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This study was designed to evaluate the mechanism by which propofol modifies leukocyte production of nitric oxide (NO) in humans. In vitro experiments used whole blood from healthy volunteers (n = 10 samples/experiment). Ex vivo experiments studied the effects of an intravenous dose of 2.5 mg propofol per kilogram body weight followed by intravenous infusion of 4 mg kg(-1) h(-1) in surgical patients in ASA class I or II (n = 20). In whole blood, neutrophils and plasma, we measured NO production and the activities of the enzymes nitric oxide synthase [inducible (iNOS) and constitutive (cNOS)] and cyclooxygenase [constitutive (COX-1) and inducible (COX-2)]. Concentrations of interleukins (IL-1beta, IL-6, and IL-10) and tumor necrosis factor-alpha (TNFalpha) were measured in plasma. In blood from healthy donors, propofol increased NO production and cNOS activity. The concentration of propofol that increased NO production by 50% (EC(50)) was 23.5 microM, and the EC(50) of propofol for cNOS was 18.6 microM. In blood from surgical patients, propofol increased NO production by 52% and cNOS activity by 57%. Propofol inhibited iNOS activity in vitro; the concentration that reduced activity by 50% (IC(50)) was 19.9 microM. In surgical patients propofol inhibited iNOS activity by 53%. COX-1 and COX-2 activities were inhibited in vitro (IC(50) 32.6 and 187 microM, respectively) and in surgical patients (53 and 81% inhibition, respectively). Plasma concentrations of IL-1beta, IL-6, and TNFalpha were significantly reduced in surgical patients (32, 23, and 21% inhibition, respectively). None of these parameters were modified in a group of patients (n = 10) anesthetized with sevoflurane. We conclude that propofol stimulated constitutive NO production and inhibited inducible NO production, possibly by curtailing the stimulation of iNOS by inflammatory mediators in surgical patients.

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.

The aim of this study was to evaluate the influence of leukocyte nitric oxide (NO) production on the antiplatelet aggregant effect of aspirin and ticlopidine. This in vitro study was done with platelets (platelet-rich plasma, PRP) and polymorphonuclear leukocytes (PMNLs) separated from samples of human blood. Collagen-induced platelet aggregation and calcium-dependent NO production by PMNL were quantified. The inhibition of NO production in PRP significantly reduced the antiaggregant affect of aspirin (IC50 2.64-fold greater), whereas it had no significant effect on the effect of ticlopidine (IC50 1.03-fold greater). Incubating PMNL in PRP increased the antiaggregant effect of both aspirin (IC50 5.09-fold lower) and ticlopidine (IC50 10.16-fold lower). The inhibition of NO production in PMNL significantly reduced the antiaggregant effect of both aspirin (IC50 2.21-fold greater) and ticlopidine (IC50 3.26-fold greater). Both drugs increased leukocyte NO production. The concentration of aspirin that raised NO production by 50% was greater than 1000 microM, whereas the concentration of ticlopidine that led to this effect was 9.14 +/- 0.87 microM. We conclude that the effect of ticlopidine on leukocyte NO production may constitute an addition mechanism to the IIb/IIIa glycoprotein complex inactivation in the inhibition of platelet activation.

The aim of this study was to investigate two factors of endothelial dysfunction and their platelet second messengers in patients with type II diabetes and different types of retinopathy. We compared 20 healthy volunteers and 117 patients with type II diabetes (34 with no signs of diabetic retinopathy, 26 with background diabetic retinopathy, 29 with ischemic-proliferative diabetic retinopathy and 28 with edematous diabetic retinopathy). The following parameters were recorded: platelet aggregometry, nitrites, 6-keto-prostaglandin-F(1alpha) and intraplatelet cAMP and cGMP. Platelet aggregation was greater in patients with diabetic retinopathy. The concentration of ADP that produced 50% maximum intensity of aggregation was 1.81 microM in patients without diabetic retinopathy, 0.92 microM in patients with background diabetic retinopathy, 0.85 microM in patients with ischemic-proliferative diabetic retinopathy and 0.44 microM in patients with edematous diabetic retinopathy. The platelets in these patients were more resistant to inhibition by SIN-1 (concentrations of SIN-1 that produced 50% inhibition of maximum intensity of collagen-induced aggregation in the four patient groups: 18.1, 13.6, 16.2 and 33.2 microM, respectively). Nitrite concentration in patients with ischemic-proliferative diabetic retinopathy was one sixth of the value in healthy controls, but there was no significant difference between the control group and patients with edematous diabetic retinopathy. In the latter group, neutrophils increased nitrite production by 68.7 +/- 3%, whereas in patients with ischemic-proliferative diabetic retinopathy, this increase was 18.7 +/- 2.0%. We conclude that nitric oxide production is higher in patients with type II diabetes and edematous retinopathy than in those with ischemic-proliferative retinopathy. This finding, together with the possibly greater production of free radicals, may explain the greater impairment of platelet function in the former patients., (Copyright 2002 S. Karger AG, Basel)

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.

The aim of this study was investigate prostacyclin and nitric oxide (NO) and their platelet second messengers cAMP and cGMP, in patients with type I diabetes with or without retinopathy. We compared 20 healthy volunteers and 97 patients with type I diabetes: 24 with no signs of diabetic retinopathy (DR), 43 with ischemicproliferative DR, and 30 with edematous DR. The following parameters were recorded: platelet aggregometry, nitrites/nitrates, 6-keto-prostaglandin-F(1alpha), and intraplatelet cAMP and cGMP. Platelet aggregation was greater in patients with edematous DR. The platelets in patients with diabetes were more resistant to inhibition by prostaglandin E(1) or sodium nitropruside. Nitrite concentration in patients with ischemic-proliferative DR was 80% lower than the value in healthy controls, but there was no significant difference between the control group and patients with edematous DR. In the latter group, stimulation of neutrophils with L-arginine increased nitrite + nitrate production by 44 +/- 3.6%, whereas in patients with ischemic-proliferative DR, the increase was 9.8 +/- 0.8%. We conclude that NO production is higher in patients with type I diabetes and edematous retinopathy than in those with ischemic-proliferative retinopathy. This finding, together with the possibly greater production of free radicals, may explain the greater impairment of platelet function in the former patients.

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.

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?µM; IC50 acetylsalicylic acid?+?vitamin E?= 12.92?±?1.10?µM, 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. [ABSTRACT FROM AUTHOR]

Ticlopidine, a thienopyridine that prevents the progression of diabetic retinopathy in humans, was recently shown to increase nitric oxide (NO) production in human neutrophils. The thienopyridine clopidogrel has been found to be clinically useful in the secondary prevention of thrombotic events. The aim of the present study was to evaluate the effect of clopidogrel on ischemic retinopathy in streptozotocin-diabetic rats and its influence on prostanoids and NO production. We compared nondiabetic rats and rats after 3 months of diabetes that were given three doses (1, 10 or 20 mg/kg per day p.o.) of ticlopidine or clopidogrel from the first day of diabetes. The variables recorded after 3 months of diabetes were platelet aggregation, thromboxane B2 (TxB2) production, 6-keto-prostaglandin F1α (stable metabolite of prostacyclin), aortic NO, plasma nitrites/nitrates, and the percentage of the 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. Ticlopidine and clopidogrel reduced the maximum extent of platelet aggregation in a dose-dependent manner: maximal inhibition with respect to untreated diabetic rats was 48.6% with ticlopidine and 66.6% with clopidogrel. Ticlopidine reduced thromboxane B2 only at a dose of 20 mg/kg per day p.o. (47.4% inhibition) and clopidogrel at doses of 10 mg/kg per day (51% inhibition) or 20 mg/kg per day (51.7% inhibition). Aortic prostacyclin production did not change after treatment with either thienopyridine. Treatment with ticlopidine reduced the inhibition of NO production in untreated rats (89.6% inhibition) to 0.9%, and clopidogrel reduced inhibition to 30%. Treatment with ticlopidine or clopidogrel reduced the retinal nonperfused area from 86.8% inhibition in untreated rats to 45.6% and 25.3%, respectively. In conclusion, the early administration of thienopyridines in streptozotocin-diabetic rats partly prevented the appearance of diabetic retinal ischemia. [ABSTRACT FROM AUTHOR]

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]

Objective.To study prospectively whether serum and urinary nitrate levels are related to lupus activity. [ABSTRACT FROM PUBLISHER]