave open the question about the use of low dose ASA in this clinical setting. [ABSTRACT FROM AUTHOR]