Dte200

Arterial blood was collected after induction of anesthesia before onset of CPB (pre-CPB), after initiation of CPB (CPB), 1 h after weaning from CPB (post-CPB), and 24 h and 72 h following surgery (+ 24 h and +  72 h, respectively) and immediately centrifuged at 4.000 G for 10 min at 4 °C. Plasma supernatant was centrifuged for another 5 min at 12.000g at 4 °C to obtain platelet-free plasma. Platelet-free plasma was snap frozen in liquid nitrogen and stored at − 80 °C. Plasma concentrations of angiopoietin-1 (DANG10), angiopoietin-2 (DANG20), and soluble Tie2 (DTE200, R&D Systems, Biotechne, Minneapolis, MN, USA) were measured using commercially available enzyme-linked immunosorbent assays in accordance with the manufacturer’s instructions.

Peripheral blood samples were collected from all participants at recruitment as part of standard care and serum was processed by the Townsville University Hospital Pathology Department (Pathology Queensland) prior to storing at − 80 °C for later analysis. Serum angpt-1, matrix-metalloproteinase-9 (MMP-9) and Tie-2 concentrations were measured using commercially available ELISAs according to manufacturer’s directions (DANG 10, DMP900 and DTE200, respectively, R&D Systems, Minneapolis, USA). Serum angiopoietin-2 (angpt-2), and vascular endothelial growth factor (VEGF)-A, -C and -D concentrations were measured using the MilliPLEX MAP Human Angiogenesis/Growth Factor Magnetic Bead Panel—Cancer Multiplex Assay kit (HAGP1MAG-12 K, Merck, Australia), using the MagPIX platform, and analyses were conducted by a researcher blinded to patient diagnosis. Samples in which the assessed biomarker fell outside of the detectable limits of the test were excluded from analysis (detailed in Supplement 1). Biomarker analysis was conducted independently of patient care and had no potential to influence clinical outcome.

Commercially available enzyme-linked immunoassays (ELISAs) were used to determine the plasma levels of soluble EPCR (MyBioSource; MBS703733; sensitivity: 1.95 ng/mL), soluble E-selectin (R&D Systems; DSLE00; sensitivity: 0.027 pg/mL), IL1β (R&D Systems; DLB50; sensitivity: 1 pg/mL), TGFβ1 (R&D Systems; DB100B; minimal detectable dose: 1.7–15.4 pg/mL), TNFα (R&D Systems; DTA00D; sensitivity: 6.23 pg/mL), Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17; Thermo Scientific; EDADAM17; sensitivity: 70 pg/mL), soluble Tie2 (R&D Systems; DTE200; minimal detectable dose: 0.001–0.066 ng/mL), and VEGF (R&D Systems; DVE00; minimal detectable dose: 5–9 pg/mL). Plasma was used undiluted (sEPCR, TNFα, VEGF) or diluted 1:2 (ADAM17), 1:10 (sE-selectin), or 1:20 (TGFβ1), respectively. Latent TGFβ1 was activated, according to the manufacturer’s instructions, immediately before the analysis. To eliminate interassay variability, plasma aliquots were thawed and assayed on the same day. All analyses were performed in duplicate. Results were calculated from a standard curve created using computer software capable of generating two-dimensional graphs using logarithmic scales (log–log), or four-parameter logistic (4-PL) curve fit for CRP values.