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Background: The protective effect of acetylsalicylic acid (aspirin) against cardiovascular events is known to be weaker in women than in men. The present study was designed to test whether this effect of aspirin differed between sexes in an experimental model of diabetes with retinal ischemia., Methods: We compared nondiabetic rats and rats after 1, 2 and 3 months of diabetes that were given 2 mg/kg/day p.o. of aspirin from the first day of diabetes. The variables recorded were platelet aggregation, production of thromboxane B(2) (TxB(2)), 6-keto-prostaglandin F(1alpha) and aortic nitric oxide, and the percentage of the retinal surface occupied by horseradish peroxidase (HRP)-permeable vessels., Results: In female rats made diabetic, TxB(2) synthesis was more markedly reduced, and the percentage of HRP-permeable retinal vessels was less markedly reduced, than in their male counterparts. The response to aspirin treatment was weaker in female than in male diabetic rats in terms of inhibition of TxB(2) synthesis, increased nitric oxide production, and prevention of the increase in the percentage of retinal surface covered by HRP-permeable vessels., Conclusion: Aspirin was less effective in preventing retinal ischemia in experimental diabetes in female than in male rats.

The aim of this study was to characterize the influence of the interaction between acetylsalicylic acid (ASA) and salicylic acid (SA) on the inhibition by ASA of platelet aggregation in platelets isolated from whole blood, and to determine whether leukocytes influence this pharmacological interaction. This in vitro study was done in human blood from which we prepared samples of whole blood, platelet-rich plasma (PRP), PRP plus mononuclear leukocytes, and PRP plus neutrophils. The variables recorded were maximum platelet aggregation intensity, thromboxane B2 (TxB2) production, and nitric oxide (NO) production (N=10 different samples in each type of experiment). Different concentrations of ASA and SA were incubated with all samples. In PRP, the concentration of ASA that inhibited maximum aggregation by 50% (IC50) (281+/-16microM) increased with increasing SA concentration to a maximum of more than 2mM when 500microM SA was used. In whole blood, the IC50 for ASA (24.9+/-1.2microM) decreased with decreasing SA concentrations to 7.9+/-0.8microM with 50microM SA and 15.6+/-0.9microM with 125microM SA, and increased to 46.2+/-2.6microM with 250microM SA and 96.3+/-7.2microM with 500microM SA. In experiments with PRP+neutrophils the IC50 of ASA increased in the presence of all concentrations of SA. The antagonistic interactions were also reflected in the changes in TxB2 production in all samples. In samples of neutrophils incubated with ASA, the curve for NO production was shifted to the right, a finding that paralleled the changes in platelet aggregation. In conclusion, the influence of the interaction between ASA and its metabolite SA on platelet aggregation difference depending on the type of sample, and was antagonistic in PRP but partially agonistic in whole blood. Nitric oxide synthesis showed an additive effect of the two compounds.

We analyze the effect of the combination of acetylsalicylic acid (2 mg/kg/day p.o.) and alpha-tocopherol (25 mg/kg/day p.o.) in a type-1-like experimental model of diabetes mellitus on platelet factors, endothelial antithrombotic factors and tissue oxidative stress. In diabetic rats, the combination of drugs had a greater inhibitory effect on platelet aggregation than in untreated control animals with diabetes (88.87%). The combination of drugs had little effect on the inhibition of thromboxane production (-90.81%) in comparison to acetylsalicylic acid alone (-84.66%), potentiated prostacyclin production (+162%) in comparison to alpha-tocopherol alone (+30.55%), and potentiated nitric oxide production (+241%) in comparison to either drug alone (acetylsalicylic acid +125%, alpha-tocopherol +142%). The combination of the two drugs improved the thromboxane/prostacyclin balance (0.145+/-0.009) in comparison to untreated diabetic animals (4.221+/-0.264) and in untreated healthy animals (0.651+/-0.045). It did not potentiate the antioxidant effect of either drug alone, but did increase tissue concentrations of reduced glutathione, especially in vascular tissue (+90.09% in comparison to untreated animals). In conclusion, in the experimental model of diabetes tested here, the combination of acetylsalicylic acid and alpha-tocopherol led to beneficial changes that can help protect tissues from thrombotic and ischemic phenomena.

Diabetes mellitus is a risk factor for cerebrovascular ischemic disease. Aspirin (acetylsalicylic acid) is the most widely used drug for the secondary prevention of thrombotic phenomena. It has been also recently demonstrated that alpha-tocopherol influenced in vitro the antiplatelet effect of aspirin. The aim of the present study is to evaluate the effects aspirin plus alpha-tocopherol on cerebral oxidative stress, prostaglandin production and the nitric oxide pathway in a model of hypoxia-reoxygenation in rat brain slices. Our results show an imbalance in brain oxidative status (reflected mainly as the increase in lipid peroxides) as a result of diabetes itself rather than a failure of the glutathione-based antioxidant system. Moreover, our results also show a higher concentration of prostaglandins in the brain of diabetic animals and a higher nitric oxide concentration, mainly through a high iNOS activity. After 180 min of post-hypoxia reoxygenation, LDH activity was 40.6% higher in animals with diabetes, in comparison to non-diabetic animals. The increase of the LDH efflux observed in non-treated rats was reduced by 31.2% with aspirin, by 34.7% with alpha-tocopherol and by 69.8% with the association aspirin-alpha-tocopherol. The accumulation of prostaglandin E2 observed in diabetic non-treated rats was reduced statistically after the treatment with aspirin (34.2% inhibition), alpha-tocopherol (19.3% inhibition) or the association aspirin-alpha-tocopherol (54.4% inhibition). Nitric oxide production after 180 min reoxygenation was significantly reduced in aspirin (36.4%), alpha-tocopherol (22.7%) and aspirin-alpha-tocopherol (77.8%) treated rats with respect to diabetic non-treated animals; this was related mainly with a reduction in iNOS activity. The association between aspirin and alpha tocopherol could protects against brain ischemic-reperfusion damage with a better profile than aspirin alone.

We analysed the in vitro interaction between acetylsalicylic acid and vitamin E on the principal antiplatelet sites of action of acetylsalicylic acid, i.e., platelet aggregation, prostanoid production in platelets and leukocytes, and nitric oxide synthesis. Aggregation was measured in whole blood and in platelet-rich plasma (PRP) with ADP, collagen or arachidonic acid as platelet inducers, and we measured the production of thromboxane B2, prostacyclin and nitric oxide. Vitamin E potentiated the antiplatelet effect of acetylsalicylic acid in both whole blood and PRP. In PRP induced with collagen the IC50 for acetylsalicylic acid alone was 339+/-11.26, and that of acetylsalicylic acid+vitamin E was 0.89+/-0.09 (P<0.05). Vitamin E did not enhance inhibition of platelet thromboxane production by acetylsalicylic acid. Vitamin E spared or even increased prostacyclin levels, and acetylsalicylic acid+vitamin E diminished the inhibition of prostacyclin synthesis by acetylsalicylic acid (IC50 acetylsalicylic acid alone=1.81+/-0.15 microM; IC50 acetylsalicylic acid+vitamin E= 12.92+/-1.10 microM, P<0.05). Vitamin E increased the effect of acetylsalicylic acid on neutrophil nitric oxide production 42-fold (P<0.05). We conclude that vitamin E potentiates the antiplatelet effect of acetylsalicylic acid in vitro, and thus merits further research in ex vivo studies.

Triflusal is a fluorinated derivative of acetylsalicylic acid (ASA) with demonstrated antithrombotic activity. Recently, evidence for a neuroprotective effect has been obtained. The aim of this study was to compare the neuroprotective effects of the main metabolite of triflusal (2-hydroxy-4-trifluoromethylbenzoic acid, HTB) and the ASA metabolite salicylic acid (SA) in an in vitro model of anoxia-reoxygenation in rat brain slices. Rat brain slices (n=10 per group) were subjected to a period of anoxia followed by 180 min reoxygenation. We measured oxidative stress parameters (lipid peroxidation, glutathione system), prostaglandins (PGE(2)), nitric oxide pathway activity (NO) (nitrites+nitrates, constitutive and inducible NO synthase activity) and LDH efflux, a biochemical marker of cell death. Various concentrations (10, 100 and 1,000 microM) of triflusal, HTB, ASA or SA were tested. Triflusal at 10, 100 and 1,000 microM decreased LDH efflux in rat brain slices after anoxia/reoxygenation by 24%, 35% and 49% respectively. This effect was proportionately greater than that of ASA (0%, 13% and 32%). The results with HTB were similar to those with triflusal, whereas SA showed a greater protective effect than ASA (13%, 33% and 35%). The antioxidant effects of HTB and SA on the biochemical mechanisms of cell damage studied here were also greater than the effects of triflusal and ASA, a finding attributable mainly to the decrease in lipid peroxidation and to the ability of HTB to also increase glutathione levels. The triflusal metabolite reduced inducible NO synthase activity by 18%, 21% and 30%, whereas SA inhibited this activity by 9%, 17% and 23%. Triflusal and HTB led to greater increases in NO synthase than ASA or AS. In conclusion, the metabolite HTB plays an important role in the neuroprotective effect of triflusal, at least in the experimental model of anoxia-reoxygenation tested here.

The aim of the present study was to analyze the relative participation of the antiplatelet and the antioxidant effects of acetylsalicylic acid (ASA) and salicylic acid (SA) after a single dose (1 or 10 mg/kg i.p.) in an in vitro model of anoxia in slices of rat brain. After 20 min of drug administration, blood and brain were obtained (n=6 rats per group). We measured: lipid peroxidation, glutathione levels and lactate dehydrogenase efflux (LDH), ASA and SA concentrations and platelet aggregation in whole blood. An increase in lipid peroxidation (80%) and in LDH efflux (520%) and a decrease in glutathione levels (35%) were observed after 120 min anoxia in saline-treated rats. SA reduced this oxidative stress and LDH efflux, but it did not modify platelet aggregation. ASA strongly inhibited platelet aggregation but exerted a poor antioxidant effect. ASA was not detectable in brain tissue. We conclude that repeated doses of ASA are necessary to obtain a tissular antioxidant effect, probably when liver generates enough SA.

Acetylsalicylic acid (ASA) reduces the incidence of ischemic stroke mainly through its antithrombotic action; however, it also has a direct neuroprotective effect. The present study was designed to evaluate the effect of ASA on oxidative stress and the activity of nitric oxide synthase (NOS) in an in vitro model of hypoxia in rat brain slices. Rat brain slices were perfused with nitrogen (hypoxia) for a maximum of 120 min, after which we measured lipid peroxidation, glutathione levels, glutathione-related enzyme activities, and constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activities. In brain tissue subjected to hypoxia, ASA reduced oxidative stress and iNOS activity (all increased by hypoxia), but only when used at higher concentrations. The effects of salicylic acid (SA) were similar but more intense than were those of ASA. After oral administration, the effect of SA was much greater than that of ASA, and the decrease in cell death with SA was seen much more clearly. In view of the greater effect of SA compared to ASA on changes in oxidative stress parameters in a model of hypoxia, and higher brain concentrations of SA when it is administered alone than when ASA is given (undetectable levels), we conclude that SA plays an important role in the cytoprotective effect in brain tissue after ASA administration., (Copyright 2003 Wiley-Liss, Inc.)

This study was designed to evaluate the effects of extended-release aspirin on platelet aggregation and the production of prostanoids and nitric oxide. The participants in this double blind, randomized and crossover study were 20 healthy volunteers. Interventions were 150 mg of plain-formulated aspirin (PFASA) and 150 mg of extended-release aspirin (ERASA). Blood samples were collected before and 10, 20, 60, 120, 240, 480 and 1440 min after the first dose; 3, 7 and 14 days after daily administration and 24 h after the last dose. The main measures were platelet aggregometry, thromboxane B2, 6-keto-prostaglandin (PG) F1alpha and nitric oxide in each control. Platelet aggregation was inhibited by 50% with ERASA, and by 77% with PFASA. No differences were found in chronic treatment. Thromboxane B2 was inhibited more by the latter (51-67%), but 90% inhibition was observed in both groups after 3 days. The levels of 6-keto-PGF1alpha was reduced by 20% with ERASA and by 58% with PFASA. Nitric oxide production increased in both groups, but after 24 h, and 7-14 days, elevated concentrations of nitric oxide were found only in the ERASA. The antiplatelet effects of ERASA provide pharmacological advantages (greater prostacyclin synthesis and prolonged increase in nitric oxide production) over those provided by the plain formulation.

Objective: The lowest dose of aspirin shown to be effective in the secondary prevention of thrombotic accidents is 75 mg/day. Presystemic acetylation of cyclooxygenase and the formation of salicylic acid in the liver are fundamental to ensure optimum antithrombotic effects of aspirin. This study was designed to compare the effects of two forms of extended-release aspirin (at 75 mg/day) on prostanoid and nitric oxide synthesis in healthy volunteers., Methods: The participants in this single-blind cross-over study (n = 6) were randomly assigned to receive one of three different formulations: plain-formulated aspirin (PF), extended-release aspirin that released acetylsalicylic acid steadily over 5 h (EX5) or an extended-release formulation that released 49% of the drug during the first 2 h after intake (EX2) and the rest of the dose during the subsequent 5 h. Laboratory analyses were done for platelet aggregation and thromboxane B2 (in whole blood), 6-keto-prostaglandin F1alpha (in leucocytes), neutrophil nitric oxide production and plasma nitrite/ nitrate levels., Results: The PF and EX2 formulations inhibited platelet aggregation by 97% with no significant difference in effect between the two. In contrast, maximum inhibition of aggregation by the EX5 formulation was only 30%. Similar effects were found for platelet thromboxane production: PF and EX2 led to 99% inhibition, whereas EX5 led to 76% inhibition (P < 0.05). The inhibition of prostacyclin production differed in all three treatments (63% for PF, 40% for EX2 and 24% for EX5). The increase in leucocyte nitric oxide production also differed in all three treatments (1.01-fold the basal value with PF, 1.4-fold with EX5 and 3.6-fold with EX2). Both extended-release formulations maintained high levels of nitric oxide production 24 h after the last dose, whereas in the PF period nitric oxide concentration had returned to basal values after this time. The changes in plasma nitrite concentrations in each period of treatment were similar to those seen for leucocyte nitric oxide., Conclusion: The pharmacodynamic profile of the extended-release formulations was better than that of plain-formulated aspirin in terms of thromboxane/prostacyclin balance and nitric oxide production. However, the EX2 formulation inhibited platelet function more effectively than did the EX5 formulation.

This study was designed to determine the influence of the combination of aspirin and dipyridamole on the interaction in vitro between neutrophils and platelets through the nitric oxide (NO) pathway. Collagen-induced platelet aggregation (impedance method) was determined in platelet-rich plasma and in platelet-rich plasma+neutrophils, and cGMP (enzyme immunoanassay) and NO levels (electrochemical method, with a ISO-200 electrode) were also measured. The 50% inhibitory concentration (IC(50)) of aspirin was 139+/-11 microM in platelet-rich plasma, 367+/-21 microM in platelet-rich plasma+L-N(G)-nitro-arginine-methyl-ester (L-NAME), and 42+/-3 microM in platelet-rich plasma+L-arginine. The IC(50) for dipyridamole in platelet-rich plasma was not affected by L-NAME or L-arginine; the combination of aspirin with 20 microM dipyridamole (which has no effect per se) led to an IC(50) of 51+/-2 microM in platelet-rich plasma, 101+/-7 microM in platelet-rich plasma+L-NAME, and 13+/-2 microM in platelet-rich plasma+L-arginine. The cGMP levels showed the greatest increases in the aspirin plus dipyridamole group. Dipyridamole and aspirin increased the leukocyte production of NO: 50% increases were obtained at concentrations of 285+/-31 microM aspirin, 110+/-9 microM dipyridamole, and 16+/-2 microM aspirin+dipyridamole. Dipyridamole alone at a concentration of 20 microM had no significant effect on NO levels. We conclude that the combination of aspirin and dipyridamole significantly increases the antiplatelet effect of leukocytes, through an increase of NO, and that this effect is further evidence of the therapeutic benefits of this combination of drugs.

Platelet hyperactivity has been one of the mechanisms implicated in the pathogenesis of diabetic retinopathy. Antiplatelet agents have been shown, in experimental models, to prevent the development of retinal vascular abnormalities when given from the first day after the onset of diabetes. We assessed the effect of aspirin plus dipyridamole (6 + 12 mg/kg daily) on the retinal vascular pattern in experimental streptozotocin-induced diabetes in rats, when the treatment was given at different intervals after the induction of diabetes, over a 3-month study period. Saline-pretreated diabetic rats showed a time-dependent increases in the platelet production of thromboxane B2 (r = 0.981, P < .0001) and a decrease in the aortic production of 6-keto-PGF1 alpha. The percentage of retinal area occupied by horseradish peroxidase-labeled vessels decreased progressively in relation to the length of time of the evolution of diabetes (r = 0.983, P < .00001) and the thromboxane/prostacyclin ratio. Treatment with aspirin plus dipyridamole caused an inhibition of the platelet production of thromboxane B2 and a decrease in the vascular synthesis of prostacyclin. Treatment with antiplatelet agents slowed down the decrease in the percentage of retinal area occupied by horseradish peroxidase-labeled vessels. These data provide further evidence to support the results of previous clinical trials in which antiplatelet agents had a beneficial effect on the evolution of retinal lesions in early diabetic retinopathy.

1. Triflusal is a salicylic derivative that inhibits platelet aggregation in human whole blood with a minimal inhibition of prostacyclin production. 2. Aspirin inhibits platelet aggregation at concentrations that reduce vascular prostacyclin production.

The antiproteinuric effect of the antiplatelet agent dipyridamole has been assessed after inhibition of thromboxane B2 (TxB2) synthesis in 8 patients with confirmed membranous glomerulonephritis. There were three study periods, each of 30 days, and 45 days apart, namely a washout period, treatment with dipyridamole 300 mg/d, and dipyridamole 225 mg/d plus aspirin 150 mg/d. On Days 1 and 30 of each study period serum and urine creatinine, 24-h excretion of protein, creatinine clearance, platelet aggregometry on whole blood and serum TxB2 were measured. Treatment with dipyridamole alone or with aspirin produced significant inhibition of platelet aggregation and a fall in 24-h protein excretion; the latter amounted to 54% with dipyridamole alone and 56% with dipyridamole plus aspirin (NS). Dipyridamole plus aspirin caused an 82% reduction in serum TxB2.

1. Some of the antiplatelet effects of the dipyridamole-aspirin association may be explained by a functional (additive) synergism, but other effects by a potentiation synergism. 2. Platelet adhesivity and aggregation induced by ADP, adrenaline and collagen, and malondialdehyde production, are tests in which dipyridamole and aspirin shows a potentiation synergism.

We performed a study to evaluate the effect of acetylsalicylic acid (ASA) plus dipyridamole on the retinal vascular pattern over 3 months in rats with experimental diabetes mellitus. Rats treated with ASA alone showed a continuous vascular bed and less tortuous vessels than untreated diabetic rats. Rats treated with ASA plus dipyridamole showed a continuous vascular bed, scarce tortuous vessels and vascular diameters similar to those in nondiabetic control rats. The findings were related to aortic prostacyclin production: treatment with ASA alone produced a reduction in prostacyclin production, whereas treatment with ASA plus dipyridamole resulted in normal prostacyclin production. ASA alone or with dipyridamole inhibited collagen-induced aggregation in whole blood by 57% compared with the untreated diabetic rats.

We have used the impedance aggregometer to study the "ex vivo" effect of acetylsalicylic acid (ASA) in whole blood (WB) versus platelet-rich plasma (PRP) in 35 male healthy volunteers after 10 days of treatment with 25, 50, 125, 250, and 500 mg/day of ASA. Percent of inhibition of platelet aggregation was determinated at the end of treatment. A greater inhibition of platelet aggregation was observed in WB than in PRP when ASA was administrated at almost all doses. Maximal differences were at 25, 50, and 125 mg/day of ASA on adrenaline, collagen and arachidonic acid induced aggregation, and with 250 and 500 mg/day of ASA when ADP was used as aggregating agent. In the "in vitro" trials, IC-50 values of ASA on ADP and collagen induced aggregation were determined in platelet aggregation by the impedance method in both WB and PRP. ASA shows a lower IC-50 in WB than in PRP. When leucocytes were incubated in PRP samples, it effect was similar to the percent of inhibition in WB.

A study has been made on the in vitro effect of triflusal, acetylsalicylic acid (ASA and their major metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB), and salicylic acid (SA), on platelet aggregation in human whole blood. SA exhibited no significant antiplatelet effects (IC50 greater than 2mM) against several inducers; the IC50 values for the other compounds were: triflusal, 140 microM against ADP and 63.2 microM against collagen; HTB, 100 microM against ADP and 260 microM against collagen; ASA 687 microM against ADP and 9.3 microM against collagen. Red blood cells potentiate the antiaggregant effect of HTB and of triflusal, and to a lesser extent, that of ASA; leukocytes primarily potentiate the effect of ASA and, to a lesser extent, that of triflusal.

We have used the impedance aggregometer to study the in vitro effect of acetylsalicylic acid (ASA) in whole blood (WB) versus platelet-rich plasma (PRP) using blood samples from 24 male and 24 female healthy volunteers. IC50 was calculated from dose-response curves of ADP-, adrenaline-, collagen- and arachidonic acid-induced aggregation. ASA inhibited platelet aggregation in WB with a lower IC50 than PRP in male and female samples; the greater differences between WB and PRP inhibitory effect of ASA were in collagen- and archidonic acid-induced aggregation. A higher ASA concentration was needed in order to produce half maximal inhibition of platelet aggregation in female than in male samples with both WB and PRP method, except when ADP was used as the aggregating agent in PRP.

A study was made on the inhibitory effect of triflusal (600 mg/d X 15) and acetylsalicylic acid (ASA, 400 mg/d X 15) on platelet aggregation in whole blood (WB) and platelet-rich plasma (PRP) induced by ADP (2.5 mumol/l), adrenaline (50 mumol/l), collagen (1 microgram/ml) and arachidonic acid (0.8 mmol/l), in 30 insulin-dependent diabetic patients without vascular complications. Determination was also made of the serum levels of thromboxane B2 (TxB2) and of the plasma levels of 6-keto-PGF1-alpha and of beta-thromboglobulin (B-TG). Both drugs exhibited higher inhibitory effects in WB than in PRP. In WB, a significant difference between triflusal and ASA was observed against ADP-induced aggregation (67% and 46% inhibition respectively, p less than 0.01). Both drugs strongly inhibit the formation of TxB2 in serum (85% and 99%, respectively). Triflusal does not significantly change the plasma levels of 6-keto-PGF1-alpha; ASA, by contrast, causes reduction of over 95% in those plasma levels. The plasma levels of B-TG were not modified by either of the drugs.

The importance in experimental diabetic retinopathy of prostacyclin and nitric oxide (NO), as well as the possible effect of acetylsalicylic acid (ASA), are well known. To investigate the effect of two doses of aspirin in the prevention of retinal ischemia in streptozotocin-diabetic rats, we compared nondiabetic rats and diabetic rats after 1, 2 and 3 months of diabetes, and diabetic rats treated with 2 mg or 10 mg ASA/kg per day p.o. from the first day of diabetes. The parameters determined after 1, 2 and 3 months of development were platelet aggregation, thromboxane B2 (TxB2) production, 6-keto-prostaglandin F1α (stable metabolite of prostacyclin), NO, plasma nitrites/nitrates, and percentage retinal surface occupied by horseradish peroxidase (HRP)-permeable vessels. In diabetic rats platelet aggregation and thromboxane concentration were increased, and prostacyclin, NO and area occupied by HRP-permeable vessels were decreased. Acetylsalicylic acid reduced platelet aggregation, and lowered thromboxane production by 82%–99%. Prostacyclin production was inhibited by 92%–95% with 10 mg ASA/kg per day, and by 8%–20% with 2 mg ASA/kg per day. In diabetic rats NO production increased after 2 and 3 months of treatment to levels seen in nondiabetic rats. The reduction in HRP-permeable retinal surface decreased from a maximum of 87% in DR to 51% after treatment with 2 mg ASA/kg per day, and to 62% after 10 mg ASA/kg per day. We conclude that ASA (2 mg/kg per day and 10 mg/kg per day) increased NO production in streptozotocin-diabetic rats and reduced the degree of retinal ischemia. [ABSTRACT FROM AUTHOR]