Fibrinolytic activity was determined in a plasminogen-free fibrin plate, as described in section 2.1. After polymerization, 3 mm holes were punctured, filled with 10 μl of purified AprE127 or urokinase (Sigma U4010) with final concentrations of 5.30, 0.60, 0.07, and 0.01 mg/ml, and incubated at 37°C for 2 h. Images of the halos in the plate were taken at different times to measure the diameter of hydrolysis. The thrombolytic activity was calculated using the equation where R is the radius of the halo, and t is the diffusion time [30 (link)]. The plasminogen activation assay was performed by reacting 71 μg purified S127e with 2.5 μg plasminogen (Sigma P7999) in fibrin plates and comparing with the same amount of urokinase. The fibrinogenolytic activity was assayed according to Salazar et al. (2007) with minor modifications [31 (link)]. Briefly, 5 μg of purified protein AprE127e was added to 50 μg of fibrinogen (Sigma F4129), suspended in 100 μl of Tris–HCl 50 mM, pH 7.4 and incubated at 37°C. Aliquots of 10 μl were taken from the reaction mixture at different time points (0, 5, 10, 20, 40, and 80 min), added to reducing Laemmli buffer, denatured at 95°C for 5 min and run in 12% SDS-PAGE.
Plasminogen
Manufactured by Merck Group
Sourced in United States, Germany, Australia
Plasminogen is a laboratory equipment product manufactured by Merck Group. It is a key component in the plasmin activation system, which plays a role in fibrinolysis and tissue remodeling. The core function of Plasminogen is to serve as a precursor for the serine protease plasmin, which is involved in the breakdown of fibrin clots and the regulation of various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Fibrinogen, by Merck Group (5 mentions)
Thrombin, by Merck Group (4 mentions)
Fibronectin, by Merck Group (4 mentions)
Plasminogen from human plasma, by Merck Group (2 mentions)
Proteinase k, by Merck Group (2 mentions)
Ab14715, by Abcam (2 mentions)
Ab66705, by Abcam (2 mentions)
Ab1791, by Abcam (2 mentions)
D val leu lys 7 amido 4 methylcoumarin, by Merck Group (2 mentions)
α2 antiplasmin, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Urokinase, by Merck Group (1 mentions)
Plasmin from human plasma, by Merck Group (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Ccl21, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
α2 antiplasmin, by Innovative Research (1 mentions)
Il 21, by Thermo Fisher Scientific (1 mentions)
35 protocols using plasminogen
1
Fibrinolytic and Plasminogen Activation Assays
2
Plasminogen Activation Assay Protocol
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Samples of the collected culture medium (20 µL) were dispensed into a
96-well microplate and mixed with 30 µL of a plasminogen working solution
(2.5 µg/well plasminogen; Sigma-Aldrich). The solution was incubated at
38° for 1 h. After incubation, 200 µL of substrate buffer [0.18 mM
Z-L-Lys-SBzl hydrochloride, 0.22 mM 5,5′-dithiobis-(2-nitrobenzoic acid),
and 0.01% Triton X-100] was added and was subsequently incubated at 38°
for 1 h. PAs activity was determined by absorbance at the wavelength of 405 nm
using a microplate reader.
96-well microplate and mixed with 30 µL of a plasminogen working solution
(2.5 µg/well plasminogen; Sigma-Aldrich). The solution was incubated at
38° for 1 h. After incubation, 200 µL of substrate buffer [0.18 mM
Z-L-Lys-SBzl hydrochloride, 0.22 mM 5,5′-dithiobis-(2-nitrobenzoic acid),
and 0.01% Triton X-100] was added and was subsequently incubated at 38°
for 1 h. PAs activity was determined by absorbance at the wavelength of 405 nm
using a microplate reader.
3
Embryo Culture Optimization Using IGF2, uPA, and Plasminogen
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The media used for embryo culture was EmbryoAssist (stage 1) and BlastAssist (stage 2) sequential media system (MediCult A/s, now ORIGI A/s Denmark). All embryos were cultured in EmbryoAssist media overnight for 20 h and assessed for development to the 2-cell stage.
The 2-cell embryos were pooled into a single dish and then randomly allocated into one of four treatment groups: (1) control, which were cultured in EmbryoAssist media alone; (2) IGF2, which were cultured in media containing 12.5 nM IGF2 (GroPep, Thebarton, SA, Australia); (3) U + P, which were cultured in media containing 10 µg/mL urokinase plasminogen activator (uPA; Sigma-Aldrich) and 5 µg/mL plasminogen (Sigma-Aldrich); or (4) IGF2 + U + P treatments, which were cultured in media containing a combination of 12.5 nM IGF2, 10 µg/mL urokinase plasminogen activator and 5 µg/mL plasminogen.
Embryos were washed and then cultured in this media for a further 23 h (Fig. 1). Cleavage stage embryos were then transferred to BlastAssist media with appropriate treatment for further 48 h. All embryo culture dishes were equilibrated to appropriate temperature and gas mix for at least 4 h before culture.
The 2-cell embryos were pooled into a single dish and then randomly allocated into one of four treatment groups: (1) control, which were cultured in EmbryoAssist media alone; (2) IGF2, which were cultured in media containing 12.5 nM IGF2 (GroPep, Thebarton, SA, Australia); (3) U + P, which were cultured in media containing 10 µg/mL urokinase plasminogen activator (uPA; Sigma-Aldrich) and 5 µg/mL plasminogen (Sigma-Aldrich); or (4) IGF2 + U + P treatments, which were cultured in media containing a combination of 12.5 nM IGF2, 10 µg/mL urokinase plasminogen activator and 5 µg/mL plasminogen.
Embryos were washed and then cultured in this media for a further 23 h (Fig. 1). Cleavage stage embryos were then transferred to BlastAssist media with appropriate treatment for further 48 h. All embryo culture dishes were equilibrated to appropriate temperature and gas mix for at least 4 h before culture.
4
Cytokine and Plasmin Regulation
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Human recombinant interleukin (IL)-4 (catalog no.: 200-04), granulocyte macrophage colony-stimulating factor (catalog no.: 300-23), 1β (catalog no.: 200-01B), IL-6 (catalog no.: 200-06), TNF-a (catalog no.: 300-01A), CCL21 (catalog no.: 300-35), IL-7 (catalog no.: 200-07), IL-12 (catalog no.: 200-12p80H), and IL-21 (catalog no.: 200-21) was purchased from PeproTech and are reported to have endotoxin levels less than 0.1 ng/μg of protein (<1 EU μg−1). All CCL21 solutions were prepared in low-bind 1.5 ml Eppendorf tubes. Prostaglandin E2 (P0409), plasmin from human plasma (catalog no.: P1867), and plasminogen (catalog no.: RSP6518) from human plasma were purchased from Sigma–Aldrich. tPA (Actilyse) was kindly donated by Boehringer Ingelheim Limited. α2-antiplasmin (catalog no.: HA2AP) was purchased from Molecular Innovations.
5
Plasminogen Purification and Characterization
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Milli-Q water was used for buffer preparation (conductivity below 0.2 μS.cm-1).
plasminogen from human plasma was purchased from Sigma-Aldrich (Sigma-Aldrich Danmark A/S, Copenhagen, Denmark) in powder form. In the majority of the experiments and unless stated differently we have used the Sigma-Aldrich plasminogen product P7999. The product was dissolved directly in 20 mM Lysine buffer pH 7.2 (Fluka, 62840) in order to make stock solutions and stored at ~-20°C in 50–100 µl aliquots until use. Prior to use the protein aliquots were slowly thawed at 4–8°C before dilution in the experimental buffer.
plasminogen concentrations were determined by Abs280nm using a molar extinction coefficient of 152200 M-1.cm-1, estimated using the bioinformatic tool ProtParam (Expasy, [39 ], entry: uniProt sequence P00747 [AA 20–810] for human plasminogen).
plasminogen from human plasma was purchased from Sigma-Aldrich (Sigma-Aldrich Danmark A/S, Copenhagen, Denmark) in powder form. In the majority of the experiments and unless stated differently we have used the Sigma-Aldrich plasminogen product P7999. The product was dissolved directly in 20 mM Lysine buffer pH 7.2 (Fluka, 62840) in order to make stock solutions and stored at ~-20°C in 50–100 µl aliquots until use. Prior to use the protein aliquots were slowly thawed at 4–8°C before dilution in the experimental buffer.
plasminogen concentrations were determined by Abs280nm using a molar extinction coefficient of 152200 M-1.cm-1, estimated using the bioinformatic tool ProtParam (Expasy, [39 ], entry: uniProt sequence P00747 [AA 20–810] for human plasminogen).
6
In Situ Proteinase Activity Assay
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The rat was transcardially perfused with PBS, followed by quick removal of the brain, freeze in OCT (Sakura Finetechnical, Tokyo, Japan) and store at −80°C. Cryosections (20 μm) were analysed for in situ proteinase activity as described previously.19 In brief, 100 μl overlays of 1% agarose in PBS containing 10 μg/mL of BODIPY TR‐X FL casein (Molecular Probes, #E6638, Invitrogen, CA, USA) and 5 mmol/L EDTA with plasminogen (#P7999; Sigma), were added to pre‐warmed tissue and sealed under glass coverslips. After 2‐hours incubation at 37°C, image of casein fluorescence was achieved with a microscope (model Axioskop; Carl Zeiss Vision).
7
Real-time SPR analysis of tPA-plasminogen-PAI-1 interactions
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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).
8
Plasma Protein Purification Protocol
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Fibrinogen, plasminogen, thrombin and fibronectin from human plasma, laminin from basement membrane Engelbreth-Holm-Swarm (EHS) murine sarcoma, and decorin from bovine articular cartilage were purchased from Sigma-Aldrich. Gelatin and Matrigel from EHS murine sarcoma were purchased from Difco and BD Biosciences, respectively, and the recombinant proteoglycans lumican (human), and biglycan (human) were purchased from R&D Systems. Rabbit anti-human Fibrinogen was from Sigma-Aldrich. Depletion of albumin from human plasma was performed according to Subramanian (1984 (link)).
9
Pancreatic Cancer Cell Line Culture
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The Panc‐1 (CRL‐1469, male), Panc10.05 (CRL‐2547, male), and HPAF‐II (CRL‐1997, male) cell lines were purchased from the American Type Culture Collection (ATCC). The AsPC‐1 (female) and Bx‐PC3 (female) cell lines were a generous gift from Dr. David Hoskin (Dalhousie University, Halifax, Nova Scotia, Canada). All cell lines tested negative for mycoplasma. Panc‐1 cells were supplemented with Dulbecco's modified Eagle's media with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Hyclone). AsPC‐1 and BxPC‐3 cells were supplemented with Roswell Park Memorial Institute with 10% fetal bovine serum and 1% pencillin/streptomycin. All cells were maintained at 37 °C with 5% CO2. Zarnestra (Tipifarnib; Cat no. S1453, Selleckchem, Houston, TX, USA) and decitabine (Cat no. A3656, Sigma Aldrich, Oakville, ON, Canada) were reconstituted in DMSO. Doxycycline (Cat no. 631311, Clontech, Mountain View, CA, USA) was reconstituted in tissue‐culture grade water. Plasminogen (Cat no. 528180, Sigma Aldrich), S2251 (Via Diapharma, Cat no. 82033239, West Chester, OH, USA), ε‐aminocaproic acid (Cat no. A2504, Sigma Aldrich), and aprotinin (Cat no. 800277, Pentapharm, Dornacherstrasse, Switzerland) were reconstituted in PBS.
10
Thymoquinone Inhibits TGF-β1-Induced MMP-9 and u-PA Activities
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786-O-SI3 cells were treated with Thymoquinone at the indicated concentrations for 24 h. Otherwise, cells were pretreated with Thymoquinone (0-20 μM) for 2 h followed by incubated with or without 10 ng/mL TGF-β1 for an additional 24 h. The activities of MMP-9 and u-PA in the cultured medium were determined by using gelatin-zymogram protease assays as previously described 21 (link). Briefly, the collected medium samples were reacted with the analysis buffer (0.01% SDS, 50 mM Tris-HCl, pH 6.8) in the room temperature for 30 min, loaded into the 8% SDS-gel containing 0.1% gelatin, and then electrophoresed at 150 V in an OWL P-1 apparatus (Alpha Multiservices, Inc., Conroe, TX, USA) for 3 h. After the electrophoresis, the gels were washed with the 2% Triton X-100 in distilled water with gentle shaking at room temperature for 30 min. Then, the washed gels were incubated with the reaction buffer (40 mM Tris-HCl, pH 8.0, containing 10 mM CaCl2 and 0.02% NaN3) at 37°C for 12 h, and stained with Coomassie brilliant blue R-250. u-PA activity was determined using the same method for MMP-2, and 0.1% gelatin was replaced with 2% casein and 20 mg/mL plasminogen (Sigma). Electrophoresis and zymography were then performed for gelatin zymography.
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