Thromboxane B2 Levels Research Articles

Suppressive effect of citalopram on plasma concentrations of thromboxane B2

Background. Citalopram, a selective serotonin reuptake inhibitor (SSRi), is widely used to treat major depression. Patients treated with SSRIs suffer more frequently from bleeding disorders caused by the antiplatelet effect of SSRIs. Methods. To investigate the potential suppressive effect of citalopram treatment on plasma thromboxane B2 levels and its possible correlation with actual plasma concentration of citalopram. Plasma concentrations of thromboxane B2 and citalopram were examined in a cohort of 77 aspirin-treated geriatric patients before and in the third week of citalopram therapy. Results. Citalopram therapy led to a significant decrease of plasma concentrations of thromboxane B2 compared to its levels before initiation of the therapy. Furthermore, we have shown negative correlation in thromboxane B2 levels and actual plasma concentration of citalopram. Actual plasma concentrations of citalopram were significantly higher compared to younger adult patients treated with similar dose. Conclusions. In this study we have shown that even short-term citalopram therapy led to a suppression of thromboxane B2 production in aspirin-treated patients. This suppressive effect correlates with actual plasma concentration of citalopram.

Metabolomic analysis revealed the role of DNA methylation in the balance of arachidonic acid metabolism and endothelial activation

Arachidonic acid (AA) metabolism plays an important role in vascular homeostasis. We reported that DNA hypomethylation of EPHX2 induced a pro-inflammatory response in vascular endothelial cells (ECs). However, the change in the whole AA metabolism by DNA methylation is still unknown. Using a metabolomic approach, we investigated the effect of DNA methylation on the balance of AA metabolism and the underlying mechanism. ECs were treated with a DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-AZA), and AA metabolic profiles were analyzed. Levels of prostaglandin D2 (PGD2) and thromboxane B2 (TXB2), metabolites in the cyclooxygenase (COX) pathway, were significantly increased by 5-AZA treatment in ECs resulting from the induction of PGD2 synthase (PTGDS) and thromboxane A synthase 1 (TBXAS1) expression by DNA hypomethylation. This phenomenon was also observed in liver and kidney cell lines, indicating a universal mechanism. Pathophysiologically, homocysteine, known to cause DNA demethylation, induced a similar pattern of the change of AA metabolism. Furthermore, 5-AZA activated ECs, as evidenced by the upregulation of adhesion molecules. Indomethacin, a COX inhibitor, reversed the effects of 5-AZA on the levels of PGD2 and TXB2, EC activation and monocyte adhesion. In vivo, the plasma levels of PGD2 and TXB2 and the expression of In vivo PTGDS and TBXAS1 as well as adhesion molecules were increased in the aorta of the mice injected with 5-AZA. In conclusion, using a metabolomic approach, our study uncovered that DNA demethylation increased AA metabolites PGD2 and TXB2 by upregulating the expression of the corresponding enzymes, which might contribute to the DNA hypomethylation-induced endothelial activation.

Contribution of oxidative stress and prostanoids in endothelial dysfunction induced by chronic fluoxetine treatment

ObjectivesThe effects of chronic fluoxetine treatment were investigated on blood pressure and on vascular reactivity in the isolated rat aorta. Methods and resultsMale Wistar rats were treated with fluoxetine (10mg/kg/day) for 21days. Fluoxetine increased systolic blood pressure. Chronic, but not acute, fluoxetine treatment increased the contractile response induced by phenylephrine, serotonin (5-HT) and KCl in endothelium-intact rat aortas. l-NAME and ODQ did not alter the contraction induced by phenylephrine and 5-HT in aortic rings from fluoxetine-treated rats. Tiron, SC-560 and AH6809 reversed the increase in the contractile response to phenylephrine and 5-HT in aortas from fluoxetine-treated rats. Fluoxetine treatment increased superoxide anion generation (O2−) and the expression of cyclooxygenase (COX)-1 in the rat aorta. Reduced expression of nNOS, but not eNOS or iNOS was observed in animals treated with fluoxetine. Fluoxetine treatment increased prostaglandin (PG)F2α levels but did not affect thromboxane (TX)B2 levels in the rat aorta. Reduced hydrogen peroxide (H2O2) levels and increased catalase (CAT) activity were observed after treatment. ConclusionsThe major new finding of our study is that chronic fluoxetine treatment induces endothelial dysfunction, which alters vascular responsiveness by a mechanism that involves increased oxidative stress and the generation of a COX-derived vasoconstrictor prostanoid (PGF2α). Moreover, our results evidenced a relation between the period of treatment with fluoxetine and the magnitude in the increment of blood pressure. Finally, our findings raise the possibility that fluoxetine treatment increases the risk for vascular injury, a response that could predisposes to cardiovascular diseases.

Does the response to aspirin and clopidogrel vary over 6 months in patients with ischemic heart disease?

Dual-antiplatelet therapy (DAPT) with aspirin and a P2Y12 inhibitor, mostly clopidogrel, is the default therapy in both acute coronary syndrome (ACS) and after intracoronary stents. It is well established that responses to antiplatelet therapy (APT), particularly clopidogrel, are subject to considerable interindividual variability. We investigated whether responses to APT in individuals vary significantly over time. Simultaneous assay with VerifyNow(™) and short thrombelastography (s-TEG) was performed before and at four time points over 6 months after hospital discharge in 40 patients receiving DAPT. Serum thromboxane B2 levels were also measured. While aspirin response units (ARU) by VerifyNow(™) and serum thromboxane B2 levels remained stable over time, arachidonic acid (AA)-mediated platelet aggregation with s-TEG (i.e. area under the curve at 15 min in AA channel, AUC15AA ) increased at 1 week compared with predischarge (P < 0.008). In addition, platelet reactivity units (PRU) by VerifyNow(™) (P = 0.046) and adenosine diphosphate (ADP)-mediated platelet aggregation with s-TEG (i.e. AUC15ADP ) also increased at 1 week compared with predischarge (P = 0.026). There were no significant changes in either platelet reactivity or rates of high on-treatment platelet reactivity while receiving clopidogrel beyond 1 week. This study demonstrates important variability in responses to APT within individuals between predischarge and 1 week but not thereafter. The use of a single early (predischarge) platelet function assay as an indicator of future response may therefore be flawed. The design of future strategies to assess individual responses for tailored therapy needs to take this into account.

Determinants of reduced antiplatelet effect of aspirin in patients with stable coronary artery disease.

BackgroundAspirin is a cornerstone in management of coronary artery disease (CAD). However, considerable variability in the antiplatelet effect of aspirin has been reported.AimTo investigate independent determinants of reduced antiplatelet effect of aspirin in stable CAD patients.MethodsWe performed a cross-sectional study including 900 stable, high-risk CAD patients. Among these, 795 (88%) had prior myocardial infarction, 250 (28%) had type 2 diabetes, and 170 (19%) had both. All patients received 75 mg aspirin daily as mono antiplatelet therapy. The antiplatelet effect of aspirin was assessed by measurement of platelet aggregation employing 1) multiple electrode aggregometry (MEA, Multiplate Analyzer) in whole blood anticoagulated with citrate or hirudin using arachidonic acid (AA) or collagen as agonists, and 2) VerifyNow Aspirin Assay. Compliance was assessed by measurement of serum thromboxane B2.ResultsPlatelet count, prior myocardial infarction, type 2 diabetes and body mass index were independent determinants of increased AA-induced MEA platelet aggregation in citrate and hirudin anticoagulated blood (p-values ≤ 0.045). Similar results were found with VerifyNow. Prior coronary artery bypass grafting, age, smoking (MEA, AA/citrate) and female gender (MEA, AA/hirudin) were also independent determinants of increased platelet aggregation (p-values ≤ 0.038). Compliance was confirmed by low serum thromboxane B2 levels in all patients (median [25%;75%]: 0.97 [0.52;1.97], range 0.02-26.44 ng/ml).ConclusionPlatelet count, prior myocardial infarction, type 2 diabetes and body mass index were independent determinants of increased platelet aggregation, indicating that these characteristics may be key factors in reduced antiplatelet effect of aspirin in stable CAD patients.

Calprotectin and platelet aggregation in patients with stable coronary artery disease.

BackgroundRecent studies suggest that the inflammation-associated protein calprotectin may be implicated in the pathogenesis of coronary artery disease (CAD). However, the impact of calprotectin levels on platelet aggregation in CAD patients has never been investigated.ObjectivesWe investigated the association between calprotectin levels and platelet aggregation in stable, high-risk CAD patients receiving aspirin as mono antiplatelet therapy. Furthermore, we aimed to investigate independent clinical and laboratory determinants of calprotectin levels.MethodsWe performed a cross-sectional study including 581 stable, high-risk CAD patients. All patients received 75 mg aspirin daily as mono antiplatelet therapy. Platelet aggregation was assessed by 1) impedance aggregometry (Multiplate Analyzer) using arachidonic acid (AA) and collagen as agonists and by 2) the VerifyNow Aspirin Assay. Low-grade inflammation was evaluated by calprotectin, high-sensitive C-reactive-protein (hs-CRP) and interleukin-6. Platelet activation was assessed by soluble P-selectin, and cyclooxygenase-1 inhibition was evaluated by serum thromboxane B2, both measured by ELISA.ResultsCalprotectin levels correlated positively with platelet aggregation according to Multiplate Analyzer (r=0.12, p=0.01). Additionally, calprotectin was positively associated with leukocytes (r=0.33, p<0.0001), hs-CRP (r=0.31, p<0.0001), interleukin-6 (r=0.28, p<0.0001), soluble P-selectin (r=0.10, p=0.02) and serum thromboxane B2 (r=0.10, p=0.02). Type 2 diabetes mellitus was an independent predictor of increased calprotectin levels (p=0.004), and trends were seen for body mass index (p=0.06) and smoking (p=0.07). Compliance with aspirin was confirmed by low serum thromboxane B2 levels in all patients (median [25%;75%]: 1.07 [0.52;1.87] ng/mL).ConclusionCalprotectin levels correlated positively, though weakly, with platelet aggregation and activation as well as serum thromboxane B2 in high-risk, stable CAD patients treated with aspirin.

Effects of alprostadil and ufinastatin on protamine-induced pulmonary injury in patients undergoing off-pump coronary artery bypass grafting

Objective To investigate the therapeutic effects of alprostadil and ulinastatin on protamine-induced pulmonary injury in patients undergoing off-pump coronary artery bypass grafting(CABG). Methods Sixty patients scheduled to undergoing off-pump CABG were randomly divided into 4 groups:alprostadil group(A,n= 15), ufinastatin group(U,n= 15), AU group receiving alprostadil and ufinastatin(n= 15),and C group(control group,n= 15)receiving normal saline of the equal volume. Plasma neutrophil(PMN), interleukin(IL)- 6, IL- 8, IL- 10, tumor necrosis factor-α(TNF-α)and plasma concentrations of thromboxane B2(TXB2)in the radial arterial blood samples were measured on 5 min before and 15 min after administration of protamine. Blood gas analysis was done and alveolar- arterial oxgen gradiant(A- aDO 2), respiratory index (RI)and oxygenation index(OI) were calculated. Results The number of PMN and levels of IL-6, IL-8, IL-10 and TNF-α at T2 in A, U and AU groups were lower than those in the control group, with the group AU being the lowest(all P< 0.05). The level of TXB2 at T2 in A, U and AU groups was significantly lower than in the control group(P< 0.05) with the group AU being the lowest[(100±17)μg/L,P< 0.05]. The OI at T2 in A, U and AU groups was significantly higher than in control group(P<0.05). The highest OI value existed in AU group[(428±28)mm-Hg,P< 0.05]. Conclusion Alprostadil combined with ufinastatin can inhibit protamine- induced pulmonary injury in patients undergoing off- pump CABG. Key words: Alprostadil; Ulinastatin; Off-pump coronary artery bypass grafting; Protamine; Pulmonary injury

Aspirin resistance in children and young adults with splenectomized thalassemia diseases

Aspirin is now recommended for splenectomized thalassemia patients with high platelet counts. However, aspirin resistance defined by arachinodic acid (ACA) induced platelet aggregation ≥20%, has never been reported in this group of patients. In this study, twenty-four splenectomized thalassemia patients (15.7±4.1years), with platelet counts ≥800x109/L, and 21 non-splenectomized severe thalassemia patients (14.3±3.2years), were enrolled. After taking aspirin (2mg/kg/day), seven patients (29.2%) displayed aspirin resistance. Serum thromboxane B2 (TXB2) levels in the aspirin responsive group decreased significantly [52.6(8.8-174.6) vs 4.0(1.6-7.3) mcg/mL, p<0.001], while no change was demonstrated in the aspirin resistant group. Having increased aspirin to 4mg/kg/day, three of the seven aspirin resistant patients responded, while one developed upper GI bleeding from esophageal varices and was withdrawn from the study. For the three remaining patients, their doses were increased to the maximum of 300mg/day, and two of the three responded. Thrombin antithrombin complex and D-dimer levels were significantly decreased after taking aspirin (2mg/kg/day), although D-dimer level was still significantly higher than that in non-splenectomized group. Therefore, aspirin dosage can be adjusted individually to reach maximum effect of platelet inhibition. In addition, aspirin can reduce the levels of coagulation markers.

Arachidonic acid-stimulated platelet tests: Identification of patients less sensitive to aspirin treatment

Serum thromboxane-B2 (TxB2), together with arachidonic acid (AA)-induced platelet aggregation, are, at the moment, the most used tests to identify patients displaying high on-aspirin treatment platelet reactivity (HAPR). Both tests are specific for aspirin action on cyclooxygenase-1. While the correlation between serum TxB2 assay and clinical outcome is established, data are conflicting with regard to aspirin treatment and a possible association with AA-stimulated platelet markers and clinical outcome. To understand such discrepancy, we performed a retrospective study to compare both assays. We collected data from 132 patients receiving a daily dose of aspirin (100 mg/day) and data from 48 patients receiving aspirin on alternate days. All Patients who received a daily dose of aspirin were studied for AA-induced platelet aggregation together with serum TxB2 levels and AA-induced TxB2 formation was also studied in 71 patients out of entire population. Consistent with recommendations in the literature, we defined HAPR by setting a cut-off point at 3.1 ng/ml for serum levels of thromboxane B2 and 20% for AA-induced platelet aggregation. According to this cut-off point, we divided our overall population into two groups: (1) TxB2 < 3.1 ng/ml and (2) TxB2 > 3.1 ng/ml. We found low agreement between such tests to identify patients displaying HAPR. Our results show that AA-induced platelet aggregation >20% identify a smaller number of HAPR patients in comparison with TxB2. A good correlation between serum TxB2 and arachidonic acid-induced TxB2 production was found (r = 0.76619).

Multiple daily doses of acetyl‐salicylic acid (ASA) overcome reduced platelet response to once‐daily ASA after coronary artery bypass graft surgery: a pilot randomized controlled trial

The efficacy of ASA for prevention of graft failure following CABG surgery may be limited by incomplete platelet inhibition due to increased post-operative platelet turnover. To determine whether acetyl-salicylic acid (ASA) 325mg once-daily or 81mg four-times daily overcomes the impaired response to ASA 81mg once-daily in post-operative coronary artery bypass graft (CABG) patients. We randomized 110 patients undergoing CABG surgery to either ASA 81mg once-daily, 81mg four times daily or 325mg once-daily and compared their effects on serum thromboxane B2 (TXB2 ) suppression and arachidonate-induced platelet aggregation. One hundred patients were included in the final analysis. Platelet counts fell after surgery, reached a nadir on day 2, and then gradually increased. Although there was near complete suppression of TXB2 on the second or third post-operative day, TXB2 levels increased in parallel with the rise in platelet count on subsequent days. This increase was most marked in patients receiving ASA 81mg once-daily and less evident in those receiving ASA four times daily. On post-operative day 4, (i) median TXB2 levels were lower with four times daily ASA than with either ASA 81mg once-daily (1.1ng/mL; Quartile(Q) Q1,Q3: 0.5, 2.4 and 13.3ng/mL; Q1,Q3: 7.8, 30.8ng/mL, respectively; P<0.0001) or ASA 325mg once-daily (3.4ng/mL; Q1,Q3: 2.0, 8.2ng/mL; P=0.002), and (ii) ASA given four times daily was more effective than ASA 81mg once-daily and 325mg once-daily at suppressing platelet aggregation. Four times daily ASA is more effective than ASA 81 and 325mg once-daily at suppressing serum TXB2 formation and platelet aggregation immediately following CABG surgery.

Dual antiplatelet response during PCI: VerifyNow P2Y12 predicts myocardial necrosis and thromboxane B2 generation confirms wide variation in aspirin response

IntroductionThere remains concern that the antiplatelet effects of aspirin and clopidogrel vary between patients and poor responders may be at increased risk of adverse events. However, the optimal method of measuring aspirin and/or clopidogrel response remains unresolved. We compared three methods of measuring clopidogrel response recommended by a recent consensus statement for the European Society of Cardiology, and investigated a novel approach to measuring aspirin response in patients established on both aspirin and clopidogrel. In addition, we investigated whether any of these assays predict peri-procedural myocardial necrosis following percutaneous coronary intervention (PCI). MethodsA cross-section of 323 patients attending for PCI was tested for clopidogrel response using VerifyNow P2Y12, VASP Platelet Reactivity Index (VASP-PRI) and whole blood impedance aggregometry (WBPA). Aspirin response was assessed by measuring the residual ability of platelets to generate thromboxane, calculated as the difference between thromboxane B2 levels in serum and plasma, [TxB2]S-P. Peri-procedural myocardial necrosis was determined by a change in troponin I >0.2μmol/l. ResultsPatients demonstrated wide variation in response to both aspirin and clopidogrel. Correlation between VerifyNow P2Y12 and VASP-PRI was good (r=0.702, p<0.001). Correlation was moderate between WBPA and VerifyNow P2Y12 (r=0.639, p<0.001) and weak for WBPA and VASP-PRI (r=0.353, p<0.001). Only VerifyNow P2Y12 predicted peri-procedural myocardial necrosis. ConclusionsThe three methods of measuring response to clopidogrel identify different patients as poor responders. Poor response to clopidogrel assessed by VerifyNow P2Y12 predicts myocardial necrosis. Measurement of [TxB2]S-P demonstrates a wide variation in aspirin response in patients taking dual antiplatelet therapy.

Relationship of TSH with atherosclerosis-related factors in subclinical hypothyroidism

Two hundred and fifty patients with subclinical hypothyroidism(SCH)and 120 euthyroid volunteers were recruited for the study, SCH patients were stratified into 2 groups according to TSH levels(group A: TSH 10 mIU/L). All subjects were examined for clinical characteristics, thyroid profile, lipid profile, and biomarkers of early atherosclerosis. Patients in group B received L-thyroxine replacement treatment to achieve euthyroidism. After 6 months of stable euthyroidism all measurements were repeated. SCH patients had higher levels of total cholesterol(TC), low-density lipoprotein-cholesterol(LDL-C), asymmetric dimethyl arginine(ADMA), carotid artery intima media thickness(CIMT), thromboxane B2(TXB2), and C-reactive protein(CRP)but with lower nitric oxide(NO)level compared with euthyroid subjects. Levels of TC, LDL-C, CIMT, TXB2, and ADMA decreased, and NO level increased significantly after 6 months of euthyroidism. TSH was positively correlated with TC, LDL-C, CIMT, ADMA, and TXB2; and negatively correlated with NO. Based on multivariate regression analysis, TSH was an independent influential factor of CIMT and NO. We conclude that raised TSH is an important risk factor of atherosclerosis in SCH. (Chin J Endocrinol Metab, 2015, 31: 133-135) Key words: Hypothyroidism; Thyroid-stimulating hormone; Atherosclerosis; L-thyroxine

Abstract T P386: The Clinical Implications of Assessing Platelet Responsiveness to Aspirin Using Whole Blood Platelet Aggregation versus Urinary 11-Dehydrothromboxane Testing

Background: Whole blood aggregometry (WBA) is a direct measure of the effects of aspirin (ASA) on platelet aggregation while the measurement of urinary thromboxane B2 (TXB2) quantitates the metabolite produced by the ASA inhibited COX-1 enzyme. The objectives of this study were to a) compare indirect urine testing to a direct measure of platelet activity, b) determine the impact on clinical decision making if only TXB2 were measured, and c) compare time to obtain results from both procedures. Methods: Patients ages 18 to 90 who were on a stable dose of ASA for at least 14 days and had a confirmed stroke/TIA in the past 90 days were included. Patients were excluded if they used any medication that altered platelet function. Once consented, as per standard of care, blood was collected for WBA as well as a urine sample for a salicylate test for compliance. As per protocol, urine was sent out to have TXB2 levels measured in pg/mg of CR. Non-responsiveness was defined as aggregation to collagen 1 ug/mL &gt;10 ohms of impedance, ohms ratio of collagen 1 to 5 ug/mL &gt;.5, or arachidonic acid (AA) &gt;6 ohms. Borderline patients were classified as either responsive or non-responsive for analysis according to the treatment decisions that were made based on clinical judgment. ABCD 2 scores were calculated and TXB2 test turnaround time was noted. Results: In 58 patients TXB2 reported only 11 as non-responsive to ASA while WBA found 18 as non-responsive and 4 as borderline. Chi square analysis found significantly different proportions of participants to be responsive or non-responsive to ASA (p=.024) through each test. ASA therapy was adjusted based on WBA results typically the next day while mean turnaround time for TXB2 was 7.29 days. ABCD 2 scores based on risk factors predicted 10.5 ischemic events while only two actually occurred. Conclusions: WBA testing reported significantly more patients to be ASA non-responsive, allowing therapy changes to be made immediately. TBX2 testing failed to identify many patients that were ASA non-responsive and results did not become available for 1 to 2 weeks.

Valsartan Decreases Platelet Activity and Arterial Thrombotic Events in Elderly Patients with Hypertension

Background:Angiotensin type 1 receptor (AT1R) antagonists are extensively used for blood pressure control in elderly patients with hypertension. This study aimed to investigate the inhibitory effects of AT1R antagonist valsartan on platelet aggregation and the occurrence of cardio-cerebral thrombotic events in elderly patients with hypertension.Methods:Two-hundred and ten patients with hypertension and aged > 60 years were randomized to valsartan (n = 140) or amlodipine (n = 70) on admission. The primary endpoint was platelet aggregation rate (PAR) induced by arachidonic acid at discharge, and the secondary endpoint was the rate of thrombotic events including brain infarction and myocardial infarction during follow-up. Human aortic endothelial cells (HAECs) were stimulated by angiotensin II (Ang II, 100 nmol/L) with or without pretreatment of valsartan (100 nmol/L), and relative expression of cyclooxygenase-2 (COX-2) and thromboxane B2 (TXB2) and both p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor-kB (NF-kB) activities were assessed. Statistical analyses were performed by GraphPad Prism 5.0 software (GraphPad Software, Inc., California, USA).Results:PAR was lower after treatment with valsartan (11.49 ± 0.69% vs. 18.71 ± 2.47%, P < 0.001), associated with more reduced plasma levels of COX-2 (76.94 ± 7.07 U/L vs. 116.4 ± 15.89 U/L, P < 0.001) and TXB2 (1667 ± 56.50 pg/ml vs. 2207 ± 180.20 pg/ml) (all P < 0.001). Plasma COX-2 and TXB2 levels correlated significantly with PAR in overall patients (r = 0.109, P < 0.001). During follow-up (median, 18 months), there was a significantly lower thrombotic event rate in patients treated with valsartan (14.3% vs. 32.8%, P = 0.002). Relative expression of COX-2 and secretion of TXB2 with concordant phosphorylation of p38MAPK and NF-kB were increased in HAECs when stimulated by Ang II (100 nmol/L) but were significantly decreased by valsartan pretreatment (100 nmol/L).Conclusions:AT1R antagonist valsartan decreases platelet activity by attenuating COX-2/TXA2 expression through p38MAPK and NF-kB pathways and reduces the occurrence of cardio-cerebral thrombotic events in elderly patients with hypertension.

Features of complement activation-related pseudoallergy to liposomes with different surface charge and PEGylation: Comparison of the porcine and rat responses

Pigs are known to provide a sensitive model for studying complement (C) activation-related pseudoallergy (CARPA), a hypersensitivity reaction to liposomal and many other nanomedicines that limits their clinical use. The utility of rats as a CARPA model has, however, not been analyzed to date in detail. The present study compared the two models by inducing CARPA with i.v. bolus injections of two reactogenic liposomes that differed from each other in surface properties: one was AmBisome, a strong anionic, free-surface small unilamellar liposome (SUV), while the other was neutral, polyethylene glycol (PEG)-grafted SUV wherein the 2kDa-PEG was anchored to the membrane via cholesterol (Chol-PEG). Both in pigs and rats AmBisome caused significant consumption of C3, indicating C activation, along with paralleling massive changes in blood pressure, white blood cell, platelet counts and in plasma thromboxane B2 levels, indicating CARPA. These processes were similar in the two species in terms of kinetics, but significantly differed in the doses that caused major hemodynamic changes (~0.01 and ~22mg phospholipid (PL)/kg in pigs and rats, respectively). Pigs responded to AmBisome with pulmonary hypertension and systemic hypotension, and the reaction was not tachyphylactic. The major response of rats was systemic hypotension, leukopenia followed by leukocytosis, and thrombocytopenia. Chol-PEG liposomes caused severe reaction in pigs at 0.1mg/kg, while the reaction they caused in rats was mild even at 300mgPL/kg. Importantly, the reaction to Chol-PEG in pigs was partly tachyphylactic. These observations highlight fundamental differences in the immune mechanisms of porcine and rat CARPA, and also show a major impact of liposome surface characteristics, determining the presence or absence of tachyphylaxis. The data suggest that rats are 2–3 orders of magnitude less sensitive to liposomal CARPA than pigs; however, the causes of these differences, the PEG-dependent tachyphylaxis and the massive reactivity of Chol-PEG liposomes remain unclear.

Younger age, higher body mass index and lower adiponectin concentration predict higher serum thromboxane B2 level in aspirin-treated patients with type 2 diabetes: an observational study.

BackgroundEvidence from the literature suggests diminished acetylsalicylic acid (ASA) treatment efficacy in type 2 diabetes (DM2). High on-aspirin platelet reactivity (HAPR) in DM2 has been linked to poor glycemic and lipid control. However, there are no consistent data on the association between HAPR and insulin resistance or adipose tissue metabolic activity. The aim of this study was to assess the relationship between laboratory response to ASA and metabolic control, insulin resistance and adipokines in DM2.MethodsA total of 186 DM2 patients treated with oral antidiabetic drugs and receiving 75 mg ASA daily were included in the analysis. Response to ASA was assessed by measuring serum thromboxane B2 (TXB2) concentration and expressed as quartiles of TXB2 level. The achievement of treatment targets in terms of glycemic and lipid control, insulin resistance parameters (including Homeostatic Model Assessment-Insulin Resistance, HOMA-IR, index), and serum concentrations of high-molecular weight (HMW) adiponectin, leptin and resistin, were evaluated in all patients. Univariate and multivariate logistic regression analyses were performed to determine the predictive factors of serum TXB2 concentration above the upper quartile and above the median.ResultsSignificant trends in age, body mass index (BMI), HOMA-IR, HMW adiponectin concentration, C-reactive protein concentration and the frequency of achieving target triglyceride levels were observed across increasing quartiles of TXB2. In a multivariate analysis, only younger age and higher BMI were independent predictors of TXB2 concentration above the upper quartile, while younger age and lower HMW adiponectin concentration were predictors of TXB2 concentration above the median.ConclusionsThese results suggest that in DM2, the most important predictor of HAPR is younger age. Younger DM2 patients may therefore require total daily ASA doses higher than 75 mg, preferably as a twice-daily regimen, to achieve full therapeutic effect. Higher BMI and lower HMW adiponectin concentration were also associated with less potent ASA effect. This is the first study to demonstrate an association of lower adiponectin concentration with higher serum TXB2 level in patients treated with ASA.

Aspirin reload before elective percutaneous coronary intervention: impact on serum thromboxane b2 and myocardial reperfusion indexes.

Microvascular obstruction seems to predict poor outcome in patients undergoing elective percutaneous coronary intervention (PCI), but the underlying mechanism is still unclear. We analyzed whether serum thromboxane B2, a stable metabolite of thromboxane A2, may be implicated in post-PCI microvascular obstruction. We enrolled 91 patients (74 males, 66±10 years) on chronic low-dose aspirin therapy (aspirin, 100 mg daily) scheduled for elective PCI and randomly assigned to receive aspirin reload (325 mg orally, n=46) or no reload (control group, n=45) ≥1 hour before elective PCI. Serum levels of thromboxane B2, reperfusion indexes (corrected Thrombolysis In Myocardial Infarction frame count and myocardial blush grade), and serum cardiac troponin I were assessed before and after PCI. Serum thromboxane B2 significantly increased after 120 minutes (P=0.0447) from PCI in control but not in aspirin reload group. After PCI, both groups showed a statistically significant reduction in corrected Thrombolysis In Myocardial Infarction frame count more evident in aspirin reload group (P=0.0023). Moreover, after PCI, 61% of patients allocated to aspirin reload and only 32% of patients allocated to control group reached normal microcirculatory reperfusion (myocardial blush grade=3); patients with myocardial blush grade=3 exhibited lower values of serum thromboxane B2 compared with those with myocardial blush grade <3 (P=0.05). Periprocedural cardiac troponin I significantly increased (F=3.64; P=0.01334) and correlated with serum thromboxane B2 (ρ=0.31; P=0.0413) in control but not in aspirin reload group. In addition, left ventricular ejection fraction significantly increased after PCI only in the aspirin reload group (P=0.0005). Aspirin loading dose before elective PCI improves myocardial reperfusion and injury indexes, suggesting a possible role of platelet thromboxane A2 in microvascular occlusion. http://www.clinicaltrials.gov. Unique identifier: NCT01374698.

Enhanced oxidative stress and platelet activation in patients with Cushing's syndrome.

Cushing Syndrome (CS) is implicated by increased cardiovascular risk (CVR) leading to increased morbidity and mortality. Oxidative stress (OS) and platelet activation (PA) are associated with increased CVR. However, scarce data of OS in CS exist. Our objective was to determine the oxidant-antioxidant balance in CS. Fourteen patients with CS at diagnosis and fourteen healthy subjects (NS) were evaluated OS by measuring plasma 15-F2t -Isoprostane (15-F2t -IsoP), PA by thromboxaneB2 levels (TXB2 ), and antioxidant reserve measuring total antioxidant capacity (TAC) and serum vitamin E. 15-F2t -IsoP and TXB2 levels were significantly higher (P<0·01) in CS, while vitamin E levels were higher in NS (P<0·03). 15-F2t -IsoP levels were significantly higher (P<0·01) in complicated vs not-complicated CS and NS and significantly higher (P<0·03) in CS not-complicated vs NS. TXB2 levels were significantly reduced (P<0·03) in NS vs complicated and not-complicated CS. A negative correlation between Vitamin E and UFC was observed in CS (P<0·05 r=-0·497). TXB2 correlated with glucose, HbA1c and T-score (P<0·05 r=0·512, P<0·03 r=0·527 and P<0·01 r=0·783, respectively) and HDL (P<0·01 r=-0·651). 15-F2t -IsoP correlated with triglicerides, HbA1c and diastolic pressure (P<0·01 r=0·650, P<0·03 r=0·571 and P<0·05 r=0·498, respectively) and HDL (P<0·03 r=-0·594). This study emphasizes the major role of OS in CS. As our findings demonstrated that enhanced OS and PA take place in this rare metabolic disorder which is associated with increased CVR, it could be suggested that these biochemical alterations can further contribute in the pathogenesis of atherosclerosis, increased CVR and mortality in CS.

Biological evaluation of liposome-encapsulated hemoglobin surface-modified with a novel PEGylated nonphospholipid amphiphile.

Traumatic injury is often associated with hemorrhagic shock. Liposome-encapsulated hemoglobin (LEH) is being developed as an artificial oxygen carrier to address post-hemorrhage oxygen and volume deficit. Here, we report a new composition of LEH based on the use of polyethylene glycol (PEG2K ) conjugated with nonphospholipid hexadecylcarbamoylmethylhexadecanoate (HDAS) to modify the surface of LEH particles. LEH was manufactured by the high-pressure homogenization method using dipalmitoylphosphatidylcholine (∼38 mol%), cholesterol (∼38 mol%), HDAS (∼20 mol%), and highly purified stroma-free human hemoglobin. HDAS-PEG2K was postinserted into the resultant LEH to generate HDAS-PEG2K -LEH. We investigated the potential immune response to HDAS-PEG2K -LEH in a mice model. At the same time, the preparation was tested in a rat model to study the effect of repeated HDAS-PEG2K -LEH injection over 4 weeks. We found that HDAS-PEG2K modification substantially reduced the circulating levels of anaphylatoxins C3a and C5a, as well as plasma levels of thromboxane B2, in mice. Repeated injections of HDAS-PEG2K -LEH in rats did not appear to alter its clearance profile after 4 weeks of treatment. No antibody response against human hemoglobin or PEG was detected in rat plasma. Histological observations of lung, liver, spleen, and kidney were not significantly different between saline-treated rats and HDAS-PEG2K -LEH-treated rats. Immunohistochemical staining for rat heme oxygenase-1 (HO-1) did not show induced expression of HO-1 in these organs. These results suggest that the new surface modification of LEH is immune-neutral and does not adversely affect histology even after repeated administration.

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

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