Recombinant Human Thrombomodulin/BDCA-3 Protein, CF

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

Recombinant Human Thrombomodulin/BDCA-3 Protein, CF Summary

• Details of Functionality: Measured by its ability to mediate thrombin activation of protein C.
• Source: Mouse myeloma cell line, NS0-derived human Thrombomodulin/BDCA-3 protein
  Ala19-Ser515 (Ala473Val), with a C-terminal 6-His tag
• Accession #: P07204
• N-terminal Sequence: Ala19
• Protein/Peptide Type: Recombinant Enzymes
• Gene: THBD
• 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: 53 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: 80-105 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 °C as supplied.
  • 3 months, -20 to -70 °C 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: TCNB (0.05 M Tris, 0.01 M CaCl₂, 0.15 M NaCl, 0.05 % Brij-35, pH 7.5)
  • Assay Buffer: 0.05 M Tris, 0.1 M NaCl, 0.01% Brij-35, pH 8.5
  • Recombinant Human Coagulation Factor II/Thrombin (rhThrombin) (Catalog # 1473-SE)
  • Recombinant Human Thrombomodulin/BDCA‑3 (rhTHBD) (Catalog # 3947-PA)
  • Recombinant Human Coagulation Factor XIV/Protein C (rhPROC) (Catalog # 3349-SE)
  • Recombinant Human Serpin C1/Antithrombin-III (rhSerpin C1) (Catalog # 1267-PI)
  • Heparin (Sigma-Aldrich, Catalog # H3393), 20 mg/mL in deionized water
  • Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhThrombin to 4 µg/mL in Activation Buffer.
  2. Dilute rhTHBD to 4 µg/mL in Activation Buffer.
  3. Dilute rhPROC to 40 µg/mL in Activation Buffer.
  4. Combine 25 µL of the diluted rhPROC, 12.5 µL of the diluted rhThrombin, and 12.5 µL of the diluted rhTHBD in a microfuge tube. Incubate for one hour at 37 °C to activate rhPROC. As a control combine 25 µL of the diluted rhPROC, 12.5 µL of the diluted rhThrombin, and 12.5 µL of Activation Buffer in a microfuge tube.
  5. Dilute rhSerpin C1 to 20 µg/mL with 32.4 µg/mL heparin in Activation Buffer.
  6. Add and mix 50 µL of the diluted rhSerpin C1/heparin solution to the reaction vials.
  7. Incubate at room temperature for 30 minutes to stop rhThrombin activity.
  8. Dilute the reaction mixture 1:5 in Assay Buffer.
  9. Dilute Substrate to 200 µM in Assay Buffer.
  10. Load in a black well plate 50 µL of diluted reaction mixture (step 8), and start the reaction by adding 50 µL of 200 µM Substrate.
  11. Read (top read) immediately in kinetic mode for 5 minutes at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
  12. Calculate specific activity:

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

  *Adjusted for control containing no rhTHBD
  **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (Sigma-Aldrich, Catalog # A9891).

  Per Well:
  • rhPROC: 0.1 μg
  • rhTHBD: 5 ng
  • 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 Thrombomodulin/BDCA-3 Protein, CF

• BDCA3
• BDCA-3
• CD141 antigen
• CD141
• Fetomodulin
• THBD
• THRM
• Thrombomodulin
• TM

Background

Encoded by the THBD gene, Thrombomodulin is also known as CD141 antigen and blood dendritic cell antigen 3 (BDCA-3). The deduced amino acid (aa) sequence of human THBD (Accession # P07204) predicts a signal peptide (aa 1-18) and a mature chain (aa 19‑575) that consists of following domains: C‑type lectin (aa 31-169), EGF-like (aa 241-281, aa 284-324, aa 325-363, aa 365‑405, aa 404‑440, and aa 441‑481), transmembrane (aa 516 to 539) and cytoplasmic (aa 540‑575). The R&D Systems recombinant human THBD consists of aa 19-515, corresponding to the extracellular portion of the type I membrane protein. Predominantly synthesized by vascular endothelial cells, THBD inhibits coagulation and fibrinolysis (1‑3). It functions as a cell surface receptor and an essential cofactor for active thrombin, which in turn activates protein C and thrombin-activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase B2 (CPB2). Activated protein C (APC), facilitated by protein S, degrades coagulation factors Va and VIIIa, which are required for thrombin activation. Activated CPB2 cleaves basic C‑terminal aa residues of its substrates, including fibrin, preventing the conversion of plasminogen to plasmin. In addition, THBD gene polymorphisims are associated with human disease and THBD plays a role in thrombosis, stroke, arteriosclerosis, and cancer (4). For example, increased serum levels of THBD, due to protease cleavage, have been associated with smoking, cardiac surgery, atherosclerosis, liver cirrhosis, diabetes mellitus, cerebral and myocardial infarction, and multiple sclerosis (5).

1. Van de Wouwer, M. et al. (2004) Arterioscler. Thromb. Vasc. 24:1374.
2. Wu, K.K. et al. (2000) Ann. Med. 32:73.
3. Li, Y.H. et al. (2006) Cardiovasc. Hematol. Agents Med. Chem. 4:183.
4. Weiler, H. and B.H. Isermann (2003) J. Thromb. Haemost. 1:1515.
5. Califano, F. et al. (2000) Eur. Rev. Med. Pharmacol. Sci. 4:59.

Publications for Thrombomodulin/BDCA-3 (3947-PA)(5)

Publications using 3947-PA

• Bhargavan, B;Kanmogne, GD; SARS-CoV-2 Spike Proteins and Cell-Cell Communication Induce P-Selectin and Markers of Endothelial Injury, NETosis, and Inflammation in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for the Pathogenesis of COVID-19 Coagulopathy International journal of molecular sciences 2023-08-09 [PMID: 37628764] (Bioassay, Human)
• A Siwiec-Koz, P Kuszmiersz, L Kasper, M Frolow, P Kozlik-Siw, T Iwaniec, J Kosalka-We, L Zareba, K Sladek, JG Bazan, S Bazan-Soch, J Dropinski Prothrombotic state, endothelial injury, and echocardiographic changes in non-active sarcoidosis patients Scientific Reports, 2022-12-09;12(1):21291. 2022-12-09 [PMID: 36494464] (Bioassay, Human)
• B Bhargavan, GD Kanmogne SARS-CoV-2 Spike Proteins and Cell-Cell Communication Inhibits TFPI and Induces Thrombogenic Factors in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for COVID-19 Coagulopathy Pathogenesis International Journal of Molecular Sciences, 2022-09-09;23(18):. 2022-09-09 [PMID: 36142345] (Bioassay, Human)
• M Hultström, K Fromell, A Larsson, B Persson, B Nilsson, SE Quaggin, C Betsholtz, R Frithiof, M Lipcsey, M Jeansson Angiopoietin-2 Inhibition of Thrombomodulin-Mediated Anticoagulation-A Novel Mechanism That May Contribute to Hypercoagulation in Critically Ill COVID-19 Patients Biomedicines, 2022-06-06;10(6):. 2022-06-06 [PMID: 35740360] (Bioassay, Human)
• C Daly, X Qian, C Castanaro, E Pasnikowsk, X Jiang, BR Thomson, SE Quaggin, N Papadopoul, Y Wei, JS Rudge, G Thurston, GD Yancopoulo, S Davis Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor Sci Rep, 2018-01-11;8(1):505. 2018-01-11 [PMID: 29323190] (Bioassay, Human)

Bioinformatics

• Gene Symbol: THBD
• Uniprot:
  • P07204(Mouse)