The lack of aspirin resistance in patients with coronary artery disease.

BACKGROUND: Aspirin resistance established by different laboratory methods is still a debated problem. Using COX1 specific methods no aspirin resistance was detected among healthy volunteers. Here we tested the effect of chronic aspirin treatment on platelets from patients with stable coronary artery disease. The expression of COX2 mRNA in platelets and its influences on the effect of aspirin was also investigated. METHODS: One hundred and forty four patients were enrolled in the study. The direct measurement of COX1 acetylation was carried out by monoclonal antibodies specific to acetylated and non-acetylated COX1 (acCOX1 and nacCOX1) using Western blotting technique. Arachidonic acid (AA) induced TXB2 production by platelets was measured by competitive immunoassay. AA induced platelet aggregation, ATP secretion and VerifyNow Aspirin Assay were also performed. COX2 and COX1 mRNA expression in platelets were measured in 56 patients by RT-qPCR. RESULTS: In 138 patients only acCOX1 was detected, in the remaining six patients nacCOX1 disappeared after a compliance period. AA induced TXB2 production by platelets was very low in all patients including the 6 patients after compliance. AA induced platelet aggregation, secretion and with a few exceptions the VerifyNow Assay also demonstrated the effect of aspirin. Smoking, diabetes mellitus and inflammatory conditions did not influence the results. The very low amount of COX2 mRNA detected in 39 % of the investigated platelets did not influence the effect of aspirin. CONCLUSIONS: No aspirin resistance was detected among patients with stable coronary artery disease. COX2 expression in platelets did not influence the effect of aspirin.

How to test the effect of aspirin and clopidogrel in patients on dual antiplatelet therapy?

Dual antiplatelet therapy with clopidogrel and aspirin is frequently used for the prevention of recurrent ischemic events. Various laboratory methods are used to detect the effect of these drugs administered in monotherapy, however their value in dual therapy has not been explored. Here, we determined which methods used for testing the effect of clopidogrel or aspirin are influenced by the other antiplatelet agent. One arm of the study included 53 ischemic stroke patients being on clopidogrel monotherapy showing effective inhibition of the P2Y12 ADP receptor. Laboratory tests routinely used for the detection of aspirin resistance (arachidonic acid (AA)-induced platelet aggregation/secretion, AA-induced thromboxane B2 (TXB2) production in platelet-rich plasma and VerifyNow Aspirin assay) were carried out on samples obtained from these patients. The other arm of the study involved 52 patients with coronary artery disease being on aspirin monotherapy. Methods used for testing the effect of clopidogrel (ADP-induced platelet aggregation and secretion, flow cytometric analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation and a newly developed P2Y12-specific platelet aggregation (ADP[PGE1] test)) were performed on samples obtained from these patients. Clopidogrel monotherapy significantly inhibited AA-induced platelet aggregation and secretion, moreover, AA-induced TXB2 production was also significantly decreased. VASP phosphorylation and AA-induced platelet aggregation showed fair correlation in patients taking clopidogrel only. Clopidogrel did not inhibit the VerifyNow Aspirin test significantly. Aspirin monotherapy influenced ADP-induced platelet aggregation and secretion, but did not have an effect on VASP phosphorylation and on the ADP[PGE1] platelet aggregation test.

Use of Complementary and Alternative Medicine Among Patients With Epilepsy and Diabetes Mellitus, Focusing on the Outcome of Treatment.

Introduction: Millions all over the world live with epilepsy, and they may require long-term drug treatment. The use and interest in complementary and alternative medicine (CAM) have grown over the previous years. Coadministration of herbal products with medicines may result in adverse drug reactions (ADRs) and/or unfavorable interactions. The aims of this study were to determine the prevalence of CAM use among patients with epilepsy, to compare the results to those of the patients with diabetes mellitus (DM), to reveal factors that may drive the use of CAM, and to measure outcomes and adherence. It was also our intent to have state-of-the-art information on CAM use in our region among patients with the two diseases above. Materials and Methods: We conducted a non-interventional study using a self-developed questionnaire. It was distributed among adult patients with either epilepsy or DM who also suffered from cardiovascular consequences. A database was compiled from the anonymous questionnaires filled in voluntarily by the patients. Basic statistics were used to analyze this database. Results: A total of 227 questionnaires were filled in by 127 patients (55.9%) with epilepsy and 100 patients (44.1%) with DM. Mean age was 54.54 ± 17.33 years. Of the patients, 50.2% were male. Average body weight was 80.3 ± 17.3 kg. Of the patients, 22 (9.7%) used CAM because they believed in CAM. Two of them reported ADRs. Among the patients with epilepsy, the ratio was only 7.9% compared to 12% among those with DM. While the number of CAM users was higher among younger patients with epilepsy, it was the elderly patients with DM who tended to use CAM. Conclusion: Attention should be paid to reliance on CAM during the follow-up. Our finding that health-conscious patients tend to use CAM more often (than the general population) may indicate it is necessary to discuss CAM usage sincerely. CAMs modulating cytochrome P450 (CYP) enzymes were the most common, leading to interactions with medication used and resulting in ADRs. This shows the importance of educating patients and treating team including clinical pharmacists in this field.

Effect of timing of clopidogrel administration on 30-day clinical outcomes: 300-mg loading dose immediately after coronary stenting versus pretreatment 6 to 24 hours before stenting in a large unselected patient cohort.

BACKGROUND: The aim of our prospective multicenter Clopidogrel Registry was to evaluate the efficacy and safety of a 300-mg loading dose of clopidogrel at the time of ad hoc stenting in patients with suspected coronary artery disease who were not pretreated with clopidogrel for any reason, and to compare the 30-day clinical event rates with the outcome of patients pretreated with a loading dose of clopidogrel 6 to 24 hours before stenting. METHODS: Between March 2002 and February 2004, 4160 consecutively included patients received a 300-mg loading dose of clopidogrel immediately after (group 1, n = 2679) or 6 to 24 hours before stenting (group 2, n = 1481). RESULTS: The primary end point (triple composite end point of acute myocardial infarction, all-cause death, and urgent repeat target vessel revascularization) at 30 days occurred in 4.74% versus 2.77% in groups 1 and 2, respectively (P = .002). The secondary end point events, the stent thrombosis, occurred significantly more frequently in group 1, with a trend toward increase in incidence of death, target vessel revascularization, or need for glycoprotein IIb/IIIa antagonists during percutaneous coronary intervention. Pretreatment with clopidogrel was associated with more major bleeding (secondary safety end point) (0.41% vs 1.35% in groups 1 and 2, respectively; P = .001). CONCLUSIONS: The results of our multicenter prospective Clopidogrel Registry demonstrate lower efficacy of a 300-mg loading dose of clopidogrel at the time of stenting compared with pretreatment 6 to 24 hours before percutaneous coronary intervention on the 30-day composite clinical end point in the large unselected patient cohort, which suggests the benefit of clopidogrel pretreatment in all incoming patients with suspected significant coronary artery disease scheduled for coronary angiography.

Evaluation of laboratory methods routinely used to detect the effect of aspirin against new reference methods.

BACKGROUND: Aspirin, a commonly used antiplatelet agent, blocks platelet thromboxane A2 (TXA2) formation from arachidonic acid (AA) by acetylating platelet cyclooxygenase-1 (COX-1). Laboratory methods currently used to detect this antiplatelet effect of aspirin provide variable results. We have reported three methods that assess platelet COX-1 acetylation (inactivation) by aspirin and its direct consequences. The first and second assays use monoclonal anti-human-COX-1 antibodies that only detect acetylated (inactivated) COX-1 and active (non-acetylated) COX-1, respectively. The third method measures platelet production of TXB2 (the stable metabolite of TXA2) in vitro in response to AA. We compared the results of these three reference methods with other routinely used methods for assessing the functional consequences aspirin treatment. METHODS: 108 healthy volunteers were treated with low-dose aspirin for 7 days. On day 7 following aspirin treatment COX-1 in the platelets was fully acetylated whereas only non-acetylated COX-1 was present in the day 0 platelets. Further, TXB2 production by day 7 platelets was completely blocked. The following tests were performed on the samples obtained from study participants before and after seven days of aspirin treatment: PFA-100 closure time with collagen/epinephrine cartridge, VerifyNow (VN) Aspirin Assay, platelet aggregation and ATP secretion using AA, ADP, epinephrine and collagen as agonists. RESULTS: Comparing the pre-treatment and day 7 values, methods that use AA as platelet agonist (AA-induced platelet aggregation/secretion and VN Aspirin Assay) showed high discriminative power. In contrast, results of the other tests showed considerable overlap between day 7 and day 0 values. CONCLUSIONS: Only assays that clearly distinguish between acetylated and non-acetylated platelet COX-1 are useful for establishing the antiplatelet effect of aspirin. The other tests are not suitable for this purpose.

New direct and indirect methods for the detection of cyclooxygenase 1 acetylation by aspirin; the lack of aspirin resistance among healthy individuals.

BACKGROUND: Aspirin is widely used in the prevention of acute atherothrombotic complications. It acetylates Ser529 residue in cyclooxygenase-1 (COX-1) and prevents thromboxane A2 (TXA2) formation from arachidonic acid (AA) in platelets. Laboratory methods used for the detection of aspirin effect provide inconsistent results. METHODS: Two new methods were developed for the direct and indirect detection of COX-1 acetylation by aspirin in 108 healthy volunteers treated daily with 100mg enteric-coated aspirin for 7days. Monoclonal antibodies were raised against acetylated and non-acetylated nonapeptides corresponding to the amino acid sequence of human COX-1 525-533 residues. Using Western blotting technique the antibodies clearly distinguished between acetylated and non-acetylated COX-1 in platelet lysate. The second method measures AA-induced TXB2 production of platelets in diluted platelet rich plasma. RESULTS: No acetylated COX-1 was detected in platelets before aspirin treatment. At the same time antibodies raised against non-acetylated peptide gave intense reaction with COX-1 on the Western blot. In contrast, after 7days of aspirin treatment, with a single exception, only acetylated COX-1 could be detected in the platelet lysate. The non-responding volunteer showed full response to aspirin after controlled drug intake. In parallel experiments aspirin treatment for 7days practically completely inhibited AA-induced TXB2 production by platelets. CONCLUSIONS: Chemical ("true") aspirin resistance, if it exists, must be a rarity among healthy individuals. The new methods could be used for detecting the acetylation of COX-1 by aspirin in patients on preventive aspirin therapy and for evaluating methods routinely used for such purpose.