To assess the pharmacologic effects of aspirin on endogenous prostacyclin and thromboxane biosynthesis, 2,3-dinor-6-keto PGF1 alpha (PGI-M) and 2,3-dinor-thromboxane B2 (Tx-M) were measured in urine by mass spectrometry during continuing administration of aspirin. To define the relationship of aspirin intake to endogenous prostacyclin biosynthesis, sequential urines were initially collected in individuals prior to, during, and subsequent to administration of aspirin. Despite inter- and intra-individual variations, PGI-M excretion was significantly reduced by aspirin. However, full mass spectral identification confirmed continuing prostacyclin biosynthesis during aspirin therapy. Recovery of prostacyclin biosynthesis was incomplete 5 d after drug administration was discontinued. To relate aspirin intake to indices of thromboxane biosynthesis and platelet function, volunteers received 20 mg aspirin daily followed by 2,600 mg aspirin daily, each dose for 7 d in sequential weeks. Increasing aspirin dosage inhibited Tx-M excretion from 70 to 98% of pretreatment control values; platelet TxB2 formation from 4.9 to 0.5% and further inhibited platelet function. An extended study was performed to relate aspirin intake to both thromboxane and prostacyclin generation over a wide range of doses. Aspirin, in the range of 20 to 325 mg/d, resulted in a dose-dependent decline in both Tx-M and PGI-M excretion. At doses of 325-2,600 mg/d Tx-M excretion ranged from 5 to 3% of control values while PGI-M remained at 37-23% of control. 3 d after the last dose of aspirin (2,600 mg/d) mean Tx-M excretion had returned to 85% of control, whereas mean PGI-M remained at 40% of predosing values. Although the platelet aggregation response (Tmax) to ADP ex vivo was inhibited during administration of the lower doses of aspirin the aggregation response returned to control values during the final two weeks of aspirin administration (1,300 and 2,600 mg aspirin/d) despite continued inhibition of thromboxane biosynthesis. These results suggest that although chronic administration of aspirin results in inhibition of endogenous thromboxane and prostacyclin biosynthesis over a wide dose range, inhibition of thromboxane biosynthesis is more selective at 20 than at 2,600 mg aspirin/d. However, despite this, inhibition of platelet function is not maximal at the lower aspirin dosage. Doses of aspirin in excess of 80 mg/d resulted in substantial inhibition of endogenous prostacyclin biosynthesis. Thus, it is unlikely that any dose of aspirin can maximally inhibit thromboxane generation without also reducing endogenous prostacyclin biosynthesis. These results also indicate that recovery of endogenous prostacyclin biosynthesis is delayed following aspirin administration and that the usual effects of aspirin on platelet function ex vivo may be obscured during chronic aspirin administration in man.

There is increasing evidence that aspirin initiates biosynthesis of novel antiinflammatory mediators by means of interactions between endothelial cells and leukocytes. These mediators are classified as aspirin-triggered 15-epi-lipoxins. Such compounds may account at least in part for aspirin's clinical benefits, which are distinct from the well appreciated action of aspirin as a platelet inhibitor. Here, we addressed whether aspirin-triggered 15-epilipoxinA4 (ATL) formation is aspirin-dependent in humans and its relationship to aspirin's antiplatelet activity. We conducted a randomized clinical trial among 128 healthy subjects allocated to placebo or to 81-, 325-, or 650-mg daily doses of aspirin for 8 weeks. Plasma thromboxane (TX)B2, an indicator of platelet reactivity, and ATL were assessed from blood collected at baseline and at 8 weeks. Plasma ATL levels significantly increased in the 81-mg aspirin group (0.25 +/- 0.63 ng/ml, P = 0.04), with borderline increases in the 325-mg group (0.16 +/- 0.71 ng/ml) and no apparent significant changes in the 650-mg group (0.01 +/- 0.75 ng/ml, P = 0.96). When ATL and TXB2 were compared, levels changed in a statistically significant and opposite direction (P < 0.01) for all three aspirin doses. These results demonstrated that low-dose aspirin (81 mg daily) initiates production of antiinflammatory ATL opposite to the inhibition of TX. Monitoring ATL may represent a simple clinical parameter to verify an individual's vascular leukocyte antiinflammatory response with low-dose aspirin treatment. These results also emphasize the importance of cell-cell interactions in the modulation of hemostatic, thrombotic, and inflammatory processes.

The capacity of platelets to generate thromboxane A2, reflected by measurement of serum thromboxane B2 (TxB2), greatly exceeds the systemic production of thromboxane in vivo. Thus, it is possible that substantial but incomplete inhibition of thromboxane formation ex vivo would still allow marked augmentation of thromboxane production in vivo. To address this hypothesis, we administered aspirin 120 mg, a selective inhibitor of thromboxane synthase (TxSl), 3-(1H-imidazol-1-yl-methyl)-2-methyl-1H-indole-1-propanoic acid (UK-38, 485) 200 mg, and a combination of both drugs to 12 healthy volunteers and measured the effects on serum TxB2 and urinary 2,3-dinor-thromboxane B2 (Tx-M), an index of endogenous thromboxane biosynthesis. Although serum TxB2 was maximally inhibited by 94 +/- 1% after aspirin and 96 +/- 2% after the TxSl, maximal depression of Tx-M was only 28 +/- 8% and 37 +/- 9%, respectively. Combination of aspirin with the TxSl resulted in a small but significant increase in inhibition of thromboxane generation ex vivo (98 +/- 1% v 94 +/- 1%; P less than 0.05), but a disproportionately greater fall in thromboxane synthesis in vivo (58 +/- 7%; P less than 0.01). Consistent with further inhibition of platelet thromboxane synthesis, addition of the TxSl abolished the transient decline in prostacyclin formation after aspirin alone. Administration of a lower dose of aspirin (20 mg) to 6 healthy subjects caused a small reduction in Tx-M (12 +/- 4%; P less than 0.05) and inhibited serum TxB2 by 48 +/- 2%. The relationship between inhibition of platelet capacity to form thromboxane ex vivo (serum TxB2) and synthesis in vivo (Tx-M) departed markedly from the line of identity. When total blockade of the capacity of platelets to generate thromboxane is approached, minor decrements in capacity result in a disproportionate depression of actual thromboxane biosynthesis. These results imply that pharmacologic inhibition of serum TxB2 must be virtually complete before thromboxane-dependent platelet activation is influenced in vivo.

Neutrophils have been implicated as central mediators in myocardial and skeletal muscle ischemia-reperfusion injury. This study tests whether these cellular elements and the chemoattractant leukotriene (LTB4) play a role in postischemic renal failure. Anesthetized rats underwent 45 min of left renal pedicle clamping. Five minutes after reperfusion, LTB4 levels were elevated to 1.42 ng/ml (P less than 0.05); thromboxane (Tx)B2 was 2,840 pg/ml, higher than 503 pg/ml in sham controls (P less than 0.05); renal artery blood flow was 67% of preclamping values at 1 min of reperfusion compared with 111% in sham (P less than 0.05). At 24 h, creatinine levels were 4.6 mg/dl (P less than 0.05). At 24 h, creatinine levels were 4.6 mg/dl (P less than 0.05); histology showed acute tubular necrosis (ATN). Neutrophil depletion by rabbit antiserum (n = 8) led during reperfusion to reduced LTB4 and TxB2 levels, 1.04 ng/ml and 1.043 pg/ml (P less than 0.05); increased renal blood flow of 174% (P less than 0.05); reduced creatinine levels of 1.8 mg/dl (P less than 0.05); and limited ATN. Pretreatment with diethycarbamazine prevented the increases in LTB4 and TxB2 (P less than 0.05), increased renal blood flow (P less than 0.05), minimized creatinine increase to 1.7 mg/dl (P less than 0.05), and reduced ATN. These data indicate that neutrophils and LTB4 play a role in ischemia-induced Tx synthesis and mediate postischemic renal injury.

In human plasma, 11-dehydrothromboxane (TX) B2 is a major long lived metabolite (t1/2 45 min) formed from infused TXB2, the hydration product of biologically active TXA2. Plasma concentrations of TXB2 itself are readily confounded by ex vivo platelet activation and, theoretically, an enzymatic derivative of this compound, not subject to formation in whole blood, would more accurately reflect TXA2 formation in vivo. To address this hypothesis, we developed a sensitive assay for both 11-dehydro-TXB2 and TXB2, using capillary gas chromatography/negative-ion chemical ionization mass spectrometry. We established that whole blood possesses a minor capacity to form 11-dehydro-TXB2, attributable to nonenzymatic formation in erythrocytes. However, the nonenzymatic formation of 11-dehydro-TXB2 was not a practical limitation to its use as an index of TX biosynthesis. Blood was drawn from healthy volunteers (i) via an indwelling catheter at the time of insertion and at 30, 60, 90, 180, and 240 min thereafter and (ii) via separate venipunctures at 0 time and at 90 and 240 min thereafter. Plasma TXB2 drawn via the catheter at baseline (66 +/- 63 pg/ml) was substantially greater than the maximal estimate of endogenous TXB2 (1-2 pg/ml) in plasma [Patrono, C., Ciabattoni, G., Pugliese, F., Perruci, A., Blair, I. A. & FitzGerald, G. A. (1986) J. Clin. Invest. 77, 590-594] and increased in magnitude and variance over time (339 +/- 247 pg/ml at 240 min). By contrast, 11-dehydro-TXB2 did not change significantly in the sequential catheter samples or in the samples drawn by separate venipuncture. Basal plasma concentrations in volunteers were depressed by pretreatment with 325 mg of aspirin. Furthermore, the range of concentrations in patients with severe atherosclerosis in whom urinary 2,3-dinor-TXB2 was increased was significantly higher (5-50 pg/ml, P less than 0.01) than in healthy subjects (0.9-1.8 pg/ml). Concentrations of 11-dehydro-TXB2 were increased in patients who had recently suffered a pulmonary embolism to a greater extent than either the 11-dehydro-13,14-dihydro-15-keto-TXB2 or the 2,3-dinor-TXB2 metabolites in plasma. These results indicate that plasma TXB2 is readily confounded by platelet activation ex vivo. Measurement of enzymatic metabolites of TXB2 minimizes this problem. The 11-dehydro metabolite is the most appropriate analytic target to detect phasic release of TXA2 in the human circulation, such as might occur in human syndromes of platelet activation.

F2-isoprostanes are bioactive prostaglandin (PG)-like compounds that are produced from arachidonic acid through a nonenzymatic process of lipid peroxidation catalyzed by oxygen free-radicals. 8-Epi-PGF2 alpha may amplify the platelet response to agonists, circulates in plasma, and is excreted in urine. We examined the hypothesis that the formation of 8-epi-PGF2 alpha is altered in patients with hypercholesterolemia and contributes to platelet activation in this setting. Urine samples were obtained from 40 hypercholesterolemic patients and 40 age- and sex-matched control subjects for measurement of immunoreactive 8-epi-PGF2 alpha. Urinary excretion of 11-dehydro-thromboxane (TX) B2, a major metabolite of TXA2, was measured as an in vivo index of platelet activation. Low-dose aspirin, indobufen, and vitamin E were used to investigate the mechanism of formation and effects of 8-epi-PGF2 alpha on platelet activation. Urinary 8-epi-PGF2 alpha was significantly (P = .0001) higher in hypercholesterolemic patients than in control subjects: 473 +/- 305 versus 205 +/- 95 pg/mg creatinine. Its rate of excretion was inversely related to the vitamin E content of LDL and showed a positive correlation with urinary 11-dehydro-TXB2. Urinary 8-epi-PGF2 alpha was unchanged after 2-week dosing with aspirin and indobufen despite complete suppression of TX metabolite excretion. Vitamin E supplementation was associated with dose-dependent reductions in both urinary 8-epi-PGF2 alpha and 11-dehydro-TXB2 by 34% to 36% and 47% to 58% at 100 and 600 mg daily, respectively. We conclude that the in vivo formation of the F2-isoprostane 8-epi-PGF2 alpha is enhanced in the vast majority of patients with hypercholesterolemia. This provides an aspirin-insensitive mechanism possibly linking lipid peroxidation to amplification of platelet activation in the setting of hypercholesterolemia. Dose-dependent suppression of enhanced 8-epi-PGF2 alpha formation by vitamin E supplementation may contribute to the beneficial effects of antioxidant treatment.

We examined herein the functional linkage of enzymes regulating the initial, intermediate, and terminal steps of PG biosynthesis to provide PGs in rat peritoneal macrophages stimulated with LPS and/or A23187. Quiescent cells stimulated with A23187 produced thromboxane B2 (TXB2) in marked preference to PGE2 within 30 to 60 min (constitutive immediate response), which was mediated by preexisting cytosolic phospholipase A2 (cPLA2), cyclooxygenase-1 (COX-1), and TX synthase. Cells treated with LPS predominantly produced PGE2 during culture for 3 to 24 h (delayed response), where cPLA2 and secretory PLA2 functioned cooperatively with inducible COX-2, which was, in turn, coupled with inducible PGE2 synthase. Cells primed for 12 h with LPS and stimulated for 30 min with A23187 produced PGE2 in marked preference to TXB2 (induced immediate response), in which three inducible enzymes, cPLA2, COX-2, and PGE2 synthase, were functionally linked. Preferred coupling of the two inducible enzymes, COX-2 and PGE2 synthase, was further confirmed by the ability of LPS-treated cells to convert exogenous arachidonic acid to PGE2 optimally at a time when both enzymes were simultaneously induced. These results suggest that distinct PG biosynthetic enzymes display segregated functional coupling following different transmembrane stimulation events even when enzymes that catalyze similar reactions in vitro coexist in the same cells.

BACKGROUND

Hypertensive patients with renovascular disease (RVD) may be exposed to increased oxidative stress, possibly related to activation of the renin-angiotensin system.

RESULTS

We measured the urinary excretion of 8-iso-prostaglandin (PG) F2alpha and 11-dehydro-thromboxane (TX) B2 as indexes of in vivo lipid peroxidation and platelet activation, respectively, in 25 patients with RVD, 25 patients with essential hypertension, and 25 healthy subjects. Plasma renin activity in peripheral and renal veins, angiotensin II in renal veins, cholesterol, glucose, triglycerides, homocysteine, and antioxidant vitamins A, C, and E were also determined. Patients were also studied 6 months after a technically successful angioplasty of the stenotic renal arteries. Urinary 8-iso-PGF2alpha was significantly higher in patients with RVD (median, 305 pg/mg creatinine; range, 124 to 1224 pg/mg creatinine) than in patients with essential hypertension (median, 176 pg/mg creatinine; range, 48 to 384 pg/mg creatinine) or in healthy subjects (median, 123 pg/mg creatinine; range, 58 to 385 pg/mg creatinine). Urinary 11-dehydro-TXB2 was also significantly higher in RVD patients compared with healthy subjects. In RVD patients, urinary 8-iso-PGF2alpha correlated with 11-dehydro-TXB2 (r(s)=0.48; P<0.05) and renal vein renin (r(s)=0.67; P<0.005) and angiotensin II (r(s)=0.65; P=0.005) ratios. A reduction in 8-iso-PGF2alpha after angioplasty was observed in RVD patients with high baseline levels of lipid peroxidation. Changes in 8-iso-PGF2alpha were related to baseline lipid peroxidation (r(s)=-0.73; P<0.001), renal vein angiotensin II (r(s)=-0.70; P<0.01) and renin (r(s)=-0.63; P<0.05) ratios.

CONCLUSIONS

Lipid peroxidation is markedly enhanced in hypertensive patients with RVD and is related to activation of the renin-angiotensin system. Moreover, persistent platelet activation triggered or amplified by bioactive isoprostanes may contribute to the progression of cardiovascular and renal damage in this setting.

Rat peritoneal macrophages were stimulated with lipopolysaccaride (LPS) for various periods and their ability to convert exogenous arachidonic acid to various prostanoids was examined. Unstimulated cells, which expressed cyclooxygenase (COX)-1 but not COX-2, produced thromboxane (TX) B2>> prostaglandin (PG) D2>> PGE2, whereas cells stimulated for 6-12 h with LPS exhibited marked increase in conversion to PGE2, which paralleled COX-2 induction, with minimal change in conversion to TXB2 and PGD2. Pharmacological studies showed that formation of PGE2 was mediated predominantly by COX-2, PGD2 by COX-1, and TXB2 by both COX-1 and COX-2 depending upon the timing of LPS stimulation. Measurement of the conversion of exogenous PGH2 to each prostanoid in cell lysates demonstrated LPS-dependent increase in PGE2 synthase activity that was degenerated by pretreatment with actinomycin D or cycloheximide. Thus, concordant induction of terminal PGE2 synthase with COX-2 leads to the preferred production of PGE2 to TXB2 and PGD2 by LPS-stimulated macrophages.

OBJECTIVE

We performed a placebo-controlled study to evaluate the effect of low-dose aspirin on serum C-reactive protein (CRP) levels.

BACKGROUND

Elevated circulating concentrations of CRP, an inflammatory marker, increase the risk of thrombotic cardiovascular diseases such as myocardial infarction (MI). Moreover, low-dose aspirin therapy has been reported to be more effective in preventing MI in men with higher CRP levels than it is in those with lower levels, raising the possibility that aspirin prevents thrombosis by reducing vascular inflammation. The effect of low-dose aspirin therapy on serum CRP levels in men has been addressed recently, but the results of the two studies conflict.

METHODS

Effects of aspirin (81 mg every day or 325, 81 or 40 mg every-third-day given for 31 days) on serum CRP, using a highly-sensitive assay, and on serum platelet-cyclo-oxygenase (COX)-1-derived thromboxane (Tx) B2 concentrations were studied simultaneously in 57 healthy volunteers (30 men and 27 women).

RESULTS

Trough platelet COX-1-derived serum Tx B2 concentrations decreased by 100% with daily aspirin and by 90%, 84% and 78% with 325, 81 and 40 mg aspirin every-third-day (p < 0.001). However, there were no significant changes in serum CRP levels from baseline with daily low-dose aspirin therapy, with any of the every-third-day aspirin regimens or with placebo treatment.

CONCLUSIONS

Low doses of aspirin that markedly inhibit platelet COX-1 activity, as manifested by a profound decline in platelet-derived serum Tx B2 concentrations, have no detectable effect on serum CRP levels in healthy men and women.

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.

OBJECTIVE

Aspirin resistance may be relatively common and associated with adverse outcome. Meta-analysis has clearly shown that 75 mg plain aspirin is the lowest effective dose; however, it is not known whether the recent increased use of enteric-coated aspirin could account for aspirin resistance. This study was designed to determine whether enteric-coated aspirin is as effective as plain aspirin in healthy volunteers.

METHODS

Seventy-one healthy volunteers were enrolled in 3 separate bioequivalence studies. Using a crossover design, each volunteer took 2 different aspirin preparations. Five aspirin preparations were evaluated, 3 different enteric-coated 75-mg aspirins, dispersible aspirin 75 mg and asasantin (25-mg standard release aspirin plus 200-mg modified-release dipyridamole given twice daily). Serum thromboxane (TX) B2 levels and arachidonic acid-induced platelet aggregation were measured before and after 14 days of treatment.

RESULTS

All other aspirin preparations tested were inferior to dispersible aspirin (P<0.001) in their effect on serum TXB(2) level. Treatment failure (<95% inhibition serum TXB2 formation) occurred in 14 subjects, none of whom were taking dispersible aspirin. Mean weight for those demonstrating treatment failure was greater than those with complete TXB2 (>99%) inhibition (P<0.001). Using logistic regression analysis an 80-kg subject had a 20% probability of treatment failure. Asasantin was the most potent preparation in terms of inhibition of platelet aggregation.

CONCLUSIONS

Equivalent doses of the enteric-coated aspirin were not as effective as plain aspirin. Lower bioavailability of these preparations and poor absorption from the higher pH environment of the small intestine may result in inadequate platelet inhibition, particularly in heavier subjects.

The present study examined the role of enhanced production of prostaglandin (PG) E2 and 6-keto-PGF1a, the stable metabolite of PGI2, by glomeruli from streptozotocin diabetic rats in the mediation of hyperfiltration. Correlative measurements of insulin clearance (CIn) and glomerular production of PGE2, 6-keto PGF1a and thromboxane (TX) B2 (the stable metabolite of TXA2) were made at two time points, nine to 15 days and 25 to 28 days after streptozotocin. CIn was elevated by 40% to 50% in diabetic rats studied at nine to 15 or 25 to 28 days compared to values in age-matched controls. Basal production of PGE2, 6-keto PGF1a and TXA2 (as reflected by TXB2) and increases in response to A23187 were elevated in glomeruli from nine to 15-day diabetic rats compared to values in control glomeruli. Exogenous arachidonate abolished these differences. Treatment of nine-day diabetic rats with indomethacin (3 mg/kg/d) rapidly (within 24 hours) and reversibly suppressed CIn without altering CIn in control rats. Indomethacin had no effect on plasma glucose in control or diabetic rats. Treatment of nine to 15-day diabetic rats with insulin (10 U/kg/d by osmotic minipump) beginning 24 hours after streptozotocin lowered plasma glucose to values that were not significantly different from control and prevented the rise in CIn. Treatment of diabetic rats with insulin or incubation of glomeruli from untreated diabetic rats with insulin (0.3 mU/mL) for two hours in vitro reduced basal and A23187 induced increases in PGE2, 6-keto PGF1a, and TXB2 to values that were not different from those in control glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of prostanoids in Crohn's disease was investigated using rectal biopsy specimens maintained in organ culture. As with ulcerative colitis increased synthesis of prostaglandin (PG)E2 was observed when the mucosa was inflamed, compared with uninflamed mucosa in Crohn's disease, and with control biopsy specimens. In contrast with ulcerative colitis differences from control specimens were observed even in the absence of inflammation. There was a raised synthesis of thromboxane (Tx)B2 (stable breakdown product of TxA2); concentrations of 6-keto PGF1 alpha (stable breakdown product of prostacyclin) were unchanged and hence the ratio of 6-keto PGF1 alpha/TxB2 was reduced. These changes might lead to an altered cytoprotective capacity or reduced suppressor cell activity, such as has previously been reported in intestinal lymphocytes in Crohn's disease.

Gh is a GTP binding protein that couples to the thromboxane receptor (TP), but also functions as tissue transglutaminase II (tTG). A transgenic mouse model was generated in which Gh was overexpressed (GhOE) in ventricular myocytes under the control of the alpha-myosin heavy chain promoter. Heart rate was elevated and both blood pressure and left ventricular ejection fraction were depressed in GhOEs. Left ventricular mass was increased, consistent with genetic and ultrastructural evidence of hypertrophy. Fibrosis and apoptosis were also augmented. Survival declined disproportionately in older GhOEs. Cardiomyocyte expression of COX-2, thromboxane synthase (TxS), and the receptors for TxA2 (the TP), PGF2alpha (the FP), and PGI2 (the IP) were upregulated and urinary 8,12-iso-iPF2alpha-VI,2,3-dinor-6-keto-PGF1alpha and 2,3-dinor-thromboxane B2 were increased in GhOEs, reflecting increased lipid peroxidation and cyclooxygenase (COX) activation. Selective COX-2 inhibition, TP antagonism, and suppression of lipid peroxidation each rescued the cardiac phenotype. Infusion of an FP agonist exacerbated the phenotype, whereas administration of an IP agonist improved cardiac function. Directed cardiac overexpression of Gh/tTG causes both TG activation and increased TP/Gh-dependent signaling. The COX-2-dependent increase in TxA2 generation augments cardiac hypertrophy, whereas formation of PGI2 by the same isozyme ameliorates the phenotype. Oxidant stress may contribute, via regulation of COX-2 expression and/or ligation of the TP and the FP by isoprostanes. Gh/tTG activation regulates expression of COX-2 and its products may differentially modulate cardiomyocyte commitment to cell death or survival.

OBJECTIVE

We investigated whether use of low-dose enteric-coated (EC) aspirin for secondary prevention of cardiovascular events has sufficient bioavailability to achieve complete platelet cyclooxygenase (COX) inhibition in all individuals.

BACKGROUND

Aspirin reduces cardiovascular morbidity and mortality in patients with pre-existing vascular disease; however, there is variability in the way individuals respond. Persistent normal platelet function despite therapy, referred to as "aspirin resistance," is associated with an increased risk of major cardiovascular events.

METHODS

We studied 131 stable cardiovascular patients between March and September 2002 who were taking 75 mg EC aspirin. Serum thromboxane (TX) B2 levels were assayed as a measure of COX activity. Mean arachidonic acid (AA)-induced platelet aggregation>> or =20% was deemed evidence of persistent platelet activity and an incomplete aspirin response.

RESULTS

Patients of median age 63 years (61% men) were enrolled. Forty-four percent of patients had elevated serum TX B2 levels (>2.2 ng/ml). Arachidonic acid-induced platelet aggregation occurred more frequently in these patients (21% vs. 3%; p = 0.004). In all cases addition of exogenous aspirin during the assay abolished platelet aggregation. Patient weight and age were significant independent predictors of an incomplete response to EC aspirin (p = 0.025 and p < 0.001, respectively). These patients were also more likely to have a history of myocardial infarction (MI) (p = 0.038).

CONCLUSIONS

Many patients who are prescribed low-dose EC aspirin for secondary prevention of cardiovascular events have persistent uninhibited platelet COX activity. Younger and heavier patients and those with a previous MI are most likely to have an inadequate response to treatment.

Normal human epidermal melanocytes became swollen and more dendritic with an increase in the amount of tyrosinase and immunoreactive b-locus protein when they were cultured for 2 days with the following arachidonic acid metabolites: prostaglandin (PG) D2, leukotriene (LT) B4, LTC4, LTD4, LTE4, thromboxane (TX) B2 and 12-hydroxy eicosatetraenoic acid (12-HETE). The effect of LTC4 was particularly strong compared to that of PGE2, about which we have previously reported. On the other hand, PGE1, PGF2 alpha and 6-ketoPGF1 alpha did not show any significant stimulatory effect. These data suggest that arachidonate-derived chemical mediators, especially LTC4, may be responsible for the induction of post-inflammatory hyperpigmentation of the skin.

Enteric coated aspirin (ECA) at doses of 325-1300 mg is an effective alternative to regular aspirin for inhibition of platelet activity while avoiding gastric irritation. The objectives of this study were to determine: (1) the lowest chronic dose of ECA providing effective inhibition of platelet activities, (2) the time course of the inhibition, and (3) the reappearance of platelet cyclo-oxygenase activity. Seven subjects were studied before and after seven daily doses of 40-325 mg ECA. Serum thromboxane (TX) B2 levels indicated that the lowest dose of ECA resulting in greater than 90% inhibition of platelet cyclo-oxygenase was 80 mg/d. Platelet aggregation and ATP release in response to collagen (1 microgram/ml) and arachidonic acid (1 mM) were abolished and bleeding times were prolonged from 6.1 +/- 1.5 min to 9.7 +/- 2.8 min (mean +/- SD, P less than 0.01). Examination of platelet cyclo-oxygenase activity on a daily basis revealed that 24 h after the first 80 mg dose serum TXB2 had decreased by approximately 60% and was suppressed by more than 90% after four doses. Recovery of platelet cyclo-oxygenase activity after a single 80 mg dose of ECA was delayed for 48-72 h indicating that aspirin reached the systemic circulation. We conclude that chronic inhibition of platelet activity may be achieved in a cumulative manner with 80 mg ECA/d.

Alveolar macrophages (AM) were obtained by bronchoalveolar lavage (BAL) from seven healthy nonallergic, nonasthmatic donors, 15 patients with allergic bronchial asthma, and six patients with aspirin-sensitive asthma. AM were purified by adherence over 2 hr and cultured for an additional 24 hr. Functional assessment of viable cells was carried out for zymosan phagocytosis and for prostaglandin (PG) E2-PGF2 alpha and thromboxane (Tx) B2 release by resting and zymosan-stimulated AM. The eosinophil count in BAL fluid from allergic asthmatics was higher than that from control subjects (3.9% +/- 1.6% vs 0.4% +/- 0.3%, p less than 0.05) and still greater in BAL from patients with aspirin-sensitive asthma (21.7% +/- 9.0%, p less than 0.01). After the 24 hr of incubation, the AM viability was inversely correlated to the percentage of eosinophils in BAL fluid (r = -0.54, n = 21, p less than 0.02). Zymosan phagocytosis was significantly lower by viable cells from both allergic asthmatics and aspirin-sensitive patients as compared with cells from normal donors (p less than 0.05). Zymosan phagocytosis induced a twofold to threefold increase in the release of PGE2, PGF2 alpha, and TxB2 from AM of normal subjects (p less than 0.01) but only a onefold to twofold increase from AM of allergic asthmatic patients. The stimulated AM from aspirin-sensitive patients released smaller quantities of each product than AM from normal subjects or allergic asthmatic patients (p less than 0.05). We conclude that the viability and functional activity of AM are impaired in asthmatic patients and that these deficits correlate with the percent eosinophilia in the BAL; it is therefore suggested that they may be due to an interaction between eosinophils and AM in the bronchoalveolar lumen.

OBJECTIVE

The goals of this study were to characterize the platelet contribution to soluble CD40 ligand (sCD40L), to correlate its formation with the extent of oxidative stress and platelet activation, and to investigate the effects of improved metabolic control and low-dose aspirin on these processes.

BACKGROUND

Inflammation, oxidative stress, and platelet activation are involved in the pathogenesis of type 2 diabetes (T2DM) and its complications. The CD40-CD40L interactions result in inflammatory and pro-thrombotic responses.

METHODS

Urinary 8-iso-prostaglandin (PG)F2alpha and 11-dehydro-thromboxane (TX)B2, in vivo markers of oxidative stress and platelet activation, respectively, plasma CD40L, and C-reactive protein (CRP) were measured in 114 T2DM patients and 114 control patients. A randomized, parallel group, 17-day study of aspirin (30, 100, or 325 mg/day) was performed in 18 T2DM patients. A similar study was performed in six healthy volunteers (aspirin, 100 mg/day). Twenty poorly controlled T2DM patients were studied before and after improved metabolic control.

RESULTS

Compared with control patients, diabetic patients showed significantly higher levels of 8-iso-PGF2alpha, 11-dehydro-TXB2, sCD40L, and CRP. On multiple regression analysis, 11-dehydro-TXB2 and 8-iso-PGF2alpha excretion rates predicted sCD40L levels. Soluble CD40L linearly correlated with 11-dehydro-TXB2 (rho = 0.67, p < 0.0001), and both were reduced after one week of aspirin (p < 0.0026), with slow recovery over 10 days after aspirin withdrawal. Improved metabolic control was associated with a reduction in sCD40L, 8-iso-PGF2alpha, and 11-dehydro-TXB2.

CONCLUSIONS

This study provides several lines of evidence for the dependence of sCD40L release on TXA(2)-dependent platelet activation in T2DM and provides novel mechanistic insight into the amplification loops of persistent platelet activation in this setting.

BACKGROUND

Cigarette smoking is highly pathogenic to the vasculature. In smokers, the biosynthesis of both thromboxane (Tx) A2 and prostacyclin is increased. We hypothesized that the excess in prostacyclin biosynthesis in smokers was derived from the inducible cyclooxygenase-2 (COX-2). We further hypothesized that if the overproduction of prostacyclin in smokers were restraining platelet activation, then inhibition of COX-2 would lead to an increase in the activation of platelets, with a corresponding increase in the biosynthesis of TxA2.

RESULTS

Smokers and nonsmokers received rofecoxib 25 mg twice daily or placebo for 1 week each in random sequence. The systemic biosynthesis of TxA2 and prostacyclin was assessed by analysis of their respective urinary metabolites, 11-dehydrothromboxane B2 (Tx-M) and 2'3-donor-6-keto-PGF(1alpha) (PGI-M). Serum TxB2 was measured as an indicator of platelet COX-1 activity. Results are expressed as mean+/-SE with median and range. The elevated PGI-M in smokers (189+/-25, median 174, range 85 to 390 pg/mg creatinine) was reduced by rofecoxib to 78+/-27, median 71.5, range 50 to 135 pg/mg creatinine (P=0.002), and in nonsmokers, PGI-M at baseline (115+/-10, median 107, range 67 to 198 pg/mg creatinine) fell to 56+/-15, median 50, range 34 to 125 pg/mg creatinine (P=0.001) with rofecoxib. The increased excretion of Tx-M in smokers (284+/-26, median 252, range 200 to 569 pg/mg creatinine) was reduced by 21% to 223+/-16, median 206, range 154 to 383 pg/mg creatinine by rofecoxib (P=0.04) but was not changed in nonsmokers. Levels of serum TxB2 were not different in smokers and nonsmokers and were unaffected by rofecoxib.

CONCLUSIONS

The increased prostacyclin biosynthesis in smokers is derived largely from the inducible COX-2. COX-2 also contributes to the increased biosynthesis of TxA2 in smokers, most likely from inflammatory cells.

1. The effects of the non-selective cyclo-oxygenase (COX) inhibitor indomethacin and the selective COX-2 inhibitors, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398), 5-methanesulphonamido-6-(2,4-difluorothio-phenyl)-1-indan one (L-745,337) and 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone (DFU), on the protection induced by the mild irritant 20% ethanol were investigated in the rat stomach. 2. Instillation of 20% ethanol (1 ml, p.o.) effectively protected against gastric mucosal injury induced by subsequent instillation of 70% or 96% ethanol (1 ml, p.o.). 3. Oral administration of indomethacin (1.25-20 mg kg[-1]) dose-dependently counteracted the protective effect of 20% ethanol (ID50: 3.5 mg kg[-1]). 4. Likewise, NS-398 (0.1-1 mg kg[-1]), L-745,337 (0.2-2 mg kg[-1]) and DFU (0.02-0.2 mg kg[-1]) inhibited the protective effect of 20% ethanol in a dose-dependent manner with ID50 values of 0.3 mg kg(-1), 0.4 mg kg(-1) and 0.06 mg kg(-1), respectively. 5. Inhibition of mild irritant-induced protection was also found when NS-398 (1 mg kg[-1]) was administered s.c. or when 96% ethanol was used to damage the mucosa. 6. Pretreatment with 16,16-dimethyl-prostaglandin (PG)E2 at 4 ng kg(-1), a dose that did not protect against ethanol (70%)-induced mucosal damage when given alone, completely reversed the effect of the selective COX-2 inhibitors on the mild irritant-induced protection. 7. Pretreatment with dexamethasone (3 mg kg(-1), 24 and 2 h before instillation of 20% ethanol) did not affect the protective activity of the mild irritant, indicating that enzyme induction is not involved. 8. Indomethacin (20 mg kg(-1), p.o.) did not prevent the protection conferred by sodium salicylate (100 mg kg[-1]), dimercaprol (30 microg kg[-1]), iodoacetamide (50 mg kg[-1]) and lithium (20 mg kg[-1]). Likewise, the protective effect of these agents was not counteracted by NS-398 (1 mg kg(-1), p.o.). 9. Whereas indomethacin (20 mg kg(-1), p.o.) near-maximally inhibited gastric mucosal formation of PGE2, 6-keto-PGF1alpha and thromboxane (TX) B2 as well as platelet TXB2 release, the selective COX-2 inhibitors were ineffective. 10. The findings show that selective COX-2 inhibitors, although lacking in ulcerogenic activity, prevent the protection conferred by a mild irritant. Prostaglandis generated by a constitutive COX-2 could thus contribute to physiological functions involved in gastric homeostasis, although at present a non-COX-2-related mechanism underlying the effect of the selective COX-2 inhibitors tested on mild irritant-induced protection cannot be completely excluded.

We have evaluated the biochemical selectivity of novel cyclo-oxygenase (COX)-2 inhibitors, etoricoxib, valdecoxib, DFU and DFP, vs rofecoxib and celecoxib, using the human whole blood assays of COX-isozyme activity, in vitro. Compounds were incubated with human whole blood samples, allowed to clot for 1 h at 37 degrees C, or stimulated with lipopolysaccharide (10 microg/ml) for 24 h at 37 degrees C. Serum thromboxane (TX) B2 and plasma prostaglandin (PG) E2 levels were measured by specific radioimmunoassays as indices of platelet COX-1 and monocyte COX-2 activity, respectively. Valdecoxib, etoricoxib, DFU and DFP inhibited platelet COX-1 and monocyte COX-2 with the following COX-1/COX-2 IC50 ratios: 61.5, 344, 660 and 1918, respectively. The reference compounds, celecoxib and rofecoxib had corresponding values of 29.6 and 272. In conclusion, a second wave of COX-2 inhibitors with higher biochemical selectivity than the existing coxibs has been developed. Whether their administration will be associated with improved clinical efficacy and/or safety vis-à-vis celecoxib and rofecoxib remains to be established.

Inflammatory chemical mediators, platelet-activating factor (PAF), thromboxane (TX) B2, and 6-keto-prostaglandin (PG)F1 alpha, were extracted from lung lavage fluid after conventional mechanical ventilation (CMV) and high frequency oscillatory ventilation (HFOV) to clarify the relation between mode of ventilation and lung injury in surfactant-depleted rabbit lungs. Anesthetized adult rabbits were tracheostomized, and surfactant depletion was induced by repeated saline lavage. Lung lavage for measurement of mediators was performed after 4 h of CMV at an FIO2 of 1.0 and a mean airway pressure of 15 cm H2O or HFOV (15 Hz) at an FIO2 of 1.0 or 0.21 and a mean airway pressure of 15 cm H2O. The number of total cells and polymorphonuclear leukocytes (PMN) and the levels of PAF, TXB2, and 6-keto-PGF1 alpha were measured by radioimmunoassay. Total respiratory compliance (Crs) was measured by the passive flow-volume curve method. The numbers of PMN, and the levels of PAF and TXB2 in lung lavage fluid were significantly greater during CMV than during HFOV. HFOV resulted in decreased production of PAF and TXB2 in a surfactant-depleted rabbit lung. Crs was significantly less during CMV than during HFOV. These results suggest that HFOV could prevent the release of such inflammatory chemical mediators and result in less lung injury than CMV.