Nt pro anp

Peripheral blood was collected in tubes (EDTA or citrate) (Vacutainer, BD Biosciences, Heidelberg, Germany) and stored in polypropylene tubes at −80 °C until use. Samples were centrifuged at 3000× g for 10 min or, in terms of biomarker of platelet activation, platelet-poor plasma was prepared. For PTF 1 + 2, TAT (both purchased from Abbexa Ltd., Cambridge, UK); thrombin activity (purchased from ASSAYPRO LLC, St. Charles, MO, USA); P-selectin, CXCL4, CXCL7, TGF-β, sST2, galectin 3, NT-pro ANP (all purchased from R&D Systems, Minneapolis, MN, USA); and PICP, PIIINP, ICTP, PINP (all purchased from Aviva Systems Biology, San Diego, CA, USA) ELISAs/assays were performed according to the manufacturer’s instructions. The intra-assay coefficient of variation (CV) was <10%, and inter-assay was CV < 10%. FIIa activity in plasma was measured with the Human thrombin Chromogenic Activity Kit (ASSAYPRO LLC, St. Charles, MO, USA). The absorbance at 405 nm was read every 30 min for 2 h. Data were presented as change in absorbance per minute (∆A/min). Routine laboratory results (accredited Labor Berlin—Charité Vivantes GmbH, Department of Laboratory Medicine) were obtained from the patient’s medical records, and these included TNF-α (chemiluminescent immunoassay), IL-6 (electrochemiluminescence immunoassay), VWF–Ag, VWF–RCo (both latex-enhanced immunoassays), and FVIII (coagulometry).

Before analysis, with the lids tightly secured, the tubes were placed in a water bath and heated at 15–20°C. After thawing, samples were centrifuged for 15 min at 1000 g (15°C) to remove precipitates if any formed. After that, the supernatant was diluted 1 : 1 by transferring 35 μl of serum into a new tube containing 35 μl of analyzed protein-deprived serum-like buffer. As a quality control, lyophilized human serum was used. Before analysis, lyophilisates and calibration standards were dissolved in distilled water. Sample analysis was performed at 22°C.
Analysis of osteogenesis/osteolysis factor serum concentrations was carried out using commercially available, high-sensitive Luminex kits: (1) DKK-1, TNF-α, OPG, OCN, OPN, and FGF-23—EMD Millipore Corporation, and (2) NT-proANP, PCSK9, TRAIL, sRANKL, TSP-2—R&D Systems. The analyses were carried out according to the manufacturer's instructions in duplicates.