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, Roberto Cangemi, Pasquale Pignatelli, Roberto Carnevale, Carmen Nigro, Marco Proietti, Francesco Angelico, Davide Lauro, Stefania Basili, Francesco Violi

Abstract

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) A(2) (P < 0.001) was inhibited in all aspirin-treated patients. In the interventional study, aspirin similarly inhibited platelet TxA(2) 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 stress-mediated platelet isoprostane overproduction is associated with enhanced platelet recruitment, an effect that mitigates aspirin-mediated TxA(2) inhibition.

Trial registration: ClinicalTrials.gov NCT01250340.

Figures

FIG. 1.
FIG. 1.
TxB2 (A) and isoprostane formation (B), platelet recruitment (REC%) (C), and sNOX2-dp (D) from arachidonic acid–activated platelets in nondiabetic and diabetic patients taking (ASA+) or not (ASA−) 100 mg acetylsalicylic acid daily in the previous month. *P < 0.001.
FIG. 2.
FIG. 2.
TxB2 (A) and isoprostane (B) formation, sNOX2-dp release (C), and ROS production (D) from arachidonic acid-activated platelets in nondiabetic and diabetic patients at baseline and after 3 and 7 days of aspirin (100 mg) treatment. E and F: Platelet recruitment (REC%) in diabetic and nondiabetic patients at baseline and after 3 and 7 days of low-dose aspirin treatment in the presence or not of SQ29548 (0.1 μmol/L). *P < 0.001.
FIG. 3.
FIG. 3.
GpIIb/IIIa activation in platelets treated with a scalar dose of 8-iso-PGF2α (25–450 pmol/L) treated or not with SQ29548 (0.1 μmol/L), ADP (0.01–10 μmol/L) treated or not with 8-iso-PGF2α (300 pmol/L), and TRAP (0.01–10 μmol/L) (values are mean of experiments performed in five healthy subjects). *P < 0.001.
FIG. 4.
FIG. 4.
Platelet sizing and leukocyte contamination in the top 75% of the PRP in nondiabetic patients (A) and in diabetic patients (B). Evaluation of TxB2 (C) and isoprostanes (D) production in the stratified two portions of the top 75% of the PRP (50% upper phase, 50% bottom phase; up and down, respectively). Values are means of experiments performed in five nondiabetic patients and five diabetic patients. *P < 0.001.

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