Journal: Bioengineering & Translational Medicine

Article Title: Micrometer‐scale tPA beads amplify plasmin generation for enhanced thrombolytic therapy

doi: 10.1002/btm2.70012

Figure Lengend Snippet: Plasmin generation of tPA beads. Plasmin generation was measured using chromogenic substrate S‐2251, glu‐plasminogen, and 2.25 mU/mL tPA activity with (a) free tPA, (b) 0.1 μm t‐PA beads, or (c) 1 μm tPA beads and its co‐factors fibrinogen or fibrin degradation product (FDP) at 37°C. Each line and shaded region represent the average and standard deviation of n = 3. (d) Slope of the reaction curves at 50% of the maximum absorbance for each condition. Bars and error bars represent the average and standard deviation of n = 3. * p < 0.001 represents the significant difference between matching tPA conditions for a given co‐factor (comparison of grouped blue, orange, and green bars). Δ p < 0.001 represents the significant difference between co‐factors for a given tPA condition (comparison of like colored bars).

Article Snippet: Purified human lys‐plasmin (cat# IHUPLMLYS1MG) and plasmin chromogenic substrate (cat# IAFPLMCGSLY25MG) were obtained from Innovative Research (Novi, MI).

Techniques: Activity Assay, Standard Deviation, Comparison

Journal: Frontiers in Medicine

Article Title: Tranexamic acid impairs plasmin generation on human mesenchymal stem cells and derived membrane microvesicles, halting pericellular proteolysis

doi: 10.3389/fmed.2025.1570395

Figure Lengend Snippet: Detection of plasminogen cleavage and plasmin formation at the MSC membrane. MSC cultured in 96-well plates were incubated with a concentration of scuPA equivalent to the Kd (5 nM), after washing plasmin generation was detect by Western blot or plasmin generation kinetics. (A) Western blot analysis of membrane lysates. Plasmin generation was detected at different time points (0–120 min) using a constant concentration of plasminogen (1 μM). The supernatant was collected and cells in the wells were lysed for further analysis. The molecular mass of plasminogen (Pg) and plasmin. (Pn) are represented at the left. (B) Kinetics of plasmin generation using varying plasminogen concentrations (0–2 μM) and 0.75 mM of the plasmin selective chromogenic substrate CBS0065. Kinetics of plasmin formation was followed during 4 h by measuring the release of p -nitroaniline. Data were fitted using the Michaelis–Menten equation and a Km = 0.5 μM was calculated.

Article Snippet: The chromogenic substrate CBS0065 (methylmalonyl)-hydroxyprolylarginine- para -nitroanilide selective for plasmin was kindly provided by G. Contant (Stago, Gennevilliers, France).

Techniques: Membrane, Cell Culture, Incubation, Concentration Assay, Western Blot

Journal: The Journal of General Virology

Article Title: Double-edged sword: flavivirus NS1 enhances human plasmin-mediated fibrinolysis while being targeted for cleavage and inactivation

doi: 10.1099/jgv.0.002253

Figure Lengend Snippet: DENV2 NS1 binds human plasminogen in a lysine-dependent manner but does not alter plasminogen activation by uPA. ( a ) Human plasminogen (Plg) or BSA (10 µg ml −1 ) were coated on microplate wells and incubated with increasing concentrations of non-glycosylated dimeric DENV2 NS1 (0–25 µg ml −1 ). Binding was detected by anti-DENV2 NS1 antibody labelling, followed by absorbance detection at 492 nm. Data are presented as mean± sd from two independent experiments. ( b ) Plasminogen was coated (100 nM) and incubated with fixed concentrations of non-glycosylated dimeric DENV2 NS1 (25 µg ml −1 ), followed by the addition of serial dilutions of the lysine analogue EACA (100–0 mM). Binding was detected by anti-DENV2 NS1 antibody labelling and absorbance detection at 492 nm. Data are presented as mean± sd from three independent experiments and asterisk indicates statistical significance relative to the control (no EACA) following one-way ANOVA with Dunnett’s post-hoc test (***, P <0.001; ****, P <0.0001). ( c ) Representation of the plasminogen-DENV2 NS1 complex by molecular docking. Docking solutions converged into a dominant cluster (114 models; ~57% of the water-refined structures) with a favourable score ( Z -score=−1.3), corroborating the lysine-dependent recruitment of the viral protein (cyan) through the kringle 1 domain (KR-1; purple) in human plasminogen (grey). ( d ) Close-up view of the predicted binding between the Lys11 of one of DENV2 monomers (cyan) with the anionic and cationic centres of the lysine-binding site (purple); amino acid side chains are shown as sticks and hydrogen bonds are coloured red. ( e) and (f ) Plasminogen and uPA were mixed with glycosylated HEK293-derived DENV2 NS1 or BSA (10 µg ml −1 ), EACA (1 mM) or PBS (untreated). Plasmin activity was measured following S-2251 addition and absorbance detection at 405 nm over 1 h. ( d ) A representative graph indicating OD variation over time is shown. ( f ) OD at 60 min post-incubation is plotted according to the different conditions. Data are presented as mean± sd from three independent experiments and asterisk indicates statistical significance following one-way ANOVA with Tukey’s post-hoc test (NS, non-significant; **, P <0.01).

Article Snippet: Afterwards, the chromogenic plasmin substrate S-2251 (Chromogenix #S820332; 200 nM) was added to a final volume of 100 μl, and the absorbance kinetics at 405 nm was detected over 1 h by the SpectraMax M5 (Molecular Devices).

Techniques: Activation Assay, Incubation, Binding Assay, Control, Derivative Assay, Activity Assay