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Prothrombin

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Prothrombin is a coagulation factor that plays a crucial role in the blood clotting process. It is a protein produced by the liver and is essential for the formation of fibrin, the main component of blood clots.

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Lab products found in correlation

Thrombin, by Enzyme Research (18 mentions) Human fxa, by Enzyme Research (18 mentions) Human fibrinogen, by Enzyme Research (9 mentions) Glu plasminogen, by Enzyme Research (9 mentions) α thrombin, by Enzyme Research (9 mentions) Spectrozyme th, by Sekisui (6 mentions) Spectrozyme fxa, by Sekisui (6 mentions) Recombinant human upa, by R&D Systems (3 mentions) Bz fvr amc, by Bachem (3 mentions) Rabbit anti β tubulin antibody, by Santa Cruz Biotechnology (3 mentions) Goat anti mouse hrp, by Jackson ImmunoResearch (3 mentions) Mouse anti rabbit hrp, by Jackson ImmunoResearch (3 mentions) Amicon, by Merck Group (3 mentions) Goat anti mouse irdye 680, by LI COR (3 mentions) Irdye 800cw goat anti rabbit, by LI COR (3 mentions) Plasmin, by Merck Group (3 mentions) Chymase, by Merck Group (3 mentions) Kallikrein, by Biosynth (3 mentions) Proteinase 3, by Merck Group (3 mentions) Matriptase, by R&D Systems (3 mentions)

9 protocols using prothrombin

1

Protease Activity Assay Protocol

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Human fibrinogen (plasminogen, von Willebrand factor, and fibronectin-depleted) and human zymogens Glu-plasminogen, α-thrombin, prothrombin, factor X zymogen, and activated factor Xa (all >95% purity) were purchased from Enzyme Research Laboratories, South Bend, IN, USA. Human plasmin and tranexamic acid (TXA) were purchased from MilliporeSigma, Burlington, MA, USA. Recombinant human pro-uPA zymogen [70 (link)] was kindly provided M. Ploug (Finsen Laboratory, Copenhagen, Denmark). Recombinant human uPA and recombinant mouse testisin proteins were from R&D Systems, Minneapolis, MN, USA. Fluorogenic substrates for thrombin (thrombin substrate III, Bz-FVR-AMC), FXa (Boc-IEGR-AMC [36 (link)]), uPA (Glt-GR-AMC [36 (link)]), plasmin (Boc-EEK-AMC [71 (link)]), and trypsin-like (Boc-QAR-AMC) proteases were from BACHEM, Bubendorf, Switzerland. Rivaroxaban (BAY 59-7939) was from Thermo Fisher Scientific, Waltham, MA, USA. Antibodies used were mouse anti-testisin antibody (MAbD9.1) [12 (link)], mouse anti-uPA antibody (IM15L, MilliporeSigma, Burlington, MA, USA), and rabbit anti-β-tubulin antibody (Santa-Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies were goat anti-mouse-HRP, mouse anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA), and donkey anti-sheep-HRP (R&D Systems, Minneapolis, MN, USA).
Buzza M.S., Pawar N.R., Strong A.A, & Antalis T.M. (2023). Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin. International Journal of Molecular Sciences, 24(11), 9306.
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2

Recombinant Human CETP Protein Expression

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cDNAs for recombinant (r) wt-human CETP, Ala373Pro-CETP, and Arg451Gln-CETP were kindly provided by Dr. Lloyd and Dr. Bamberger (Pfizer). rCETPs were expressed in HEK239 cells as previously described23 ). Prothrombin was from Enzyme Research Laboratories (South Bend, IN), and bovine serum albumin (BSA) was from Calbiochem-Novabiochem Corp (San Diego, CA). Factor Va, factor Xa, biotinylated (B)-EGR-factor Xa, Gla-domainless factor Xa (DG-factor Xa), BEGR-DG-factor Xa, factor IXa, and BEGR-chloromethylketone were purchased from Hematologic Technologies Inc. (Essex Junction, VT). BEGR-factor IXa was prepared as described28 ). Streptavidin chips (Sensor Chip SA) were obtained from GE Healthcare Bio-Sciences (Pittsburgh, PA).
Deguchi H., Banerjee Y., Elias D.J, & Griffin J.H. (2016). Elevated CETP Lipid Transfer Activity is Associated with the Risk of Venous Thromboembolism. Journal of Atherosclerosis and Thrombosis, 23(10), 1159-1167.
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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

Purification and Crystallization of Thrombin-XC-43 Complex

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Prothrombin (Enzyme Research Laboratories) activation was performed in 20 mM Tris, 0.1 M NaCl, 1 mM EGTA, 10 mM CaCl2 pH 7.5, and 1% of Oxyuranus scutellatus venom at 37 °C for 40 min. Activated thrombin (α-thrombin) was purified using a HiTrap heparin column pre-equilibrated with 20 mM Tris, 0.1 M NaCl, 1 mM EGTA, pH 7.5. Proteins were eluted with 20 mM Tris, 1 M NaCl, 1 mM EGTA pH 7.5 and concentrated in 10 mM HEPES pH 7.4 using a 10 kDa Amicon (Merck). The XC-43-thrombin complex was assembled in 10 mM HEPES pH 7.4 with a 1:1.5 M ratio (thrombin: XC-43) at room temperature for 1 h. The complex was concentrated to 11.4 mg/ml and crystallized using the hanging drop vapor diffusion method in 0.2 M magnesium acetate, 9% PEG 8000 (measured pH ∼ 7) at room temperature. After growth, crystals were flash cooled in liquid nitrogen in 0.2 M magnesium acetate, 12% PEG 8000, 15% glycerol.
Lu S., Tirloni L., Oliveira M.B., Bosio C.F., Nardone G.A., Zhang Y., Hinnebusch B.J., Ribeiro J.M, & Andersen J.F. (2021). Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis. The Journal of Biological Chemistry, 297(5), 101322.
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5

Coagulation Factors Protocol Synthesis

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Human FXa, thrombin, prothrombin, and FX were from Enzyme Research Laboratories (South Bend, IN). Human FV was from Paula B. Tracy or Kathleen M. Brummel-Ziedins (University of Vermont, Burlington, VT) and activated with FXa (FVaXa) or thrombin (FVaIIa).12 (link) FV810, a recombinant form of FV that retains the TFPIα-binding acidic region,3 (link),5 (link) and FV810 containing the Leiden Arg506→Gln substitution (FVL810) were from Rodney M. Camire (University of Pennsylvania, Philadelphia, PA). TF (Dade Innovin) was from Siemens (Washington, DC). Human FVIIa and a mouse antibody against the TFPI K2 domain (anti-K2) were obtained as described.24 (link) A rabbit polyclonal antibody that recognizes the final 12 amino acids of TFPIα (anti-CTP) was produced by Abcam (Burlingame, CA). Goat anti-mouse IRDye680 and goat anti-rabbit IRDye800CW were from LI-COR Biosciences (Lincoln, NE).
Wood J.P., Petersen H.H., Yu B., Wu X., Hilden I, & Mast A.E. (2017). TFPIα interacts with FVa and FXa to inhibit prothrombinase during the initiation of coagulation. Blood Advances, 1(27), 2692-2702.
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6

Preparation of Coagulation Factors and Lipid Vesicles

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Plasma-derived human FXa, PZ, and prothrombin were from Enzyme Research Laboratories (South Bend, IN). Human FVa and sheep anti-human PZ polyclonal antibody (PZAb) were from Haematologic Technologies (Essex Junction, VT). Recombinant human WT and Y387A/K239A mutant (ZPI-2A) ZPIs (expressed in E. Coli) and NBD fluorophore labeled K239C were prepared as described [10 (link), 12 (link)]. Small unilamellar phospholipid vesicles (SUVs) made from porcine brain extract(PBE) (Polar extract, 18.5% phosphatidylserine (PS)/33% phosphatidylethanolamine(PE)/13% phosphatidylcholine (PC)) or from 20% synthetic dioleoyl PS and 20%PE/60%PC from bovine heart (Avanti, Alabaster, AL) were prepared as described[8 (link), 13 (link), 14 (link)].
, & Huang X. (2019). Engineering a protein Z-dependent protease inhibitor (ZPI) mutant as a novel antagonist of ZPI anticoagulant function for hemophilia treatment. Journal of thrombosis and haemostasis : JTH, 17(10), 1655-1660.
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7

Thrombin and Coagulation Factor Purification

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α-Thrombin was purchased from Sigma, Haematologic Technologies or purified after activation of prothrombin (Enzyme Research Laboratories) using Oxyuranus scutellatus venom. α-Chymotrypsin, plasmin and chymase were purchased from Sigma; β-tryptase was purchased from Promega, FXa was purchased from EMD Biosciences, FV, FX, FXI, FXIIa, γ-thrombin was purchased from Haematologic Technologies and from Enzyme Research Labs, kallikrein was purchased from Fitzgerald Industries International, elastase was purchased from Elastin Products, cathepsin G, FXIa, uPA, and tPA were purchased from Molecular Innovations, matriptase was from R&D Systems, proteinase 3 was from Merck and sequencing-grade trypsin was purchased from Roche. PT and APTT reagents were purchased from Stago Inc. Fibrinogen was purchased from Sigma-Aldrich. Polyphosphate, High MW (P700), was purchased from KeraFast.
Jablonka W., Kotsyfakis M., Mizurini D.M., Monteiro R.Q., Lukszo J., Drake S.K., Ribeiro J.M, & Andersen J.F. (2015). Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin. PLoS ONE, 10(8), e0133991.
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8

Anticoagulant Reagents and Enzyme Characterization

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UFH (MW ~12000–16000Da) was from Sigma Aldrich (St. Louis, MO), fondaparinux (MW = 1728Da) from Apotex (Weston, FL), enoxaparin (MW ~5000Da) from Sandoz (Princeton, NJ) and dalteparin (MW ~5000Da) from Eisai (Woodcliff Lake, NJ). ODSH (Fryer, et al 1997 (link)) was a gift from J.A. Voynow (Virginia Commonwealth University). FV810QQ, an altered form of FVa in which the B-domain residues 811–1490 are absent and the thrombin cleavage sites at Arg709 and Arg1545 have been mutated to Gln (Bos and Camire 2012 (link)), was a gift from R.M. Camire (University of Pennsylvania). Human FXa and prothrombin were from Enzyme Research Laboratories (South Bend, IN). Thrombin, the thrombin inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amine (DAPA) and corn trypsin inhibitor (CTI) were from Haematologic Technologies (Essex Junction, VT). Recombinant TFPIα was as described (Lockett and Mast 2002 (link)), and an inhibitory monoclonal antibody directed against the second Kunitz domain of TFPIα (anti-K2) was a gift from Novo Nordisk (Bagsvaerd, Denmark). TF (Dade Innovin) was from Siemens (Washington, DC). Thrombin and FXa chromogenic substrates (Spectrozyme TH and Spectrozyme FXa, respectively) were from Sekisui Diagnostics (Lexington, MA).
Wood J.P., Baumann Kreuziger L.M., Desai U.R, & Mast A.E. (2016). Blocking Inhibition of Prothrombinase by Tissue Factor Pathway Inhibitor alpha: A Procoagulant Property of Heparins. British journal of haematology, 175(1), 123-132.
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9

Recombinant CETP Protein Expression

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cDNAs for recombinant (r) wt-human CETP, Ala373Pro-CETP, and Arg451Gln-CETP were kindly provided by Dr. Lloyd and Dr. Bamberger (Pfizer). rCETPs were expressed in HEK239 cells as previously described (23 ). Prothrombin was from Enzyme Research Laboratories (South Bend, IN), and bovine serum albumin (BSA) was from Calbiochem-Novabiochem Corp (San Diego, CA). Factor Va, factor Xa, biotinylated (B)-EGR-factor Xa, Gla-domainless factor Xa (DG-factor Xa), BEGR-DG-factor Xa, factor IXa, and BEGR-chloromethylketone were purchased from Hematologic Technologies Inc. (Essex Junction, VT). BEGR-factor IXa was prepared as described (28 ). Streptavidin chips (Sensor Chip SA) were obtained from GE Healthcare Bio-Sciences (Pittsburgh, PA).
Deguchi H., Banerjee Y., Elias D.J, & Griffin J.H. (2016). Elevated CETP Lipid Transfer Activity is Associated with the Risk of Venous Thromboembolism. Journal of Atherosclerosis and Thrombosis, 23(10), 1159-1167.
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