Thromboxane Metabolites Research Articles

Reference interval establishment and correlation research of urine thromboxane metabolites: Unveiling new possibilities in platelet activation

BackgroundThromboxane metabolites could indirectly reflect platelet activation, among which 11-dehydro-thromboxane B2 (11dhTxB2) and 11-dehydro-2, 3-dinor thromboxane B2 (11dh23dinorTxB2) are two stable metabolites that are abundant in urine, and both are closely related to disease progression and drug use. However, most clinical application studies have focused on the single indicator of 11dhTxB2. We propose an LC-MS/MS method suitable for routine clinical screening with simultaneous determination of both metabolites and conduct preliminary studies in different populations. Methods and ResultsThe thromboxane metabolites were extracted by liquid–liquid extraction and determined by LC-MS/MS. Reference intervals (RI) were established in 333 healthy adults and validated in 25 patients with coronary atherosclerosis (CA). This LC-MS/MS method was over a wide quantitative range (0.1-10 μmol/L), the imprecision and accuracy were 5.2 %-11 % and 89.3 %-106.5 %, and was suitable for clinical routine quantitative screening. The 95th percentile RI of unire 11dhTxB2 was 1220 (95 % CI: 1048, 1376) pg mg Cr -1, for 11dh23dinorTxB2, RI was 908 (95 % CI: 821, 1102) pg mg Cr -1. For the first time, we found a significant correlation between 11dhTxB2 and 11dh23dinorTxB2 in both healthy adults (r = 0.67, P < 0.001) and CA patients (r = 0.77, P < 0.001). ConclusionThe establishment of RI provides a reference for diseases related to platelet activation and the use of drugs, and the first discovery of the correlation between 11dhTxB2 and 11dh23dinorTxB2 in urine provides a new possibilitie for the diagnostic and prognostic of cardiovascular diseases.

Adjustment for Renal Function Improves the Prognostic Performance of Urinary Thromboxane Metabolites.

Systemic thromboxane A2 generation, assessed by quantifying the concentration of stable thromboxane B2 metabolites (TXB2-M) in the urine adjusted for urinary creatinine, is strongly associated with mortality risk. We sought to define optimal TXB2-M cutpoints for aspirin users and nonusers and determine if adjusting TXB2-M for estimated glomerular filtration rate (eGFR) in addition to urinary creatinine improved mortality risk assessment. Urinary TXB2-M were measured by competitive ELISA in 1363 aspirin users and 1681 nonusers participating in the Framingham Heart Study. Cutpoints were determined for TXB2-M and TXB2-M/eGFR using log-rank statistics and used to assess mortality risk by Cox proportional hazard modeling and restricted mean survival time. Multivariable models were compared using the Akaike Information Criterion (AIC). A cohort of 105 aspirin users with heart failure was used for external validation. Optimized cutpoints of TXB2-M were 1291 and 5609 pg/mg creatinine and of TXB2-M/eGFR were 16.6 and 62.1 filtered prostanoid units (defined as pg·min/creatinine·mL·1.73 m2), for aspirin users and nonusers, respectively. TXB2-M/eGFR cutpoints provided more robust all-cause mortality risk discrimination than TXB2-M cutpoints, with a larger unadjusted hazard ratio (2.88 vs 2.16, AIC P < 0.0001) and greater differences in restricted mean survival time between exposure groups (1.46 vs 1.10 years), findings that were confirmed in the external validation cohort of aspirin users. TXB2-M/eGFR cutpoints also provided better cardiovascular/stroke mortality risk discrimination than TXB2-M cutpoints (unadjusted hazard ratio 3.31 vs 2.13, AIC P < 0.0001). Adjustment for eGFR strengthens the association of urinary TXB2-M with long-term mortality risk irrespective of aspirin use.

Changes in the hemocoagulation system in pregnant women, after pre-gravid preparation before the DRT program, with a history of sexually transmitted infections

The objective: to analyze the dynamics of laboratory parameters of the functioning of the thermocoagulation system in pregnant women with a history of sexually transmitted infections(STIs) after pre-gravid preparation before cycles of assisted reproductive technologies (ART). Materials and methods. An analysis of the functioning of the thermocoagulation system was carried out in women with infertility who had a history of STIs after the DRT program: GroupI – 56 pregnant women received our proposed treatment and prevention measures in the pregravidperiod; II group – 55 pregnant women received generally accepted medical and preventive measures. The assessment of the state of the hemostasis system was determined by the following indicators: fibrinogen concentration (Fg), activated recalcification time (ACR), activated partial thromboplastin time (ATP), integrative indicator «Thrombodynamic Potential Index» (TIP), the concentration of fibrin and fibrinogen degradation products (FRP). and the level of more stable, but less biologically active metabolites of prostacyclin and thromboxane – 6-keto-PGF1α and T×B2. Statistical processing of research results was carried out using standard programs «MicrosoftExcel 5.0» and «Statistica 8.0». Results. In the period of the final formation of the placental barrier, in pregnant women of the Igroup, the APTC slowly lengthened (31.3±1.6 s in the II trimester, 34.3±2.9 s in the III trimester)with an increase in gestational age and probably differed from the indicators in the II group(27.6±3.0 s and 30.2±1.7 s respectively; p&lt;0.05); there was a slow reduction of AChR during the II and III trimesters of pregnancy in pregnant women of the I group (63.1±2.8 s, 59.3±2.8 s); gradual decrease in the level of PDFF (5.8±0.27×10-2 g/l, 5.1±0.22×10-2 g/l) in contrast to the level of PDFF in the II group, where a gradual increase of this indicator was observed (9.4 ±0.17×10-2 g/l, 11.6±0.27×10-2 g/l) (р&lt;0.01). The level of a stable metabolite of thromboxane (T×B2) in the trimesters decreased by 2 times and was found to be lower (p&lt;0.05) than in the II study group; the level of a stable metabolite of prostacyclin (6-keto-PGF1α) also increased in the III trimester(p&lt;0.05). This led to an increase in the PgI2/T×A2 balance in group I from 0.34±0.02 to 1.16±0.03, which corresponded to the physiological needs of systemic and organ hemodynamics during pregnancy. Conclusions. The development and implementation of effective pre-gravid preparation before cycles in women with a history of STIs and medication correction during pregnancy contribute to increasing the adaptive compensatory and adaptive potential of the mother’shemocoagulation homeostasis and perinatal protection of the fetus.

Зміни в гемокоагуляційній системі після прегравідарної підготовки перед програмою ДРТ у вагітних з інфекціями, що передаються статевим шляхом, в анамнезі

Purpose - to analyze the dynamics of laboratory parameters of haemocoagulation system functioning after pregravid preparation before assisted reproductive technologies (ART) cycles in pregnant women with a history of sexually transmitted infections (STIs). Materials and methods. The functioning of the haemocoagulation system after the ART program in women with infertility and a history of STIs was analyzed: the Group 1 - 56 pregnant women received the treatment and preventive measures proposed by us in the pregravid period; the Group 2 - 55 pregnant women received conventional treatment and preventive measures. The state of the hemostasis system was assessed by the following indicators: fibrinogen concentration, activated recalcification time, activated partial thromboplastin time (APTT), integrative «index of thrombodynamic potential» (ITP), concentration of fibrin and fibrinogen degradation products (FDP) and the level of more stable but less biologically active metabolites of prostacyclin (6-keto-PGF1α) and thromboxane (T×B2). The statistical processing of the study results was performed using standard software «Microsoft Excel 5.0» and «Statistica 8.0». Results. During the period of final formation of the placental barrier in pregnant women of the Group 1, the APTT slowly lengthened (31.3±1.6 s - in the trimester ІІ; 34.3±2.9 s - in the trimester III) with increasing gestational age and significantly differed from the indicators in the Group 2 (27.6±3.0 s and 30.2±1.7 s, respectively; p&lt;0.05); activated recalcification time slowly decreased during the trimesters II and III of gestation in pregnant women of the Group 1 (63.1±2.8 s and 59.3±2.8 s, respectively); gradually decreased the level of FDP (5.8±0.27×10-2 g/l and 5.1±0.22×10-2 g/l), in contrast to the level of FDP in the Group 2, in which this indicator gradually increased (9.4±0.17×10-2 g/l and 11.6±0.27×10-2 g/l); (p&lt;0.01). The level of stable T×B2 in the trimester II decreased by 2 times and was lower (p&lt;0.05) than in the Group 2; the level of stable 6-keto-PGF1α increased in the trimester III (p&lt;0.05). This resulted in an increase in the PgI2/T×A2 balance in the Group 1 from 0.34±0.02 to 1.16±0.03, which corresponded to the physiological needs of systemic and organ hemodynamics during pregnancy. Conclusions. The development and implementation of effective pregravid preparation before ART cycles in women with a history of STIs and medical correction during pregnancy contribute to the increase of the adaptive compensatory and adaptive potential of maternal haemocoagulation homeostasis and perinatal fetal care. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

The beneficial effect of sulforaphane on platelet responsiveness during caloric load: a single-intake, double-blind, placebo-controlled, crossover trial in healthy participants.

As our understanding of platelet activation in response to infections and/or inflammatory conditions is growing, it is becoming clearer that safe, yet efficacious, platelet-targeted phytochemicals could improve public health beyond the field of cardiovascular diseases. The phytonutrient sulforaphane shows promise for clinical use due to its effect on inflammatory pathways, favorable pharmacokinetic profile, and high bioavailability. The potential of sulforaphane to improve platelet functionality in impaired metabolic processes has however hardly been studied in humans. This study investigated the effects of broccoli sprout consumption, as a source of sulforaphane, on urinary 11-dehydro-thromboxane B2 (TXB2), a stable thromboxane metabolite used to monitor eicosanoid biosynthesis and response to antithrombotic therapy, in healthy participants exposed to caloric overload. In this double-blind, placebo-controlled, crossover trial 12 healthy participants were administered 16g of broccoli sprouts, or pea sprouts (placebo) followed by the standardized high-caloric drink PhenFlex given to challenge healthy homeostasis. Urine samples were collected during the study visits and analyzed for 11-dehydro-TXB2, sulforaphane and its metabolites. Genotyping was performed using Illumina GSA v3.0 DTCBooster. Administration of broccoli sprouts before the caloric load reduced urinary 11-dehydro-TXB2 levels by 50% (p = 0.018). The amount of sulforaphane excreted in the urine during the study visits correlated negatively with 11-dehydro-TXB2 (rs = -0.377, p = 0.025). Participants carrying the polymorphic variant NAD(P)H dehydrogenase quinone 1 (NQO1*2) showed decreased excretion of sulforaphane (p = 0.035). Sulforaphane was shown to be effective in targeting platelet responsiveness after a single intake. Our results indicate an inverse causal relationship between sulforaphane and 11-dehydro-TXB2, which is unaffected by the concomitant intake of the metabolic challenge. 11-Dehydro-TXB2 shows promise as a non-invasive, sensitive, and suitable biomarker to investigate the effects of phytonutrients on platelet aggregation within hours. [https://clinicaltrials.gov/], identifier [NCT05146804].

Стан імунної та гемокоагуляційної систем у динаміці вагітності після ДРТ у жінок з інфекціями, що передаються статевим шляхом, в анамнезі

Purpose - to analyze the dynamics of laboratory parameters of functioning of the immune and haemocoagulation systems in pregnant women with a history of sexually transmitted infections (STIs) after pregravid preparation before cycles of assisted reproductive technologies (ART). Materials and methods. The functioning analysis of the immune and hemocoagulation systems of 200 pregnant women with a history of STIs after ART has been conducted. Group I (main) consisted of 100 pregnant women with pregravid preparation before the ART cycle, obstetric and perinatal support and delivery in accordance with the medical and organizational algorithms developed by our team, prognostic methods and treatment and preventive schemes; Group II (main) - 100 pregnant women who were treated using generally accepted prognostic, therapeutic and preventive measures. The control group is 100 practically healthy pregnant women with a favorable reproductive history and an uncomplicated course of pregnancy. The women underwent an in-depth immunological examination. Statistical processing of research results was carried out with the use of programs «Microsoft Excel 5.0» and «Statistica 8.0». Results. It is demonstrated a significant decrease (p&lt;0.05) in lymphocyte levels with properties of natural killers (CD56+) during the gestational period in pregnant women of Group I against Group II; the level of total hemolytic activity of the classical complement activation pathway (CH50) in Group I of pregnant women increased steadily with increasing gestational age; at the end of the II trimester, there was a significant decrease in CH50; in the III trimester of pregnancy, the level of CH50 in Group II was 123±6.7, but in the Group I this figure remained higher. Throughout the II and III trimesters of gestation, there was a probable decrease (p&lt;0.05) of the immunoregulatory coefficient in Group I of pregnant women, due to adequate correction of derivative immunological disorders, compared with pregnant women of Group II. During the residual formation of the placental barrier in Group I pregnant women the active partial thromboplastin time increased significantly during the gestational period and differed significantly from the indicators of Group II; there was a general decrease in the active recalcification time during the II and III trimesters of pregnancy in women of the Group I; a gradual decrease in the level of fibrin and fibrinogen degradation products, unlike the analogous indicator in the Group II, which showed a gradual increase. The level of stable thromboxane metabolite (TxB2) in the II trimester decreased twofold and was lower than in Group II; the level of stable prostacyclin metabolite (6-keto-PGF1α) increased in the III trimester. This led to an increase of PgI2/TxA2 balance in Group I, which corresponded to the physiological requirements of systemic and organ hemodynamics during pregnancy. Conclusions. Development and implementation of effective pregravid preparation before ART cycles in women with a history of STIs and medical correction during pregnancy helps to increase an adaptive compensatory and an adaptive potential in the immune and haemocoagulatory homeostasis of the mother and perinatal protection of the fetus. The study was carried out in accordance with the principles of the Helsinki Declaration. The research protocol was approved by the Local Ethics Committee of the institution specified in the article. The study received informed consent from the women. No conflict of interests was declared by the author.

Nonplatelet thromboxane generation is associated with impaired cardiovascular performance and mortality in heart failure.

Nonplatelet thromboxane generation, stimulated largely by oxidative stress, is a novel mortality risk factor in individuals with coronary artery disease. Though inversely associated with left ventricular ejection fraction (LVEF), a potential role in the pathobiology of heart failure (HF) remains poorly defined. Nonplatelet thromboxane generation and oxidative stress were assessed by measuring urine thromboxane-B2 metabolites (TXB2-M) and 8-isoPGF2α by ELISA in 105 subjects taking aspirin and undergoing right heart catheterization for evaluation of HF, valve disease, or after transplantation. Multivariable logistic regression and survival analyses were used to define associations of TXB2-M to invasive measures of cardiovascular performance and 4-year clinical outcomes. TXB2-M was elevated (>1,500 pg/mg creatinine) in 46% of subjects and correlated with HF severity by New York Heart Association (NYHA) functional class and brain natriuretic peptide level, modestly with LVEF, but not with HF etiology. There was no association of oxidative stress to HF type or etiology but a trend with NYHA functional class. Multiple invasive hemodynamic parameters independently associated with TXB2-M after adjustment for oxidative stress, age, sex, and race with pulmonary effective arterial elastance (Ea pulmonary), reflective of right ventricular afterload, being the most robust on hierarchical analysis. Similar to Ea pulmonary, elevated urinary TXB2-M is associated with increased risk of death (adjusted HR = 2.15, P = 0.037) and a combination of death, transplant, or mechanical support initiation (adjusted HR = 2.0, P = 0.042). Nonplatelet TXA2 thromboxane generation is independently associated with HF severity reflected by invasive measures of cardiovascular performance, particularly right ventricular afterload, and independently predicted long-term mortality risk.NEW & NOTEWORTHY Nonplatelet thromboxane generation in heart failure is independently associated with risk of death, transplant, or need for mechanical support. Measurement of urine thromboxane metabolites using a clinically available assay may be a useful surrogate for invasive measurement of cardiovascular hemodynamics and performance that could provide prognostic information and facilitate tailoring of therapy in patients with heart failure. Inhibiting thromboxane generation or its biological effects is a potential strategy for improving cardiovascular performance and outcomes in heart failure.

MO403: The Association Between Urinary 11-Dehydrothromboxane B2 and Laboratory Parameters in Aspirin-Treated Patients With Cardiorenal Syndrome

Abstract BACKGROUND AND AIMS Evaluation of urinary levels of the 11-dehydrothromboxane B2 is one of the methods for assessing platelet reactivity while taking aspirin. High levels of 11-dehydrothromboxane B2 in urine indicate continued thromboxane generation which is associated with an increased risk of atherothrombotic cardiovascular events. The present study aimed to evaluate the association between 11-dehydrothromboxane B2 in urine and laboratory parameters in aspirin-treated patients with the cardiorenal syndrome in subgroups from quartile with the highest and the lowest levels of thromboxane metabolite. METHOD A total of 82 patients with stable coronary artery disease (CAD) and CKD (stages 2–4) were included in the study. Urinary levels of 11-dehydrothromboxane B2 (a stable metabolite of thromboxane A2) were assessed using ELISA kit by Enzo Life Sciences (Switzerland). Statistical analysis was performed using the chi-squared test and Spearman's rank correlation coefficient. RESULTS In the subgroup of patients from the quartile with the highest levels of urinary 11-dehydrothromboxane B2, a history of ischemic stroke (P = 0.032) and a history of myocardial infarction (P = 0.048) were more common. There were significant correlations between urinary 11-dehydrothromboxane B2 and platelet count (rs = 0.457, P = 0.043), platelet crit (rs = 0.481, P = 0.037), pulmonary artery systolic pressure (rs = 0.847, P = 0.016) and left ventricular ejection fraction (rs = −0.719, P = 0.045) in the same subgroup of patients. In the subgroup of patients from the quartile with the lowest levels of 11-dehydrothromboxane B2 in urine statistically significant correlations between thromboxane metabolite and fasting plasma glucose (rs = 0.581, P = 0.007) and hematocrit (rs = 0.441, P = 0.003) were found. CONCLUSION Our study identified the relationships between the urinary concentration of 11-dehydrothromboxane B2 and laboratory and echocardiography parameters in aspirin-treated patients with cardiorenal syndrome. Accordingly, the received results represent the varying response to aspirin in patients with stable CAD and CKD. Additional research is needed to clarify the impact of platelet indices and the association between platelet reactivity while taking aspirin and pulmonary hypertension on behalf of the possibility of personalization of antiplatelet therapy.

Nephroprotective Effect of the Virgin Olive Oil Polyphenol Hydroxytyrosol in Type 1-like Experimental Diabetes Mellitus: Relationships with Its Antioxidant Effect

The aim of this study was to determine whether hydroxytyrosol administration prevented kidney damage in an experimental model of type 1 diabetes mellitus in rats. Hydroxytyrosol was administered to streptozotocin-diabetic rats: 1 and 5 mg/kg/day p.o. for two months. After hydroxytyrosol administration, proteinuria was significantly reduced (67–73%), calculated creatinine clearance was significantly increased (26–38%), and the glomerular volume and glomerulosclerosis index were decreased (20–30%). Hydroxytyrosol reduced oxidative and nitrosative stress variables and thromboxane metabolite production. Statistical correlations were found between biochemical and kidney function variables. Oral administration of 1 and 5 mg/kg/day of hydroxytyrosol produced an antioxidant and nephroprotective effect in an experimental model of type 1-like diabetes mellitus. The nephroprotective effect was significantly associated with the systemic and renal antioxidant action of hydroxytyrosol, which also influenced eicosanoid production.

Résistance à l'aspirine : l'ennemi de mon ami est mon ennemi

Low dose aspirin is an efficient antiplatelet agent to decrease the risk of occlusive arterial events, however it is not infallible. Aspirin resistance describe its inability to block the formation of thromboxane A2 in platelets and/or to produce an inhibitory effect on platelet aggregation. Detection of aspirin resistance relies on the results of various platelet function tests or on blood and urinary thromboxane metabolites concentrations, but these methods show very low correlation and reproducibility. Moreover, light-transmission aggregometry using arachidonic acid, known as the reference functional assay, requires technical expertise. The incidence rate of aspirin resistance amoung populations suffering from cardiovascular diseases is about 25%, however there is a wide variability depending on the specificity of the used test and the clinical features of the considered population.Aspirin resistance is associated with the recurrence of arterial occlusive events: the odds ratio is about 4 all tests combined, therefore it could be considered as a risk marker. Evidence is lacking regarding the relevance of these tests to resort an intensification of the antithrombotic treatment, and experts recommend to reserve their use for high-risk situations. Nevertheless several studies have explored the effect of dose increases or intake frequency increases, and revealed encouraging results regarding pharmacodynamic endpoints. The reasons for aspirin resistance are numerous, often remain debate, and can accumulate to result in poor response to aspirin.

Persistent long-term platelet activation and endothelial perturbation in women with Takotsubo syndrome

BackgroundTakotsubo (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. AimThis 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. MethodsTwenty-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 LCMS/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. ResultsIn 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. ConclusionsOur 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.

Thromboxane metabolite excretion is associated with serious vascular events in diabetes mellitus: a sub-study of the ASCEND trial

Abstract Background Platelet activation plays a major role in the atherothrombotic complications of diabetes. Thromboxane (TX)A2 is a pro-thrombotic prostanoid, synthesized via cyclooxygenase-1 and released by activated platelets. The metabolism of TXA2 in vivo leads to a major stable end-product, 11-dehydro-TXB2 (TXM), measurable in urine and reflecting the whole-body rate of TXA2 biosynthesis. In two large trials of high-risk, aspirin-treated (mostly, without diabetes) patients, (CHARISMA and HOPE trials), the baseline rate of urinary TXM excretion was an independent predictor of future cardiovascular events. Purpose The aim of the ASCEND (A Study of Cardiovascular Events in Diabetes) TXM sub-study was to investigate the association between baseline urinary TXM and future serious vascular events or revascularization (SVE-R), major bleeds and incident cancer independent of other risk factors and treatment, in people with diabetes and no manifest cardiovascular disease at trial entry. Methods Urinary TXM was measured by a previously GC/MS-validated, immunoassay in 6,487 participants with eligible baseline samples. Analyses excluded 539 participants using NSAIDs. TXM appeared log-normally distributed, so analyses were by quintiles and per SD (=0.622) of continuous loge TXM. The association of loge TXM with outcome was adjusted by basic factors (age, sex, sample volume and randomized treatment allocation) and by the predictors of log TXM (smoking, type 2 diabetes treated with insulin or oral hypoglycaemics, HDL cholesterol, body mass index, urinary albumin/creatinine ratio, eGFR). The association of log TXM with non-vascular, non-cancer MedDRA outcomes was investigated to determine whether TXM had a general effect on outcome. During a mean of 6.6 years follow-up there were 618 SVE-Rs, 206 bleeds and 700 cancers among these patients. Results Log TXM correlated significantly with SVE-R, hazard ratio (HR) per 1 SD of log TXM: 1.13 (1.04–1.23), p=0.003 (Figure 1, panel a), non-significantly with major bleeds [HR 1.15 (1.00–1.32), p=0.055] (Figure 1, panel b), and marginally significantly with cancer [HR 1.09 (1.01–1.17), p=0.03] (Figure 1, panel c). There was no association of log TXM with non-vascular non-cancer MedDRA outcomes (HR per 1 SD, 0.99; 99% CI, 0.94–1.05). Conclusion The rate of urinary TXM excretion, a non-invasive biomarker of TXA2-mediated platelet activation in vivo, is log-linearly associated with serious vascular events independent of other risk factors in people with diabetes. Its potential association with cancer must be viewed as hypothesis-generating and needs confirmation. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): IMI1: Surrogate markers for micro- and macro-vascular hard endpoints for innovative diabetes tools (SUMMIT).

Annexin A7 is a critical regulator of Ca2+ mobilization and lipid metabolism during platelet activation and arterial thrombosis

Abstract Background Platelet activation after contact to subendothelial collagen following atherosclerotic plaque rupture can lead to arterial thrombosis with acute thrombotic vascular occlusion. Annexin A7 (AnxA7) is an intracellular Ca2+- and phospholipid-binding protein that participates in the regulation of prostaglandin production in inflammatory diseases, but also in cell survival and tumor growth. Objective In the present study, we aimed to determine the role of AnxA7 for platelet Ca2+ signaling and lipid metabolism in platelet activation and arterial thrombosis in gene-targeted mice lacking annexin A7 (Anxa7−/−). Results AnxA7 is strongly expressed in platelets of platelet-rich human coronary thrombi aspirated from patients with acute ST elevation myocardial infarction. Functionally, platelet aggregation and dense granule secretion were significantly abrogated in Anxa7−/− platelets as compared to wildtype platelets (Anxa7+/+) after activation with collagen or collagen-related peptide (CRP), a specific agonist of the major platelet collagen receptor glycoprotein VI (GPVI). Further, in vitro thrombus formation on a collagen-coated surface under high arterial shear rates was significantly diminished in Anxa7-deficient platelets, and thrombotic vascular occlusion after FeCl3-induced injury in vivo was blunted in Anxa7−/−bone marrow chimeric mice, but no prolongation of bleeding time was observed. Moreover, Anxa7−/− platelets showed a significant reduction of IP3 production due to an abolished phospholipase C (PLC) gamma2 phosphorylation resulting in an abolished increase of [Ca2+]i after platelet activation with CRP. Moreover, we could show by quantitative lipidomics analysis that annexin A7 critically affects platelet oxylipid metabolism following activation of GPVI-dependent platelet signalling since Anxa7−/− platelets showed a significant reduction of the bioactive metabolites thromboxane A2 and 12(S)-hydroxy-eicosatetraenoic acid (12(S)-HETE) levels as well as significantly reduced levels of several other prostaglandins following stimulation with collagen or CRP. Finally, defective PLCgamma2 phosphorylation, IP1 production and blunted increase of [Ca2+]i in Anxa7−/− platelets could be rescued by exogenous addition of 12(S)-HETE indicating that AnxA7 is a critical regulator of the platelet oxygenase 12-lipoxygenase (12-LOX) in GPVI-dependent platelet Ca2+ signalling during arterial thrombosis following activation by collagen. Conclusions The present study reveals annexin A7 as a critical regulator of oxylipid metabolism and Ca2+ signaling in GPVI-dependent platelet activation. Anxa7-deficiency further results in decreased in vitro and in vivo thrombus formation, but does not affect bleeding time. In conclusion, annexin A7 plays an important role in platelet signaling during arterial thrombosis and thus, may reflect a promising target for novel antiplatelet strategies. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (Deutsche Forschungsgemeinschaft, DFG)

A randomized double-blind trial of 3 aspirin regimens to optimize antiplatelet therapy in essential thrombocythemia

AbstractEssential 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).

Unique Effect of Aspirin Therapy on Biomarkers in Aspirin-exacerbated Respiratory Disease. A Prospective Trial.

Rationale: Daily high-dose aspirin therapy benefits many patients with aspirin-exacerbated respiratory disease but provides no benefit for aspirin-tolerant patients with asthma. Type 2 inflammation characterizes aspirin-exacerbated respiratory disease.Objectives: To determine whether high-dose aspirin therapy changes biomarkers of type 2 inflammation in aspirin-exacerbated respiratory disease.Methods: Forty-two subjects with aspirin-exacerbated respiratory disease underwent an aspirin desensitization and were placed on high-dose aspirin (1,300 mg daily). Fifteen aspirin-tolerant subjects with asthma were also placed on high-dose aspirin. Biologic specimens and clinical parameters were collected at baseline and after 8 weeks on aspirin. Urinary eicosanoids, plasma tryptase and cytokine levels, platelet-leukocyte aggregates, and granulocyte transcripts were assessed.Measurements and Main Results: Eight weeks of high-dose aspirin decreased nasal symptoms and urinary prostaglandin E metabolite (P < 0.05) and increased urinary leukotriene E4 (P < 0.01) levels in subjects with aspirin-exacerbated respiratory disease, but not in those with aspirin-tolerant asthma. Urinary prostaglandin D2 and thromboxane metabolites decreased in both groups. Only in subjects with aspirin-exacerbated respiratory disease, exhaled nitric oxide (P < 0.05), plasma tryptase (P < 0.01), and blood eosinophil (P < 0.01) and basophil (P < 0.01) counts increased and plasma tryptase correlated with eosinophil counts (Pearson r = 0.514; P < 0.01) on aspirin. After correction for eosinophil counts, aspirin-induced changes in blood granulocyte transcripts did not differ between groups. Aspirin had no effect on platelet-leukocyte aggregates, platelet activation markers, or plasma cytokines in either group.Conclusions: High-dose aspirin therapy for 8 weeks paradoxically increases markers of type 2 inflammation in subjects with aspirin-exacerbated respiratory disease, despite reducing nasal symptoms. This effect of aspirin is unique to aspirin-exacerbated respiratory disease and not observed in subjects with aspirin-tolerant asthma.

The impact of cyclooxygenase‐2 selective and non‐isoform selective NSAIDs on the gut microbiota

Nonsteroidal anti‐inflammatory drugs (NSAIDs) exert their analgesic and anti‐inflammatory effects by inhibiting the cyclooxygenase (COX)‐1 and/or COX‐2, enzymes involved in biotransformation of arachidonic acid into prostanoids. It was previously reported that some NSAIDs alter populations of bacteria in the gut and modify the way that NSAIDs work in the body. Here we directly compared the impact of NSAIDs, selective (celecoxib) and non‐selective (naproxen) for COX‐2 inhibition, on the composition of the gut microbiota in mice. We conducted a longitudinal study in adult male C57BL/6 mice that were fed ad libitum with a regular chow or with a diet containing celecoxib or naproxen for three weeks. Naproxen caused significant COX‐1 inhibition as reflected by a ~70% inhibition of urinary thromboxane metabolite (TxM), an in vivo index of COX‐1 activity, and a 60% inhibition of a COX‐1/COX‐2 urinary prostaglandin (PG)E2 metabolite (PGEM), as measured by LC‐MS/MS. The treatment with celecoxib caused a significant 40% reduction of PGEM, but had no significant effect on TxM. Fecal samples were collected on day zero and day 21, DNA purified, 16S rRNA gene segments amplified using V1V2 primers, and amplicons sequenced. Community structure was compared using UniFrac, which generates distances between all pairs of samples as shared distances on a common phylogenetic tree, allowing assessment of clustering and gradients in community composition. For the unweighted analysis, the control group did not show a significant change over time (p=0.34; Permutational Multivariate Analysis of Variance Using Distance Matrices (‘Adonis’ function in R)), while a strong change was seen for naproxen (p=0.007; Adonis), and a lesser change was seen for celecoxib (p=0.023; Adonis). For the weighted analysis, the control group again did not change (p=0.31; Adonis), naproxen showed a trend (p= 0.089; Adonis), and no change was seen with celecoxib (p= 0.23; Adonis). Analysis of specific lineages showed strong longitudinal changes in some sequence clusters (operational taxonomic units), including Bacteroidetes S24‐7 and Clostridiales both p values &lt; 10−4). Moreover, we observed that naproxen caused gastroenteropathy in mice. The stomach of naproxen‐treated mice showed pyloric mucosal erosion and ulcers while the small intestine showed ulceration, inflammation in the lamina propria and epithelium, and thickening and blunting of the villi. Significantly fewer and less severe lesions were observed in mice treated with celecoxib both in the stomach and in the intestine. Thus, both NSAIDs have an impact on the composition of the gut microbiota, with the stronger effects seen for naproxen versus celecoxib. Effects were most evident in the unweighted analysis, indicating a greater effect on community membership than on their relative proportions. These data are consistent with the hypothesis that microbial dysbiosis is involved in the NSAID‐induced enteropathy that is more common with non‐isoform selective than COX‐2 selective NSAIDs.Support or Funding InformationThis work was supported by NIH grant U54 HL117798 to Garret A. FitzGerald.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

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

The Effects of Exercise, Aspirin, and Celecoxib in an Atherogenic Environment.

Optimal vascular function is a hallmark of cardiovascular health. Specifically, the balance of vasoconstricting and vasodilating substances is recognized as a marker of vascular health. One of the greatest challenges to vascular health and vasodilatory balance is tumor necrosis factor alpha (TNFα)-mediated inflammation. Uncovering effective strategies that maintain a vascular environment that is more vasodilatory and antithrombotic in the face of an inflammatory challenge is favorable. To test the ability of various antithrombotic and provasodilatory treatments, as well as combinations thereof, to prevent unfavorable changes in markers of endothelial dysfunction in human umbilical vein endothelial cells when presented with an inflammatory challenge. Human umbilical vein endothelial cells were pretreated with exercise-like levels of laminar shear stress (LSS), aspirin, celecoxib, and their combination before a TNFα challenge. Western blot analysis as well as colorimetric assays were used to determine levels of endothelial nitric oxide synthase (eNOS) and prostacyclin (6-keto PGF1α)/thromboxane (TXB2) metabolite ratio, respectively. Neither aspirin nor celecoxib were effective in preventing TNFα-induced reduction in eNOS. Further, aspirin was unable to maintain baseline levels of prostacyclin/thromboxane ratio in the face of the inflammatory challenge. Laminar shear stress, aspirin/LSS combination, and celecoxib/LSS combination were all able to prevent TNFα-induced alterations in eNOS levels and prostacyclin/thromboxane ratio. Effective strategies to maintain a healthy endothelium, and therefore resistance vessel health, need to include exercise-levels of shear stress to be effective.

Perioperative urinary thromboxane metabolites and outcome of coronary artery bypass grafting: a nested case-control study

ObjectiveAs a marker of in vivo thromboxane generation, high-level urinary thromboxane metabolites (TXA-M) increase the occurrence of cardiovascular events in high-risk patients. To investigate whether perioperative urinary TXA-M level is...

Flipping the cyclooxygenase (Ptgs) genes reveals isoform-specific compensatory functions ,

Two prostaglandin (PG) H synthases encoded by Ptgs genes, colloquially known as cyclooxygenase (COX)-1 and COX-2, catalyze the formation of PG endoperoxide H2, the precursor of the major prostanoids. To address the functional interchangeability of these two isoforms and their distinct roles, we have generated COX-2>COX-1 mice whereby Ptgs2 is knocked in to the Ptgs1 locus. We then “flipped” Ptgs genes to successfully create the Reversa mouse strain, where knock-in COX-2 is expressed constitutively and knock-in COX-1 is lipopolysaccharide (LPS) inducible. In macrophages, flipping the two Ptgs genes has no obvious impact on COX protein subcellular localization. COX-1 was shown to compensate for PG synthesis at high concentrations of substrate, whereas elevated LPS-induced PG production was only observed for cells expressing endogenous COX-2. Differential tissue-specific patterns of expression of the knock-in proteins were evident. Thus, platelets from COX-2>COX-1 and Reversa mice failed to express knock-in COX-2 and, therefore, thromboxane (Tx) production in vitro and urinary Tx metabolite formation in COX-2>COX-1 and Reversa mice in vivo were substantially decreased relative to WT and COX-1>COX-2 mice. Manipulation of COXs revealed isoform-specific compensatory functions and variable degrees of interchangeability for PG biosynthesis in cells/tissues.