Pai 1

The interaction of tPA (tPA‐WT or tPA‐Y471H) with plasminogen (Sigma–Aldrich) and PAI‐1 (Sino Biological) was analyzed in real time by SPR using an Open SPR (Nicoya).⁴⁰ Briefly, the ligand (tPA‐WT or tPA‐ Y471H, 20 μg/mL) was immobilized onto COOH‐sensor chips (Nicoya) by EDC/NHS chemistry. Analytes serially diluted in buffer (140 mM NaCl,10 mM N‐2‐hydroxyethylpiperazine‐N9‐2‐ethanesulfoic acid, pH 7.4) were then injected and captured at a flow rate of 20 μL/min for 240 s. Kinetic parameters for the binding reactions were calculated and analyzed using Trace Drawer software (Ridgeview Instruments AB).

Chemotaxis experiments were performed in a chemotaxis slide (Ibidi, Martinsried, Germany). Microscopy was performed at 37°C and 5% CO₂ on an inverted microscope Eclipse Ti (Nikon, Duesseldorf, Germany), a 4 x objective and a CCD camera. Images were obtained over a total time of 20 hours every ten minutes.
HUVECs (Human umbilical vein endothelial cells from Promocell, Heidelberg, Germany) were cultured in growth medium (ECGM, Promocell, Heidelberg, Germany) containing 10% fetal calf serum. After reaching confluency, cells were trypsinised, embedded into 30% Matrigel (Corning, Amsterdam, Netherlands), diluted with basal medium (ECBM, Promocell, Heidelberg, Germany) and finally filled into the chemotaxis chambers at a density of 3 x 10⁶ cells/mL. ECGM was used as attractant-free medium, while ECGM supplemented with 30 ng/mL recombinant human tPA, 20 ng/mL uPA, or 1 μg/mL PAI-1 (Sino Biological, Beijing, China) was used to analyze the chemotactic effect of these proteins on migration of HUVECs.
Cell tracking was performed using ImageJ software (National Institutes of Health, Bethesda, USA) plugin “Manual Tracking”. 30 cells per experiment were tracked and each experiment was repeated independently three times. For further analysis and evaluation of chemotactical processes “Chemotaxis and Migration Tool” (ibidi, Martinsried, Germany) was used.