Plasmin fluorescent substrate came from SensoLyte AFC Plasmin Activity Assay Kit *Fluorimetric* (Anaspec) and tPA Spectrofluor 444FL substrate was from American Diagnostica (ADF Biomedical, Neuville-sur-oise, France). Hygromycin B, bovine thrombin, carboxyfluorescein succinidimyl ester (CFSE), EDTA and lipopolysaccharide (LPS 0111:B4) were from Sigma-Aldrich (L'Isle d'Abeau, France). Bovine fibrinogen used for fibrin-agar zymography came from Bovogen Labs (Australia). Plasminogen used for plasmin substrate activity studies was from Enzyme Research Laboratories (ERL, UK). Sc-uPA, purified from human cell lines, was from Biopool AB (UK). The uPA standards for fibrin-agar zymographies were obtained using ACTOSOLV® (Eumedica). Lipofectamine 2000, fetal bovine sera were from Invitrogen (Cergy Pontoise). tPA (Actilyse) was from Boehringer-Ingelheim (Paris, France). Human PAI-1 (N-terminal cysteine, active fraction) came from Gentaur. Sheep antiserum raised against human tPA was prepared at the national institute for agronomic research (INRA, Clermont-Theix, France). Tumor Necrosis Factor-α was purchased from PeproTech (Rocky Hill, NJ). Recombinant annexin-V came from Abcam (France). Aprotinin (Trasylol) was a gift from Bayer HealthCare AG.
Plasminogen
Manufactured by Enzyme Research
Sourced in United Kingdom
Plasminogen is a lab equipment product used in research activities. It is a glycoprotein that serves as the inactive precursor to the enzyme plasmin, which plays a crucial role in the breakdown of blood clots and the degradation of the extracellular matrix. The core function of Plasminogen is to provide a key component for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fibrinogen, by Enzyme Research (18 mentions)
Plasminogen, by Stratech Scientific (6 mentions)
Pai 1, by Merck Group (6 mentions)
Prism 7, by GraphPad (6 mentions)
Fibrinogen, by Sekisui (6 mentions)
Bovine thrombin, by Merck Group (3 mentions)
Hygromycin b, by Merck Group (3 mentions)
Tumor necrosis factor α, by Thermo Fisher Scientific (3 mentions)
Recombinant annexin 5, by Abcam (3 mentions)
Aprotinin trasylol, by Bayer (3 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Biacore 3000, by GE Healthcare (3 mentions)
Fibrinogen, by Thermo Fisher Scientific (3 mentions)
8 protocols using plasminogen
1
Fluorometric Plasmin Activity Assay with Diverse Reagents
2
Fibrinogen Labeling and Biomaterial Characterization
3
SPR Analysis of Polysaccharide-Plasminogen Interactions
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To examine the interaction between polysaccharide fractions and plasminogen, SPR analysis was performed using a Biacore 3000 (GE Healthcare, Milwaukee, WI).
plasminogen (human; Enzyme Research Laboratories, South Bend, IN) was diluted with 10 mM sodium acetate buffer (pH 5.0) to a concentration of c. 20 μg mL−1 and immobilized on a standard sensor chip (CM 5) using an amine-coupling kit according to the manufacturer's instructions. Running buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20 was used. The flow rate was maintained at 10 μL min−1 for immobilization and 20 μL min−1 for analysis.
plasminogen (human; Enzyme Research Laboratories, South Bend, IN) was diluted with 10 mM sodium acetate buffer (pH 5.0) to a concentration of c. 20 μg mL−1 and immobilized on a standard sensor chip (CM 5) using an amine-coupling kit according to the manufacturer's instructions. Running buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20 was used. The flow rate was maintained at 10 μL min−1 for immobilization and 20 μL min−1 for analysis.
4
Fibrin Formation and Lysis Kinetics
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Thrombin (5 nM final; Sekisui Diagnostics) was mixed with fibrinogen (2-9 g/L final; ThermoFisher Scientific, Waltham, MA), preincubated with either FXII or FXIIa (10-40 μg/mL final; both from Sekisui Diagnostics) in a total volume of 25 μL of 0.1 M imidazole buffer in a clear, flat-bottomed, 96-well plate. Fibrin formation was initiated by the addition of 20 μL of 20 mM CaCl2. To measure fibrinolysis, tPA (0.1 µg/mL final) and plasminogen (20 µg/mL final; Enzyme Research Laboratories) were added to the clotting solution. Turbidity was monitored as described earlier. In some experiments, FXIIa (40 μg/mL final) was preincubated with corn trypsin inhibitor (CTI; 0.01 U/mL final; Sekisui Diagnostics) before mixing with fibrinogen. FXII and FXIII contaminations were not detected in fibrinogen preparation by means of western blot analysis and enzyme-linked immunosorbent assay.
5
Magnesium-Dependent Modulation of PAI-1 Fibrinolysis
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To test the influence of the interaction between Mg
2+and PAI-1 on lysis time, turbidimetric fibrin clot lysis assays were performed in a purified system that included exogenous PAI-1. A buffer (50 mM Tris, 100 mM NaCl, pH 7.4) was added to the wells of a 96-well plate, followed by fibrinogen (depleted in plasminogen, von Willebrand factor and fibronectin; Enzyme Research Laboratories, Swansea, UK). MgCl
2was then added followed by PAI-1 (Sigma-Aldrich; Gillingham, UK), then plasminogen (Stratech; Ely, UK) and tPA, and finally CaCl
2and thrombin. Final concentrations were 0.5 mg/mL fibrinogen, 2.5 mM CaCl
2, 0.05 U/mL thrombin, 3.12 µg/mL plasminogen, 39 ng/mL t-PA, 0 or 200 ng/mL PAI-1, and 0 to 3.2 mM MgCl
2. Absorbance at 340 nm was read with a Multiskan FC plate reader every 12 seconds at 37°C. Clot lysis was calculated from the resultant data using Prism 7.0 (GraphPad Software).
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To test the influence of the interaction between Mg
2+and PAI-1 on lysis time, turbidimetric fibrin clot lysis assays were performed in a purified system that included exogenous PAI-1. A buffer (50 mM Tris, 100 mM NaCl, pH 7.4) was added to the wells of a 96-well plate, followed by fibrinogen (depleted in plasminogen, von Willebrand factor and fibronectin; Enzyme Research Laboratories, Swansea, UK). MgCl
2was then added followed by PAI-1 (Sigma-Aldrich; Gillingham, UK), then plasminogen (Stratech; Ely, UK) and tPA, and finally CaCl
2and thrombin. Final concentrations were 0.5 mg/mL fibrinogen, 2.5 mM CaCl
2, 0.05 U/mL thrombin, 3.12 µg/mL plasminogen, 39 ng/mL t-PA, 0 or 200 ng/mL PAI-1, and 0 to 3.2 mM MgCl
2. Absorbance at 340 nm was read with a Multiskan FC plate reader every 12 seconds at 37°C. Clot lysis was calculated from the resultant data using Prism 7.0 (GraphPad Software).
6
Fibrinolysis Rates of Fibrin Clots
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Fibrinolysis rates of fibrin clots formed in the presence of full‐length or truncated recombinant FXIII‐A were measured by the use of non‐fluorescence confocal microscopy as previously described 32 . Clots were formed in triplicate, with the same concentrations of reactants as used for the turbidity experiments. Lysis was then initiated with 280 μg mL−1 plasminogen (Enzyme Research Laboratories) and 1 μg mL−1 tissue‐type plasminogen activator (t‐PA) (Technoclone, Vienna, Austria). The clot was visualized under low magnification every 20 s with a Leica TCS SP‐2 laser scanning 1072 confocal microscope (Leica Microsystems, Heidelberg, Germany), and the time taken for the lysis front to migrate from a fixed point was measured. The lysis front velocity was determined and used to calculate the mean overall lysis rate in μm s−1.
7
Mg2+ and PAI-1 Influence on Fibrin Clot Lysis
8
Fibrin Clot Lysis under Flow
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Fibrinolysis rates of fibrin clots formed in the presence of FXIII‐A were also measured under flow with a Chandler loop system as previously described 32 , 33 . Clots were formed by the use of FXIII‐depleted fibrinogen containing 5% Alexa Fluor 488‐conjugated FXIII‐depleted fibrinogen. The same concentrations of reactants as used for the turbidity experiments were used. After 2 h, clots were retained within the tubing and washed with Tris‐buffered saline. Lysis was initiated with 28 μg mL−1 plasminogen (Enzyme Research Laboratories) and 0.1 μg mL−1 t‐PA (Technoclone, Vienna, Austria).
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