Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: a mass spectrometric and isotope labeling study

Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called "aspirin resistance". We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to "aspirin resistance" in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the α-chain: αK191, αK208, αK224, αK429, αK457, αK539, αK562, in the β-chain: βK233, and in the γ-chain: γK170 and γK273. Glycations were found at βK133 and γK75, alternatively γK85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [(14)C-acetyl]salicylic acid and [(14)C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 μM aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of fibrinogen.     

Successful therapy of the prostaglandin defect "Wien-Hietzing" (lack of platelet high-affinity binding sites for prostaglandin I2)

Some years ago we detected a lack of platelet high-affinity PGI2 binding sites in a 10 year-old girl who presented to the outpatient unit with clinical symptoms and signs of acute popliteal artery occlusion. We named this new defect in the prostaglandin system "Wien-Hietzing". Acute surgery was successful. On the basis of earlier findings that aspirin is able to sensitize platelets to the action of PGI2 and produce beneficial changes in platelet sensitivity, we decided to treat this girl with a daily dosage of 20 mg aspirin orally. Repeated control examinations during the total follow-up period of about 6 years revealed normalized platelet sensitivity and normalized receptor behaviour. The girl is symptom-free to date. It is concluded that this prostaglandin defect may be successfully treated with long-term, low-dose aspirin administration.     

Aspirin enhances evaporation in hydrated and dehydrated rats

The effect of acetysalicylic acid (aspirin) on thermoregulation in a warm environment was studied in hydrated and dehydrated adult rats to test the hypothesis that dehydration hyperthermia can be modified by an antipyretic drug. Metabolic rate (MR), evaporative water loss (EWL), and deep body temperature (Tb) were measured during 2 h of exposure to an ambient temperature of 36 degrees C after the rats had received an oral pellet of aspirin (100 mg.kg-1) or placebo. The dehydrated placebo group had a higher Tb and lower EWL than the hydrated placebo group. Aspirin increased MR and EWL in both hydrated and dehydrated animals. Aspirin did not affect Tb in hydrated rats, but reduced Tb by 0.2 degree C in dehydrated rats during the heat exposure. The elevation in EWL appears to be a thermoregulatory response to increased heat production in both hydrated and dehydrated animals after aspirin treatment. The possibility that aspirin may act in dehydrated animals to restore central thermosensitivity toward hydrated levels needs to be tested further.     

Do Zinc and Selenium Prevent the Antioxidant, Hepatic and Renal System Impairment Caused by Aspirin in Rats?

Aspirin is widely used as an antiinflammatory drug especially in children with rheumatic fever arthritis. The diminishing effects of aspirin on antioxidant enzymes and hepato-renal systems at high doses are well-known. It is now evident that the damage at antioxidant system worsens the clinical picture of the disease and prolongs the treatment time. Thus, we investigated the effect of antioxidant enzyme cofactors-zinc and selenium-supplementation on superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels (erythrocyte and liver) and hepato-renal toxicity during aspirin treatment at therapeutic doses. The rats were divided into five groups. The first and second groups were given aspirin 75 mg/kg/day and aspirin plus selenium (Selenium 200, selenium 200 mg tablet as selenium yeast, GNC) and zinc (Zinc 100, zinc 100 mg tablet as zinc gluconate, GNC), respectively, the third and fourth take 50 mg/kg/day aspirin and aspirin plus selenium and zinc twice a day, respectively. The fifth group was control. The rats were treated with aspirin for 5 weeks as in the treatment of rheumatic fever arthritis in children. Erythrocyte SOD and MDA levels were preserved with supplementation, whereas there was no change for GSH-Px levels. Liver SOD, GSH-Px, and MDA levels were not changed. In zinc- and selenium-supplemented groups, the levels of serum alanine aminotransferase, uric acid, and direct bilirubin levels were found statistically decreased compared with nonsupplemented groups. There was no significant histopathologic change in specimens of hepatic and renal tissues. Trace element supplementation may prevent free radical damage and shorten treatment time in children using long-term aspirin treatment.     

血小板膜糖蛋白（GP）ⅢaPLA、Ⅰa807C/T基因多态性与阿司匹林抵抗的相关性研究

目的：探讨青岛地区汉族人群阿司匹林抵抗与血小板膜糖蛋白GPⅢaPLA、Ⅰa807C/T基因多态性之间的关系。方法：筛选150例动脉粥样硬化患者服用阿司匹林（100mg/d）至少14d以上,根据血小板聚集功能测定将其分为阿司匹林抵抗（AR）组、阿司匹林半抵抗（ASR）组,阿司匹林敏感（AS）组。用PCR-RFLP法确定各组GPⅢaPLA、GPⅠa 807C/T基因型。结果：仅于ASR组检出1例PLA1/A2基因型,其余均为PLA1/A1基因型,未发现PLA2/A2基因型,差异无统计学意义（P〉0.005）;GPⅠa807C/T基因位点AR组、ASR组的T等位基因频率均显著高于AS组,有统计学意义（P〈0.005）。结论：GPⅢaPLA2基因可能不是阿司匹林抵抗的遗传危险因素。而GPⅠa 807C/T基因位点的T等位基因与阿司匹林抵抗的发生相关联,可能是阿司匹林抵抗遗传易感因素。     

A comparison of chronic aspartame exposure to aspirin on inflammation, hyperalgesia and open field activity following carrageenan-induced monoarthritis.

The purpose of the present study was to investigate whether chronic aspartame exposure possesses analgesic and anti-inflammatory actions in the carrageenan-induced monoarthritis model similar to those properties of aspirin. Prior research demonstrated that aspartame can reduce second phase formalin pain and increase motor activity in arthritic patients. Fifty-eight male Sprague-Dawly rats were treated with aspartame (25, 50, 100 mg/kg) or saline for six days. An additional group of animals received daily injections of saline and on the sixth treatment day, received a 150-mg/kg dose of aspirin 30-minutes prior to behavioral testing. On Day 6, animals received an intra-articular (i.a.) injection of 2% lambda carrageenan (CARR) or an equal volume of saline and were tested four hours later on threshold to mechanical and thermal stimuli, open field activity, and knee joint diameter. Aspirin-treated arthritic animals exhibited significantly less mechanical hyperalgesia and knee joint inflammation compared with vehicle treated arthritic animals. However, aspirin did not reverse thermal hyperalgesia or increase motor activity to control levels. Aspartame did not reduce inflammation, increase motor activity, or attenuate thermal allodynia, but at 50 mg/kg did attenuate mechanical allodynia compared with vehicle treated arthritic animals. The anti-hyperalgesic effect on mechanical hyperalgesia was not seen at 25 mg/kg or 100 mg/kg aspartame. These results suggest that a certain amount of aspartame may provide relief of arthritic pain to a similar degree as aspirin in some individuals. The specific effect of aspartame and aspirin on mechanical hyperalgesia should be considered when these agents are used for the therapeutic treatment of arthritic conditions.     

The role of aspirin-triggered lipoxins in the mechanism of action of aspirin

Few drugs have treated so many diseases, provided us with so much understanding of their pathogenesis, and tested our scientific creativity over the last 100 years as much as aspirin. Originally, the beneficial effects of aspirin were shown to stem from its inhibition of cyclooxygenase (COX 2)-derived prostanoids, fatty acid metabolites that modulate host defense and regulate the cardiovascular system. However, the inhibition of COX 2 enzyme activity and prostaglandin synthesis has never fully explained aspirin's repertoire of anti-inflammatory effects, leaving many questions pertaining to its true mechanism of action unanswered. Here, data from a series of comparatively recent experiments exploring aspirin's unique ability to acetylate the active site of inducible COX 2 and generate a family of lipid mediators called the epi-Lipoxins will be discussed in light of their ability to exert profound modulatory effects on the innate and adaptive immune systems.     

Aspirin as a quantitative acetylating reagent for the fatty acid oxygenase that forms prostaglandins.

A selective acetylation of the prostaglandin-forming fatty acid oxygenase (part of the prostaglandin "synthetase" system) occurs with 100 muM concentrations of aspirin (acetylsalicylic acid). The amount of acetylation, measured by counting the [3H]acetyl-protein formed, was proportional to the amount of active, functional oxygenase in a sample. When samples were aged to allow spontaneous inactivation of the oxygenase, the amount of acetylation was proportional to the remaining measurable activity rather than the initial amount of oxygenase protein in the sample. Diethyl dithiocarbamate inhibited the oxygenase activity, but did not interfere with the subsequent acetylation by aspirin. Indomethacin, on the other hand, appeared to inactivate the oxygenase in a manner that interfered only partially with the action of aspirin as an acetylating reagent. The amount of acetylation appeared to be dependent upon the amount of native, undenatured enzyme. The results suggest that the acetylation may be dependent upon an essential functional group or conformation of groups in the catalytic peptide chain(s) that can be destroyed during spontaneous inactivation of the oxygenase, and altered by indomethacin.     

The effect of inhibition of prostaglandin synthesis on ovarian hypertrophy in the rat.

Aspirin and indomethacin, inhibitors of prostaglandin biosynthesis, were utilized to determine the role of prostaglandins (PGs) in ovarian weight gain in rats following unilateral ovariectomy or treatment with PMSG. After unilateral ovariectomy, the compensatory ovarian hypertrophy was 185-0% compared with 139-8% and 97-5% in rats treated with indomethacin and aspirin, respectively. The adrenal weights in rats treated with aspirin were also reduced significantly. Administration of PGE2 or PGF2alpha with aspirin reversed the effect of aspirin on the adrenals but had no effect on the ovarian weight. Indomethacin and aspirin treatment of animals injected with PMSG also reduced the ovarian weight gain. If 100 mug PGE2 were given twice daily, this effect was reversed in both groups but thrice daily administration had no effect on rats receiving aspirin. In PMSG-treated rats, 100 mug PGF2alpha twice daily did not reverse the effect of indomethacin and aspirin, and actually enhanced the effect of aspirin.     

Aspirin as a quantitative acetylating reagent for the fatty acid oxygenase that forms prostaglandins

A selective acetylation of the prostaglandin-forming fatty acid oxygenase (part of the prostaglandin “synthetase” system) occurs with 100 μM concentrations of aspirin (acetylsalicylic acid). The amount of acetylation, measured by counting the [³H]acetyl-protein formed, was proportional to the amount of active, functional oxygenase in a sample. When samples were aged to allow spontaneous inactivation of the oxygenase, the amount of acetylation was proportional to the remaining measurable activity rather than the initial amount of oxygenase protein in the sample.Diethyl dithiocarbamate inhibited the oxygenase activity, but did not interfere with the subsequent acetylation by aspirin. Indomethacin, on the other hand, appeared to inactivate the oxygenase in a manner that interfered only partially with the action of aspirin as an acetylating reagent.The amount of acetylation appeared to be dependent upon the amount of native, undenatured enzyme. The results suggest that the acetylation may be dependent upon an essential functional group or conformation of groups in the catalytic peptide chain(s) that can be destroyed during spontaneous inactivation of the oxygenase, and altered by indomethacin.     

Failure of Intravenous Aspirin to Increase Gastrointestinal Blood Loss

Studies of the effect of intravenous sodium acetylsalicylate (aspirin) on gastrointestinal blood loss with ⁵¹Cr-labelled red cells were made on 15 healthy male volunteers. After a control period of five days 1 g. of sodium acetylsalicylate was infused over a period of 100 minutes twice daily for three days. Faecal blood loss was not increased.In a further six subjects 3 g. of sodium acetylsalicylate was infused over a period of 120 minutes. No salicylate or acetylsalicylate was detected in saliva or gastric washings from these six subjects. Hence gastrointestinal blood loss induced by aspirin may be explained by a local effect on mucosa and not by any systemic effect.     

Ingestion of chilli pepper (Capsicum annuum) reduces salicylate bioavailability after oral asprin administration in the rat.

The bioavailabilities of aspirin (acetylsalicylic acid) and of salicylic acid were studied in male Wistar rats after acute and chronic administration of a Capsicum annuum extract, containing 100 mg of capsaicin per gram. With a single administration of 100 mg/kg of the extract, aspirin blood levels remained unchanged, but salicylic acid bioavailability was reduced in 44% compared with control animals. With a single administration of 300 mg/kg of the extract, aspirin blood levels were undetectable while salicylic acid bioavailability was reduced in 59%. Chronic administration once daily for 4 weeks of 100 and 300 mg/kg of the extract resulted in undetectable aspirin blood levels, while salicylic acid bioavailability was reduced in 63 and 76%, respectively, compared with controls. Results show that Capsicum ingestion reduces oral drug bioavailability, likely as a result of the gastrointestinal effects of capsaicin.     

Effect of aspirin on blood-lipid interaction and lipid peroxidation phenomena in relation to partition coefficient and biological activity

Considering the lipophilicity of aspirin (log P = -1.15), a significant contributor to its action mechanism, interaction of the drug with the whole lipids of goat blood have been investigated using phospholipid binding and lipid peroxidation phenomena as the parameters under investigation. The lipid content change along with the peroxidation induced by aspirin and its suppression with ascorbic acid had been quantitatively measured. Significant loss in phospholipid was observed after incubation of whole blood with aspirin in varying periods of time. This may be ascribed to binding affinity of aspirin with lipid constituents in blood, which may have potential role in its therapeutic effect. Lipid peroxidation induction potential of aspirin caused significant extent of peroxidation. Ascorbic acid, an antioxidant could significantly reduce aspirin induced lipid peroxidation.     

冠心病患者阿司匹林抵抗发生率与正常人的比较

目的:观察冠心病患者阿司匹林抵抗的发生率是否与正常人群有差异。方法:心内科住院的冠心病病人50例和正常对照组30例每天服用阿司匹林100mg,7天后用二磷酸腺苷(ADP)、花生四烯酸(AA)作为血小板聚集诱导剂检测定血小板聚集率。结果:在50例冠心病患者中,阿司匹林完全抵抗发生率为12%(6/50);阿司匹林半抵抗发生率为26(13/50),阿司匹林敏感率为62%(31/50)。在30例正常对照组中,阿司匹林完全抵抗发生率为13.3%(4/30);阿司匹林半抵抗发生率为26.6%(8/30),阿司匹林敏感率为60.1%(18/30)。结论:冠心病患者阿司匹林抵抗的发生率是与正常人群没有差异。     

Case report: Immunoglobulin A deficiency in patients with juvenile rheumatoid arthritis treated with aspirin

In three patients with juvenile rheumatoid arthritis, serum IgA concentrations were within the normal limit at the onset of disease and before aspirin administration. After aspirin administration, their serum IgA levels gradually decreased. After discontinuation of aspirin, their serum IgA levels gradually increased. These results suggest that IgA deficiency may be due to aspirin administration in such patients. The IgA production in vitro of peripheral blood mononuclear cells from a patient taken 3 months of discontinuation of aspirin was markedly inhibited by preincubation with aspirin. Since the patients' serum IgG and IgM levels hardly changed the heavy chain class switching may be influenced by aspirin through some undefined mechanisms.Abbreviations CH heavy chain constant region - ³H-TdR ³H-thymidine - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - JRA juvenile rheumatoid arthritis - MEM minimum essential medium - PBMCs peripheral blood mononuclear cells - PBS phosphate buffered saline - PFCs plaque forming cells - PWM pokeweed mitogen - SI stimulation index     

Eicosanoids in preeclampsia

Preeclampsia is characterized by an imbalance between two cyclooxygenase metabolites of arachidonic acid, thromboxane and prostacyclin, that favors thromboxane. Because of the biologic actions of these two eicosanoids, this imbalance might explain major clinical symptoms of preeclampsia, such as hypertension, platelet aggregation and reduced uteroplacental blood flow. In the maternal circulation, this imbalance is primarily manifested by decreased production of prostacyclin by endothelial cells. Platelet thromboxane synthesis is only increased in severe preeclampsia. In the placenta and in leukocytes, the imbalance is exacerbated by increased production of thromboxane coupled with decreased production of prostacyclin in both mild and severe preeclampsia. Longitudinal measurements of urinary metabolites of thromboxane and prostacyclin reveal that the thromboxane/prostacyclin imbalance predates the onset of clinical symptoms of preeclampsia. The imbalance between thromboxane and prostacyclin is most likely caused by oxidative stress, which is manifest in preeclampsia by increased lipid peroxidation and decreased antioxidant protection. Oxidative stress may drive this imbalance because lipid peroxides activate the cyclooxygenase enzyme to increase thromboxane synthesis, but at the same time they inhibit prostacyclin synthase to decrease prostacyclin synthesis. Low-dose aspirin therapy (50-150 mg/day) has been considered for the prevention of preeclampsia because it selectively inhibits thromboxane synthesis. Several studies reported dramatic decreases in the incidence of preeclampsia with low-dose aspirin therapy. However, two large multicenter studies reported only modest decreases, which dampened enthusiasm. The two large studies were "intent to treat" studies which included patients who were noncompliant and who discontinued the use of aspirin. In one of the studies for which compliance statistics were available only 53% of the aspirin group had a compliance rate greater than 75%, which raises a question as to whether the effectiveness of aspirin was being tested. Low-dose aspirin therapy should not yet be dismissed for the prevention of preeclampsia, but be reconsidered with emphasis on compliance using doses of aspirin in the range of 100-150 mg/day combined with antioxidants.     

Equivalent inhibition of in vivo platelet function by low dose and high dose aspirin treatment

In vitro platelet function was inhibited in healthy volunteers by two different doses of aspirin, as confirmed by measurement of maximum serum production of thromboxane B2 (TXB2) by platelets. 75 mg aspirin did not fully inhibit serum TXB2 production after 24 hours, whereas 300 mg aspirin did. Inhibition of platelet function in vitro was maintained by both 75 mg/day aspirin or 300 mg/alternate day aspirin. In contrast, in vivo production of TXB2, measured as urinary levels of the 11-keto-TXB2 metabolite, was inhibited similarly by both doses of aspirin throughout the study. These findings suggest that 75 mg/day aspirin may be sufficient adequately to inhibit platelet aggregation in vivo.     

Effect of aspirin in "aspirin sensitive" patients.

Eighteen patients with a history of urticaria or asthma, or both, induced by aspirin were studied before and after provocation of symptoms with aspirin. The plasma prostaglandin F2 alpha concentration, which was characteristically raised before challenge, fell significantly at the time of adverse reactions. Repeated administration of aspirin up to a dose of 650 mg daily induced tolerance in most of the patients, and several developed bronchodilator responses to aspirin. Although median total IgE concentrations may be raised in patients with aspirin sensitivity, it appears likely that pharmacological rather than immunological mechanisms are chiefly responsible for the phenomena of aspirin sensitivity and desensitisation.