Recombinant human upa

Human fibrinogen (plasminogen, von Willebrand factor, and fibronectin-depleted) and human zymogens Glu-plasminogen, α-thrombin, prothrombin, factor X zymogen, and activated factor Xa (all >95% purity) were purchased from Enzyme Research Laboratories, South Bend, IN, USA. Human plasmin and tranexamic acid (TXA) were purchased from MilliporeSigma, Burlington, MA, USA. Recombinant human pro-uPA zymogen [70 (link)] was kindly provided M. Ploug (Finsen Laboratory, Copenhagen, Denmark). Recombinant human uPA and recombinant mouse testisin proteins were from R&D Systems, Minneapolis, MN, USA. Fluorogenic substrates for thrombin (thrombin substrate III, Bz-FVR-AMC), FXa (Boc-IEGR-AMC [36 (link)]), uPA (Glt-GR-AMC [36 (link)]), plasmin (Boc-EEK-AMC [71 (link)]), and trypsin-like (Boc-QAR-AMC) proteases were from BACHEM, Bubendorf, Switzerland. Rivaroxaban (BAY 59-7939) was from Thermo Fisher Scientific, Waltham, MA, USA. Antibodies used were mouse anti-testisin antibody (MAbD9.1) [12 (link)], mouse anti-uPA antibody (IM15L, MilliporeSigma, Burlington, MA, USA), and rabbit anti-β-tubulin antibody (Santa-Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies were goat anti-mouse-HRP, mouse anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA), and donkey anti-sheep-HRP (R&D Systems, Minneapolis, MN, USA).