Thromboxane B2

The thromboxane biosynthesis study was undertaken at 28 UK centres. Participants provided 6-ml urine samples at trial entry/baseline (off aspirin), after the 8-week run-in period of aspirin 100 mg daily (24 h after the previous aspirin dose), and a third sample between 3 and 6 months after randomisation to blinded treatment (aspirin 100 mg, aspirin 300 mg or matched placebo) (Fig. 1) using a minimisation algorithm with a random element.Fig. 1

Urine samples were collected at trial entry (off aspirin), after an 8-week run-in period of aspirin 100 mg daily for all participants and 3–6 months after random allocation to aspirin 100 mg, aspirin 300 mg or matched placebo daily.

End of run-in, and post-randomisation, urine samples were only included in this analysis if the participant had remained on allocated treatment. Adherence was assessed by participant self-reporting and review of used blister packs/completion of diary cards at the end of the run-in period. Based on these assessments participants were included if they had taken 6–7 tablets per week and had not discontinued treatment prior to the urine sample being obtained.
Urine samples were sent by post from trial centres to one of two trial biobanks in the UK within 24 h of collection. Aliquoted into 2-ml cryo-vials, frozen at −20 °C or below and transferred on dry ice to the Institute of Pharmacology at the Catholic University School of Medicine (Rome, Italy). The extraction procedure was performed on 1 ml urine samples as described in detail in Supplementary Appendix S1 and [36 (link)]. U-TXM was measured with a standard Enzyme-Linked Immunosorbent Assay (ELISA) [36 (link)–38 (link)]; 96-well plates were coated with commercial monoclonal anti-rabbit IgG antibodies (Cayman Chemical, Ann Arbor, MI, USA) and U-TXM assessed with a standard acetylcholinesterase ELISA immunometric method using a specific rabbit polyclonal antibody. The cross-reactivity of the anti-11-dehydro-TXB₂ antibody against other prostanoids that can be measured in urine, namely PGE_2, TXB₂, 6-keto PGF_1alpha, and the isoprostane 8-iso-PGF_2alpha was <0.05% [39 (link)]. We also tested cross-reactivity with 2,3-dinor-TXB2 which was <1.5% (data not shown). Results were expressed per milligram of urinary creatinine measured with a commercial kit (Creatinine Colorimetric Detection Kit; Enzo Life Sciences, Farmingdale, NY, USA). The authors involved in U-TXM measurements (DH, GP and BR) were blinded to all clinical details and aspirin/placebo allocation.

A validated commercially available ELISA Kit for Human Thromboxane B₂ measurement was used. First, the reagents were brought to room temperature. Fifty microliters of the standard liquid belonging to the kit itself was poured into each tube, then 100 microliters of the standard solution was added to the first tube, and 100 microliters was removed from the first tube and added to the second tube. Fifty microliters was taken from the second tube and added to the third tube and this process continued until the end. After removing the blank, 40 microliters of the sample was added to the sample tubes and mixed gently. Then, the solutions were incubated at 37 °C for 30 min. After incubation, the washing solution was diluted 30 times. In the next step, a washing solution was added to the samples and they were incubated for the second time for 30 min at the same temperature. After the incubation, the washing step was performed again. In the next step, 50 U of chromogen A and chromogen B were added to each tube except the blank, and the tubes were prevented from light exposure for 30 min. Then, 50 microliters of stopping solution was added and after 15 min, the optical absorption of the solutions was measured using a spectrophotometer at a wavelength of 450 nm. The measurement range was 1000–500 pg/mL.

The anticoagulated blood sample was centrifuged at 220×g for 20 min, and the platelet-rich plasma (PRP) was collected and transferred to a new tube for centrifugation at 480×g for 20 min at RT. The supernatant (plasma) was collected and stored at − 80 °C for further ELISA assay to detect the concentration of TXB2 (ADI-900-002, Enzo Life Sciences). Then, suspend the pellet (pure platelet) with 10 volumes of Thermo Scientific M-PER Mammalian Protein Extraction Reagent containing protease/phosphatase inhibitor Cocktail (Cat:5872, Cell Signal). The suspension was subjected to three cycles of sonication of 5 s each and centrifuged at 13,000×g at 4 °C for 10 min. 50 µg total protein per lane was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated in blocking buffer (5% w/v nonfat dry milk in Tris-buffered saline, 0.05% Tween-20 [TBS-T]) for 1 h at RT, and then incubated with primary antibodies overnight at 4 °C. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody at RT for 1 h.