Sodium citrate

Blood was drawn from healthy volunteers after the approval of the Ethics Committee of Ulm University (ethic decision number 459/18) and after obtaining written informed consent from the blood donors. Subjects were healthy males or females aged between 18 and 35 years without any signs of infection or other medical problems and under no medication. Human blood was drawn by peripheral venous puncture as described by the World Health Organization guidelines in phlebotomy [44 ] in monovettes containing 3.2% sodium citrate (Sarstedt, Nürnbrecht, Germany).
Neutrophils were isolated from human blood using the Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation and subsequent dextran sedimentation as described previously [14 (link),15 (link),17 (link),18 (link)]. Following hypotonic lysis of the remaining erythrocytes and resuspension in Hank’s balanced salt solution with calcium and magnesium (HBSS, Gibco Thermo Fisher, Darmstadt, Germany) that was titrated to a pH of 7.3, the cell concentration was adjusted to 2 × 10⁶ per milliliter.

Blood samples were collected within the first hour of patient admission (before coronary angiography) or during the visit of volunteers without CVD. All participants provided written informed consent prior to the collection of biological samples. The utilization of blood samples for research purposes was approved by the by the Interuniversity Committee of Ethics of Moscow State University of Medicine and Dentistry. We collected peripheral blood by intravenous withdrawal in vacuum tubes with sodium citrate as anticoagulant (Sarstedt, Nuembrecht, Germany, cat#05.1071.001). The first tube with blood was discarded because it contained platelets activated during venepuncture and skin fibroblasts. We prepared platelet-poor plasma less than 1 h after blood collection by double centrifugation at 3000 g for 15 min. Then, plasma was aliquoted to 300 μl, frozen at − 80 °C, and shipped on dry ice to the Section on Intracellular Interactions of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (NICHD/NIH/DHHS) (Bethesda, MD, USA) for cytokine measurement.

To measure coagulation activity in terms of the prothrombotic factors D-dimer and fibrinogen, venous blood was drawn into polypropylene tubes containing 3.8% sodium citrate (Sarstedt, Nümbrecht, Germany). Citrate tubes were immediately centrifuged for 20 min at 4°C at 2,000 g, and plasma was pipetted into aliquots. D-dimer was analyzed using a particle-enhanced immunoturbidimetric assay for the quantitative determination of D-dimers in human plasma (INNOVANCE® D-Dimer, Siemens Healthcare GmbH, Erlangen, Germany) on a Sysmex CS-5100 (Sysmex Europe, Norderstedt, Germany). Plasma fibrinogen levels were determined by a routine clotting assay applying standard quality procedures following the Clauss method. The intra- and inter-assay CVs were ≤ 7.9%.