Study Design

An explorative interventional trial investigating the effect of DPI on monocyte-driven inflammation was initiated by and conducted in the Radboud University Medical Center (Nijmegen, the Netherlands). Approval of the Medical Research Ethics Committee Oost-Nederland (file number 2021-13291) and the local institutional review board was obtained. This study was conducted in accordance with the latest revision of the Helsinki Declaration of 1964 and Good Clinical Practice regulations and was registered at ClinicalTrials.gov on January 27, 2022 (registration number NCT05210725). Written informed consent was obtained from all participants.

Participants

Patients with stable CAD and an indication for single antiplatelet therapy according to the leading international guidelines [9] were recruited at our outpatient clinic. Patients were considered eligible when they were (1) currently treated with aspirin (80–100 mg once daily) monotherapy, (2) at high risk of developing recurrent vascular events based on a SMART risk score ≥ 20%, (3) at least 1 year after myocardial infarction or suffering from multivessel CAD, and (4) aged ≥ 16 years. Exclusion criteria were concomitant use of immunosuppressant/anti-inflammatory therapies and known contraindications to rivaroxaban including hypersensitivity, at significant risk for major bleeding, severe hepatic disease, severe kidney failure (estimated glomerular filtration rate < 15 ml/min or requiring dialysis), severe heart failure (ejection fraction < 30% or New York Heart Association class III or IV symptoms), or concomitant treatment with medication with a strong pharmacokinetic interaction with rivaroxaban.

Procedures

Eligible patients visited the hospital three times, once at baseline and twice during follow-up (after 4 and 12 weeks of DPI treatment, respectively).

Baseline

At baseline (T0), written informed consent was obtained. Data regarding demographics, lifestyle, medical history, and medication use (including recent vaccinations, at most 1 month before screening) were recorded using standardized case report forms and electronic patient files. Measurement of blood pressure, height, weight, and hip-waist circumference took place. Venous blood samples were collected. Finally, participants were prescribed rivaroxaban 2.5 mg twice daily for a 12-week period and follow-up visits were scheduled.

Follow-up

Patients were scheduled for follow-up after 4 weeks (T1) and 12 (T2) weeks of DPI treatment. During follow-up visits, adverse events were reported, and medication adherence was evaluated by interview. If medication adherence was below 80%, rivaroxaban was discontinued, and participants were excluded from further study participation. Venous blood samples were collected.

Whole Blood Stimulation

A 100-µl sample of whole blood was added to each round-bottomed well of a 96-well plate along with 400 µl RPMI 1640 Medium (“Dutch modification” containing 11 mM glucose; Thermo-Fischer, Waltham, MA, USA) supplemented with 10 μg/ml gentamicin, 2 mM GlutaMAX and 1 mM pyruvate, or 400 µl culture medium supplemented with 12.5 ng/ml for a final concentration of 10 ng/ml of Escherichia coli lipopolysaccharide (LPS) (serotype 055:B5 Sigma-Aldrich, St. Louis, MO). Blood was incubated at 37 °C and 5% CO2 for 24 h, after which supernatants were collected and stored at − 80 °C until cytokine assessment.

Peripheral Blood Mononuclear Cell Isolation and Stimulation

Peripheral blood mononuclear cells (PBMCs) were isolated from blood of study participants by dilution in phosphate-buffered saline (PBS) and density-gradient centrifugation using Ficoll-Paque (GE healthcare, Chicago, IL, USA). PBMCs were washed three times with cold PBS and resuspended in RPMI 1640 medium. For stimulations experiments, 5 × 105 PBMCs were seeded in each round-bottomed well of a 96-well plate (Corning, NY, USA) and stimulated for 24 h with either culture medium or culture medium supplemented with 10 ng/ml of E. coli LPS (serotype 055:B5 Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO2. After incubation for 24 h, supernatants were stored after plate centrifugation at − 80 °C until cytokine assessment.

Cytokine Measurements

Levels of tumor necrosis alpha (TNFα) and interleukin-6 (IL-6) were measured in supernatants using the IL-6 and TNFα DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA).

Immune Cell Measurements via Sysmex

Complete blood counts were performed on EDTA whole blood and PBMC fractions after Ficoll isolation, on the Sysmex XN-450 hematology analyzer (Sysmex America Inc, Lincolnshire, IL, USA).

Olink Analysis in Patients with PAD

An additional cohort of 33 patients with PAD, a subset of the DUAL-PAD study (NCT04218656) [10], were included and assessed for circulating plasma protein expression. Circulating plasma protein expression both at baseline and after 3 months of DPI was assessed using the commercially available multiplex proximity extension assay from Olink® Proteomics AB (Uppsala, Sweden). The Target 96 Inflammation Panel was run in which 96 inflammatory proteins were measured.

Statistical Analysis

Since this is exploratory research, detailed sample size calculation is not appropriate. We aimed to include 15–20 patients based on the average LPS-induced TNFα production in patients with symptomatic atherosclerosis [11], and an expected 20% increase in cytokine production capacity from baseline (aspirin) to 3 months of DPI (aspirin with rivaroxaban). Statistical testing was performed by using the Wilcoxon matched pairs signed rank test. For volcano plots, data were analyzed by Mann–Whitney test; the Benjamini–Hochberg procedure was employed to correct multiple testing errors. False discovery rate (FDR)-adjusted p values smaller than 0.05 were considered statistically significant. Statistical analysis and data visualization were performed with Graphpad Prism v9.3.1 (GraphPad software, La Jolla, CA) or R/Bioconductor (https://www.R-project.org/).