Thromboxane biosynthesis and future events in diabetes: the ASCEND trial.

BACKGROUND AND AIMS: Thromboxane (TX) A2, released by activated platelets, plays an important role in atherothrombosis. Urinary 11-dehydro-TXB2 (U-TXM), a stable metabolite reflecting the whole-body TXA2 biosynthesis, is reduced by ∼70% by daily low-dose aspirin. The U-TXM represents a non-invasive biomarker of in vivo platelet activation and is enhanced in patients with diabetes. This study assessed whether U-TXM is associated with the risk of future serious vascular events or revascularizations (SVE-R), major bleeding, or cancer in patients with diabetes. METHODS: The U-TXM was measured pre-randomization to aspirin or placebo in 5948 people with type 1 or 2 diabetes and no cardiovascular disease, in the ASCEND trial. Associations between log U-TXM and SVE-R (n = 618), major bleed (n = 206), and cancer (n = 700) during 6.6 years of follow-up were investigated by Cox regression; comparisons of these associations with the effects of randomization to aspirin were made. RESULTS: Higher U-TXM was associated with older age, female sex, current smoking, type 2 diabetes, higher body size, urinary albumin/creatinine ratio of ≥3 mg/mmol, and higher estimated glomerular filtration rate. After adjustment for these, U-TXM was marginally statistically significantly associated with SVE-R and major bleed but not cancer [hazard ratios per 1 SD higher log U-TXM (95% confidence interval): 1.09 (1.00-1.18), 1.16 (1.01-1.34), and 1.06 (0.98-1.14)]. The hazard ratio was similar to that implied by the clinical effects of randomization to aspirin for SVE-R but not for major bleed. CONCLUSIONS: The U-TXM was log-linearly independently associated with SVE-R in diabetes. This is consistent with the involvement of platelet TXA2 in diabetic atherothrombosis.

Stability of urinary thromboxane A2 metabolites and adaptation of the extraction method to small urine volume.

BACKGROUND: Thromboxane (TX) A2 is a pro-thrombotic prostanoid synthesized by activated platelets, biotransformed into 11-dehydro-TXB2, measurable in urines. Eleven-dehydro-TXB2 excretion is increased in high risk cardiovascular diseases; however, this cardiovascular biomarker awaits validation in large trials. The need of large urine volume (8 - 10 mL) and the unknown stability of 11-dehydro-TXB2 in urine after collection might limit its implementation. METHODS: We scaled the original method for urine extraction and 11-dehydro-TXB2 measurement down to 1 mL, and assessed its stability at 4 degrees C or 25 degrees C up to 6 days after collection. The sensitivity of the 1 mL procedure was also tested in aspirin-treated patients with low 11-dehydro-TXB2 excretion RESULTS: The 1 mL adapted method was highly correlated with the original assay (rho = 0.98, p < 0.001, n = 33). Both methods showed similar recoveries in samples spiked with exogenous 11-dehydro-TXB2. Urinary 11-dehydro-TXB2 values in samples immediately frozen were comparable and highly correlated to values in samples at 4 degrees C (day 6: rho = 0.99, p > 0.001, n = 8) or 25 degrees C (day 6: rho = 0.98, p < 0.001, n = 23) up to 6 days in controls and patients. CONCLUSIONS: Eleven-dehydro-TXB2 can be measured in small urine volumes and is relatively stable for a few days after collection, even at 25 degrees C. These data allow the validation of this non-invasive cardiovascular biomarker in large studies.

Influence of simvastatin on the thromboxane and prostacyclin pathways, in vitro and in vivo.

3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) are believed to exert beneficial effects against cardiovascular disease beyond correction of dyslipidemia. The aim of this combined in vitro and in vivo study was to investigate the influence of the commonly used simvastatin on prostacyclin and thromboxane A2, 2 prostaglandins with different cardiovascular effects, normally in homeostatic balance in the circulatory system. Single-dose administration of simvastatin significantly decreased urinary prostacyclin excretion of healthy volunteers (P < 0.01) and increased the ratio between thromboxane A2 and prostacyclin (2-fold increase, P < 0.01), as assessed by enzyme immunoassays of the corresponding metabolites in urine. Human vascular endothelial cells, exposed to corresponding concentrations of simvastatin and assayed in the same way, reduced the release of prostacyclin about 40% (P < 0.05), altered the transcriptional expression of cyclooxygenase and prostacyclin synthase as analyzed by real-time polymerase chain reaction, and reduced the prostacyclin synthase promoter activity by 50% (P < 0.05), evaluated in a luciferase reporter system. We speculate that simvastatin shifts the balance between thromboxane A2 and prostacyclin in favor of the thromboxane pathway in vivo, and after exposure to clinically relevant concentrations in vitro. This may have pathophysiological implications by promoting a prothrombotic state in the blood vessels.

Thromboxane A(2) generation, in the absence of platelet COX-1 activity, in patients with and without atherothrombotic myocardial infarction.

BACKGROUND: Aspirin's therapeutic action is via inhibition of platelet cyclooxygenase 1 (COX-1) thromboxane A2 (TxA2) production. The aim of this study was to evaluate TxA2 production, in the absence of platelet COX-1 activity, in coronary atherosclerotic heart disease patients with and without atherothrombotic myocardial infarction (MI). METHODS AND RESULTS: TxA2 production, in the absence of platelet COX-1 activity, was evaluated in 44 patients taking aspirin on 3 commercially available assays that detect metabolites of TxA2 in the urine. Two assays measure urine 11-dehydro-thromboxane B2 (TxB2) alone and 1 measures urine 11-dehydro-TxB2 plus 11-dehydro-2,3-dinor-TxB2. Platelet COX-1 inhibition was confirmed on <10% platelet aggregation in response to ≥1 mmol/L arachidonic acid. Median urine 11-dehydro-TxB2 was no different in those with and without a diagnosis of atherothrombotic MI (325 vs. 311 pg/mg creatinine, P=0.59 via polyclonal ELISA) and (312 vs. 244 pg/mg creatinine, P=0.11 via LC-MS/MS). Median urine 11-dehydro-TxB2 plus 11-dehydro-2,3-dinor-TxB2, however, was higher in those with vs. those without a diagnosis of atherothrombotic MI (1,035 vs. 606 pg/mg creatinine, P=0.03 via monoclonal ELISA). CONCLUSIONS: Differences in TxA2 production, in the absence of platelet COX-1 activity, between those with vs. without atherothrombotic MI were not observed when TxA2 generation was assessed on 11-dehydro-TxB2 production alone (polyclonal ELISA or LC-MS/MS), but differences were observed when TxA2 generation was assessed using 11-dehydro-TxB2 plus 11-dehydro-2,3-dinor-TxB2 (monoclonal ELISA). These findings highlight important differences between different commercially available assays for TxA2 generation and suggest that 11-dehydro-2,3-dinor-TxB2 may be critical to the biology of atherothrombosis.

The contribution of cyclooxygenase-1 and -2 to persistent thromboxane biosynthesis in aspirin-treated essential thrombocythemia: implications for antiplatelet therapy.

We tested whether cyclooxygenase 2 (COX-2) expression and unacetylated COX-1 in newly formed platelets might contribute to persistent thromboxane (TX) biosynthesis in aspirin-treated essential thrombocythemia (ET). Forty-one patients on chronic aspirin (100 mg/day) and 24 healthy subjects were studied. Platelet COX-2 expression was significantly increased in patients and correlated with thiazole orange-positive platelets (r = 0.71, P < .001). The rate of TXA(2) biosynthesis in vivo, as reflected by urinary 11-dehydro-TXB(2) (TXM) excretion, and the maximal biosynthetic capacity of platelets, as reflected by serum TXB(2), were higher in patients compared with aspirin-treated healthy volunteers. Serum TXB(2) was significantly reduced by the selective COX-2 inhibitor NS-398 added in vitro. Patients were randomized to adding the selective COX-2 inhibitor, etoricoxib, or continuing aspirin for 7 days. Etoricoxib significantly reduced by approximately 25% TXM excretion and serum TXB(2). Fourteen of the 41 patients were studied again 21 (+/- 7) months after the first visit. Serum TXB(2) was consistently reduced by approximately 30% by adding NS398 in vitro, while it was completely suppressed with 50 microM aspirin. Accelerated platelet regeneration in most aspirin-treated ET patients may explain aspirin-persistent TXA(2) biosynthesis through enhanced COX-2 activity and faster renewal of unacetylated COX-1. These findings may help in reassessing the optimal antiplatelet strategy in ET.

Targeting the urine and plasma determinants of thromboxane A2 metabolism in detection of aspirin effectiveness.

Detection of reduced aspirin effectiveness has gained significant importance since clinical consequences of aspirin resistance were reported. Nevertheless, due to differentiated molecular basis of aspirin resistance, the conflicting choice of referential method for detection of acetylsalicylic acid ineffectiveness has become troublesome. This study, using a rat model of antiplatelet therapy, examines the aptitude of selected TXB2 metabolism-based methods in the detection of acetylsalicylic acid effectiveness. We hypothesized that ex-vivo whole blood spontaneous TXB2 generation assay could be, contrary to basal TXB2 and urine 11-dTXB2, a novel surrogate measure for impaired acetylsalicylic acid-dependent inhibition of thromboxane synthesis. To address this hypothesis, we evaluated the sensitivity of TXB2 generation assay in hirudinized whole blood to detect acetylsalicylic acid-mediated inhibition of cyclooxygenase activity in healthy rats and diabetic rats treated with acetylsalicylic acid. In diabetic and control animals, both acetylsalicylic acid drenches in the dose-independent manner contributed to significant attenuation of basal plasma TXB2 and urinary 11-dTXB2 formation. Urinary concentrations of 11-dTXB2 were, contrary to basal TXB2, significantly higher, regardless of acetylsalicylic acid dose, among all diabetic groups, compared with corresponding control groups. Determination of TXB2 generation in whole blood enabled sensitive detection of dose-related acetylsalicylic acid effect in both groups, as well as increased TXB2 formation in diabetes. We showed for the first time that evaluation of spontaneous generation of TXB2 in hirudinized whole blood enables, contrary to basal plasma TXB2 and urine 11-dTXB2 concentrations, to sensitively determine the acetylsalicylic acid effect in healthy and diabetic subjects.

A comparison of hormone therapies on the urinary excretion of prostacyclin and thromboxane A2.

OBJECTIVE: To evaluate the effect of estradiol, estradiol and norethisterone acetate (NETA), raloxifene and tibolone on the prostacyclin (PGI(2))/thromboxane A2 (TxA(2)) ratio in postmenopausal women after 8 weeks of treatment. DESIGN: This was a randomized, double-blind, cross-over study. Each patient took 8-week courses of estradiol 2 mg, estradiol 2 mg + NETA 1 mg, tibolone 2.5 mg, and raloxifene 60 mg; there was an 8-week placebo wash-out between each different intervention. All volunteers took all four treatment options and were randomized to one of three possible sequences. Urine was collected and frozen at each visit. Urinary metabolites of PGI(2) and TxA(2) were then assessed at the end of the study. RESULTS: The ratio of PGI(2)/TxA(2) was significantly increased for raloxifene. No other treatments showed statistically significant changes. CONCLUSIONS: The relationship between cardiovascular risk and hormone replacement therapy remains poorly understood. Raloxifene may have additional cardioprotective effects that the other treatments did not demonstrate, and none of the treatments statistically worsened the PGI(2)/TxA(2) ratio. This ratio may be under-utilized as a marker of net effect on cardiovascular health, but more research is needed to link it to health outcomes.

Digoxin and Platelet Activation in Patients With Atrial Fibrillation: In Vivo and In Vitro Study.

Background Digoxin use was shown to be associated with an increased risk of cardiovascular events in atrial fibrillation ( AF ). We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation. Methods and Results Post hoc analysis of a prospective study of anticoagulated patients with AF . Patients were divided into 2 groups balanced for age, sex, and cardiovascular risk factors: digoxin users (n=132) and nonusers (n=388). Urinary excretion of 11-dehydro-thromboxane B2 (TxB2), a marker of platelet activation, and serum digoxin concentration ( SDC ) were measured. In vitro experiments were performed on platelets from healthy subjects and AF patients, which were incubated with scalar doses of digoxin (0.6-2.4 ng/mL) with or without prestimulation with a sub-threshold of collagen. Median 11-dehydro-TxB2 was 105.0 ( interquartile range, 60.0-190.0) ng/mg creatinine, and median SDC was 0.65 ( interquartile range, 0.40-1.00) ng/mL. Urinary 11-dehydro-TxB2 and SDC were correlated ( rs=0.350, P<0.001). Patients in the upper tertile of SDC showed higher 11-dehydro-TxB2 compared with non-digoxin users ( P=0.019). In vitro study showed an increased basal platelet activation in patients with AF compared with healthy subjects . Digoxin (2.4 ng/mL) induced calcium mobilization, PAC -1 (procaspase-activating compound 1) and platelet aggregation in AF patients but not in healthy subjects . After pretreatment with a sub-threshold of collagen, digoxin dose-dependent induced calcium mobilization, arachidonic acid release, TxB2 biosynthesis, PAC -1 and soluble platelet selectin expression, and platelet aggregation, which were inhibited by antibody against digoxin. Conclusions We found a significant in vivo correlation between SDC and platelet activation. Supratherapeutic SDC increased in vitro platelet aggregation via calcium-related phospholipase A2 phosphorylation. Our findings may have clinical implications for AF patients treated with digoxin.

Urinary excretion of degradation products of prostacyclin and thromboxane is increased in patients with gestational choriocarcinoma.

Gestational choriocarcinoma metastasizes rapidly, in which process the vasoactive prostanoids may be significant. We therefore compared the urinary excretion of prostacyclin and thromboxane A2 (TxA2) metabolites in 19 women with gestational choriocarcinoma and 20 healthy age-matched women by assessing spot urine samples for 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and 2,3-dinor-6-keto-prostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha) (degradation products of prostacyclin) as well as for thromboxane B2 (TxB2) and 2,3-dinor-TxB2 (degradation products of TxA2) by high-pressure liquid chromatography, followed by radioimmunoassay; the data were related to urinary creatinine concentration. The urinary output of 6-keto-PGF1 alpha [29.56 +/- 7.0 versus 25.08 +/- 3.91 ng/mmol creatinine (SE)] in patients with choriocarcinoma was normal, but that of 2,3-dinor-6-keto-PGF1 alpha in cancer patients was higher than in controls (24.44 +/- 5.20 versus 14.84 +/- 1.94, P less than 0.02), as was that of TxB2 (22.72 +/- 4.69 versus 9.69 +/- 1.52, P less than 0.001) and 2,3-dinor-TxB2 (114.21 +/- 30.81 versus 51.81 +/- 10.40, P less than 0.01). The ratio of net prostacyclin output (6-keto-PGF1 alpha plus 2,3-dinor-6-keto-PGF1 alpha) to the net TxA2 output (TxB2 plus 2,3-dinor-TxB2) in cancer patients [0.52 +/- 0.1 (SE)] was lower (P less than 0.03) than in the controls (0.83 +/- 0.1), and in an inverse relation (r = -0.54, P less than 0.05) to the scoring index of poor prognosis for the disease. We conclude that the prostanoid excess in gestational trophoblastic disease, as evidenced for the first time in this study, may originate from choriocarcinoma cells, or may be a paraneoplastic phenomenon, and we conclude also that TxA2 excess may contribute to the tumor growth and/or formation of metastases.

Simultaneous quantification of PGI2 and TXA2 metabolites in plasma and urine in NO-deficient mice by a novel UHPLC/MS/MS method.

The balance between vascular prostacyclin (PGI2) generated mainly via cyclooxygenase-2 (COX-2) and its physiological antagonist platelet-derived thromboxane A2 (TXA2) formed by cyclooxygenase-1 (COX-1) determines cardiovascular homeostasis. In the present work, a novel bioanalytical method for simultaneous quantification of stable plasma and urinary metabolites of PGI2 (6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α) and TXA2 (TXB2, 2,3-dinor-TXB2) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was developed. The method was validated using artificial plasma and urine and linearity range, intra- and inter-day precision and accuracy, recovery of analytes, relative and absolute matrix effect and stability of analytes were determined. The use of artificial biofluids improved the method sensitivity as it eliminated the contribution of endogenous metabolites present in mice plasma and urine to validation procedure. The newly developed and validated method allowed to quantify 6-keto-PGF1α and TXB2 in mice plasma as well as 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 in urine samples with high sensitivity and accuracy. The calibration range was established from 0.1 to 100ng/mL for all analytes using artificial biofluids and the recoveries were greater than 89.9%. All validated parameters met the criteria of acceptance specified in FDA and EMA guidance. This method was successfully employed for profiling of the changes in PGI2 and TXA2 generation in NO-deficient mice. This work demonstrated that NO-deficiency induced by L-NAME, evidenced by a fall in nitrite in plasma and urine, was associated with platelet activation, robust increase in TXB2 and mild increase in 6-keto-PGF1α concentration in plasma. Changes in 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 concentration in urine were less evident suggesting that the measurements in plasma better reflect modest changes in PGI2/TXA2 homeostasis than measurements in urine.

In Vivo Platelet Activation and Aspirin Responsiveness in Type 1 Diabetes.

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.

Lipid peroxidation and platelet activation in murine atherosclerosis.

BACKGROUND: Lipid peroxidation and platelet activation are thought to be important contributors to the pathogenesis of atherosclerosis. The relevance of their interaction in vivo, however, is unknown. METHODS AND RESULTS: LDL receptor-deficient (LDLR(-/-)) mice on a high-fat diet developed extensive atherosclerosis and had increased urinary levels of 8,12-iso-isoprostane (iP) F(2alpha)-VI and 2,3-dinor-thromboxane (Tx) B(2), markers of in vivo lipid peroxidation and platelet activation, respectively. Vitamin E supplementation suppressed 8,12-iso-iPF(2alpha)-VI biosynthesis and reduced atherosclerosis (65%) without having a significant effect on lipid levels or TxB(2) biosynthesis. Addition of the platelet inhibitor indomethacin to vitamin E simultaneously suppressed 8,12-iso-iPF(2alpha)-VI and TxB(2), significantly reduced soluble intercellular adhesion molecule-1 and monocyte chemoattractant protein-1, and remarkably, further reduced atherosclerosis (80%). CONCLUSIONS: These results indicate that in vivo lipid peroxidation and platelet activation coexist in murine atherosclerosis and that lipid peroxidation does not contribute to platelet activation and reflects the oxidant component of the inflammatory response. Our findings suggest that oxidant stress and platelet activation represent 2 distinct therapeutic targets in atherogenesis. We propose that a combination of antioxidants and platelet inhibitors might be rationally evaluated in the prevention of progression of human atherosclerosis.

Renal hemodynamics and urinary excretion of 6-keto-prostaglandin F1 alpha and thromboxane B2 in newly diagnosed type I diabetic patients.

We have studied the functional importance of renal eicosanoids in renal hemodynamics of seven newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients by treatment with two structurally unrelated inhibitors of cyclooxygenase (i.e., piroxicam and sulindac). Glomerular filtration rate (GFR), renal plasma flow (RPF), daily urinary excretion of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha, the stable hydrolysis product of prostacyclin), and thromboxane B2 (TXB2, the stable hydrolysis product of thromboxane A2) were measured before, during, and after piroxicam (all patients) or sulindac (3 patients) treatment. Urinary excretion of 6-keto-PGF1 alpha was significantly increased (P less than .01) in diabetic patients compared with seven healthy subjects, whereas urinary excretion of TXB2 was unchanged. The baseline value of GFR was significantly (P less than .01) higher in diabetic compared with normal volunteers, whereas baseline RPF was comparable in both groups. Piroxicam (20 mg/day) reduced urinary excretion of 6-keto-PGF1 alpha and TXB2 by 65.7 +/- 26 and 64.6 +/- 33%, respectively. These biochemical changes were temporally associated with the approximately 19% decrease in GFR (P less than .01). A week after discontinuation of the drug, GFR and urinary excretion of 6-keto-PGF1 alpha were still significantly (P less than .05) reduced, whereas urinary excretion of TXB2 returned to control values. In contrast, urinary excretion of eicosanoids and renal function were not affected by sulindac (0.4 g/day) treatment. No functional changes were detected in healthy subjects despite a similar suppression of renal cyclooxygenase activity when they were treated with piroxicam.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidant stress but not thromboxane decreases with epoprostenol therapy.

Epoprostenol has improved the outcome of patients with primary pulmonary hypertension (PPH); however, its mechanism of action remains poorly understood. Isoprostanes are easily measured markers of oxidant stress and can activate platelets leading to increased thromboxane A2 (TxA2) production. We hypothesized that oxidant stress is associated with increased TxA2 synthesis and that epoprostenol decreases oxidant stress and TxA2 production in patients with PPH. Morning urine samples were obtained from 19 patients with PPH. We measured urinary metabolites of the isoprostane, 8-iso-PGF2alpha (F2-IsoP-M), and of TxA2 (Tx-M) before and after treatment with epoprostenol in patients with PPH. Mean (+/-SE) levels of F2-IsoP-M were elevated at baseline in our patients, 863 +/- 97 pg/mg creatinine. During treatment with epoprostenol, values decreased to 636 +/- 77 pg/mg creatinine (P = 0.011), and there was a strong correlation between the change in F2-IsoP-M and follow-up pulmonary vascular resistance (R2 = 0.69, P < 0.001). Tx-M levels were markedly elevated at baseline and were unchanged with therapy. These results indicate that oxidant stress decreases with epoprostenol therapy and is associated with hemodynamic and clinical improvement. The failure of Tx-M to decrease with therapy suggests that epoprostenol does not exert a beneficial effect through inhibition of TxA2 production in patients with PPH.

Optimal suppression of thromboxane A(2) formation by aspirin during percutaneous transluminal coronary angioplasty: no additional effect of a selective cyclooxygenase-2 inhibitor.

OBJECTIVES: We examined the contribution of cyclooxygenase (COX)-1 and -2 to the generation of prostacyclin, thromboxane (Tx) A(2), and 8-epi prostaglandin (PG) F(2alpha) during percutaneous transluminal coronary angioplasty (PTCA). BACKGROUND: Both TxA(2) and 8-epi PGF(2alpha) activate platelets and are mitogenic, whereas prostacyclin is a platelet inhibitor, and therefore may influence the outcome of PTCA. METHODS: Twenty-one patients undergoing PTCA while receiving aspirin 300 mg daily or aspirin plus the selective COX-2 inhibitor nimesulide were compared with 13 patients treated only with fradafiban, a glycoprotein IIb/IIIa antagonist. Urine was analyzed for the metabolites of TxA(2) (Tx-M) and prostacyclin (PGI-M) and for the isoprostane, 8-epi PGF(2alpha). RESULTS: In the fradafiban group, there was a marked increase in Tx-M during PTCA (mean, 1973; 95% confidence interval [CI] 112 to 3834 rising to mean 7645; 95% CI 2,009 to 13281 pg/mg creatinine, p = 0.018). The Tx-M excretion was similarly reduced by aspirin and the combination of aspirin and nimesulide. In contrast, the combination of nimesulide and aspirin inhibited PGI-M excretion to a greater extent than aspirin (p = 0.001). Urinary 8-epi PGF(2alpha) excretion was elevated following PTCA compared with normal subjects (p = 0.002) and appeared to be unaffected by any of the treatments. CONCLUSIONS: The increase in TxA(2) during PTCA is primarily COX-1 dependent, and aspirin alone is effective in suppressing its formation. In contrast, prostacyclin generation is both COX-1 and COX-2 dependent. The inhibition of COX-1 and COX-2 did not prevent the production of 8-epi PGF(2alpha), suggesting that this is not enzymatically derived. The persistent generation of 8-epi PGF(2alpha) may contribute to the thrombosis and restenosis that complicate PTCA.

Nicotine effects on eicosanoid formation and hemostatic function: comparison of transdermal nicotine and cigarette smoking.

OBJECTIVES: The purpose of this study was to examine the possible role of nicotine in enhancing coagulation and to assess the potential cardiovascular toxicity of transdermal nicotine therapy for smoking cessation. BACKGROUND: Cigarette smoking increases the risks of acute coronary events. A likely contributing mechanism is activation of coagulation. The role of nicotine in enhancing coagulability has not been resolved. METHODS: We compared in a crossover study the effects of cigarette smoking, transdermal nicotine and placebo transdermal nicotine, each for 5 days, in 12 healthy smokers. RESULTS: Cigarette smoking increased the urinary excretion of 11-dehydro-thromboxane B2 (reflecting thromboxane A2 generation) and increased plasma concentration of the platelet alpha-granule constituents, platelet factor 4 and beta-thromboglobulin, compared with placebo treatment, indicating in vivo platelet activation. Cigarette smoking was also associated with higher levels of fibrinogen in plasma. Transdermal nicotine produced plasma levels of nicotine in the same range as those during smoking but had no effect on thromboxane A2 metabolite excretion, platelet alpha-granule release or plasma fibrinogen, compared with placebo. Excretion of 2,3-dinor-6-keto-PGF1 alpha (reflecting prostacyclin generation) was not significantly influenced by any treatment. These results suggest that nicotine as such is not responsible for the platelet activation or elevation of plasma fibrinogen seen in smokers. However, we cannot exclude the possibility that intermittent bolus-like dosing of nicotine from cigarettes could have different effects from those produced by continually released transdermal nicotine. Other findings were that cigarette smoking and transdermal nicotine treatment were both associated with a higher white blood cell count compared with the placebo patch condition, suggesting a direct effect of nicotine to increase circulating white cells. Factor VII coagulant activity (VIIc) was significantly lower during cigarette smoking, than during either nicotine or placebo patch conditions. CONCLUSIONS: Transdermal nicotine has less effect on platelet activation and catecholamine release than does cigarette smoking, and its use in smoking cessation treatment of patients with coronary heart disease is likely to be safer than cigarette smoking.

Attenuation of ciclosporin-induced nephrotoxicity by dietary supplementation of seal oil in Sprague-Dawley rats.

Fish oil, rich in omega-3 (n-3) polyunsaturated fatty acids (PUFAs), has been reported to attenuate nephrotoxicity induced by ciclosporin (cyclosporine A). Harp seal oil is a rich source of n-3 PUFAs. This study investigated the ability of dietary seal oil to reduce nephrotoxicity caused by ciclosporin. Sprague-Dawley rats were maintained on a standard diet (with sunflower oil as lipid, SFO) or a diet enriched with seal oil (with 85% seal oil and 15% sunflower oil as lipid, SO) for four weeks before and four weeks after intravenous administration of ciclosporin (15 mg kg(-1) daily). Kidney function was assessed by measuring blood urea nitrogen, creatinine clearance, urinary N-acetyl-1-beta-D-glucosaminidase, 6-keto-prostaglandin F(1alpha), thromboxane B(2) and malondialdehyde. Systolic blood pressure (SBP) was monitored. Ciclosporin concentrations in blood were measured using liquid chromatographytandem mass spectrometry (LC-MS/MS). The fatty acid compositions of the diets and erythrocyte membranes were analysed by gas chromatography (GC). The results showed that nephrotoxicity was induced by ciclosporin in rats maintained on both SO and SFO diets. However, rats fed on SO diet endured less toxicity than those on SFO diet. The n-3 and n-6 PUFAs in the erythrocyte membrane of rats maintained on SO diet were found to be 10.79% and 11.93%, while those in rats maintained on SFO diet were found to be 1.67% and 22.71%, respectively. In conclusion, dietary supplementation of seal oil was found to reduce ciclosporin-induced nephrotoxicity in rats.

Altered excretion of prostaglandin and thromboxane metabolites in pregnancy-induced hypertension.

The renal and systemic metabolites (the latter as 2,3-dinor derivatives) of prostacyclin and thromboxane A2 were measured, along with renal prostaglandin E2 and kallikrein, in the urine of 15 patients with pregnancy-induced hypertension, 15 normotensive pregnant women matched for both age and gestational age, and 15 normotensive nonpregnant control women. Urinary excretion of all prostaglandin and thromboxane metabolites studied proved significantly higher in normotensive pregnant women than in controls. Prostaglandin E2, 6-keto-prostaglandin F1 alpha, and 2,3-dinor-6-keto-prostaglandin F1 alpha were significantly lower in pregnancy-induced hypertensive women than in normotensive pregnant women, whereas thromboxane B2 and 2,3-dinor-thromboxane B2 showed no significant differences in the two groups. A significant negative correlation (r = -0.636, p less than 0.01) was found between urinary 2,3-dinor-6-keto-prostaglandin F1 alpha and mean blood pressure in the two groups of pregnant women taken as a whole. These data indicate that, in pregnancy-induced hypertension, there is an imbalance between vasodilator and vasoconstrictor factors, not only in the kidneys, but also at the systemic vascular level. This imbalance, which may in itself produce vasoconstriction, may also potentiate the hypertensive effect of catecholamines and angiotensin II.

Pharmacokinetics of CGS 12970 and inhibition of thromboxane synthesis after oral administration in healthy adults.

The pharmacokinetics and pharmacodynamics of biochemical effect of a selective thromboxane synthase inhibitor, CGS 12970, were studied in healthy male volunteers after a dosing scheme of either 200 mg once daily or 100 mg twice a day for 6 days. The peak plasma concentration appeared 1 to 2 hours after administration, followed by a biexponential decline with half life values of about 1 and 7 hours, respectively. The mean oral clearance was 16 L/hr. Biochemically, the capacity of the platelets to form thromboxane A2 ex vivo (serum) was inhibited greater than 90% at both doses. In contrast to the short plasma half-life, the suppression of ex vivo serum thromboxane production was maintained greater than 70% to 80% at 48 hours after dosing. Inhibition of the thromboxane production in vivo (urine) was also substantial, but incomplete at both doses (200 mg daily; thromboxane B2, 75%; 2,3-dinor-thromboxane B2, 83%; 11-dehydrothromboxane B2, 90%). The urinary excretion, however, returned to the predose level at the end of a 1-week follow-up period after the last dosing. In conclusion, CGS 12970 is an orally active, reversible inhibitor of thromboxane synthase with a prolonged duration of action in humans.

A vitamin E concentrate rich in tocotrienols had no effect on serum lipids, lipoproteins, or platelet function in men with mildly elevated serum lipid concentrations.

BACKGROUND: Tocotrienols, lipid-soluble antioxidants with vitamin E activity, have been reported to lower LDL-cholesterol concentrations and platelet aggregation in men, but results are contradictory. OBJECTIVE: To examine in detail the effects of a vitamin E concentrate rich in tocotrienols on serum lipoproteins and on platelet function in men at risk for cardiovascular disease. DESIGN: In this randomized, double-blind, placebo-controlled parallel trial, 20 men received daily for 6 wk 4 capsules, each containing 35 mg tocotrienols and 20 mg alpha-tocopherol; 20 other men received 4 capsules daily, each providing 20 mg alpha-tocopherol. All men had concentrations of serum total cholesterol between 6.5 and 8.0 mmol/L or lipoprotein(a) concentrations > 150 mg/L. RESULTS: Compliance was confirmed by changes in serum tocopherol and tocotrienol concentrations. Serum LDL cholesterol in the tocotrienol group was 4.80 mmol/L before and 4.79 mmol/L after intervention, and increased from 4.70 to 4.86 mmol/L in the placebo group (95% CI for the difference: -0.54, 0.19 mmol/L; P = 0.333). Also, changes in HDL cholesterol, triacylglycerol, lipoprotein(a), and lipid peroxide concentrations did not differ between the groups. After adjustment for differences in initial values, no effects were found on collagen-induced platelet aggregation velocity, maximum aggregation, or thromboxane B2 formation in citrated whole blood. ATP release, however, was lower in the tocotrienol group. Urinary thromboxane B2 and 11-keto-thromboxane B2 concentrations and coagulation and fibrinolytic measures did not change. CONCLUSION: The tocotrienol supplements used had no marked favorable effects on the serum lipoprotein profile or on platelet function in men with slightly elevated lipid concentrations.