Aspirin Pretreatment Research Articles

Hydrogen Peroxide as Trigger of Platelet Aggregation

In order to verify if H2O2 affects platelet function, platelet-rich plasma and human washed platelets were incubated with subthreshold concentrations (STC) of collagen or arachidonic acid or ADP and/or with 75-150 microM H2O2. While H2O2 alone did not affect platelet aggregation, it amplified platelet aggregation response in samples stimulated with STC of arachidonic acid and collagen but not in samples stimulated with STC of ADP. When platelets were preventively treated with aspirin, a cyclooxygenase inhibitor, the platelet activation by H2O2 was not observed. Thromboxane A2 (TxA2) was not produced by human washed platelets stimulated with STC of arachidonic acid, collagen or by H2O2 alone. On the contrary, when STC of agonists were tested on platelets supplemented with H2O2 an evident TxA2 production was seen. This effect was prevented by aspirin pretreatment or by the addition of catalase, an enzyme which destroys H2O2. This study suggests that H2O2 triggers the activation of platelets exposed to STC of collagen and arachidonic acid, via the cyclooxygenase pathway.

Cyclooxygenase inhibitors antagonize the rate-depressant effects of ethanol on fixed-ratio responding

Prostaglandin synthetase inhibitors (PGSIs), which inhibit actions of the enzyme complex cyclooxygenase, significantly antagonize the effects of alcohols but not other sedative-hypnotics across a broad range of measures. This suggests that certain actions of alcohols, in particular ethanol, are mediated at least partially through a prostaglandin-related pathway. This study examined changes in the rate-depressant effects of ethanol in an operant task following pretreatment with either of two PGSIs, indomethacin (INDO) or aspirin (ASP). Water reinforcement maintained the responding of male Sprague-Dawley (SD) rats under a Fixed-Ratio (FR) 32 schedule. Saline, INDO or ASP had no significant effect on control rates of responding. INDO pretreatment significantly antagonized the rate-depressant effects of 1.0 and 1.5 g/kg ethanol. ASP pretreatment also resulted in significant antagonism of ethanol's rate-decreasing effects. INDO was more potent than ASP in antagonizing the rate-depressant effects of ethanol. These results provide further support for the hypothesis that alcohols, by fluidizing neuronal membranes and mobilizing the release of arachidonate, serve to increase prostaglandin production, resulting in some of the behavioral and physiological sequellae of ethanol administration.

Regulation of Prostaglandin H Synthase mRNA Levels and Prostaglandin Biosynthesis by Platelet-derived Growth Factor

Stimulation of serum-starved NIH-3T3 cells with 20 ng/ml recombinant platelet-derived growth factor BB (rPDGF) results in the synthesis of prostaglandin E2 (PGE2) that is detectable within 10 min and which peaks after 2 h. Inhibition of translation with 36 microM cycloheximide inhibits rPDGF-stimulated PGE2 synthesis (greater than 90%), suggesting that de novo synthesis of prostaglandin H synthase (PGHS) is required for growth factor stimulation of PGE2 synthesis. Paradoxically, the addition of 10 microM exogenous arachidonate to the serum-starved cells resulted in 2-fold more PGE2 synthesis, and maximal synthesis occurred at 30 min compared to 2 h following rPDGF treatment. These data suggest that the serum-starved cells have ample biosynthetic capacity to support maximal rPDGF-stimulated PGE2 synthesis without de novo enzyme synthesis. Kinetic analysis of PGHS mRNA levels showed that although rPDGF did elevate the steady-state level of PGHS mRNA, the increase occurred after maximal PGE2 synthesis was achieved. Quantification of PGHS levels by immunoblot showed that there was no significant increase in enzyme levels following rPDGF stimulation even under conditions where PGHS mRNA levels were elevated. Irreversible inactivation of PGHS by treatment with aspirin and subsequent stimulation with rPDGF synchronized the maximal elevation of PGHS mRNA levels and PGE2 synthesis; both peaked at 3 h. Interestingly, aspirin pretreatment increased both the rate and amplitude of rPDGF-stimulated PGHS mRNA induction. The enhanced induction was not simply the result of a loss in PGE2 production since treatment with exogenous PGE2 also induced PGHS mRNA levels. Since rPDGF-stimulated PGE2 synthesis is blocked by both transcription and translation inhibitors but arachidonate-stimulated PGE2 synthesis is unaffected, the requirement for protein synthesis does not appear to involve de novo PGHS synthesis. The synthesis of a protein required for coupling of the rPDGF receptor to phospholipase activation and arachidonic acid mobilization is a more likely interpretation. In summary, our data suggest that rPDGF can induce PGHS mRNA levels, but that rPDGF-stimulated PGE2 synthesis is not dependent upon de novo PGHS synthesis.

Lipoxygenase metabolites mediate increased airways responsiveness to histamine after acute platelet activating factor exposure in the guinea-pig.

Platelet activating factor (Paf, 0.02 micrograms/kg, i.v. bolus) caused an acute increase in airways responsiveness to histamine in anaesthetized guinea-pigs prepared for recording airways resistance (RL) and dynamic compliance (Cdyn). Aspirin pretreatment (10 mg/kg, i.v.) attenuated the return of airways responsiveness to prechallenge levels. Pretreatment with the combined cyclooxygenase/lipoxygenase inhibitors BW 755C (20 mg/kg, i.v.) and ETYA (20 mg/kg, i.v.), or with the putative cysteinyl-containing leukotriene antagonist FPL 55712 (0.25 mg/kg/min, i.v.), or a Paf antagonist SRI 63441 (2.5 mg/kg, i.v.), prevented Paf-induced increased airways responsiveness. Inhibitors of leukotriene synthesis, BW 755C and ETYA, or action, FPL 55712, had variable effects on Paf-induced bronchoconstriction. These data suggest that lipoxygenase metabolites, possibly leukotrienes, may mediate an acute increase in airways responsiveness to histamine after Paf exposure.

111In-platelet and 125I-Fibrinogen deposition in the lungs in experimental acute pancreatitis

An experimental model of acute pancreatitis in rats has been used to study intrapulmonary 125I-fibrinogen and 111In-platelet deposition. Pancreatitis caused a significant increase in wet lung weight compared to normal, and this could be abolished by heparin or aspirin pretreatment. 125I-fibrinogen was deposited in the lungs of animals to a significantly greater degree than in controls ( P < 0.01). 125I-fibrinogen deposition was reduced to control levels by pretreatment with aspirin or heparin ( P < 0.05). The uptake of radiolabeled platelets was greater in pancreatitis than in controls ( P < 0.001). Pancreatitis appears to be responsible for platelet entrapment in the lungs. Platelet uptake was reduced by heparin treatment but unaffected by aspirin therapy.

Coronary vasoconstrictor response to cold pressor test in variant angina: Lack of relation to intracoronary thromboxane concentrations

To test the hypothesis that intracoronary concentrations of thromboxane (Tx)A 2 could influence the response to cold pressor test (CPT) in variant angina, great cardiac vein blood flow (by thermodilution) and the concentration of TxB 2 (the stable metabolite of TxA 2) in the great cardiac vein and aorta were measured under control conditions and during CPT in 14 patients with angina at rest associated with transient ST-segment elevation in the anterior leads. In seven patients pretreated with aspirin (intravenous administration of 3.6 mg/kg lysine salt of acetylsalicylic acid, corresponding to 2 mg/kg aspirin), TxB 2 baseline concentrations were lower in both the great cardiac vein (47 ± 19 vs 176 ± 88 pg/ml; p < 0.005) and the aorta (45 ± 16 vs 109 ± 56 pg/ml, p < 0.02) than in seven patients who were not taking cyclooxygenase inhibitors. In the two groups, great cardiac vein flow and anterior region coronary resistance were similar under control conditions. During CPT anterior region coronary resistance increased in patients pretreated with aspirin (from 1.97 ± 0.99 to 2.22 ± 1.11 mm Hg/ml/min; p < 0.02) and in patients without aspirin pretreatment (from 1.94 ± 0.43 to 2.06 ± 0.34 mm Hg/ml/min; p < 0.05), and the difference between the two groups was not statistically significant. Therefore the vasoconstrictor response of coronary vessels to CPT in variant angina is not influenced by the intracoronary TxB 2 concentrations and is not modified by aspirin pretreatment.

Induction of cyclo-oxygenase by interleukin-1 in rheumatoid synovial cells

The ability of interleukin-1 (IL-1) to stimulate prostaglandin E 2 (PGE 2 production by human rheumatoid adherent synovial cells was found to be time-dependent and sensitive to protein synthesis inhibitors. Cells incubated with exogenous arachidonic acid (10 μM) showed no increase in PGE 2 production. However, with IL-1 (2.5 U ml ) and exogenous arachidonic acid there was a marked increase, with levels reaching twice that for cells incubated with IL-1 alone. Aspirin pre-treatment studies and the use of [ acetyl- 14C]aspirin showed that IL-1 increased PGE 2 production through the induction of cyclo-oxygenase.

Teratogenic effects on the CD-1 mouse embryo exposed to concurrent doses of ethanol and aspirin.

Human fetal alcohol syndrome characteristics have been seen in the mouse fetus by several investigators who dosed the dam with only one or two doses of alcohol. The purpose of this study was to determine if the fetal effects of acute doses of alcohol (ethanol) are altered by aspirin. CD-1 mice were given two IP doses of a 25% v/v solution of 95% ethanol/saline (2.5 hours apart) and intubated with 250 mg/kg aspirin. The treatment regimen, begun at 8 days, 4 hours gestation, consisted of either aspirin pretreatment 1 hour before or posttreatment 1 hour after the ethanol. Control animals were treated similarly and included vehicle only, ethanol/vehicle, and aspirin/vehicle groups. One group was untreated. On gestational day 18, the dams were killed and the uterine horns were examined for live, dead, and resorbed fetuses. The live were weighed and examined for external malformations and either skeletal or visceral abnormalities. With the litter as the unit of analysis, no significant difference was found in the number of dead and resorbed among groups. There was a significant difference (P less than .01) in average fetal weight in the aspirin-pretreated group. When the total number of fetuses affected was considered, the aspirin pretreatment group showed significantly (P less than .05) more external and visceral malformations. The skeletal examination revealed a significant (P less than .05) difference in anomalies plus delayed ossification in both groups treated with the aspirin/ethanol combination. No significant differences were seen in any category in the groups receiving aspirin alone or ethanol alone. These results indicate an additive effect of aspirin and ethanol on the developing CD-1 mouse fetus.

Blood-induced arterial segmental spasm.

Blood-induced arterial segmental spasm has been noticed clinically, and is thought to result from contact between a main feeding artery and extravasating blood from one of its side branches. This localized spasm can persist long enough to compromise the survival of a replantation or flap transfer. A partially amputated rat hind limb model was used to investigate this phenomenon. Bleeding from a side branch was shown to cause severe vasoconstriction and reduced limb perfusion only on direct contact with the main artery. Pretreatment with heparin and minimization of endothelial damage resulted in decreased vasospasm, but aspirin pretreatment had no effect on the degree of spasm and resulting reduced blood flow. This study demonstrates the clinical importance of careful ligation of side branches to reduce vasospasm that may compromise the survival of a replanted part or tissue flap.

CHARACTERIZATION OF SUBSTANCE P‐INDUCED BRONCHOCONSTRICTION IN THE GUINEA‐PIG: A COMPARISON WITH ACETYLCHOLINE

Substance P (0.5-8.0 micrograms/kg, i.v.) induced bronchoconstriction in anaesthetized, mechanically-ventilated guinea-pigs, comprising increases in airways resistance and decreases in dynamic compliance. These bronchoconstrictor responses were unaffected by bilateral vagotomy, pretreatment with pheniramine (2 mg/kg, i.v.) or by pretreatment with atropine (100 micrograms/kg, i.v.). Acetylcholine-induced (4-32 micrograms/kg, i.v.) bronchoconstriction was prevented by atropine pretreatment, whereas bilateral vagotomy inhibited responses to acetylcholine. Ganglionic blockade using hexamethonium (20 mg/kg, i.v.) potentiated both substance P and acetylcholine on airways resistance and dynamic compliance. Indomethacin (1 or 5 mg/kg, i.v.) did not affect substance P-induced bronchoconstriction, whereas the higher dose enhanced acetylcholine-induced increases in airways resistance. In addition, aspirin pretreatment (20 mg/kg, i.v.) did not alter the bronchoconstrictor potency for either substance P or acetylcholine. On the other hand, the combined cyclo-oxygenase/lipoxygenase inhibitors eicosatetraynoic acid (ETYA, 20 mg/kg, i.v.) and BW755C (20 mg/kg, i.v.) potentiated both acetylcholine and substance P on airways resistance and dynamic compliance. The results suggest that substance P-induced bronchoconstriction may be modulated by the sympathetic nervous system and does not appear to be influenced by vagal or histaminergic mechanisms. The failure of indomethacin or aspirin to affect substance P-induced bronchoconstriction, together with the enhancing effects of ETYA and BW755C pretreatments, provide evidence consistent with the existence of a bronchodilator mechanism which may be inhibited by compounds inhibiting lipoxygenase enzymes.

Aspirin enhances the sensitivity of human platelet 12-lipoxygenase to inhibition by 15-hete, an endogenous regulator

Human platelets metabolize arachidonic acid via cyclooxygenase (E.C. 1.14.99.1) to thromboxane A 2 and the 12-lipoxygenase to 12-hydroxyeicosatetraenoic acid (12-HETE). Aspirin inhibits cyclooxygenase while the neutrophil product 15-Hydroxyeieosatetraenoic acid (15-HETE) is a selective inhibitor of platelet 12-lipoxygenase. The unexpected observation was made that the platelet 12-lipoxygenase of individuals who had ingested aspirin showed up to a twenty-fold increase in sensitivity to inhibition by 15-HETE. This observation was confirmed in platelets treated with aspirin in vitro . Aspirin pretreatment consistently resulted in a decrease in the I 50 for 15-HETE from an average of 21.5 μM to only 5.2 ± 1.5 μM, indicating a probable interaction between the cyclooxygenase and lipoxygenase pathways.

Myocardial ischaemia produced by ergonovine-induced vasoconstriction during preexisting coronary stenosis: experimental conditions for the geometric theory.

Experiments were designed to determine the contribution of active vasomotor tone of a large coronary artery during a preexisting coronary stenosis to the production of myocardial ischaemia. The quantitative relations between ergonovine dose and systemic and coronary haemodynamic and electrocardiographic responses during various degrees of coronary stenosis were evaluated in 55 anaesthetised open-chest dogs. In the absence of coronary stenosis, intracoronary infusion of ergonovine (0.04 to 4 micrograms X min-1) had no systemic or coronary haemodynamic effects. In dogs with coronary stenosis created with intraluminal microballoon occluder, ergonovine produced marked decreases in coronary blood flow and distal coronary pressure followed by a decline in left ventricular dP/dt and ST-elevation in epicardial electrogram in the presence of moderate (28 +/- 1.1 mmHg in pressure gradient) and severe (41 +/- 1.4 mmHg), but not mild (15 +/- 0.9 mmHg) stenosis. These detrimental effects of ergonovine were dependent on its dose as well as the severity of preexisting coronary stenosis. Interventions such as aspirin pretreatment or endothelial denudation did not attenuate the coronary vasomotor response or ergonovine, but pretreatment with nifedipine (3 micrograms X kg-1 iv) prevented this response. Intravenous injection of ergonovine (4 to 15 micrograms X kg-1) in doses relevant to clinical usage during intraluminal obstruction resulted in similar changes in coronary haemodynamics as those of intracoronary ergonovine. In contrast, in dogs with various degrees of coronary stenosis produced with an externally applied constrictor device, ergonovine did not affect systemic and coronary haemodynamics. These experiments demonstrate that normal vasomotion superimposed on moderate and severe pliable coronary stenosis can cause transient myocardial ischaemia, which helps to clarify the conditions to produce myocardial ischaemia according to geometric theory.

Alterations in ocular and optic nerve blood flow during intraocular surgery in aspirin pretreated rabbits.

The effect of intraocular surgery on ocular and optic nerve blood flow was determined in rabbits either untreated or pretreated with aspirin. Surgery was either extracapsular lens extraction through a 100 degrees corneal incision or a sham-operation in which a 45 degrees corneal incision was performed and the lens left in place. In both cases, the incisions were not sutured and the intraocular pressure remained zero for one hour. A (85Sr) radioactive microsphere technique was used to measure blood flow in the iris, scraped ciliary processes, retina, choroid, and optic nerve. In rabbits not receiving aspirin pretreatment, blood flow was statistically increased in all portions of the eye, except the retina and optic nerve, studied for sham-operated eyes, and in the iris and optic nerve in the lens extraction group. In rabbits pretreated with aspirin, blood flow remained at normal levels. These results are consistent with the hypothesis that prostaglandins released during ocular surgery cause increased ocular tissue blood flow.

Endothelial cell prostacyclin production induced by activated neutrophils.

A bovine aortic endothelial cell (EC) line released prostacyclin (greater than 1 pmol/10(+5) EC cells) when incubated with fMet-Leu-Phe (FMLP)-stimulated rat and human neutrophils (PMNs). This prostaglandin (PG) I2 was shown to come from the ECs and not from the PMNs by radioactive, high-performance liquid chromatography, and immunochemical criteria. Both FMLP-stimulated rat peritoneal and human peripheral PMNs as well as their stimulated cell-free supernatants and unstimulated sonicates could elicit the release of PGI2 from ECs. Since phorbol myristate acetate stimulated PMN adherence but elicited little PGI2 release from ECs, the PGI2 stimulation in ECs is unrelated to PMN adhesion. The addition of catalase and superoxide dismutase to FMLP-stimulated PMNs enhanced rather than reduced PGI2 formation, indicating that activated oxygen products of the PMN are not responsible for the induction of PGI2. Incubation of ECs with leukotriene (LT) B4, LTC4, or LTD4 did not trigger PGI2 release nor did aspirin pretreatment of the PMNs reduce the PGI2 induction. These data suggest that arachidonic acid metabolites of the PMNs were not responsible for the PGI2 induction. Available data indicates that the PMN factor that stimulates PGI2 from ECs is either released concomitantly with the azurophilic granules or is closely related to this event.

Aspirin reduces alcohol-induced prenatal mortality and malformations in mice.

Oral alcohol administration (5.8 g/kg) on gestation day 10 resulted in an increase in both prenatal mortality and birth defects as well as decreased fetal weight in C57BL/6J mice. Aspirin pretreatment (150 mg/kg subcutaneously) significantly reduced the number of malformed pups and prevented the increase in prenatal mortality produced by alcohol. The mechanism of action remains to be elucidated.

Maternal aspirin administration inhibits pulmonary arachidonic acid metabolism in fetal rabbits

Pregnant rabbits were treated with aspirin (100 mg/kg/day) for ten consecutive days during the last third of the pregnancy. The ability of isolated perfused fetal rabbit lungs to metabolize arachidonic acid (AA) was studied on the 31 st day of the pregnancy. After the infusion of 14C-AA (100 nmol) into the pulmonary circulation about 10 % of the radioactivity was found in the nonrecirculating perfusion effluent and about 80 % was incorporated into the lung lipids. Aspirin pretreatment of the rabbits inhibited the formation of AA metabolites in the lungs of their fetuses. The inhibition was clear when the metabolites of AA were extracted from the perfusion effluent at pH 7.4 (mainly lipoxygenase products) but a slight inhibition was also seen in the amounts of some metabolites extracted at pH 4.5 (cyclo-oxygenase products). When aspirin (1 mM) was infused simultaneously with AA into the pulmonary circulation the inhibition of AA metabolism was nearly complete. Aspirin pretreatment of the pregnant rabbits caused a slight increase in the amount of 14C-AA incorporated into some of the phospholipid and neutral lipid fractions of the perfused fetal lungs. Aspirin pretreatment clearly inhibited the TXB 2 formation during clotting in the blood of maternal rabbits but not significantly in the blood of their fetuses.

Plasmatic TXB 2 and 6-keto-PGF 1α levels during charcoal hemoperfusion in chronic renal failure patients

6 patients with end-stage renal disease underwent hemoperfusion with charcoal colums, for 60 min. Blood samples anticoagulated with 2% EDTA/aspirin solution were obtained from arteriovenous fistulas in the basal condition, 5 min after a bolus injection of heparin (7,500 U), at the end of hemoperfusion, and 30 min after. The study was repeated few days later, in the same patients, two hours after 100 mg aspirin by mouth. TXB 2 and 6-keto-PGF 1α were assayed with RIA in unextracted (U) and extracted (E) and chromatographed platelet poor plasma (PPP). Platelet counts before and after hemoperfusion were also performed. Low levels of the two prostaglandins were found in plasma; this could be related to the procedures for collection and processing of plasma samples; no significant differences were observed between extracted and unextracted samples: there were slightly higher levels of 6-keto-PGF 1α in unextracted samples. After charcoal hemoperfusion there was only a slight and not significant increase of TXB 2 and 6-keto-PGF 1α; low dose aspirin did not modify significantly plasma levels of the two prostaglandins before hemoperfusion but it reduced TXB 2 and 6-keto-PGF 1α levels after charcoal hemoperfusion. The platelet count fell (−22%) after charcoal hemoperfusion with heparin alone and in a similar manner after lowdose aspirin pretreatment (−24,7%).

Effects of aspirin and prostacyclin on arrhythmias resulting from coronary artery ligation and on infarct size.

1 The effects of pretreatment with aspirin, and of prostacyclin (PGI(2)) infusions, on responses to myocardial ischaemia and infarction produced by ligation of a coronary artery were investigated in conscious rats.2 Surgical preparation, under halothane anaesthesia, consisted of implanting exteriorized aortic and jugular cannulae, ECG leads and a polypropylene/polyethylene occluder for the left anterior descending coronary artery. Ligation of the coronary artery was performed six to nine days after surgery.3 Aspirin pretreatment consisted of 100 mg/kg given intravenously 1 or 36 h before ligation. PGI(2) infusions (10-400 ng kg(-1) min(-1), i.v.) were begun 2 min before ligation and continued for 4 h afterwards.4 ECG, blood pressure, heart rate and arrhythmias were recorded starting 30 min before, and continuing for 4 h after, ligation. Twenty-four hours after ligation, in surviving animals, the heart was removed for estimation of occluded and infarcted zones.5 Some treatments provided antiarrhythmic and other protection in the first 30 min post-ligation. By 4 and 24 h post-ligation, protective effects were lost.6 Both aspirin pretreatment and low doses of prostacyclin reduced arrhythmias occurring within 30 min of ligation. The highest dose of prostacylin (400 ng kg(-1) min(-1)) was arrhythmogenic.7 None of the treatments influenced the amount of cardiac tissue occluded or infarcted by ligation.8 The conclusions from this study in conscious rats were that acute aspirin pretreatment and low doses of infused prostacyclin have limited beneficial actions which are mainly confined to the earliest post-ligation period.

Does Carbon Dioxide Play a Role in Retrolental Fibroplasia?

The role of carbon dioxide tensions in the evolution of acute and/or chronic retrolental fibroplasia (RLF) is a subject of much current interest. Flower and Blake1 have produced cicatricial disease for the first time in an animal model by inhibiting prostaglandin-mediated retinal vasoconstriction. Similar vascular alterations were induced in this puppy model by either aspirin pretreatment or by hypercarbia exposure—both resulting in vasodilator predominance and scarring disease. This suggests that "vasodilation" may be a pathologic process, rather than the oxygen-induced "vasoconstriction," which has previously been incriminated in the pathogenesis of this disease.

The effect of aspirin pretreatment on the fate of arachidonic acid in hamster isolated lungs

The fate of exogenous arachidonic acid (AA) was investigated in isolated perfused lungs from hamsters, which were pretreated with aspirin drinking water (0, 5, 50 or 500 mg/1) for one week. When 40 nmol of 14C-AA was infused in two minutes into the pulmonary circulation, the perfusion pressure increased. This pressor response was decreased by 50 mg/l aspirin and was negligible after 500 mg/1. The amount of radioactivity was increased by aspirin pretreatment in the perfused lungs and decreased in the nonrecirculating perfusion effluent, which was collected for 6 minutes after the beginning of the AA infusion. The amount of unmetabolized free arachidonate was not changed significantly in the perfusion effluent or in the perfused lungs. The perfusion effluent was extracted with ethyl acetate first at pH 7.4 to extract unmetabolized AA, metabolites of lipoxygenase and HHT and then at pH 3.5 for prostaglandins and thromboxanes. The amounts of all arachidonate metabolites were decreased rather similarly by aspirin pretreatment In the perfused lung tissue the amount of 14C-AA was increased by aspirin in phospholipids and in the triacylglycerol fraction of neutral lipids.