The turbidity assay was performed as described previously
25 (link)
26 (link)
at 37°C in a 96-well plate (Corning) on a SpectraMax Paradigm plate reader (Molecular Devices, Sunnyvale, California, United States). Assays were performed in quadruplicate. Turbidity was monitored once a minute at a 350 nm wavelength and calculated as the mean value (
n = 4) in a volume of 100 μL HBS (HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-buffered saline solution) at pH 7.4.
For fibrin gel formation, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl
2(Wako Pure Chemical), 0.01% Tween 80 (Sigma-Aldrich), and 0.5 NIH unit/mL thrombin (Sigma-Aldrich) were added to 10 μg/mL 1101 mAb or 3 U/mL anti-thrombin (SLS Behring K) as antithrombin III (AT III).
For the lysis of the fibrin gel, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl
2(Wako), 0.01% Tween 80 (Sigma-Aldrich), 0.5 NIH unit/mL thrombin (Sigma-Aldrich), 0.2 μM PLG (Enzyme Research Laboratories), and 0.3 nM tPA (Alteplase, Kyowa Hakko Kirin) were added to 10 μg/mL 1101 mAb or a mixture of 0.10 μM α2-PI (Hematologic Technologies) and 2.0 ng/mL PAI-1 (ProSpec), and used as a negative control.
