De novo synthesis of cyclooxygenase-1 counteracts the suppression of platelet thromboxane biosynthesis by aspirin

Aspirin affords cardioprotection through the acetylation of serine529 in human cyclooxygenase-1 (COX-1) of anucleated platelets, inducing a permanent defect in thromboxane A2 (TXA2)-dependent platelet function. However, heterogeneity of COX-1 suppression by aspirin has been detected in cardiovascular disease and may contribute to failure to prevent clinical events. The recent recognized capacity of platelets to make proteins de novo paves the way to identify new mechanisms involved in the variable response to aspirin. We found that in washed human platelets, the complete suppression of TXA2 biosynthesis by aspirin, in vitro, recovered in response to thrombin and fibrinogen in a time-dependent fashion (at 0.5 and 24 hours, TXB2 averaged 0.1+/-0.03 and 3+/-0.8 ng/mL; in the presence of arachidonic acid [10 micromol/L], it was 2+/-0.7 and 25+/-7 ng/mL, respectively), and it was blocked by translational inhibitors, by rapamycin, and by inhibitors of phosphatidylinositol 3-kinase. The results that COX-1 mRNA was readily detected in resting platelets and that [35S]-methionine was incorporated into COX-1 protein after stimulation strongly support the occurrence of de novo COX-1 synthesis in platelets. This process may interfere with the complete and persistent suppression of TXA2 biosynthesis by aspirin necessary for cardioprotection.     

A high level of cyclooxygenase-2 inhibitor selectivity is associated with a reduced interference of platelet cyclooxygenase-1 inactivation by aspirin

Both nonsteroidal anti-inflammatory drugs, such as ibuprofen, and the prototypical selective cyclooxygenase (Cox)-2 inhibitors DuP-697 and NS-398 block the inhibition of Cox-1 by aspirin in vitro. However, clinical studies have shown that the Cox-2 selective drugs (or coxibs) rofecoxib and etoricoxib, at therapeutic doses, do not interfere with the antiplatelet effect of aspirin, in contrast to ibuprofen. Here, we have evaluated the relative potential of ibuprofen and various coxibs to interfere with the inactivation of Cox-1 by aspirin by using purified enzyme and calcium ionophore-activated human platelets. The irreversible inactivation of Cox-1 by aspirin can be antagonized by ibuprofen and coxibs, albeit with widely different potencies. The rank order of potencies for this process (ibuprofen > celecoxib > valdecoxib > rofecoxib > etoricoxib) parallels that obtained for the inhibition of Cox-1-mediated thromboxane B(2) production by calcium ionophore-stimulated platelets. The antagonism of aspirin therefore likely involves a competition at the enzyme active site. The EC(50) value for the antagonism against 10 microM aspirin for each drug is approximately 10- to 40-fold lower than the corresponding IC(50) value for inhibition of platelet Cox-1 activity, consistent with the much weaker initial binding of aspirin to Cox-1 as compared with arachidonic acid. These results show that a low affinity for Cox-1 and a high degree of Cox-2 selectivity confers a low potential to block aspirin inhibition of platelet Cox-1, consistent with the results of clinical studies.     

Heterogeneity in platelet cyclooxygenase inhibition by aspirin in coronary artery disease

BackgroundPlatelets, long believed to be incapable of de novo protein synthesis, may retain their ability to form the cyclooxygenase (COX) enzyme once it has been inactivated by aspirin. This may explain the inefficacy of the drug to induce sustained platelet inhibition in certain patients. We evaluated the stability of platelet inhibition following once-daily enteric-coated aspirin administration.MethodsPlatelet responsiveness to aspirin was evaluated in 11 stable coronary artery disease patients on chronic aspirin therapy before and 1, 3, 8, and 24 h after observed ingestion of 80-mg enteric-coated aspirin. Inhibition of the COX pathway was measured pharmacologically through plasma thromboxane (Tx) B₂ levels, and functionally by light transmission aggregometry in response to arachidonic acid. COX-independent platelet activity was measured in response to adenosine diphosphate, epinephrine and collagen.ResultsPlasma TxB₂ levels showed profound inhibition of TxA₂ formation, which was stable throughout 24 h, in all but 1 subject. This subject had optimal response to aspirin (inhibition of platelet TxA₂ production within 1 h), but recovered the ability to synthesize TxA₂ within 24 h of aspirin ingestion. Arachidonic acid-induced platelet aggregation closely mirrored TxB₂ formation in this patient, portraying a functional ability of the platelet to aggregate within 24 h of aspirin ingestion. COX-independent platelet aggregation triggered TxA₂ production to a similar extent in all patients, likely through signal-dependent protein synthesis.ConclusionsCOX-dependent platelet activity is recovered in certain individuals within 24 h of aspirin administration. Further research should consider increasing aspirin dosing frequency to twice daily, to allow sustained inhibition in such subjects.     

老年代谢综合征患者阿司匹林抵抗与平均血小板体积和收缩压的相关性研究

目的探讨老年代谢综合征患者阿司匹林抵抗与平均血小板体积的关系。方法选择老年代谢综合征患者220例,根据血栓弹力图结果分为阿司匹林抵抗组58例和阿司匹林敏感组162例,所有患者口服阿司匹林(75~100mg)≥1个月。血栓弹力图法检测血小板聚集率。结果与阿司匹林敏感组比较,阿司匹林抵抗组收缩压≥140mm Hg(1mm Hg=0.133kPa)、CD62P、P选择素、TC、平均血小板体积及服用血管紧张素转换酶抑制剂/血管紧张素受体拮抗剂比例显著升高,差异有统计学差异(P0.05,P0.01)。logistic回归分析显示,收缩压≥140mm Hg(OR=2.705,95%CI:1.127~6.490,P=0.026)和平均血小板体积(OR=1.935,95%CI:1.306~2.867,P=0.001)为阿司匹林抵抗的危险因素。多元线性回归分析显示,花生四烯酸诱导的血小板聚集率与TC(β=0.172,P=0.019)和平均血小板体积(β=0.327,P=0.000)呈正相关。结论收缩压≥140mm Hg和平均血小板体积水平升高的老年代谢综合征患者阿司匹林抵抗发生率增加。     

The lack of augmentation by aspirin of inhibition of platelet reactivity by ticlopidine

A decreased threshold for platelet activation apparently contributes to the risk of cardiovascular events, such as acute myocardial infarction. To evaluate the impact of specific agents, we characterized platelet reactivity in 9 healthy subjects before and after 5 days of ingestion of 4 commonly prescribed regimens, 81 mg of aspirin daily, 325 mg of aspirin daily, ticlopidine 250 mg twice daily, and ticlopidine plus 325 mg of aspirin daily. Platelet reactivity was assessed with (1) aggregometry induced by 4 microM adenosine diphosphate (ADP) and collagen (0.19 mg/ml) and performed in platelet-rich plasma; and (2) flow cytometric determination of ADP-induced (0.2, 0.8, and 1.5 microM) P-selectin expression in whole blood. Because anticoagulants alter platelet reactivity, results were obtained with 3 anticoagulants, citrate, enoxaparin, or corn trypsin inhibitor (CTI, a specific inhibitor of factor XIIa without effect on other coagulation factors). Ingestion of aspirin did not alter platelet activation as assessed with flow cytometry. Inhibition of the second phase of aggregation was seen with ADP-induced aggregation in platelet-rich plasma anticoagulated with citrate but not enoxaparin or CTI. Ingestion of ticlopidine led to inhibition of ADP-induced aggregation and P-selectin expression. Inhibition of platelet reactivity after the combination of aspirin and ticlopidine did not differ from ticlopidine alone. Marked interindividual variability in platelet reactivity was seen after ingestion of ticlopidine. The results indicate that assessment of effects of specific pharmacologic regimens with accurate and readily available assays of platelet reactivity may facilitate effective prophylaxis and treatment of high-risk subjects with antiplatelet regimens designed to optimally diminish platelet reactivity.     

Management of metabolic syndrome: aspirin.

Cardiovascular disease (CVD), which includes myocardial infarction(MI), stroke, and peripheral vascular disease, remains the leading cause of death in the United States and in most developed countries. In the United States today, 25% of patients have metabolic syndrome-including those who have had a prior occlusive vascular disease event, those who are having an acute MI or ischemic stroke, and finally, the largest segment of the population,namely those who have not yet experienced a clinical CVD, but whose risks are substantial (10-year risk 10%). This article reviews the totality of evidence for aspirin in the treatment and prevention of CVD and emphasizes its importance as adjunctive therapy for patients with metabolic syndrome.     

Effect of platelet antigen polymorphism on platelet inhibition by aspirin, clopidogrel, or their combination.

OBJECTIVES: We studied the modifier effect of platelet antigen polymorphism (PlA2) on platelet inhibition by acetylsalicylic acid (ASA, i.e., aspirin), clopidogrel, or their combination in patients with coronary heart disease. BACKGROUND: Clopidogrel, when administered with ASA, was shown to significantly improve the outcome of patients with acute coronary syndromes compared with patients receiving only ASA. We have shown previously that the effect of ASA on platelets is modified by the glycoprotein IIIa single nucleotide polymorphism PlA2. Hence, an important pharmacogenetic question remains whether the antiplatelet effect of clopidogrel is uniform for all patients or, like acetylsalicylic acid, more selective. METHODS: Thirty PlA1/A1 and 30 PlA1/A2 patients were assigned randomly to ASA 325 mg/day, clopidogrel 75 mg/day, or both. After 10 days, platelet function was studied. RESULTS: Clopidogrel provided stronger platelet inhibition than ASA with adenosine diphosphate as the agonist, and combination therapy resulted in greater inhibition than either inhibitor used alone (p < 0.0001). The use of ASA resulted in greater inhibition compared with clopidogrel with epinephrine (p < 0.0001) and collagen as agonists (p < 0.0001). With collagen as the agonist, platelets from PlA1/A2 donors were markedly and significantly less inhibited by ASA (p = 0.005). In contrast, with clopidogrel, no significant difference could be detected between inhibition of Pl(A1/A1) and Pl(A1/A2) platelets. CONCLUSIONS: The combination of ASA and clopidogrel appears superior to either agent alone in inhibiting platelet function. Pl(A2) functions as an important modifier for platelet responsiveness to ASA but not to clopidogrel. These findings could have significant impact on the future design of pharmacogenetic antithrombotic strategies for patients with coronary heart disease.     

Very-low-dose twice-daily aspirin maintains platelet inhibition and improves haemostasis during dual-antiplatelet therapy for acute coronary syndrome

Higher aspirin doses may be inferior in ticagrelor-treated acute coronary syndrome (ACS) patients and reducing bleeding risk whilst maintaining antithrombotic benefits could improve outcomes. We characterized the pharmacodynamics of a novel dual-antiplatelet-therapy regimen consisting of very-low-dose twice-daily (BD) aspirin with standard-dose ticagrelor. A total of 20 ticagrelor-treated ACS patients entered a randomized crossover to take aspirin 20 mg BD (12-hourly) during one 14-day period and 75 mg once-daily (OD) in the other. After 14 days of treatment, serum thromboxane (TX)B₂ and light-transmittance aggregometry were assessed pre- and 2 h post-morning-dose, bleeding time was measured post-dose, and TXA₂ and prostacyclin stable metabolites were measured in urine collected 2 h post-morning-dose. Data are expressed as mean ± SD. After 14 days treatment, serum TXB₂ levels were significantly greater 2 h post-dosing with aspirin 20 mg BD vs. 75 mg OD (3.0 ± 3.6 ng/mL vs. 0.8 ± 1.9 ng/mL; p = 0.018) whereas pre-dosing levels were not significantly different (3.5 ± 4.1 ng/mL vs. 2.5 ± 3.1 ng/mL, p = 0.23). 1-mmol/L arachidonic acid-induced platelet aggregation was similarly inhibited by both regimens pre-dose (8.5 ± 14.3% vs. 5.1 ± 3.6%, p = 0.24) and post-dose (8.7 ± 14.2% vs. 6.6 ± 5.3%; p = 0.41). Post-dose bleeding time was shorter with 20 mg BD (680 ± 306 s vs. 834 ± 386 s, p = 0.02). Urinary prostacyclin and TX metabolite excretion were not significantly different. In conclusion, compared to aspirin 75 mg OD, aspirin 20 mg BD provided consistent inhibition of platelet TXA₂ release and aggregation, and improved post-dose hemostasis, in ticagrelor-treated ACS patients. Further studies are warranted to assess whether this regimen improves the balance of clinical efficacy and safety.     

代谢综合征患者阿司匹林抵抗的临床研究

目的探讨代谢综合征患者阿司匹林抵抗的发生率和临床特征。方法对2005年5月至6月北京首钢社区人群中221例病情稳定的代谢综合征患者,口服阿司匹林200mg/d共10d后,应用血小板聚集仪测定花生四烯酸(AA)诱导的血小板聚集率。以0.5g/LAA诱导的血小板平均聚集率≥20%为阿司匹林抵抗。结果阿司匹林抵抗发生率为17.6%(39/221)。阿司匹林抵抗(AS)组患者的纤维蛋白原水平显著高于阿司匹林敏感(AR)组的患者[(2.6±0.4)g/L对(2.4±0.4)g/L,P=0.017)]。两组患者的血压、年龄、空腹血糖、血脂以及体重指数等差异均无统计学意义;性别、吸烟、既往心梗或脑梗病史的分布也无统计学意义。进一步根据患者性别进行分层分析发现,在男性患者中心梗病史是阿司匹林抵抗的预测因素(50%对14.5%,P=0.020),在女性患者中舒张压高于85mmHg(1mmHg=0.133kPa)是阿司匹林抵抗的预测因素(34.0%对15.5%,P=0.043)。结论研究人群中阿司匹林抵抗的发生率为17.6%,高纤维蛋白原是阿司匹林抵抗的危险因素,心梗病史和较高的舒张压可能分别是男性和女性阿司匹林抵抗的预测因素。     

Selective inhibition of platelet cyclooxygenase with controlled release, low-dose aspirin

The hypothesis that slow administration of low doses of aspirin may selectively inhibit platelet cyclooxygenase and thromboxane A₂ formation was evaluated using controlled release aspirin formulations. In the first study, doses of either 50, 100, 325 and 1,300 mg of these formulations and 300 mg soluble aspirin were ingested daily by healthy volunteers for one week. In the second study, doses of 5, 10, 25 and 50 mg controlled release aspirin, 50 mg soluble aspirin and 100 mg aspirin and glycine formulation were ingested daily for ten days. Platelet function and urinary prostaglandin production were assessed immediately before and on the seventh day of dosing in both studies and in the second study, repeated on the tenth day of dosing. Platelet function and serum thromboxane B₂ production were fully inhibited by all formulations of 50 mg aspirin and above, but not by doses of controlled release aspirin below 50 mg doses. The excretion of urinary 6-keto-PGF₁α (a major metabolite of prostacyclin) was significantly reduced at controlled release aspirin doses above 100 mg and at all doses of rapidly absorbed aspirin tested. As no significant reduction in the urinary 6-keto-PGF₁α production was observed at doses of controlled release aspirin of 50 and 100 mg and below, it appeared that these doses did not inhibit the systemic vascular cyclooxygenase. These data are consistent with a selective inhibition of platelet function by daily doses of 50 and 100 mg of the controlled release formulation of aspirin.     

Enhancement of platelet inhibition of ticlopidine plus aspirin vs aspirin alone given prior to elective PTCA

Background In many patients today, elective percutaneous transluminalcoronary angioplasty is followed by implantation of coronarystents to achieve optimal results. The current medical strategyto prevent early reocclusion is the inhibition of platelet aggregationby administration of ticlopidine, in addition to aspirin, immediatelyafter the procedure. In order to inhibit platelet aggregationas early as possible, many centres begin to treat patients withadditional ticlopidine the day before elective coronary intervention.The aim of this study was to determine the effect of this strategyon platelet aggregation before angioplasty. Method Fifty-two consecutive patients admitted to hospital forelective balloon angioplasty were prospectively randomized toreceive either standard oral aspirin 100mg per day or standardtherapy plus 250mg ticlopidine at the time of admission andthe morning before angioplasty. Adenosine diphosphate-, collagen-and epinephrine-induced platelet aggregation was measured immediatelybefore the procedure by an investigator who was blinded concerningthe arm of therapy. Results The two groups of patients were comparable in termsof age, sex, body mass index, anginal state, time interval betweenapplication of study drug and coronary intervention. Patientson aspirin and ticlopidine had a mean maximal platelet aggregationof 36±12% with adenosine diphosphate as agonist. Forthe control group, 54±12% was measured (P<0·001).Myocardial infarction or emergency coronary bypass graftingdid not occur in either group. Local haemorrhagic complicationsat the arterial access site occurred in five (aspirin) and six(aspirin and ticlopidine) patients (P=ns) none of them requiringblood transfusion. Conclusion The additional application of ticlopidine to chronicaspirin therapy the day before elective coronary balloon angioplastyleads to a significantly higher inhibition of platelet aggregationat the time of the intervention. It seems to be safe comparedto the standard procedure.     

Effect of statins on platelet PAR-1 thrombin receptor in patients with the metabolic syndrome (from the PAR-1 inhibition by statins [PARIS] study)

We investigated whether, in primary prevention patients with metabolic syndrome, statins affect the platelet protease-activated receptor-1 (PAR-1) thrombin receptor by performing serial measurements of its activity and the antigen expression level by flow cytometry before and during treatment. Recent data from randomized trials of statins are compatible with the possibility of clinically relevant pleiotropic effects. The use of statins is associated with a reduced thrombosis burden and diminished platelet activity, as shown in animal models and in vitro studies. Seventy patients with the metabolic syndrome who were not taking antiplatelet agents were assigned consecutively at starting doses at the discretion of the responsible clinician to 1 of 6 statins (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, or simvastatin) or to a no-statin group for 6 weeks. Platelet expression of intact (SPAN12 antibody) and cleaved (WEDE15) PAR-1 thrombin receptors were assessed by flow cytometry at baseline and at weeks 4 and 6 of treatment. At baseline, no difference was found in receptor expression. However, after 4 weeks of treatment, all statins had significantly inhibited (46% to 55%) the activated epitope of PAR-1 expression. After 6 weeks, inhibition remained, despite a slight rebound (22% to 37%). Also, a delayed pattern of inhibition of the intact PAR-1 receptor epitope was found. In conclusion, all statins inhibited the activity and antigen level of the platelet PAR-1 thrombin receptor, which has a major role in regulating platelet activity and thrombin formation. These observational data offer a plausible mechanism for the recently demonstrated pleiotropic effects of statins that may contribute to early clinical benefit.     

Profile and prevalence of aspirin resistance in patients with metabolic syndrome

Objective Aspirin has been used extensively in primary and secondary prevention of cardiovascular disease,particularly for subjects at high risk such as metabolic syndrome.However,the responsiveness to aspirin treatment may vary among individuals.The present study was conducted to investigate the profile and prevalence of aspirin resistance in patients with metabolic syndrome.Methods In 221 consecutive patients,platelet aggregation induced by arachidonic acid (0.5mg/ml) was assessed after 10 days of aspirin treatment (200mg/d).Aspirin resistance was defined as mean optical platelet aggregation =20%.Results Aspirin resistance occurred in 39 patients (17.6%).Serum fibrinogen level was higher in patients with than in those without aspirin resistance (2.6_+0.4g/l vs 2.4±0.4g/L,P=0.017).The 2 groups,aspirin resistance group and no aspirin resistance group,did not differ significantly,with regard to gender,age,body mass index,waist-hip ratio,blood pressure level,serum cholesterol level and history of myocardial or cerebral infarction.Multivariate logistic regression analysis revealed that only serum fibrinogen level entered the model (odds ratio 2.973,p=0.023).Subgroup analysis further showed that aspirin resistance occurred more in male patients with myocardial infarction (50% vs14.5%,P=0.02) and in female patients with diastolic blood pressure=85mmHg (34% vs 15.5%,P=0.043).But after multifactor logistic regression,in women blood pressure=85mmHg was not a predictor any more.Conclusions In patients with metabolic syndrome,aspirin resistance is not uncommon,especially for men with history of myocardial infarction.Patients with aspirin resistance have an increased serum fibrinogen level.(J Geriatr Cardio12008;5:7-10)     

Antiplatelet activity of deferiprone through cyclooxygenase-1 inhibition

Abstract

Thalassemia patients are susceptible to both iron overload and thromboembolism. Deferiprone is an iron chelator that shows an antiplatelet activity and thus may alleviate platelet hyperactivation in thalassemia. Therefore, this study aimed to characterize the inhibitory effects and mechanisms of deferiprone on normal human platelets. The results illustrated that deferiprone inhibited platelet aggregation at the iron chelating concentrations (0.08–0.25 mmol/l). Deferiprone inhibited human platelet aggregation stimulated by arachidonic acid and ADP more potently than epinephrine and collagen, with the IC₅₀ of 0.24 mmol/l and 0.25 mmol/l vs. 3.36 mmol/l and 3.73 mmol/l, respectively. Interestingly, deferiprone significantly inhibited COX-1 activity, with the IC₅₀ of 0.33 mmol/l, and slightly increased cAMP level at the high concentration of 4 mmol/l. Moreover, the results from molecular docking showed that deferiprone interacted closely with key residues in the peroxidase active site of COX-1. These results suggested that deferiprone possessed antiplatelet activity mainly through the inhibition of COX-1 activity.