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Thrombin

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Thrombin is a serine protease enzyme that plays a crucial role in the blood coagulation process. It is responsible for catalyzing the conversion of fibrinogen to fibrin, which is the primary component of blood clots. Thrombin is an essential tool for researchers and scientists studying hemostasis, thrombosis, and other related areas of blood and vascular biology.

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Lab products found in correlation

Prothrombin, by Enzyme Research (18 mentions) Human fibrinogen, by Enzyme Research (18 mentions) Factor xiia, by Enzyme Research (9 mentions) Fibrinogen, by Enzyme Research (9 mentions) Human fxa, by Enzyme Research (9 mentions) Corn trypsin inhibitor, by Enzyme Research (9 mentions) Turbidometric platelet aggregometer, by Chrono-Log (6 mentions) Aypgkf, by GL Biochem (6 mentions) Sfllrn, by GL Biochem (6 mentions) Adenosine diphosphate adp, by Merck Group (6 mentions) Fitc conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions) Anti actin, by Cell Signaling Technology (3 mentions) Protein a g bead, by Santa Cruz Biotechnology (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Kallikrein, by Werfen (3 mentions) Versamax reader, by Molecular Devices (3 mentions) Corn trypsin inhibitor, by Prolytix (3 mentions) Tautomycetin tc, by Bio-Techne (3 mentions) Fluorescein isothiocyanate fitc labeled goat anti rabbit igg, by Jackson ImmunoResearch (3 mentions) Okadaic acid oa, by Enzo Life Sciences (3 mentions)

33 protocols using thrombin

1

Detailed Reagent Sourcing for Cell Signaling Studies

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Human r-Ang-1 was purchased from R&D Systems (Minneapolis, MN). Thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). Rabbit monoclonal anti-PTPN-2 antibody was from OriGene Technologies, Inc (Rockville, MD, USA). Rabbit polyclonal anti-Claudin-5, mouse monoclonal anti-PY-20 (clone 4G10) antibody was purchased from Millipore (Billerica, MA). Mouse monoclonal anti-ZO-1, rabbit polyclonal anti-Occludin antibody and DAPI were purchased from Invitrogen (Life Technologies, Grand Island, NY, USA). Rabbit polyclonal anti-Tie-2 antibody and protein A/G beads were purchased from Santa Cruz Biotechnology Inc (Dallas, TX, USA). Mouse anti-Na+-K+ ATPase β3 was from B.D. Biosciences (San Jose, CA). Rabbit anti phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), anti- p44/42 MAPK (Erk1/2) and anti actin was from Cell Signaling Technology (Danvers, MA). FITC conjugated secondary antibody and affinipure goat anti rabbit polyclonal light chain specific secondary antibody was from Jackson ImmunoResearch (West Grove, PA). ON-TARGET plus human PTPN-2 siRNA (ID: J-008969-05-0005) and mismatch control, transfection reagents were purchased from Dharmacon. All other chemicals were obtained from Sigma-Aldrich (St Louis, MI, USA).
Siddiqui M.R., Mayanil C.S., Kim K.S, & Tomita T. (2015). Angiopoietin-1 Regulates Brain Endothelial Permeability through PTPN-2 Mediated Tyrosine Dephosphorylation of Occludin. PLoS ONE, 10(6), e0130857.
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2

Amidolytic activity of blood coagulation factors

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In a 96-well plate, thrombin (Enzyme Research Laboratories, South Bend, IN, USA, 1 U/ml) was mixed with Biophen thrombin chromogenic substrate (Nodia, 1 mM) in Tris-buffered saline. Immediately after mixing, optical density at 405 nm was read every 10 seconds for 3 minutes in a VERSAmax reader (Molecular devices, San Jose, CA, USA) at 37°Celcius. The initial slope of the time vs optical density curve reflects the amidolytic activity of thrombin. Similarly, the activity of recombinant factor XIa (purified as described,
33 (link)
10 nM), factor XIIa (Enzyme Research Laboratories, South Bend, IN, 19,5 µM), kallikrein (Stago, 2 nM), and activated protein C (a generous gift from the late Dr. Walter Kisiel, University of Albuquerque, New Mexico, 10 nM) were measured toward S2302 (kallikrein and FXIIa) or S2366 (XIa and activated protein C) at a final concentration of 0.5 mM (Chromogenix, Molndal, Sweden). The initial slope of the reactions without inhibitory compounds present was set at 100.
Lisman T., Adelmeijer J., Huskens D, & Meijers J.C. (2021). Aprotinin Inhibits Thrombin Generation by Inhibition of the Intrinsic Pathway, but is not a Direct Thrombin Inhibitor. TH Open: Companion Journal to Thrombosis and Haemostasis, 5(3), e363-e375.
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3

Chromogenic Assay for Protein C Activity

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Thrombin and proThrombin were purchased from Enzyme Research Laboratories (South Bend, IN), factor Va and factor Xa from Hematologic Technologies (Burlington VT), rabbit-lung TM from American Diagnostica, Inc. (Greenwich, CT), chromogenic substrate H-D-lysyl (g-Cbo)-prolyl-argininyl-p-nitroanilide (Pefachrome PCa) from Centerchem Inc. (Norwalk, CT), and chromogenic substrate CBS 34–47 from American Bioproducts (Parsippany, NJ). Phospholipid vesicles (80 % phosphatidylcholine, 20 % phosphatidylserine) were prepared as described[9 (link)]. Recombinant PC antigen concentration was determined using the Asserachrom Protein C ELISA assay from American Bioproducts (Parsippany, NJ).
Alsultan A., Gale A.J., Kurban K., Khalifah M., Albadr F.B, & Griffin J.H. (2016). Activation-Resistant Homozygous Protein C R229W Mutation Causing Familial Perinatal Intracranial Hemorrhage and Delayed Onset of Thrombosis. Thrombosis research, 143, 17-21.
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4

Plasma Deficiency Reagents Protocol

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Human prothrombin–, FVIII-, and FX-deficient plasma was purchased from George King Bio-Medical (Overland Park, KS). Human FV-deficient plasma and thrombin were purchased from Enzyme Research Laboratories (South Bend, IN). Hirudin was from Calbiochem (La Jolla, CA).
Joseph B.C., Miyazawa B.Y., Esmon C.T., Cohen M.J., von Drygalski A, & Mosnier L.O. (2022). An engineered activated factor V for the prevention and treatment of acute traumatic coagulopathy and bleeding in mice. Blood Advances, 6(3), 959-969.
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5

Purification and Characterization of Coagulation Factors

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Plasma purified prothrombin, thrombin, and FXa were obtained from Enzyme Research Laboratories (South Bend, IN, USA). Hirudin was from Calbiochem (La Jolla, CA, USA), and corn trypsin inhibitor was obtained from Haematologic Technologies (Essex Junction, VT, USA). Substrates Pefachrome TH and Z-Gly-Gly-Arg-AMC were from Centerchem (Norwalk, CT, USA) and Bachem (Torrance, CA, USA), respectively. Human FVIII- or FV-deficient plasma was purchased from George King Bio-Medical (Overland Park, KS, USA). Phospholipid vesicles (80% phosphatidylcholine, 20% phosphatidylserine) were prepared as described previously [17 (link)].
Von Drygalski A., Cramer T.J., Bhat V., Griffin J.H., Gale A.J, & Mosnier L.O. (2014). Improved hemostasis in hemophilia mice by means of an engineered factor Va mutant. Journal of thrombosis and haemostasis : JTH, 12(3), 363-372.
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6

Clot Formation Measurement in Secretions

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Clot formation was measured after incubating 10 μL of secretions (final concentration 50 μg/mL) or H2O as a control with 90 μL of citrated plasma (prepared from blood collected in 1/10th volume 3.2% sodium citrate) for 10 and 30 min at room temperature (RT). The activated partial thromboplastin time (APTT) was measured after addition of 100 μL of APTT reagent (Kordia Life Sciences, Leiden, The Netherlands) and 100 μL of 25 mM Ca2+ to the secretions/plasma mixture. The proThrombin time (PT) measurement was started by adding 200 μL of Thromborel S (Dade Behring BV, Leusden, The Netherlands) to the mixture whereas the Thrombin time (TT-test) was initiated by 25 μL of Thrombin (100 U/ml; Enzyme Research Laboratories Inc, South Bend, IN, USA). The time needed for clot formation was measured at 37°C.
van der Plas M.J., Andersen A.S., Nazir S., van Tilburg N.H., Oestergaard P.R., Krogfelt K.A., van Dissel J.T., Hensbergen P.J., Bertina R.M, & Nibbering P.H. (2014). A Novel Serine Protease Secreted by Medicinal Maggots Enhances Plasminogen Activator-Induced Fibrinolysis. PLoS ONE, 9(3), e92096.
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7

Multicolor Immunofluorescence Microscopy

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Antibodies: anti-FcγRI, either unlabeled rabbit IgG1 (clone EPR4624; Abcam) or Alexa Fluor 488 (AF488)- or Alexa Fluor 647 (AF647)-labeled (mIgG1 clone 10.1; Biolegend); eFluor450-labeled anti-CD14 (61D3; eBioscience); Fluorescein isothiocyanate (FITC)-labeled goat-anti-rabbit IgG (Jackson ImmunoResearch); goat-anti-rabbit IgG HRP conjugated (Pierce); rabbit IgG-anti-dinitrophenyl (anti-DNP IgG, polyclonal; Vector Labs). Anti-DNP IgG was biotinylated and subsequently conjugated to Qdot655 (QD655) (Invitrogen) as described for IgE (20 (link)). Anti-DNP IgG and anti-trinitrophenyl (TNP) human IgG1 (38 (link)) were fluorescently labeled with AF647 or Cyanine 3B (Cy3B) (Life Technologies) following the same protocol as for biotinylation. Where indicated, 1 μM okadaic acid (OA; Enzo Life Sciences) or 1–1000 nM tautomycetin (TC; Tocris) was added for 30 min or 0.1 μg/mL latrunculin A (LatA; Life Technologies) was added 10 min before IL-3 stimulation. Phos-tag Acrylamide was from Wako-Chem. The anti-human CD20 antibody rituximab (Roche) was purchased from the pharmacy of UMC Utrecht. Eight-well Lab-Tek chambers (Nunc, Rochester, NY) were coated with poly-l-lysine (1 μg/mL in 10% 1× PBS, 90% water) for 30 min at room temperature (RT). Thrombin and fibrinogen were both from Enzyme Research Laboratories.
Brandsma A.M., Schwartz S.L., Wester M.J., Valley C.C., Blezer G.L., Vidarsson G., Lidke K.A., Broeke T.T., Lidke D.S, & Leusen J.H. (2018). Mechanisms of inside-out signaling of the high affinity IgG-receptor FcγRI. Science signaling, 11(540), eaaq0891.
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8

Platelet Aggregation Assay Procedure

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Example 5

Platelet aggregation assays were carried out as follows: Platelet aggregation and secretion was measured in a turbidometric platelet aggregometer (Chronolog) at 37° C. with stirring (1000 rpm). Washed platelets (3×108/ml) in modified Tyrode's buffer were stimulated with thrombin (Enzyme Research Laboratories). For talin knockdown platelet aggregation assay stimulated with manganese and ADP, manganese and ADP was mixed prior to experiment to achieve final concentration of 1 mM manganese and 5 μM ADP in reaction tube. Aggregation traces shown are representative of at least three independent experiments.

US10011634B2. Inhibitors of beta integrin-G protein alpha subunit binding interactions (2018-07-03). THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOIS [US]. Inventors: Xiaoping Du [US].
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9

Platelet Aggregation and Morphology Assay

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To measure aggregation, platelets were stimulated with thrombin (Enzyme Research Laboratories), PAR4-AP (PAR4-activating peptide; AYPGKF; GL Biochem), or PAR1-AP (SFLLRN; GL Biochem) in a Chrono-Log lumi-aggregometer, and light transmittance was monitored in real time for 6 minutes at 37°C under stirring conditions (1100 rpm). To quantify platelet shape change, platelets were treated with EGTA (2 mmol/L) for 2 minutes before agonist stimulation in a lumi-aggregometer.
Tourdot B.E., Stoveken H., Trumbo D., Yeung J., Kanthi Y., Edelstein L.C., Bray P.F., Tall G.G, & Holinstat M. (2018). Genetic Variant in Human PAR (Protease-Activated Receptor) 4 Enhances Thrombus Formation Resulting in Resistance to Antiplatelet Therapeutics. Arteriosclerosis, Thrombosis, and Vascular Biology, 38(7), 1632-1643.
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10

Platelet activation signaling assays

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Thrombin was obtained from Enzyme Research Laboratories (South Bend IN, USA). Thrombin receptor-activating peptide 6 (SFLLRN, TRAP6) was purchased from Bachem (Bubendorf, Switzerland); cross-linked collagen-related peptide (CRP-XL) was from the University of Cambridge (Cambridge, UK); the stable ADP analogue methylthio-adenosine-diphosphate (Me-S-ADP) [8 (link)] came from Santa Cruz Biotechnology (Dallas TX, USA). Fura-2 acetoxymethyl ester and human fibrinogen were obtained from Invitrogen (Carlsbad CA, USA); Pluronic F-127 from Molecular Probes (Eugene OR, USA). Integrin αIIbβ3 inhibitor tirofiban [9 (link)] and cAMP-elevating agent iloprost [2 (link)] were from Sigma-Aldrich (St. Louis MI, USA); the selective Syk kinase inhibitor PRT-060318, 2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide (Syk-IN) [10 (link)] came from Bio-Connect (Huissen, The Netherlands).
Zou J., Wu J., Roest M, & Heemskerk J.W. (2021). Long-term platelet priming after glycoprotein VI stimulation in comparison to Protease-Activating Receptor (PAR) stimulation. PLoS ONE, 16(3), e0247425.
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