EMPs are able to activate plasminogen into plasmin. (A) Plot of plasmin generated versus time at varying EMP amounts per 50 μL/well (○ = 106; □ = 105; ◇ = 104; x = 103; +‖= control without EMPs) and fixed final concentrations of plasminogen (1 μM) and a plasmin-selective chromogenic substrate (0.75 mM). Representative graph of 4 independent experiments. (B) Similar experiment as in panel A expressed as change in absorbance at 405 nm per minute versus EMP amount per well (S: EMP last washing supernatant used as control). (C) Plasmin generated at varying plasminogen concentrations (0-5 μM) and a fixed amount (2.105/50 uL) of EMPs was detected with a chromogenic substrate as in panel A. Raw data (○) were fitted to the Michaelis-Menten equation allowing calculation of nonspecific activity (◇) and a Km = 0.122 μM for specific plamin generation (□). (D) Effect of various inhibitors on the generation of plasmin by EMPs (2.105/50 μL) at 0.5 μM plasminogen (α2AP = α2-antiplasmin; ϵ-ACA = ϵ-amino-caproïc acid; CPB = carboxypeptidase B; antibodies to uPA and tPA αvβ3 and α-enolase as compared with an isotype control IgG1). Results are the mean ± SD of 3 independent experiments.
