Aernoud Fiolet

271 Colchicine reduces extracellular vesicle NLRP3 inflammasome protein levels Platelet activation Healthy controls were recruited through the Minidonor service, an institutional ReviewBoard approved biobank at theUniversityMedical CenterUtrecht (biobank protocol number 18-774). All donors gave written informed consent in accordance with the declaration of Helsinki. Whole blood was collected in vacutainer tubes containing 1/10th volume trisodium citrate (104 mM) and was subsequently centrifuged at 160g for 15 minutes with break 1 to obtain platelet rich plasma (PRP). PRP was transferred to fresh tubes and subsequently incubated with either 10 µM colchicine or an equal volume of placebo for 30 minutes at 37°C. Next, PRP samples were transferred to an aggregometer (Chrono-log, Havertown, PA, USA) and stimulated with 25 µM Thrombin Receptor Activating Peptide-6 (TRAP-6; SFLLRN; Bachem, Bubendorf, Switzerland) to allow full aggregation. Platelet poor plasma (obtained by centrifugation at 2000g for 10 minutes) was used as blanco. Platelet aggregation was monitored for 30 minutes to allow release of EVs. Finally, supernatant was collected, centrifuged twice at 2000g for 15 minutes to remove platelet aggregates and plasma was stored at -80°C for vesicle isolation. Study design and population We conducted a pre-defined biomarker substudy of the second Low-Dose Colchicine for the secondary prevention of cardiovascular events (LoDoCo2) trial (ACTRN12614000093684). The methods and main results have been published before. 5,20 In short, patients were eligible for participation if they were between 35 and 82 years old, had evidence of coronary disease and were clinically stable. All patients entered an open label run-in phase and, if they perceived no side effects of the drug and were willing to continue, were randomized to colchicine 0.5mg once daily or placebo, on top of regular care. Randomization was performed in a double-blind manner with the use of a computerized algorithm. Main exclusion criteria were an impaired renal function, defined as an estimated glomerular filtration rate (eGFR<50 ml/min/1,73m2) or a serum creatinine >150 µmol/L, the necessity to take colchicine for any other indication, or concomitant drug use of strong CYP3A4 inhibiting drugs (i.e. verapamil, azithromycin, clarithromycin). 20 Participants of this substudy were included in three random participating Dutch hospitals with laboratory facilities (Northwest Clinics, Alkmaar; Meander Medical Center, Amersfoort and Treant Hospital, Emmen, The Netherlands). The study protocol was approved by a medical ethics committee (MEC-U, Nieuwegein, The Netherlands). All participants provided written informed consent prior to