Mpai kt

At the time of sacrifice, blood was obtained by cardiac puncture with a heparinized syringe, and then centrifuged at 5000 rpm for 5 minutes. Plasma was isolated and stored at −80°C until time of assay. The total cholesterol levels were determined using a commercial colorimetric assay (T-Cho, Wako Chemicals). A PAI-1 ELISA kit (MPAI KT, Molecular Innovations) was utilized to measure plasma levels of total PAI-1. D-dimer (D2D, USCN Life Science Inc.), thrombin-antithrombin (TAT) complexes (IRAPKT175, Innovative Research) and fibrinogen levels (MFBGNKT, Molecular Innovations) were measured using commercially available ELISA kits.

Total cholesterol was quantified in the murine plasma using an enzymatic determination (Pointe Scientific, Cat# C7510). The lipoprotein profile of the murine plasma samples was assessed running a fast-phase liquid chromatography (FPLC), in brief 100 µL of plasma (from one animal or a pool of several samples of the same conditions) were loaded to a Superose 6 column (GE HealthCare, Cat# 29-0915-96) at a flow rate of 0.5 mL/min in running buffer (0.15 M NaCl, 0.01 M Na2HPO4 and 1 mM EDTA). Fractions were collected every minute for 41 min and analyzed for cholesterol concentrations as described above. Murine plasma PCSK9 levels were determined using an ELISA kit (MBL International, Cat# CY-8078). Murine PAI-1 levels were quantified using an ELISA kit (Molecular Innovations, Cat# MPAIKT-TOT). Murine PAI-1 activity levels were quantified by ELISA (Molecular Innovations, Cat# MPAIKT). Human PAI-1 (Molecular Innovations, Cat# HPAIKT-TOT), PCSK9 levels (R&D Systems, Cat# DPC900), and FGF21 (R&D, DF2100) were quantified by ELISA.