Platelet Isoprostane Overproduction in Diabetic Patients Treated With Aspirin

Roberto Cangemi Pasquale Pignatelli Roberto Carnevale Carmen Nigro Marco Proietti Francesco Angelico Davide Lauro Stefania Basili Francesco Violi Aspirin modestly influences cardiovascular events in patients with type 2 diabetes mellitus (T2DM), but the reason is unclear. The aim of the study was to determine whether in T2DM patients aspirin enhances platelet isoprostanes, which are eicosanoids with proaggregating properties derived from arachidonic acid oxidation by platelet NOX2, the catalytic subunit of reduced NAD phosphate oxidase. A cross-sectional study was performed comparing T2DM patients, treated (n = 50) or not treated (n = 50) with 100 mg/day aspirin, with 100 nondiabetic patients, matched for age, sex, atherosclerosis risk factors, and aspirin treatment. A short-term (7 days) treatment with 100 mg/day aspirin also was performed in 36 aspirin-free diabetic and nondiabetic patients. Higher platelet recruitment, platelet isoprostane, and NOX2 activation was found in diabetic versus nondiabetic patients and in aspirin-treated diabetic patients versus nontreated patients (P , 0.001). Platelet thromboxane (Tx) A2 (P , 0.001) was inhibited in all aspirin-treated patients. In the interventional study, aspirin similarly inhibited platelet TxA2 in diabetic and nondiabetic patients (P , 0.001). Platelet recruitment, isoprostane levels, and NOX2 activation showed a parallel increase in diabetic patients (P , 0.001) and no changes in nondiabetic patients. These findings suggest that in aspirin-treated diabetic patients, oxidative stressmediated platelet isoprostane overproduction is associated with enhanced platelet recruitment, an effect that mitigates aspirinmediated TxA2 inhibition. Diabetes 61:1626-1632, 2012 - A type 2 diabetes mellitus (T2DM). Thus, patients ccelerated atherosclerosis is a typical feature of with T2DM have a two- to fourfold increased risk of cardiovascular diseases (coronary artery disease) and a two- to sixfold increased risk of stroke (13). Platelets play a major role in the etiology of atherosclerotic disease, as shown by the significant decrease of cardiovascular events in patients treated with aspirin, an inhibitor of cyclooxygenase (COX1) that prevents platelet thromboxane (Tx) A2 formation (4,5). Platelet TxA2 overproduction, combined with a significant decrease after aspirin administration, has been demonstrated in diabetic patients (6). Despite this, interventional trials with aspirin in diabetic patients failed to show a beneficial effect in primary prevention; reasons for this lack of response still are uncertain (7). Isoprostanes are a family of eicosanoids derived from arachidonic acid interaction with reactive oxidant species (ROS). Thus, ROS generated by NOX2, the catalytic subunit of reduced NAD phosphate (NADPH) oxidase, play a crucial role in platelet isoprostane formation (8). In contrast to TxA2, isoprostanes are chemically stable compounds that serve to propagate platelet activation, amplifying platelet response to common agonists via glycoprotein (Gp)IIb/IIIa activation (9). Accordingly, patients with hereditary deficiency of NOX2 showed impaired isoprostane formation and GpIIb/IIIa activation, as well as a subnormal propagation of platelet thrombus (9). It previously has been demonstrated that COX1 inhibition determines a shift in arachidonic acid metabolism toward other pathways, such as the lipooxygenase system (10). We speculated that COX1 inhibition also could be associated with an increased conversion of arachidonic acid to isoprostanes in platelets. The increase of platelet isoprostanes would counterbalance the inhibition of TxA2, therefore hampering the antiplatelet effect of aspirin. To explore this hypothesis, we performed a cross-sectional study comparing the behavior of platelet isoprostanes and TxA2 and their interplay with platelet NOX2 in diabetic and nondiabetic patients treated or not with aspirin. Platelet activation tests, including arachidonic acidinduced platelet aggregation, which is dependent upon TxA2 formation (11), and platelet recruitment, which is dependent upon ROS and isoprostane formation (9), were determined. Analysis of these variables was repeated in a prospective, short-term study of diabetic and nondiabetic patients treated for 7 days with low-dose aspirin. RESEARCH DESIGN AND METHODS Study design Cross-sectional study. The study was performed in consecutive T2DM patients attending our metabolic outpatient clinic who were taking (n = 50) or not taking (n = 50) low-dose (100 mg/day) aspirin. T2DM was diagnosed ac cording to the American Diabetes Association definition (12). As a control group, we selected nondiabetic outpatients taking (n = 50) or not taking (n = 50) low-dose aspirin who were matched to the diabetic group in terms of age, sex, and history of vascular disease. Low-dose aspirin treatment was defined as a self-reported daily intake of 100 mg acetylsalicylic acid at least in the previous month. Exclusion criteria were 1) recent history (,3 months) of acute vascular events, 2) clinical diagnosis of type 1 diabetes (diagnosis of diabetes and insulin use before the age of 35 years), 3) serum creatinine level .2.5 mg/dL, 4) active infection or malignancy, 5) cardiac arrhythmia or congestive heart failure, and 5) use of nonsteroidal anti-inflammatory drugs, vitamin supplements, or other antiplatelet drugs, such as clopidogrel, in the previous 30 days. All participants provided written informed consent. The local ethical committee approved the study protocol (approval no. Prot. 403/09-Rif. 1621/07.05.09). Diabetic patients received different antidiabetes treatments: metformin (n = 57), subcutaneous insulin (n = 25), sulfonylureas (n = 14), glinides (n = 6), glitazones (n = 3), and dipeptidyl peptidase-4 inhibitor (n = 16). Interventional study. We tested the effect of short-term treatment with 100 mg/day aspirin in diabetic (n = 18) and nondiabetic (n = 18) patients, not currently under aspirin treatment and with no clinical history of vascular diseases, who were matched for sex, age, and atherosclerotic risk factors. Aspirin was given after dinner between 8:00 and 8:30 P.M., and adherence was assessed by the pill-count method. Blood samples were collected before aspirin ingestion and after 3 and 7 days of treatment. The study was registered in August 2010 at clinicaltrials.gov (clinical trial reg. no. NCT01250340). Laboratory analyses. All materials were from Sigma-Aldrich, unless otherwise specified. Blood analyses were performed in a blinded manner. All blood samples were taken after a 12-h fast. Between 8:00 and 9:00 A.M., subjects underwent routine biochemical evaluations, including fasting total cholesterol and glucose. Serum and platelet-poor plasma sampling. After overnight fasting (12 h) and supine rest for at least 10 min, blood samples were taken into tubes containing either 3.8% sodium citrate (ratio 9:1) or anticoagulant-free tubes and centrifuged at 300g (...truncated)