Soluble p selectin

Fasting venous blood was drawn during anticoagulant therapy with direct oral anticoagulants (DOAC) and time since the last dose of DOAC was noted. Plasma samples from patients, in whom plasma DOAC levels were > 30 ng/ml, were treated with the DOAC-Stop (Haematex Research, Sydney, Australia) prior to coagulation assessment to eliminate potential residual anticoagulant effects. Blood was drawn from the antecubital vein into citrated tubes and centrifuged at 2500 g at 20 °C for 20 min or into serum tubes and centrifuged at 1600 g at 4 °C for 10 min. Aliquots were stored at -80 °C. Blood cell count, creatinine, fibrinogen, lipid profile, glucose, and CRP were assayed by routine laboratory techniques. ELISA kits were used to quantify citrullinated histone H3 (citH3), as a circulating stable NETosis marker (Cayman Chemical, Ann Arbor, MI, USA), plasminogen activator inhibitor-1 (PAI-1) antigen, thrombin activatable fibrinolysis inhibitor (TAFI) (both Hyphen-Biomed, Neuville-Sur-Oise, France), soluble P-selectin (R&D, Minneapolis, MN, USA), plasma 3-nitrotyrosine, a marker of oxidative stress (OxiSelect, Cell Biolabs Inc., San Diego, USA), and plasma NAP-2 (Invitrogen, Thermo Fisher, Waltham, MA, USA).

At the end of studies, mice were deprived of any food for 8 hours prior to sacrifice. After anesthesia with isoflurane, blood samples were withdrawn from the left femoral artery. The numbers of White Blood Cells (WBC), neutrophils, lymphocytes, and platelets were measured by complete blood count. The fasting glucose level was then measured using a hand-held Accucheck glucometer (Roche Diagnostics, Indianapolis, IN, USA). The plasma levels of insulin (Shibayagi, Gunma, Japan), leptin (R&D Systems, Minneapolis, MN, USA), soluble P-selectin (R&D Systems, Minneapolis, MN, USA), TGF-β1 (R&D Systems, Minneapolis, MN, USA), and glutamine (Abcam, Cambridge, MA, USA) were determined using enzyme immunosorbent assay (ELISA) kits, following the procedures provided by the respective manufacturers. Insulin resistance was evaluated according to the Homeostasis Model Assessment (HOMA) [32 (link)]. The HOMA Insulin Resistance (HOMA-IR) index was calculated as [fasting insulin (μU/mL) × fasting glucose (mmol/L)]/22.5.