Differential suppression of thromboxane biosynthesis by indobufen and aspirin in patients with unstable angina

BACKGROUND: We have previously reported aspirin failure in suppressing enhanced thromboxane (TX) biosynthesis in a subset of episodes of platelet activation during the acute phase of unstable angina. The recent discovery of a second prostaglandin H synthase (PGHS-2), inducible in response to inflammatory or mitogenic stimuli, prompted us to reexamine TXA2 biosynthesis in unstable angina as modified by two cyclooxygenase inhibitors differentially affecting PGHS-2 despite a comparable impact on platelet PGHS-1. METHODS AND RESULTS: We randomized 20 patients (15 men and 5 women aged 59+/-10 years) with unstable angina to short-term treatment with aspirin (320 mg/d) or indobufen (200 mg BID) and collected 6 to 18 consecutive urine samples. Urinary 11-dehydro-TXB2 was extracted and measured by a previously validated radioimmunoassay as a reflection of in vivo TXA2 biosynthesis. Metabolite excretion averaged 102 pg/mg creatinine (median value; n=76) in the aspirin group and 55 pg/mg creatinine (median value; n=99) in the indobufen group (P<.001). There were 16 samples (21%) with 11-dehydro-TXB2 excretion >200 pg/mg creatinine among patients treated with aspirin versus 6 such samples (6%) among those treated with indobufen (P<.001). In vitro and ex vivo studies in healthy subjects demonstrated the capacity of indobufen to largely suppress monocyte PGHS-2 activity at therapeutic plasma concentrations. In contrast, aspirin could only inhibit monocyte PGHS-2 transiently at very high concentrations. CONCLUSIONS: We conclude that in unstable angina, episodes of aspirin-insensitive TXA2 biosynthesis may reflect extraplatelet sources, possibly expressing the inducible PGHS in response to a local inflammatory milieu, and a selective PGHS-2 inhibitor would be an ideal tool to test the clinical relevance of this novel pathway of arachidonic acid metabolism in this setting.     

Management of patients with hereditary defects predisposing to thrombosis including pregnant women

F2-isoprostanes are prostaglandin (PG) F2-like compounds that are formed in vivo directly by free radical-catalyzed lipid peroxidation. One of the compounds that can be produced in abundance by such mechanism is 8-epi-PGF2 alpha, a potent vasoconstrictor. We have developed an enzyme immunoassay and a radioimmunoassay for measuring urinary concentrations of 8-epi-PGF2 alpha by raising antibodies against this compound. The antisera presented high titers (> 1/300,000) and provided highly sensitive assays (IC50, 8 and 24 pg/ml, for EIA and RIA, respectively); cross-reactivity with other PG was negligible. The interassay reproducibility of EIA was assessed by measuring the same urine stored frozen in aliquots after solid phase extraction and thin-layer chromatography (17%, n = 13). Measurements of urinary 8-epi-PGF2 alpha by immunoassays were validated using different antisera and by comparison with gas chromatography/mass spectrometry. Healthy volunteers excreted 25 +/- 12 ng of 8-epi-PGF2 alpha/mmol creatinine (n = 19), with no circadian variation over three consecutive 8-hr collection periods (n = 10); preliminary results showed that excretion increased as a function of age. Urinary excretion of 8-epi-PGF2 alpha was unchanged by treatment with two nonsteroidal antiinflammatory drugs, Ibuprofen at 1.2 g/day for 4 days (n = 4) or aspirin as a single administration of 1 g (n = 6). In contrast, the urinary excretion of 11-dehydro-thromboxane B2, a platelet cyclooxygenase-derived metabolite was reduced by more than 80% after aspirin administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced lipid peroxidation in patients positive for antiphospholipid antibodies

The mechanism leading to the formation of antiphospholipid antibodies (aPL) is still unknown. Because an in vitro study suggested that aPL may derive from pro-oxidant conditions, we sought a relationship between aPL and isoprostanes, indices of lipid peroxidation in vivo. Thirty patients with systemic lupus erythematosus have been studied. Seventeen (56.6%) were positive for aPL because they had lupus anticoagulant and/or high titer of anticardiolipin antibodies (aCL). Plasma levels of tumor necrosis factor (TNF ) and urinary excretion of two isoprostanes, 8-epi-PGF2alpha and IPF2alpha -I, free radical catalyzed oxidation products of arachidonic acid, were measured. Patients with systemic lupus erythematosus had higher urinary excretion of 8-epi-PGF2alpha and IPF2alpha -I than controls; urinary excretion of the two isoprostanes was highly correlated (Rho = 0.74, P < .0001). Urinary 8-epi-PGF2alpha was highly correlated with both aCL titer (Rho = 0. 70, P < .0001) and TNF (Rho = 0.84, P < .0001), a measure of disease severity. Excretion of this isoprostane was also higher in those patients who exhibited aPL (P < .0001). Comparable correlations were observed with the isoprostane IPF2alpha -I. No difference of 8-epi-PGF2alpha was observed between patients with and without previous history of thrombosis. This study, showing the existence of a close association between aPL and increased in vivo lipid peroxidation, supports the hypothesis that these antibodies may result from pro-oxidative conditions and suggests that inflammation may play an important role.     

Identification and measurement of endogenous beta-oxidation metabolites of 8-epi-Prostaglandin F2alpha

F2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F2alpha, a major component of the F2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo. Measurement of major metabolites of endogenous 8-epi-PGF2alpha, in addition to the parent compound, may be useful to better define its formation in vivo. 2,3-Dinor-5,6-dihydro-8-epi-PGF2alpha is the only identified metabolite of 8-epi-PGF2alpha in man, but its endogenous levels are unknown. In addition to this metabolite, we have identified another major endogenous metabolite, 2,3-dinor-8-epi-PGF2alpha, in human and rat urine. The identity of these compounds, present at the pg/ml level in urine, was proven by a number of complementary approaches, based on: (a) immunoaffinity chromatography for selective extraction; (b) gas chromatography-mass spectrometry for structural analysis; (c) in vitro metabolism in isolated rat hepatocytes; and (d) chemical synthesis of the enantiomer of 2,3-dinor-5, 6-dihydro-8-epi-PGF2alpha as a reference standard. In humans, the urinary excretion rate of both dinor metabolites is comparable with that of 8-epi-PGF2alpha. Both metabolites increase in parallel with the parent compound in cigarette smokers, and they are not reduced during cyclooxygenase inhibition. Another beta-oxidation product, 2, 3,4,5-tetranor-8-epi-PGF2alpha, was identified as a major product of rat hepatocyte metabolism. In conclusion, at least two major beta-oxidation products of 8-epi-PGF2alpha are present in urine, which may be considered as additional analytical targets to evaluate 8-epi-PGF2alpha formation and degradation in vivo.     

The F2-isoprostane 8-epiprostaglandin F2alpha increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.     

Reduction by indobufen of neutrophil activation in peripheral arterial occlusive disease

We evaluated the effectiveness of indobufen administration in reducing neutrophil activation in a clinical model of ischemia-reperfusion. Thirty stable patients with intermittent claudication due to occlusive peripheral arterial disease of the leg were randomly assigned to two groups. Patients in group I were treated with indobufen [200 mg orally twice daily (p.o. b.i.d.) for a week]; patients in group II received a placebo. Both groups of patients were submitted to standardized treadmill exercise until onset of claudication. Plasma levels of thromboxane B2 (TxB2) and 6-keto-prostaglandin F1alpha(6-k-PGF1alpha) neutrophil filterability, and neutrophil activation (by nitro-blue tetrazolium test) were assessed in blood samples from the femoral vein draining the ischemic leg. The values were obtained at rest and 5, 30, and 60 min after onset of claudication. Urinary albumin excretion was measured at rest and 1 h after onset of claudication. Plasma levels of TxB2 and 6-k-PGF1alpha increased significantly in the placebo group 5 min after onset of claudication, whereas only a slight nonsignificant increase was observed in the indobufen-treated group at the same timepoint.     

Selective cyclooxygenase-2 inhibition by nimesulide in man

Prostaglandins are generated through two isoforms of the enzyme cyclooxygenase, the constitutively expressed cyclooxygenase (Cox)-1 and Cox-2, which is induced at sites of inflammation. Selective inhibition of Cox-2 is desirable as this may avoid the gastropathy and platelet inhibition seen with nonselective agents. Moreover, these agents will allow us to examine the relative contribution of the two isoforms to prostaglandin formation in man. We examined the activity of nimesulide, a Cox-2 selective nonsteroidal antiinflammatory drug, in vitro against purified enzymes and in vivo in man. Nimesulide 100 mg twice daily or aspirin 300 mg three times daily were administered randomly for 14 days to 20 subjects complaining of musculoskeletal pain. Serum thromboxane B2 was determined as an index of Cox-1 activity and endotoxin-induced prostaglandin E2 formation in whole blood as an index of Cox-2 activity. Urinary excretion of prostaglandin metabolites was determined by GC/MS. Nimesulide was highly selective against ovine Cox-2, so that at concentrations attained in vivo, it had no effect on Cox-1 but completely suppressed Cox-2. Aspirin markedly inhibited serum thromboxane B2 (181.92 +/- 19.77 to 2.83 +/- 0.96 ng/ml, P <. 002), whereas nimesulide had very little effect (207.53 +/- 47.30 to 181.15 +/- 54.59 ng/ml). In contrast, nimesulide suppresses endotoxin-induced prostaglandin E2 formation (35.03 +/- 8.73 to 2.62 +/- 0.95 ng/ml, P =.002). As expected, aspirin reduced TX metabolite excretion, whereas nimesulide had no significant effect. In contrast, both compounds suppressed PGI2 formation to the same extent. The findings suggest that TX is largely Cox-1 derived. Moreover, Cox-2 is expressed in man and generates prostaglandin I2.     

Generation of the isoprostane 8-epi-prostaglandin F2alpha in vitro and in vivo via the cyclooxygenases

F2-isoprostanes are isomers of the prostaglandin PGF2alpha. At least one compound of this group, 8-epi-PGF2alpha, exhibits biological activity, and therefore special interest is focused on the mechanism of isoprostane formation: enzyme catalyzed or radical mediated. We analyzed the formation of isoprostanes in vitro and in vivo. In both systems, purified cyclooxygenase isoenzymes and cell models specific for the cyclooxygenase isoenzymes, 8-epi-PGF2alpha formation could be totally suppressed by cyclooxygenase inhibitors. Indomethacin inhibited concentration-dependent 8-epi-PGF2alpha formation in platelets stimulated with calcium ionophore, arachidonic acid or thrombin. Nordihydroguaiaretic acid, an antioxidant, blocked isoprostane formation with a similar IC50 value as thromboxane B2 synthesis, pointing toward cyclooxygenase as the primary target of inhibition. Based on the turnover number, cyclooxygenase-2 formed higher levels of 8-epi-PGF2alpha than cyclooxygenase-1. Endogenous 8-epi-PGF2alpha production in rat mesangial cells correlated well with the mRNA and protein expression of cyclooxygenase-2 during interleukin-1 induction. However, in contrast to human platelets, which produced different forms of isoprostanes, rat mesangial cells appeared to form only 8-epi-PGF2alpha. Further, this indicates that mesangial cells may represent a cellular origin for renal 8-epi-PGF2alpha formation. Next, we analyzed the formation of isoprostanes in humans. A direct correlation was observed between indomethacin treatment and the decrease in 8-epi-PGF2alpha and isoprostane levels, but compared with other prostanoids the inhibition was less pronounced. In summary, based on the in vitro studies, a clear cyclooxygenase-dependent formation of isoprostanes, especially 8-epi-PGF2alpha, was observed. However, in vivo additional formation via cyclooxygenase enzyme-independent mechanisms is likely.     

Milk production of Holstein heifers fed either alfalfa or corn silage diets at two rates of daily gain

OBJECTIVES: L-arginine exerts anti-atherosclerotic effects in hypercholesterolaemic rabbits via modulating endogenous NO production. We investigated whether L-arginine inhibits thromboxane formation in vivo and platelet aggregation ex vivo in this animal model. METHODS: The urinary excretion rates of 2,3-dinor-6-keto-PGF1 alpha (major urinary metabolite of PGI2) and 2,3-dinor-TXB2 (major urinary metabolite of thromboxane A2) were used as indicators of platelet-endothelial cell interactions in vivo. Rabbits were fed 1% cholesterol (Cholesterol group, N = 8), 1% cholesterol plus 2.25% L-arginine (Cholesterol + L-arginine, N = 8), or normal rabbit chow (Control, N = 4) for 12 weeks. Urine samples were collected in weekly intervals. At the end of the study period platelet aggregation ex vivo and endothelium-dependent and -independent vascular function of isolated aortic rings in vitro was assessed. RESULTS: Urinary 2,3-dinor-TXB2 excretion significantly increased in the cholesterol group (p < 0.05), and endogenous NO formation (measured as urinary nitrate excretion) decreased (p < 0.05). Both parameters were significantly correlated with each other (R = 0.48, p < 0.01). L-arginine partly restored urinary nitrate excretion and significantly reduced TXA2 production to values even below those in the control group (p < 0.001). Urinary 2,3-dinor-6-keto-PGF1 alpha excretion increased in early hypercholesterolaemia and returned to control values in the second half of the study period. The early increase in urinary 2,3-dinor-6-keto-PGF1 alpha excretion was attenuated by L-arginine. Platelet aggregation was significantly enhanced in cholesterol-fed rabbits and attenuated by dietary L-arginine. L-arginine also improved the impaired endothelium-dependent relaxations to ADP, and normalized the vasoconstrictor effects of 5-HT in isolated aortic rings. CONCLUSIONS: Cholesterol-feeding enhances platelet aggregation and TXA2 formation, and stimulates platelet-endothelial cell interaction in rabbits. These effects are probably due to impaired NO elaboration, as indicated by decreased urinary nitrate excretion. Chronic dietary supplementation with L-arginine elevates systemic NO elaboration and significantly increases the PGI2/TXA2 ratio. It thus beneficially influences the homeostasis between vasodilator and vasoconstrictor prostanoids in vivo.     

Marketing mix in a radiology department: challenges for future radiologists in management

Prostaglandin (PG)I2 is the primary eicosanoid synthesized by human lymphatics and 8-epi-PGF2 alpha, an isoprostane formed during free radical catalyzed peroxidation, is the most potent stimulator of lymphatic contraction tested thus far. We now examine the respective concentrations in the lymphatic wall of both human and porcine lymphatics and lymph fluid using specific immunoassays. Although both compounds are detectable in the lymphatic wall and lymph fluid, PGI2- (via its main metabolite 6-oxo-PGF1 alpha) is greater in the lymphatic wall whereas 8-epi-PGF2 alpha dominates in lymph fluid. Because inflammation is associated with oxidative injury, which in turn stimulates release of isoprostane, eicosanoid derivatives may modulate lymphatic tone during acute tissue reaction.     

Enhanced thromboxane biosynthesis in patients with chronic obstructive pulmonary disease. The Chronic Obstructive Bronchitis and Haemostasis Study Group

Thrombotic complications of pulmonary circulation occur in patients with chronic obstructive pulmonary disease (COPD). In the present study, we sought to evaluate in vivo platelet activation through the measurement of 11/dehydro-thromboxane (Tx) B2 TxA2 major metabolite in the urine, in 29 patients with COPD, compared with 29 sex- and age-matched healthy subjects. The urinary excretion of 11-dehydro-TxB2 was significantly higher in patients with COPD than in control subjects: median (range), 753 (277-4,409) and 275 (129-612) pg/mg creatinine, respectively; p < 0.0001). Moreover, 11-dehydro-TxB2 excretion was inversely related with arterial oxygen tension (rho = -0.46; p = 0.0145). In five of the 29 patients a short-term therapeutic course with oxygen supplementation induced a significant decrease of urinary 11-dehydro-TxB2 excretion: median range, 941 (452-2,640) to 445 (166-1,560) pg/mg creatinine. Moreover, selective inhibition of platelet cyclooxygenase activity by low-dose aspirin was associated with more than 90% inhibition of thromboxane metabolite excretion, demonstrating its being of platelet origin. Plasma levels of prothrombin fragment F1 + 2 were higher in patients than in control subjects (2.6 +/- 1.5 versus 0.9 +/- 0.4 nM, p = 0.0001). No relation between 11-dehydro-TxB2 excretion and plasma F1 + 2 levels was found. We conclude that platelet TxA2 biosynthesis is enhanced in patients with COPD and may be influenced by arterial oxygen tension changes.     

Characterization of signal transduction events stimulated by 8-epi-prostaglandin(PG)F2 alpha in rat aortic rings

One of the most abundant F2 isoprostanes formed under pathological conditions is 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha), a potent vasoconstrictor. The purpose of this study was to determine the signal transduction events initiated by 8-epi-PGF2 alpha-induced vasoconstriction. Isolated arterial rings from male Sprague-Dawley rats were suspended in tissue baths containing Krebs-Henseleit salt solution, stretched to optimal resting tension and stimulated. 8-epi-PGF2 alpha induced concentration-dependent contractions in pulmonary arteries (EC50: 7.7 +/- 2.1 microM; n = 3) and aortas (EC50: 0.9 +/- 0.1 microM; n = 4) which were blocked by the TXA2 receptor antagonists SQ29548, L657925 and L657926. The contractile response to 8-epi-PGF2 alpha was significantly (*p < 0.05; n = 4) diminished by: 1) indomethacin and ibuprofen; 2) Ca++ free media; 3) verapamil, a voltage gated Ca++ channel blocker; 4) flunarizine, a T-type Ca++ channel blocker; and 5) calphostin C, a protein kinase C inhibitor. These data suggest that the contractile response to 8-epi-PGF2 alpha is: 1) mediated via activation of TXA2 receptors; 2) partially dependent on the synthesis and release of other cyclooxygenase derived products; 3) dependent on an influx of extracellular Ca++ possibly via Ca++ channels; and 4) may be PKC dependent.     

Interaction of a thrombin inhibitor and a platelet GP IIb/IIIa antagonist in vivo: evidence that thrombin mediates platelet aggregation and subsequent thromboxane A2 formation during coronary thrombolysis

We examined the effect of a specific thrombin inhibitor, Ro 46-6240, alone and combined with an antagonist of the platelet GP IIb/IIIa, Ro44-9883, on the response to tissue-type plasminogen activator in a canine model of thrombolysis. Platelet activity was determined by measuring the excretion of 2,3-dinorthromboxane (TX)B2, an enzymatic metabolite of TXA2. Ro 46-6240 administered before tissue-type plasminogen activator induced a dose-dependent prolongation of the activated partial thromboplastin time and prothrombin time. The time to reperfusion decreased dose-dependently (P < .01) to 10 +/- 6 min vs. 52 +/- 5 min in controls. Ro 46-6240 also prevented reocclusion, which occurred in every case in control experiments. Urinary excretion of 2,3-dinor-TXB2 increased from 3 +/- 1 to 37 +/- 9 ng/mg creatinine in controls after reperfusion. This increase was reduced in a dose-dependent fashion by Ro 46-6240, such that at the highest dose, urinary 2,3-dinor-TXB2 after reperfusion was 5.6 +/- 1 ng/mg creatinine. Similar functional and biochemical effects were seen when a subthreshold dose of Ro 46-6240 was combined with Ro 44-9883. At the dose used, Ro 44-9883 alone abolished platelet aggregation ex vivo but failed to modify the response to tissue-type plasminogen activator or the excretion of 2,3-dinor-TXB2 after reperfusion (51 +/- 6 ng/mg creatinine, n = 3). However, the combination of Ro 44-9883 and Ro 46-6240 reduced the time to reperfusion (40 +/- 8 vs. 68 +/- 15 min; n = 7, P < .05), prevented reocclusion and abolished the rise in urinary 2,3-dinor-TXB2 (5 +/- 1 ng/mg creatinine, n = 4). These findings suggest that thrombin mediates platelet activation during coronary thrombolysis. The increased platelet activity results in platelet aggregation and a subsequent increase in TXA2 formation.     

Acute cholestasis-induced renal failure: effects of antioxidants and ligands for the thromboxane A2 receptor

BACKGROUND: Acute biliary obstruction is associated with the development of renal impairment and oxidative stress. The F2-isoprostanes, formed during oxidant injury, are renal vasoconstrictors acting via thromboxane (TX)-like receptors. We determined whether the formation of F2-isoprostanes is increased in experimental cholestasis and whether thiol containing antioxidants or ligands for the TXA2 receptor could improve renal function. METHODS: The effects on renal function of acute bile duct ligation (BDL) in the rat were studied for two days. The consequences of administration of N-acetylcysteine (NAC), alpha-lipoic acid (LA), the TX receptor antagonist (TXRA) BAYu3405, or placebo were then examined. RESULTS: BDL caused a reduction in creatinine clearance from 1.10 +/- 0.05 to 0.55 +/- 0.05 ml/min and sodium excretion from 52 +/- 3 to 17 +/- 3 micromol/hr. Urinary F2-isoprostanes increased from 14 +/- 2 to 197 +/- 22 pg/ml following BDL. Renal functional changes were ameliorated by NAC (creatinine clearance 0.73 +/- 0.05 ml/min), LA (0.64 +/- 0.03 ml/min), and a TXRA (0.90 +/- 0.15 ml/min); P < 0.05. Similarly, sodium excretion was increased from 17 +/- 3 micromol/hr (placebo) to 34 +/- 3 micromol/hr (NAC), 29 +/- 3 micromol/hr (LA), and 38 +/- 5 micromol/hr (TXRA); P < 0.005. Hepatic glutathione concentrations increased from 6.5 +/- 0.3 micromol/g (normal liver) to 8.8 +/- 0.5 micromol/g (NAC) and 7.7 +/- 0.3 micromol/g (LA), P < 0.01. However, only LA markedly inhibited F2-isoprostane formation (197 +/- 22 to 36 +/- 11 pg/ml creatinine clearance; P < 0.05). Urinary TXB2 excretion was elevated after BDL (2.2 +/- 0.5 to 111.1 +/- 20.3 pg/min) but was unaffected by NAC and LA. CONCLUSION: NAC, LA, and TXRA can partially prevent renal dysfunction in experimental cholestasis. The effects of the antioxidants are independent of their ability to inhibit lipid peroxidation or TX synthesis.     

Effects of very low dose and enteric-coated acetylsalicylic acid on prostacyclin and thromboxane formation and on bleeding time in healthy subjects

OBJECTIVE: Low dose acetylsalicylic acid (ASA) is widely used as an anti-aggregatory agent in the primary and secondary prevention of cardiovascular diseases. In an effort to spare prostacyclin formation and to reduce gastrointestinal side-effects, both very low doses and enteric-coated formulations of ASA have been introduced. However, it still remains unclear whether these different formulations and dosages are equally effective with respect to inhibition of platelet aggregation and thromboxane A2 (TXA2) formation. METHODS: In a randomized study, we therefore investigated the effects of 100 mg ASA plain (p), 100 mg ASA enteric-coated (ec) and 40 mg ASA (p) to 36 healthy male subjects given for 7 days on platelet aggregation and endogenous prostanoid formation rates. Platelet aggregation and platelet TXB2 release in platelet rich plasma (PRP) and serum TXB2 and 6-keto-PGF1alpha levels were determined at baseline and after 7 days of each medication. The urinary metabolites of TXA2 (2,3-dinor-TXB2) and prostacyclin (2,3-dinor-6-keto-PGF1alpha) were measured by gas chromatography/tandem mass spectrometry in 24-h-urines at baseline and on day 7 of each medication. RESULTS: Collagen-induced platelet aggregation was 73.1+/-1.6% of maximal aggregation at baseline. It was inhibited by 68.9%, 58.6% and 24.0% by ASA 100 mg plain, 100 mg enteric-coated, and 40 mg plain on day 7, respectively. Platelet TXB2 release was 11592.0+/-367.5 pg x ml(-1) PRP. It was inhibited by 90.1%, 86.5%, and 55.2% by ASA 100 mg plain, 100 mg enteric-coated, and 40 mg plain, respectively. Serum TXB2 was almost completely reduced on day 7 by 100 mg ASA, but not by 40 mg ASA; serum 6-keto-PGF1alpha was slightly, but significantly reduced in all three groups. Urinary 2,3-dinor-TXB, excretion was 196.0+/-41.5 pg x mg(-1) creatinine at baseline. It was reduced by 80.3% and 79.1% by ASA 100 mg plain and enteric-coated, respectively (each P < 0.05 versus baseline), but only by 55.4% by ASA 40 mg plain (P < 0.05 versus both formulations of ASA 100 mg). CONCLUSIONS: Our present data show that the plain and enteric-coated formulations of 100 mg ASA are equally effective in inhibiting platelet aggregation, platelet thromboxane production, and urinary 2,3-dinor-TXB2 excretion rates. In contrast, a very low dose of 40 mg ASA was significantly less effective in inhibiting these indices of platelet activation in healthy human subjects. ASA enteric-coated 100 mg may be a useful alternative to 100 mg ASA (p) in patients with gastrointestinal side-effects, whereas 40 mg ASA (p) may be too low to inhibit sufficiently platelet activity in patients with cardiovascular diseases in whom platelet activity is increased.     

Ovarian sensitivity to exogenous gonadotrophins in phenobarbital treated unilaterally ovariectomized albino rats

To evaluate platelet activity in patients with non-insulin-dependent diabetes mellitus (NIDDM), we measured the mean platelet volume (MPV) and 24-hour urinary excretion of 11-dehydro-thromboxane B2 (11-dTXB2) and 6-keto-prostaglandin F1 alpha (6-kPGF1 alpha), stable metabolites of thromboxane A2 and prostacyclin, respectively. The MPV of the 103 subjects in the NIDDM group were 10.72 +/- 0.82 fl for males and 10.52 +/- 1.01 fl for females (mean +/- SD), significantly higher than those of normal controls (9.95 +/- 0.75 fl for males and 9.84 +/- 0.72 fl for females). The MPV of patients with NIDDM showed positive correlations with fasting plasma glucose level and HbA1c (r = 0.234, P < 0.05; r = 0.267, P < 0.01, respectively). The urinary excretion of 11-dTXB2 was greater in the NIDDM group (7.58 +/- 4.42 micrograms/day for males and 5.65 +/- 2.38 micrograms/day for females) than in the normal controls (4.61 +/- 2.31 and 3.83 +/- 1.60, respectively), suggesting that the synthesis of thromboxane A2 by platelets may be accelerated in vivo in patients with NIDDM. The urinary 6-kPGF1 alpha was not different between the NIDDM group and normal controls among the males, but was greater in the NIDDM group among the females. As MPV showed a positive correlation (r = 0.364, P < 0.05) with urinary excretion of 11-dTXB2, MPV may be related to platelet activity. These findings suggest that the platelets of patients with NIDDM may be in a hyperactive state.     

Increased formation of distinct F2 isoprostanes in hypercholesterolemia

BACKGROUND: F2 isoprostanes are stable, free radical-catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo. METHODS AND RESULTS: Specific assays were developed by use of mass spectrometry for the F2 isoprostanes iPF2alpha-III and iPF2alpha-VI and arachidonic acid (AA). Urinary excretion of the 2 F2 isoprostanes was significantly increased in hypercholesterolemic patients, whereas substrate AA in urine did not differ between the groups. iPF2alpha-III (pmol/mmol creatinine) was elevated (P<0.0005) in homozygous familial hypercholesterolemic (HFH) patients (85+/-5. 5; n=38) compared with age- and sex-matched normocholesterolemic control subjects (58+/-4.2; n=38), as were levels of iPF2alpha-VI (281+/-22 versus 175+/-13; P<0.0005). Serum cholesterol correlated with urinary iPF2alpha-III (r=0.41; P<0.02) and iPF2alpha-VI (r=0. 39; P<0.03) in HFH patients. Urinary excretion of iPF2alpha-III (81+/-10 versus 59+/-4; P<0.05) and iPF2alpha-VI (195+/-18 versus 149+/-20; P<0.05) was also increased in moderately hypercholesterolemic subjects (n=24) compared with their controls. Urinary excretion of iPF2alpha-III and iPF2alpha-VI was correlated (r=0.57; P<0.0001; n=106). LDL iPF2alpha-III levels (ng/mg arachidonate) were elevated (P<0.01) in HFH patients (0.32+/-0.08) compared with controls (0.09+/-0.02). The concentrations of iPF2-III in LDL and urine were significantly correlated (r=0.42; P<0.05) in HFH patients. CONCLUSIONS: Asymptomatic patients with moderate and severe hypercholesterolemia have evidence of oxidant stress in vivo.     

Relationship of blood thromboxane-B2 (TxB2) with lipid peroxides and effect of vitamin E and placebo supplementation on TxB2 and lipid peroxide levels in type 1 diabetic patients

OBJECTIVE: To study the effect of vitamin E supplementation on platelet hyperaggregability in type 1 diabetic patients. RESEARCH DESIGN AND METHODS: Written informed consent according to the Institutional Review Board on Human Experimentation guidelines was obtained from diabetic patients (n = 29) and their age-matched normal siblings (n = 21) to participate in this study. Diabetic patients were supplemented with DL-alpha-tocopherol (vitamin E) capsule (orally, 100 IU/day) or placebo for 3 months in a double-blind clinical trial. Alternate diabetic patients were assigned to vitamin E or placebo during regular visits to the clinic. Fasting blood was collected from each diabetic patient before the start and after the vitamin E or placebo supplementation. Platelet aggregability was assessed by competitive enzyme-linked immunosorbent assay of the blood TxB2 (a stable thromboxane metabolite). Plasma vitamin E and MDA (malondialdehyde, a product of lipid peroxidation) was assessed by high-performance liquid chromatography. Data were analyzed statistically on 12 diabetic patients on vitamin E and 12 on placebo supplementation. RESULTS: Diabetic patients (n = 29) had 62% higher (P < 0.05) levels of TxB2 and 15% higher levels (P < 0.05) of MDA in comparison to normal subjects (n = 21). Plasma TxB2 levels had a significant correlation with MDA levels (r = 0.45, P < 0.02) but not with the HbA1 (r = -0.08), glucose (r = -0.13), duration of diabetes (r = -0.04), or age (r = 0.12) of diabetic patients. Vitamin E supplementation lowered MDA levels by 30% (P < 0.04), TxB2 levels by 51% (P < 0.03), and triglyceride levels by 22% (P < 0.04) in diabetic patients. There were no differences in these parameters before versus after placebo supplementation. CONCLUSIONS: The elevated blood level of TxB2 (hyperaggregability of platelets) is significantly related to the level of lipid peroxidation products (oxidative stress) in type 1 diabetic patients. Supplementation of modest doses of vitamin E (100 IU/day) significantly lowers blood TxB2 and lipid peroxidation products levels in type 1 diabetic patients.     

Effect of acetate on molecular and physiological aspects of Clostridium beijerinckii NCIMB 8052 solvent production and strain degeneration

OBJECTIVES: The purpose of this study was to test the hypothesis that long-term supplementation with Vitamin E improves endothelium-dependent relaxation in hypercholesterolemia patients and/or chronic smoking, two risk factors that have been shown to be associated with increased radical formation. BACKGROUND: Experimental evidence suggests that oxidized low density lipoprotein (LDL) impairs endothelium-dependent relaxation, and vitamin E, a lipid-soluble antioxidant, reduces the oxidation of LDL. METHODS: Thirteen subjects with hypercholesterolemia, 14 smokers and 15 hypercholesterolemic smokers were enrolled in a double-blind, placebo-controlled study. After baseline measurements of plasma autoantibodies against oxidized LDL and assessment of endothelium-dependent relaxation using intra-arterial forearm infusions of acetylcholine, participants within each group were randomly assigned in a 1:2 fashion to receive either placebo or vitamin E for 4 months, when plasma levels of autoantibodies against oxidized LDL and vascular function were reassessed. RESULTS: Vitamin E significantly augmented endothelium-dependent relaxation in hypercholesterolemic smokers but not in patients with either hypercholesterolemia or chronic smoking. At baseline, hypercholesterolemic smokers had significantly higher autoantibody levels against oxidized LDL (compared with the other two groups), which were significantly reduced after 4 months of vitamin E supplementation. There was a significant relationship between improvement in acetylcholine-induced vasodilation and the change in autoantibody titer against oxidized LDL (r = -0.59; p = 0.002). CONCLUSIONS: Long-term vitamin E supplementation improves endothelium-dependent relaxation in forearm resistance vessels of hypercholesterolemic smokers, which are characterized by increased levels of autoantibodies against oxidized LDL. These findings may suggest that the beneficial effect of vitamin E is confined to subjects with increased exposure to oxidized LDL.     

Cross-resistance to methotrexate and metals in human cisplatin-resistant cell lines results from a pleiotropic defect in accumulation of these compounds associated with reduced plasma membrane binding proteins

Platelet hyperactivity in vitro is found in patients with isolated hypercholesterolemia. It is, however, less well established if platelet activity in vivo is enhanced, and if there are differences between various types of hyperlipoproteinemia. Platelet function in vivo was studied at rest and during mental stress in men with isolated hypercholesterolemia (phenotype IIa; n = 21) or combined hyperlipidemia (phenotype IIb; n = 29), and age-matched normolipidemic controls (n = 41). The urinary excretion of 11-dehydrothromboxane B2 was elevated in patients compared to controls (IIa, p <0.05; IIb, p <0.001), and higher in type IIb than in IIa patients (p <0.05). Platelet secretion, assessed as plasma beta-thromboglobulin levels, was higher in type IIb patients compared to controls (p <0.01) and type IIa patients (p <0.05) during mental stress. The urinary excretion of beta-thromboglobulin was also elevated in type IIb patients compared to controls (p <0.05). Platelet aggregability at rest, as measured by filtragometry ex vivo was, however, reduced in both patient groups compared to controls (p <0.05). No correlations were found between plasma lipoprotein levels and markers of platelet function in vivo. Type IIb patients had higher plasma fibrinogen levels and higher leukocyte counts than controls (p <0.05 and p <0.001) and type IIa patients (p <0.05 and p = 0.06). Thromboxane excretion was positively related to fibrinogen levels and leukocyte counts (p <0.01 for both). Preliminary data regarding serum TNF-alpha also indicated an elevation of this inflammatory cytokine in type IIb patients (p <0.05 vs controls). In conclusion, thromboxane generation and platelet secretion in vivo are enhanced in patients with hypercholesterolemia, and particularly so among patients with concomitant elevation of plasma triglycerides. The mechanism is unknown, but inflammatory mediators may be involved. The present findings are of interest in relation to the role of triglycerides in coronary artery disease.