Recombinant Human Coagulation Factor III/Tissue Factor, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Coagulation Factor III/Tissue Factor, CF Summary

• Details of Functionality: Measured by its ability to activate Coagulation Factor VII in cleaving a fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The AC 50 is <4.0 μg/mL, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Coagulation Factor III/Tissue Factor protein Gly34-Glu251, with a C-terminal 6-His tag
• Accession #: P13726
• N-terminal Sequence: Gly34
• Protein/Peptide Type: Recombinant Enzymes
• Gene: F3
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 26 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 41 kDa and 43 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  6 months from date of receipt, -20 to -70 degreesC as supplied. 3 months, -20 to -70 degreesC under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, 2.5 mM CaCl₂, pH 8.5
  • Recombinant Human Coagulation Factor III/Tissue Factor (rhTF) (Catalog # 2339-PA)
  • Recombinant Human Coagulation Factor VII (rhF7) (Catalog # 2338-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 600 mM Stock in DMSO
  • Substrate: BOC-Val-Pro-Arg-AMC (R&D Systems, Catalog # ES011), 100 mM in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhF7 to 100 µg/mL in Activation Buffer containing 6.25 µg/mL Thermolysin.
  2. Incubate at 37 °C for 30 minutes to activate rhF7.
  3. Stop reaction by adding 1,10 phenanthroline to a final concentration of 10 mM in Activation Buffer.
  4. Incubate for 5 minutes at room temperature.
  5. Prepare a dilution curve of rhTF in Assay Buffer. Make serial dilutions of: 71.04, 35.52, 17.76, 8.88, 4.44, 2.22, 1.11, 0.555, 0.278, and 0.139 µg/mL.
  6. Dilute Thermolysin-activated rhF7 to 32 µg/mL in Assay Buffer.
  7. Load 25 µL of each dilution from the dilution curve to a plate. Include two controls containing 25 µL Assay Buffer.
  8. Add 25 µL of 32 µg/mL Thermolysin-activated rhF7 to all the wells.
  9. Incubate at 37 °C for 5 minutes.
  10. Dilute Substrate to 200 µM in Assay Buffer.
  11. Add 50 µL of 200 μM Substrate to all wells.
  12. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  13. Derive the 50% activating concentration (AC₅₀) of rhTF by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  14. The specific activity for rhF7 at each point may be determined using the following formula (if needed):

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard 7-amino, 4-methyl coumarin (Sigma, Catalog # A-9891).

  Per Well:
  • rhF7: 0.800 µg
  • rhTF: 17.76, 8.88, 4.44, 2.22, 1.11, 0.556, 0.278, 0.139, 0.0695, and 0.0348 µg/mL
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Coagulation Factor III/Tissue Factor, CF

• CD142 antigen
• CD142
• coagulation factor III (thromboplastin, tissue factor)
• Coagulation Factor III
• F3
• FLJ17960
• TF
• TFA
• Thromboplastin
• Tissue Factor

Background

Coagulation Factor III/Tissue Factor (TF), also known as thromboplastin and CD142, is an integral membrane protein found in a variety of cell types. It functions as a protein cofactor/receptor of Coagulation Factor VII, which is synthesized in the liver and circulated in the plasma (1). Upon binding of TF, the inactive factor VII is rapidly converted into activated VIIa. The resulting 1:1 complex of VIIa and TF initiates the coagulation pathway and has also important coagulation-independent functions such as angiognesis (2). Synthesized as a 295 amino acid precursor, TF consists of a signal peptide (residues 1 to 32) and the mature chain (residues 33 to 295). As a type I membrane protein, it contains a transmembrane region (residues 252 to 274) and a cytoplasmic tail (residues 275 to 295) (3-6). The purified rhTF corresponds to the ectodomain (residues 34 to 251) and is potent in activating thermolysin-processed Recombinant Human Coagulation Factor VII (Catalog # 2338-SE) under the conditions described in Activity Assay Protocol.

1. Morrissey, J.H. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. (ed) San Diego, Academic Press, p. 1659. 
2. Versteeg, H.H. et al. (2003) Carcinogenesis 24:1009. 
3. Scarpati, E.M. et al. (1987) Biochemistry 26:5234.
4. Fisher, K.L. et al. (1987) Thromb. Res. 48:89.
5. Morrissey, J.H. et al. (1987) Cell 50:129.
6. Spicer, E.K. (1987) Proc. Natl. Acad. Sci. USA 84:5148.

Publications for Coagulation Factor III/Tissue Factor (2339-PA)(6)

Publications using 2339-PA

• Suzuki, A;Hamada, S;Oono, A;Hasegawa, Y;Yamauchi, Y;Okishige, K;Hirao, K;Sasano, T; Edoxaban eliminates hypercoagulability evoked by transient temperature changes in human whole blood Journal of arrhythmia 2023-12-01 [PMID: 38045446] (Bioassay, Human)
• Rees, PA;Castle, J;Clouston, HW;Lamb, R;Singh, U;Duff, SE;Kirwan, CC; The effects of coagulation factors and their inhibitors on proliferation and migration in colorectal cancer Cancer medicine 2023-07-17 [PMID: 37455592] (Bioassay, Human)
• Y Feng, J Ma, LV Tang, W Lin, Y Tao, Z Cheng, Y Hu Characterization of a Missense Mutation in the Catalytic Domain and a Splicing Mutation of Coagulation Factor X Compound Heterozygous in a Chinese Pedigree Genes, 2021-09-27;12(10):. 2021-09-27 [PMID: 34680916] (Bioassay, N/A)
• RG Holt, D Luo, N Gruver, DB Khismatull Quasi-static acoustic tweezing thromboelastometry J. Thromb. Haemost., 2017-06-09;0(0):. 2017-06-09 [PMID: 28453901] (Bioassay, Human)
• Conroy AL, Liles WC, Molyneux ME Performance characteristics of combinations of host biomarkers to identify women with occult placental malaria: a case-control study from Malawi. PLoS ONE, 2011-12-12;6(12):e28540. 2011-12-12 [PMID: 22174834] (ELISA (Standard))
• Amara U, Flierl MA, Rittirsch D, Klos A, Chen H, Acker B, Bruckner UB, Nilsson B, Gebhard F, Lambris JD, Huber-Lang M Molecular intercommunication between the complement and coagulation systems. J. Immunol., 2010-09-24;185(9):5628-36. 2010-09-24 [PMID: 20870944] (Enzyme Assay, Human)

FAQs for Coagulation Factor III/Tissue Factor (2339-PA). (Showing 1 - 1 of 1 FAQs).

1. Have you tested any of your anti-tissue factor antibodies against pig? I need one for western blot.
   • Unfortunately, none of our Tissue Factor antibodies have yet been tested with pig samples, so we cannot guarantee that any of them will work in pig. Here is a link to Tissue Factor antibodies that have been validated for use in Western blot. If you are interested in testing any of these antibodies against pig samples, you could become eligible for our Innovator's Reward program.

Bioinformatics

• Gene Symbol: F3
• Uniprot:
  • P13726()