Factor Va, Xa, prothrombin, and thrombin were obtained from Haematologic Technologies (Essex Junction, VT). The aPTT assay reagent and PT reagents were purchased from Fisher Diagnostics (Middletown, VA). Argatroban was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Inhibitors predicted by high-throughput virtual screening were purchased from ChemBridge (San Diego, CA). All other reagents were of the highest commercially available grade.
Prothrombin
Manufactured by Prolytix
Sourced in United States
Prothrombin is a laboratory equipment product used in the analysis of blood coagulation. It measures the concentration of prothrombin, a key protein involved in the blood clotting process.
Automatically generated - may contain errors
Lab products found in correlation
Thrombin, by Prolytix (6 mentions)
Argatroban, by Santa Cruz Biotechnology (2 mentions)
Factor va, by Prolytix (2 mentions)
Factor xa, by Prolytix (2 mentions)
Mouse igg1 igg2a clone x40 x39, by BD (2 mentions)
α thrombin, by Prolytix (2 mentions)
S 2238, by Diapharma (2 mentions)
Tnf α, by Abnova (2 mentions)
S 2765, by Diapharma (2 mentions)
Fluo 3 am, by Biotium (1 mentions)
Megamix beads, by Biocytex (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Recombinant hirudin, by Hyphen Biomed (1 mentions)
Novoeight, by Novo Nordisk (1 mentions)
Fviia, by Prolytix (1 mentions)
Rvv x, by Prolytix (1 mentions)
Human factor xa, by Prolytix (1 mentions)
Prothrombin, by Merck Group (1 mentions)
13 protocols using prothrombin
1
Coagulation Factors and Inhibitor Screening
2
Llama-derived VHH Phage Display Libraries
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For the construction of VHH phage display libraries, a llama was immunized intramuscularly three times with three‐week intervals as previously described 28 using 200 sμg purified, amidated m. pasudotox per immunization. One week after the second and third injections, peripheral blood lymphocytes were isolated for RNA extraction and preparation of cDNA as described by Adams et al. 29 . The gene fragments encoding the VHH fragments were amplified by PCR and the resulting fragments of about 350 bp in size were cloned in a phagemid vector and used for transformation to the E. coli strain TG1 29 . The same approach was taken for the construction of the anti‐prothrombin VHH phage display library, except a llama was immunized with purified, fully gamma‐carboxylated human plasma‐derived prothrombin from Haematologic Technologies Inc. (Essex Junction, VT, USA).
3
Microparticle-Stimulated Thrombin Assay
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Microparticle prothrombotic activity was measured in a microparticle capture assay using published methods.30 We treated MΦMP samples with the coagulation factor inhibitors Phe‐Pro‐Arg‐chloromethylketone (50 µmol/L) and Glu‐Gly‐Arg‐chloromethylketone (50 µmol/L), and a 50‐µL microparticle aliquot was added to wells of an annexin V–coated 96‐well plate (StreptaWell plate; Roche, San Francisco, CA; biotinylated annexin V, 0.36 ng/µL coating for 30 minutes; Biovision, Milpitas, CA). After 30‐minute incubation and 3 wash steps, we added prothrombin (1.3 µmol/L), factor Va (2.5 nmol/L) and factor Xa (2.5 nmol/L) (Haematologic Technologies, Inc, Essex Junction, VT) in calcium‐containing Tris buffer (25 mmol/L Tris, 2.5 mmol/L calcium) to the microparticle‐containing wells. Following 30‐minute incubation at 37°C, EDTA addition (0.1M) halted the prothrombinase reaction, and we added Chromozym TH chromogenic thrombin substrate (0.57 mmol/L, Roche) to quantify thrombin activity. The assay measured microparticle‐stimulated thrombin production in reference to a standard curve in a multiplate reader (405 nm optic diameter at 1 minute).
4
Quantification of Coagulation Factors
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Indoxyl sulfate (IS), indole-3-acetic acid (IAA) and EGTA were obtained from Sigma (Saint Quentin Fallavier, France). Megamix beads (mix of 0.5, 0.9 and 3.0 μm beads) were from Biocytex (Marseille, France). Trucount Tube (Cat. No. 340334), Purified CD235a (clone GA-R2) and mouse IgG1/IgG2a (clone X40/X39) were from Becton Dickinson Biosciences (San Jose, CA, USA), and were labeled in our laboratory with Alexa Fluor 647 (Invitrogen, Eugene, OR, USA). Bovine lactadherin was purified as described previously Hvarregaard et al. (1996) [30 (link)] and were labeled in our laboratory with Alexa Fluor 488 (Invitrogen). Fluo 3/AM was from Biotium (Hayward, CA, USA). Human factors Va, VIIa, IXa, X, Xa, prothrombin and thrombin were all from Haematologic Technologies (Burlington, VT, USA). Recombinant human factor VIII was from American Diagnostica Inc. Chromogenic substrates S-2765 and S-2238 were from Instrumentation Laboratory Company (MA, USA).
5
Coagulation Factor Variant Characterization
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The proteins hFX-R15Q and hFXa-S195A were gifted by Dr. Camire (Children's Hospital of Philadelphia, Philadelphia, United States). TFPI was a gift from Dr. van't Veer (Amsterdam University Medical Centers, Amsterdam, the Netherlands). The human plasma-derived coagulation proteins FVIIa, FIXa, factor XIa (FXIa), prothrombin, α-thrombin, antithrombin, and RVV-X were from Haematologic Technologies. NovoEight was from Novo Nordisk (Plainsboro, New Jersey, United States). Human TF (Innovin) was from Siemens (Newark, New York, United States), and recombinant hirudin was from Hyphen Biomed (Neuville-sur-Oise, France). Recombinant wild-type hFX and venom-derived P. textilis FX and FXa (v-ptFX and v-ptFXa) were prepared, purified, and characterized as described. 15 Recombinant constitutively active B-domainless human factor V (FV-810; hFV) and venomderived P. textilis FV (v-ptFV) were prepared, purified, and characterized as described. 12, 16 The extinction coefficients (E 0.1%, 280 nm ) of the newly generated hFX-ptAP and ptFX-hAP variants were assumed to be similar to hFX and v-ptFX (1.16). 16, 17 The molecular weights of the various proteins used were taken as follows: hFX 58.0 kDa; hFX-ptAP 55.0 kDa; v-ptFX 55.0 kDa; ptFX-hAP 58.0 kDa.
6
Erythrocyte-Mediated Prothrombin Activation
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prothrombin activation by eryptotic erythrocytes was determined using a previously described assay 30 . Briefly, untreated (Control) and pyocyanin‐treated erythrocytes (4.5% haematocrit) incubated for 48 hrs were treated with human factor Xa (2 nM; Haematologic Technologies, Essex Junction, VT, USA), human factor Va (0.2 nM; Haematologic Technologies) and 2 mM CaCl2 for 3 min. at 37°C. The erythrocytes were then treated with prothrombin (1.4 μM; Haematologic Technologies) for 5 min. and the reaction was stopped by the addition of 10 mM ethylenediaminetetraacetic acid. The samples were then centrifuged (3 min. at 400 × g), diluted fivefold and kinetically evaluated at 405 nm following addition of the chromogenic substrate S2238 (100 μM; Diapharma, West Chester, OH, USA). As a negative control, the coagulation factors and other agents were added to the solution in the absence of erythrocytes.
7
Lectin Pathway Activation Prothrombin Cleavage
8
Protein Reagents for Coagulation Assays
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9
Anticoagulant and Platelet Aggregation Assays
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Heparin was purchased from Sigma (St. Louis, MO, USA) and TNF-α was purchased from Abnova (Taipei, Taiwan). The molecular weight of Heparin used as positive control in our experiments is 3500. Anti-tissue factor (TF) antibody, rivaroxaban, and argatroban were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Thromboxane A2 (TXA2) analog and U46619 were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA, USA). Prothrombin, thrombin, Factor VIIa, X, and Xa were purchased from Haematologic Technologies (Essex Junction, VT, USA) and APTT-XL and thromboplastin-D were purchased from Fisher Diagnostics (Middletown, VA, USA). S-2222 and S-2238 were purchased as substrate of FXa and thrombin from ChromogenixAB (Mölndal, Sweden). Aggregometer (Chronlog, Havertown, PA, USA), thrombotimer (Behnk Elektronik, Norderstedt, Germany), microplate reader (Tecan Austria GmbH, Grödig, Austria), and spectrophotometer (TECAN, Männedorf, Switzerland) were used.
10
Thrombin Production in Platelets
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