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.

Thromboxane A2/thromboxane A2 receptor axis facilitates hepatic insulin resistance and steatosis through endoplasmic reticulum stress in non-alcoholic fatty liver disease.

BACKGROUND AND PURPOSE: Defective insulin signalling and dysfunction of the endoplasmic reticulum (ER), driven by excessive lipid accumulation in the liver, is a characteristic feature in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Thromboxane A2 (TXA2 ), an arachidonic acid metabolite, is significantly elevated in obesity and plays a crucial role in hepatic gluconeogenesis and adipose tissue macrophage polarization. However, the role of liver TXA2 /TP receptors in insulin resistance and lipid metabolism is largely unknown. EXPERIMENTAL APPROACH: TP receptor knockout (TP-/- ) mice were generated and fed a high-fat diet for 16 weeks. Insulin sensitivity, ER stress responses and hepatic lipid accumulation were assessed. Furthermore, we used primary hepatocytes to dissect the mechanisms by which the TXA2 /TP receptor axis regulates insulin signalling and hepatocyte lipogenesis. KEY RESULTS: TXA2 was increased in diet-induced obese mice, and depletion of TP receptors in adult mice improved systemic insulin resistance and hepatic steatosis. Mechanistically, we found that the TXA2 /TP receptor axis disrupts insulin signalling by activating the Ca2+ /calcium calmodulin-dependent kinase II γ (CaMKIIγ)-protein kinase RNA-like endoplasmic reticulum kinase (PERK)-C/EBP homologous protein (Chop)-tribbles-like protein 3 (TRB3) axis in hepatocytes. In addition, our results revealed that the TXA2 /TP receptor axis directly promoted lipogenesis in primary hepatocytes and contributed to Kupffer cell inflammation. CONCLUSIONS AND IMPLICATIONS: The TXA2 /TP receptor axis facilitates insulin resistance through Ca2+ /CaMKIIγ to activate PERK-Chop-TRB3 signalling. Inhibition of hepatocyte TP receptors improved hepatic steatosis and inflammation. The TP receptor is a new therapeutic target for NAFLD and metabolic syndrome.

Modeling of Acute Pulmonary Arterial Hypertension in Pigs Using a Stable Thromboxane A2 Analogue (U46619): Dose Adjustment and Assessment of Hemodynamic Reactions.

U46619, a synthetic analogue of thromboxane A2 was used for modeling acute stable and reversible pulmonary arterial hypertension. Administration of U46619 in high doses led to vascular collapse and inhibition of cardiac function. The doses of U46619 were empirically selected that allow attaining the target level of pulmonary hypertension without systemic hemodynamic disturbances. The possibility of attaining the target level of pulmonary hypertension and reversibility of changes after termination of U46619 infusion make this model attractive for evaluation of the efficiency of different therapeutic methods of treatment of pulmonary hypertension in large animals.

Weight as an assay-independent predictor of poor response to enteric aspirin in cardiovascular patients.

Aspirin non-response is associated with poor outcome but there is no agreement between the different methods to asses it. Weight has been shown to be a predictor of poor response but only using one method. In this study, we determine the effects of weight on different assays of platelet function. The response to aspirin was determined in 138 cardiology patients using serum thromboxane, arachidonic acid-induced platelet aggregation and VerifyNow©. Twenty-five percent of patients showed an inadequate response to aspirin in at least one assay on the initial test. After ensuring patient compliance only 5% of patients were considered to be non-responders. Only 9% of non-responders were non-responsive in all three assays. When switched to plain aspirin, only 2% of patients were non-responsive. All patients responded adequately to 150 mg aspirin. The non-responders were significantly heavier than responders (78.5 kg ± 14.0 (SD); BMI: 28.4 kg/m2± 4.4 v's 102.6 kg ± 20.6, P = .0016; BMI: 38.3 kg/m2 ± 7.6, P= .0015). A rule-based approach of using plain aspirin in patients over 90 kg or BMI 32 along with patient education to ensure compliance will ensure that all patients respond to their aspirin without the need for testing.

The role of the central arachidonic acid-thromboxane A2 cascade in cardiovascular regulation during hemorrhagic shock in rats.

The aim of the current study was to elucidate the underlying central mechanism(s) of the cardiovascular effects evoked by centrally injected melittin and arachidonic acid (AA) in hemorrhaged hypotensive condition, specifically, from central AA release from the cell membrane under the influence of phospholipase A(2) (PLA(2)) to central thromboxane A(2) (TXA(2)) signaling via the cyclooxygenase (COX) pathway. As the main control of the study, melittin (3 µg) or AA (150 µg) was injected intracerebroventricularly (i.c.v.) after the hemorrhage procedure, which was performed by withdrawing a total volume of 2.2 ml of blood/100g body weight over a period of 10 min. Both treatments generated a pressor response and abolished the hypotension-induced hemorrhage. Pretreatment with the PLA(2) inhibitor mepacrine (500 µg; i.c.v.) completely blocked the pressor response to melittin in the hemorrhagic hypotensive state. Pretreatments with the nonselective COX inhibitor indomethacin (200 µg; i.c.v.) or the TXA(2) synthesis inhibitor furegrelate (250 or 500 µg; i.c.v.) were made to test the role of central COX activity and, subsequently, the TXA(2) signaling pathway in the melittin- or AA-mediated reversal of hemorrhagic hypotension. Indomethacin completely prevented the pressor response to melittin and AA in the hemorrhaged, hypotensive state, but furegrelate did so only partially. In conclusion, these findings suggest that central COX activity and, subsequently, the central TXA(2) signaling pathway, are, at least in part, involved in the melittin- or AA-induced reversal effect during hemorrhagic shock.

Antiplatelet drugs.

Platelets play a major role in thromboembolic diseases, and so antiplatelet therapy remains crucial in treatment and prophylaxis. Upon vascular injury, platelets rapidly adhere to the exposed subendothelial matrix, after which they become activated, resulting in the recruitment of additional platelets from the circulation to eventually form a stable arterial platelet plug. Although controlled plug formation is desired for the prevention of excessive blood loss and for promoting wound healing, several pathological conditions may result in the formation of occlusive thrombi leading to severe clinical complications, including myocardial infarction and ischaemic stroke. Many antiplatelet approaches have been investigated, interfering with one or more of the different stages in thrombus formation. This review discusses antiplatelet agents that interfere with the three principal phases in thrombus formation: platelet adhesion, amplification of platelet activation and platelet aggregation. For each stage, novel experimental targets and clinically established antiplatelet strategies will be reviewed. Limitations and possible benefits will be discussed for each target.

Postinjury thromboxane receptor blockade ameliorates acute lung injury.

BACKGROUND: Acute lung injury is associated with pulmonary hypertension, intrapulmonary shunting, and increased microvascular permeability, leading to altered oxygenation capacity. Thromboxane A2 has been found to be a central mediator in the development of septic and oleic acid (OA)-induced acute lung injury. Our previous study demonstrated a beneficial effect of preinjury thromboxane A2 receptor blockade. The current study examines the efficacy of postinjury receptor blockade on oxygenation capacity and pulmonary hemodynamics in an isolated lung model of OA-induced acute lung injury. METHODS: Four groups of rabbit heart-lung preparations were studied for 60 minutes in an ex vivo perfusion-ventilation system. Saline control lungs received saline solution during the first 20 minutes of study. Injury control lungs received an OA-ethanol solution during the first 20 minutes. Two treatment groups were used: T10, in which the thromboxane receptor antagonist, SQ30741, was infused 10 minutes after the initiation of OA infusion; and T30, in which the thromboxane receptor antagonist was infused 30 minutes after OA infusion. RESULTS: Significant differences were found in oxygenation (oxygen tension in T10 = 62.6 +/- 11.7 mm Hg, T30 = 68.2 +/- 21.2 mm Hg; injury control = 40.2 +/- 9.0 mm Hg, saline control = 123.5 +/- 16.01 mm Hg; p < 0.001) and percentile change in pulmonary artery pressure (T10 = 1.1% +/- 19.4% increase, T30 = 11.2% +/- 7.3% increase; injury control = 47.6% +/- 20.5%, saline control = 4.2% +/- 6.81%; p < 0.001). CONCLUSIONS: This study demonstrates that blockade of the thromboxane A2 receptor, even after the initiation of acute lung injury, eliminates pulmonary hypertension and improves oxygenation.

Improvement of renal function with a selective thromboxane A2 synthetase inhibitor, DP-1904, in lupus nephritis.

OBJECTIVE: To examine abnormalities of prostanoid metabolism in lupus nephritis, which may affect renal function, and the effects of 4 day dosing of a selective thromboxane A2 (TXA2) synthetase inhibitor, DP-1904, on prostanoid metabolism. METHODS: Urinary levels of various prostanoids, thromboxane B2(TXB2), 11-dehydro-TXB2, 6-keto-prostaglandin F1 alpha, 2,3-dinor-6-keto-PGF1 alpha, and prostaglandin E2 were determined. In a randomized crossover study, 8 patients with biopsy proven lupus nephritis were given 4 days' oral administration of DP-1904 (400 mg/day bid) or indomethacin (50 mg/day bid). The effects of DP-1904 on prostanoid metabolism were studied. RESULTS: Urinary excretion of TXB2, which reflects the renal production of TXA2, was significantly increased in patients with lupus nephritis compared with non-renal systemic lupus erythematosus (SLE)(p < 0.05); enhanced production of TXA2 was also estimated in patients with lupus nephritis. The urinary TXB2/6-keto-PGF1 alpha ratio was also increased in lupus nephritis compared with non-renal SLE (p < 0.01), indicating a prostanoid imbalance that may lead to impaired renal function and subsequent pathology. During administration of DP-1904, the urinary excretion of TXB2 was significantly decreased after 1 to 2 days. An increase in creatinine clearance as a measure of renal function was observed. In contrast, during the administration of indomethacin, urinary excretion of both TXB2 and 6-keto-PGF1 alpha decreased and there were no significant changes in the urinary TXB2/6-keto-PGF1 alpha ratio or creatinine clearance. Hemodynamic changes were associated with a slight increase in sodium excretion, but with no change in arterial blood pressure. No side effects were elicited during the 4 days of treatments. CONCLUSION: The abnormal prostanoid metabolism observed in lupus nephritis could aggravate renal function, which was mediated hemodynamically, and the altered metabolism was reversible and at least partially corrected by a TXA2 synthetase inhibitor, DP-1904.

Thromboxane receptor blockade improves oxygenation in an experimental model of acute lung injury.

BACKGROUND: Adult respiratory distress syndrome remains a major cause of morbidity and mortality. We investigated the role of thromboxane receptor antagonism in an experimental model of acute lung injury that mimics adult respiratory distress syndrome. METHODS: Three groups of rabbit heart-lung preparations were studied for 30 minutes in an ex vivo blood perfusion/ventilation system. Saline control (SC) lungs received saline solution during the first 20 minutes of study. Injury control (IC) lungs received an oleic acid-ethanol solution during the first 20 minutes. Thromboxane receptor blockade (TRB) lungs received the same injury as IC lungs, but a thromboxane receptor antagonist (SQ30741) was added to the blood perfusate just prior to study. Blood gases were obtained at 10-minute intervals, and tidal volume, pulmonary artery pressure, and lung weight were continuously recorded. Oxygenation was assessed by measuring the percent change in oxygen tension over the 30-minute study period. Tissue samples were collected from all lungs for histologic evaluation. RESULTS: Significant differences were found between SC and IC lungs as well as TRB and IC lungs when comparing pulmonary artery pressure (SC = 33.1 +/- 2.2 mm Hg, TRB = 35.4 +/- 2.1 mm Hg, IC = 60.4 +/- 11.1 mm Hg; p < 0.02) and percent change in oxygenation (SC = -20.6% +/- 10.3%, TRB = -24.2% +/- 9.5%, IC = -57.1% +/- 6.2%; p < 0.03). None of the other variables demonstrated significant differences. CONCLUSIONS: Thromboxane receptor blockade prevents the pulmonary hypertension and the decline in oxygenation seen in an experimental model of acute lung injury that mimics adult respiratory distress syndrome.

Thromboxane receptor blockade does not attenuate pulmonary pressor response in porcine single lung transplantation.

BACKGROUND: The ischemia-reperfusion lung injury is characterized by increased pulmonary vascular resistance, edema, and subsequent deterioration of oxygenation. Other models of acute lung injury suggest that thromboxane A2 may contribute to the pulmonary hypertension after transplantation. METHODS: We studied the effects of the selective thromboxane A2 receptor antagonist SQ 30741 on pulmonary hemodynamics and gas exchange in porcine single lung transplantation using extracorporeal circulation (right heart bypass) with separate cannulations of the right and left pulmonary arteries. Pulmonary vascular resistance was measured at equal and constant flow to each lung. Flow distribution between the lungs was registered at equal pulmonary artery pressures. Twelve pigs (weight 17 to 23 kg) were studied. At the onset of reperfusion a bolus dose of the drug (5 mg/kg) was injected into both pulmonary arteries followed by an infusion (5 mg/kg/hr) for 1 hour (SQ group, n = 6). The control group (n = 6) received an equal amount of vehicle. The systemic and pulmonary hemodynamics and blood gas values were registered during 2 hours of reperfusion. RESULTS: The pulmonary vascular resistance of the transplanted lung was significantly higher compared with the native lung (p < 0.001). Administration of SQ 30741 failed to ameliorate the pulmonary pressor response of the graft in comparison with the control group. No difference was found in the systemic arterial oxygen tension between the two groups. CONCLUSIONS: Thromboxane does not seem to be among the principal mediators in the pulmonary hypertension after transplantation.

The pharmacological prevention of pre-eclampsia.

The disparate results reported in the literature on the effects of low dose aspirin in preventing pre-eclampsia might be caused by non-compliance in the more recent large trials in low-risk patients. All the earlier small trials were done on identified high-risk patients who consider themselves as patients, as do their doctors. Compliance in these patients will be very high. In fact, the only study in healthy subjects in which aspirin intake was controlled for (Hauth et al 1993) showed a marked reduction in the incidence of pre-eclampsia. However, the recent large trials have demonstrated, without any doubt, that low dose aspirin is not a miracle drug. The combined literature points at a 25% reduction in the incidence of pre-eclampsia in association with the use of aspirin (Collins, 1994). The correct indication for the use of low-dose aspirin appears to be the patient that is at very high risk of developing early-onset (less than 32 weeks gestation) pre-eclampsia. Since early-onset pre-eclampsia can begin at any time after 20 weeks gestation, it is necessary to initiate low-dose aspirin therapy early in pregnancy, preferably at 10-14 weeks gestation. The results of the recent large trials emphasize the need for a reliable, sensitive method of predicting or detecting pre-eclampsia at a very early gestational age (Dekker and Sibai, 1991). Valensise et al (1993) recently confirmed earlier studies (McParland et al, 1990) on the useful combination of uteroplacental Doppler flow velocimetry and aspirin in low-risk primigravidae. Results from current large-scale trials, such as the ECPPA, the BLASP, the WHO Jamaica and the second NICHHD studies, will be available in the near future. The results of especially the second NICHHD study on low-dose aspirin, in more than 2000 high-risk women (previous pre-eclampsia/eclampsia, chronic hypertension, class B to F diabetes or multiple gestation), will hopefully give us a more definitive picture on the potential benificial effects of low-dose aspirin in high-risk patients. The effect of aspirin on placental TXA2 deserves further studies. It might be that the optimal level to inhibit placental TXA2 and lipid peroxide production is actually higher than the minimal effective doses of aspirin that are needed to inhibit platelet TXA2 production (Walsh, 1994). Low-dose aspirin appears to be safe for the fetus and neonate. If there is an increased risk of abruptio placentae, this risk appears to be minimal. The final word on the use of low-dose aspirin has not yet been reached; however, we may be getting closer to profiling patients for whom the therapy may be efficacious and beneficial to both mother and fetus. Further studies are also necessary on combinations of aspirin and other antithrombotic drugs, such as heparin or ketanserin (Tanaka et al, 1993; Bolte et al, 1994; North et al, 1994). North et al (1994) demonstrated that treatment of women with severe renal disease with heparin plus aspirin reduced the prevalence of superimposed pre-eclampsia, compared with no treatment or aspirin alone. Next to low-dose aspirin, there appear to be several new and promising pharmaceutical approaches for reducing the consequences of EC dysfunction. Among these are selective TXA2-synthetase or TXA2-receptor antagonists, Serotonin2-receptor blockers, stable PGI2 analogues and NO donors.

Prevention of complement-induced pulmonary hypertension and improvement of right ventricular function by selective thromboxane receptor antagonism.

The effect of complement activation on the pulmonary vascular system and on right ventricular function was studied in sheep (n = 12) by injection of cobra venom factor. Animals were instrumented for measurement of pulmonary flow, mean pulmonary artery pressure, right ventricular stroke work, arterial blood gases, and systemic vascular resistance. Blood was sampled from the left atrium and pulmonary artery to measure thromboxane B2, the metabolite of thromboxane A2, by radioimmunoassay. After baseline measurements, animals were randomly assigned to receive a selective thromboxane receptor antagonist SQ30741 as a 10 mg/kg bolus with an infusion of 10 mg/kg per hour or else to receive vehicle. Cobra venom factor was then injected (30 U/kg) in all animals, and data were recorded at 15, 30, 60, 90, and 120 minutes. In control animals there was a 2.4-fold increase in mean pulmonary artery pressure and a 76% increase in right ventricular stroke work at 15 minutes from baseline (p < 0.05); these values remained elevated for 30 minutes and returned to baseline by 1 hour with no change in systemic vascular resistance. Arterial oxygenation decreased by 124% at 15 minutes and remained depressed through the experiment, but in treated animals oxygen tension remained unchanged from baseline. Thromboxane B2 increased 95% from baseline in the control group and 1.5 fold in treated animals and followed a similar time course as the functional measurements (p < 0.05). A pulmonary vascular thromboxane B2 gradient of approximately 1000 pg/ml was measured at 15 and 30 minutes in both control and treated groups. (p < 0.05) We conclude that after complement activation in this model pulmonary hypertension and decreased oxygen tension are mediated by thromboxane release from the pulmonary vascular bed. This increased afterload causes a stress on the right ventricle as demonstrated by the increased right ventricular stroke work. Selective thromboxane receptor antagonism may be a beneficial therapy for pulmonary hypertension in patients after cardiopulmonary bypass.

[Experimental study of the effectiveness of ECMO (extracorporeal membrane oxygenation) and PGI2 derivative against severe respiratory failure].

Severe respiratory failure was made on twenty dogs with intratracheal administration of the linoleic acid to study the effectiveness of ECMO. The dogs were divided into four groups: First group (GI); respiratory failure, second group (G II); respiratory failure with pumpless A-V bypass, third group (G III); respiratory failure with V-A bypass, fourth group (G IV); respiratory failure with V-A bypass and administration of OP-41483 (PGI2 derivative). The effectiveness of ECMO was studied with different concentration of Prostaglandin I2 (PGI2) and Thromboxane A2 (TxA2). This experiment showed significant prolongation of mean survival time in dogs of respiratory failure treated with the use of ECMO. In GI and G II, TxB2, metabolic product of TxA2, increased and the ratio of 6-ketoPGF1 alpha, metabolic product of PGI2, versus TxB2, decreased progressively. In G III and G IV, the TxB2 was increased during the first two or three hours after intratracheal administration of linoleic acid and then reached a steady level. In G3, the ratio of 6-ketoPGF1 alpha versus TxB2 was approximately 0.5 after the linoleic acid administration. However, in the case of G IV with combined application of PGI2 derivative, the ratio decreased to 0.59 +/- 0.08 in one or two hours after the administration, then increased progressively and reached a value of 1.04 +/- 0.27 in eight hours after the linoleic acid administration. In conclusion, this study demonstrates that therapeutical effects of ECMO against respiratory failure could be elevated by using PGI2 derivative.

Thromboxane A2 receptor blockade improves contractile function following cardiopulmonary bypass in dogs and pigs.

Thromboxane A2/prostaglandin endoperoxide (TP) receptor antagonists have been reported to decrease the extent of myocardial damage after coronary ligation. The purpose of this study was to determine if the TP antagonist SQ 30,741 can protect myocardial tissue during cardiac arrest and cardiopulmonary bypass (CPB) in dogs and pigs. In the first part of the study, anesthetized dogs were subjected to normothermic CPB (37.5 degrees C) at a flow rate of 2 liters/m2/min. Dogs were treated with either 5 mg/kg + 5 mg/kg/hr SQ 30,741 or vehicle starting before CPB. The aorta was cross-clamped for 25 min and then released to allow reperfusion. In another study, pigs had hypothermic (28 degrees C) CPB but with arrest for 1 hr. Myocardial recovery was assessed by segment shortening as determined by sonomicrometry. Canine hearts treated with SQ 30,741 had a significantly improved reperfusion contractile function such that at 60 min postreperfusion, segmental shortening returned to 96% of pre-bypass levels vs 70% in vehicle-treated controls (P less than 0.05). In pigs, 70% of vehicle-treated pigs could not be weaned off CPB and died. All six pigs treated with SQ 30,741 survived. SQ 30,741 prevented platelet loss in dogs, but did not in pigs. Thus, SQ 30,741 significantly improved reperfusion function in hearts subjected to CPB.

The combined antiischemic effects of the thromboxane receptor antagonist SQ 30,741 and tissue-type plasminogen activator.

The thromboxane-receptor antagonist, SQ 30,741, may be used as adjuvant therapy for thrombolysis and has also been shown to have antiischemic activity that is independent of its thrombolytic activity. Since tissue-type plasminogen activator (t-PA) and SQ 30,741 may be administered simultaneously, we determined whether the antiischemic effects of SQ 30,741 can be potentiated by t-PA. This was accomplished by combining doses of t-PA and SQ 30,741, which alone were not cardioprotective. Anesthetized dogs were subjected to left circumflex coronary artery occlusion for 90 minutes and reperfusion for 5 hours. The dogs were treated during reperfusion with a dose of t-PA that caused approximately a 30% reduction in plasma fibrinogen alone or in combination with 1.5 mg/kg + 0.4 mg/kg/hr SQ 30,741, which started 10 minutes after initiation of ischemia. At these doses, neither t-PA nor SQ 30,741 alone significantly reduced infarct size (57% +/- 6%, 50% +/- 10%, 57% +/- 6% of the left ventricular area at risk for vehicle controls, t-PA, and SQ 30,741 respectively); however, combination treatment resulted in a significant reduction in infarct size (37% +/- 5% of the left ventricular area at risk). Higher doses of t-PA and SQ 30,741 alone significantly reduced infarct size. The protective effects of t-PA and SQ 30,741 occurred without altering peripheral hemodynamic status. No differences in collateral or reperfusion blood flow were observed between groups. Thus although SQ 30,741 may act to improve the efficacy of thrombolysis, t-PA may in turn enhance the antiischemic activity of SQ 30,741 or at least reduce the threshold dose.