Glu- and Lys-plasminogen, plasmin, low molecular weight urokinase and single or double chain t-PA were from Enzyme Research Laboratories (South Bend, IN) or Innovative Research (Novi, MI). Activated partial thromboplastin time (aPTT; STA-PTT Automate) and prothrombin time (PT; Neoplastine CI Plus) reagents were from Diagnostica Stago (Asnières, France). S-2251 (H-D-Valyl-L-leucyl-L-lysinep-Nitroaniline dihydrochloride) was obtained from Diapharma (West Chester, OH). ε-Aminocaproic acid (ε-ACA), 4-mercaptophenylacetic acid, trifluoroacetic acid (TFA), triisopropylsilane (TIS), diisopropylcarbodiimide (DIC), 3,6-Dioxa-1,8-octane-dithiol (DODT), 4-methylpiperidine, methyl t-butyl ether (MTBE) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were from Sigma (Saint Louis, MO). HBTU, N,N-diisopropylethylamine (DIPEA) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) were from Chem-Impex International (Wood Dale, IL). Fmoc-amino acid for peptide synthesis were from Midwest Biotech Inc (Fishers, IN). Dimethylformamide (DMF) and N-methylpyrrolidone (NMP) were from AGTC Bioproducts (Framingham, MA).
Plasmin
Manufactured by Enzyme Research
Sourced in United States
Plasmin is a laboratory enzyme used for specific protein degradation. It functions as a serine protease, cleaving peptide bonds in certain protein substrates.
Automatically generated - may contain errors
Lab products found in correlation
α thrombin, by Enzyme Research (9 mentions)
Kallikrein, by Enzyme Research (9 mentions)
Fibrinogen, by Enzyme Research (9 mentions)
Activated protein c, by Enzyme Research (9 mentions)
Factor xa, by Enzyme Research (9 mentions)
Triisopropylsilane, by Merck Group (3 mentions)
Diisopropylcarbodiimide, by Merck Group (3 mentions)
Neoplastine ci plus, by Diagnostica Stago (3 mentions)
Sta ptt automate, by Diagnostica Stago (3 mentions)
T butyl methyl ether, by Merck Group (3 mentions)
Tris 2 carboxyethyl phosphine hydrochloride tcep, by Merck Group (3 mentions)
Trifluoroacetic acid, by Merck Group (3 mentions)
Af6426, by R&D Systems (3 mentions)
Thrombin cleancleave kit, by Merck Group (3 mentions)
Sephacryl s 200, by GE Healthcare (3 mentions)
Plasmin, by GE Healthcare (3 mentions)
Hydrocortisone, by Thermo Fisher Scientific (3 mentions)
Glutamine, by Thermo Fisher Scientific (3 mentions)
Trypsin edta 0.25, by Thermo Fisher Scientific (3 mentions)
Mcdb131, by Thermo Fisher Scientific (3 mentions)
8 protocols using plasmin
1
Peptide Synthesis Reagents and Assays
2
Antithrombin Activity of Albumin Fusion Proteins
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To determine if albumin fusion proteins possessed latent antithrombin activity, a two-stage assay was employed as previously described [14 (link), 19 (link)]. Briefly, albumin fusion proteins (400 nM) were combined with 300 nM purified protease in PPNE buffer (20 mM sodium phosphate, pH 7.4, 0.1% polyethylene glycol 8000, 100 mM NaCl, 0.1 mM EDTA) for varying times. Purified proteases included FXIa, Factor Xa (FXa), Factor XIIa (FXIIa), plasmin (Enzyme Research Laboratories (South Bend, IN, USA)) and recombinant FVIIa (Niastase, Novo Nordisk Canada, Mississauga, ON, Canada). First-stage reaction products were combined 1:1 with 10 nM thrombin in PPNE for 1 min, and then diluted 10-fold into 100 μM S2238 chromogenic substrate (Chromogenix, Lexington, MA, USA). Colour generation from the amidolysis reaction was followed for 5 min to determine the reaction velocity. In some reactions HEPES-buffered saline (HBS; 25 mM HEPES, pH 7.4, 100 mM NaCl) supplemented with 5 mM calcium chloride was substituted for PPNE.
3
Characterization of Fibrinolytic Factors
4
Cathepsin-Mediated Fibrin Degradation
5
Purification and Characterization of Fibrinogen D Fragment
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Fg (Enzyme Research Laboratories) was digested with plasmin (10 μg/15 mg of Fg) (Enzyme Research Laboratories) in TBS containing 10 mM CaCl2 for 4 h at 37°C as previously described (47 (link)) with modifications. Following plasmin digestion, Fg D fragment was obtained by column gel filtration on a Sephacryl S-200 (GE Healthcare) column with a molecular weight cutoff of 30 K, followed then by filtration in a column with a 50-K cutoff to remove plasmin. Purity of purified D fragments was analyzed by SDS-PAGE and appeared as a single band with a molecular mass of 85 kDa. Human Fg E fragment (Fg-E) was purchased from Haematologic Technologies (HCI-0150E).
6
Microvascular Endothelial Cell Culture
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MCDB 131 (microvascular endothelial cell medium), epidermal GF, hydrocortisone, glutamine, fetal calf serum, penicillin/streptomycin, trypsin-EDTA (0.25%), and trypsin neutralizer solution were purchased from Life Technologies (Carlsbad, CA). CnT-07 epithelial proliferation medium was obtained from CellnTec Advanced Cell Systems (Bern, Switzerland). Purified human a-thrombin, g-thrombin, factor XIa, factor XIIa-a, factor XIIa-b, kallikrein, activated protein C, plasmin, factor Xa, and fibrinogen were sourced from Enzyme Research Laboratories (South Bend, IN). Human neutrophil elastase, porcine pancreatic elastase, human urokinase plasminogen (PLG) activator, human tissue-type PLG activator, and human cathepsin G were purchased from Molecular Innovations (Novi, MI). Human chymase, bovine a-chymotrypsin, porcine trypsin, bivalirudin, human antithrombin III, Glu-type human PLG, [Tyr(SO3H)]63]-Hirudin fragment 54-65, human plasma, E coli O111:B4 lipopolysaccharide, and FITC-Dextran 70,000 were procured from Sigma-Aldrich/EMD Millipore (Saint Louis, MO). A complete list of all chromogenic substrates and their suppliers is provided in Supplementary Table S1. All other chemicals were of analytical grade and were commercially available.
7
Coagulation Factors and DNA Isolation
8
Plasmin Concentration and Fibrin Fiber Lysis
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To test the effect of Plasmin concentration on the lysis of bead-labeled fibers, trials at varying Plasmin concentrations were conducted. Samples were made as described previously and labeled with a bead solution at 4.55x109 beads/μL concentration. After labeling, samples were visually searched using 480 nm light (corresponding to the ALEXA-488 wavelength) to find areas with individual fibers while minimally exciting the beads. The wavelength was changed to 553 nm (corresponding to the fluorescent bead excitation wavelength (580 nm)), Plasmin was added, and images were collected every 30 s for one hour. Plasmin (Enzyme Research Labs, 11.03 U/mL, stock concentration) was aliquoted and frozen (-20° C) at concentrations that were double the desired final concentration. During an experiment, one aliquot was thawed and added to the fibrin-network containing solution at equal volumes (final total volume of 20 μL), thereby halving the Plasmin concentration. Five trials were conducted at each of the following final Plasmin concentration, 0.7 U/mL, 1.0 U/mL, and 2.0 U/mL.
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