H d val leu lys afc

Plasmin activity assays were performed as previously described with minor modifications^{18 (link)}. E. faecalis were grown overnight in tryptone yeast media. OD₆₀₀ was normalized by dilution to 0.1, and samples were diluted 10x in the final reaction. Bacteria were incubated with 250nM human glu-plasminogen (Haematologic technologies) with or without 10mM TXA (Fisher) for two hours at 37°C. A final concentration of 4nM uPA or pro-uPA (Biovision) were added and incubation proceeded for 20 minutes. A final concentration of 6μM plasmin-specific fluorogenic substrate (H-D-Val-Leu-Lys-AFC, AnaSpec) was added immediately prior to a 30-minute kinetic fluorescent read at 380/500nm. For assays of uPA activity, PLG was omitted and a substrate specific to uPA (Z-Gly-Gly-Arg-AMC, Bachem) was utilized. When assays were performed in human plasma, fluorescence was read for 120 minutes total. Plasmin or uPA activity is expressed as initial reaction velocity calculated from change in fluorescence over time during the initial phase of the reaction, when pseudo-first order kinetics determine the rate.

To measure plasmin activation in a clot-dependent manner using bacterial plasmin activator streptokinase (SK), patient plasma samples and control pooled plasma (George King Bio-Medical Inc., Overland Park, KS) were diluted 1:5, 1:10, and 1:20 in HEPES buffer. Increasing concentrations of TXA (Pfizer, New York, NY) were added to control plasma or purified plasminogen (Haemtech, Essex Junction, VT). Diluted plasma (or purified plasminogen) and fluorogenic substrate (H-D-Val-Leu-Lys-AFC, Anaspec, Fremont, CA) were added in triplicate to a 96-well plate. Plasmin activation was initiated by the addition of 0.5 U/μL bacterial streptokinase (SK) (MilliporeSigma, Burlington, MA) and fluorescence was measured every 30 s for 1 h using a Synergy 2 plate reader (Biotek, Winooski, VT). Rates of plasmin generation were calculated based on change in fluorescence over time and were reported as a percent of control pooled plasma [9 (link)].