Thrombin (alpha)

Proteolytic Activation of Prothrombin
The serine protease α-thrombin is produced by proteolytic activation of the zymogen, prothrombin. The enzyme complex, prothrombinase, catalyzes the proteolysis of two peptide bonds in prothrombin, which gives rise to an NH2-terminal derived F1.2 region and the heterodimer, α-thrombin. Alpha-thrombin is composed of an “A” chain (Mr=6000) which is covalently linked to a “B” chain (Mr=31,000) through a single disulfide bond.

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  • Human alpha-Thrombin

    HCT-0020

    $54.00 – $166.00

    SKU: HCT-0020 Category:
    Price:$166.00/1mg, $54.00/100 µg
    Sample Data Sheet  |  Safety Data Sheet
    Size 1mg, 100 µg
    Formulation 50% glycerol/water (v/v)
    Storage -20°C
    Shelf Life 12 months
    Purity >95% by SDS-PAGE
    Activity Determination Fibrinogen clotting or chromogenic assay

  • Human alpha-Thrombin, DFP active site blocked

    HCT-DFP

    $71.00 – $417.00

    SKU: HCT-DFP Category:
    Price:$417.00/1 mg, $71.00/100 µg
    Safety Data Sheet
    Size 1 mg, 100 µg
    Formulation 20 mM Hepes, 150 mM NaCl, pH 7.4
    Storage -80°C
    Shelf Life 12 months
    Purity >95% by SDS-PAGE
    Activity Determination Fibrinogen clotting or chromogenic assay

Alpha-thrombin is a highly specific serine protease generated by proteolytic activation of the zymogen prothrombin (1). During coagulation, thrombin cleaves fibrinogen to form fibrin, leading to the ultimate step in coagulation, the formation of a fibrin clot. Thrombin is also responsible for feedback activation of the procofactors factor V and factor VIII. Thrombin has also been reported to activate factor XIII and platelets, and also functions as a vasoconstrictor protein. The procoagulant activity of thrombin is arrested in two ways: 1) inhibition by either heparin cofactor II or the antithrombin III/heparin complex; or 2) complex formation with thrombomodulin. Formation of the thrombin/thrombomodulin complex results in the inability of thrombin to cleave fibrinogen and activate factors V and VIII, but increases the efficiency of thrombin for activation of the anticoagulant, protein C.

Thrombin is a two chain enzyme composed of an NH2-terminal “A” chain (Mr=6,000) and a COOH-terminal “B” chain (Mr=31,000) which remain covalently associated through a single disulfide bond. Human thrombin is 13 amino acids shorter than the bovine thrombin due to a thrombin cleavage site on the human protein that is not present in the bovine protein.

Thrombin is also utilized for site specific cleavage of fusion proteins expressed in bacteria (9-11). A thrombin sensitive site is incorporated between the recombinant protein of interest and peptides or proteins which facilitate purification and/or expression. The target protein is released from the expressed hybrid by cleavage with thrombin. Thrombin can then be easily removed by affinity chromatography.

Human, bovine and mouse thrombin are prepared from purified prothrombin using a modification of the Lundblad procedure (1) as described by Nesheim et al. (2). Thrombin is supplied in 50% (vol/vol) glycerol/H2O and should be stored at -20oC. Purity is determined by SDS-PAGE analysis and activity is measured in a thrombin specific clotting assay, and compared to standardized NIH thrombin. Thrombin is also available with the active site blocked with either DFP, FPRck, or biotinlyated FPRck.

Cleavage of Fusion Proteins
In addition to its broad application in coagulation research thrombin can be used for site specific cleavage of fusion proteins. A thrombin sensitive site is incorporated between the recombinant protein of interest and peptides or proteins which facilitate purification and/or expression. The target protein is released from the expressed hybrid by cleavage with thrombin. Thrombin can then be easily removed by affinity chromatography. Lot to lot consistency ensures reproducible results every time. For experiments involving cell cultures, please contact us to discuss custom, low endotoxin lots designated for cell culture use.

Sample gel image
GelNovex 4-12% Bis-Tris
LoadHuman Beta & Gamma Thrombin, 1 µg per lane
BufferMES
StandardSeeBluePlus 2; Myosin (188 kDa), Phosphorylase B (98 kDa), BSA (62 kDa), Glutamic Dehydrogenase (49 kDa), Alcohol Dehydrogenase (38 kDa), Carbonic Anhydrase (28 kDa), Myoglobin Red (17 kDa), Lysozyme (14 kDa), Aprotinin (6 kDa), Insulin, B chain (3 kDa).
LocalizationPlasma
Mode of actionSerine protease which cleaves fibrinogen to form fibrin; also responsible for activation of protein C, platelet activation and feedback activation of the procofactors, factor V and factor VIII
Molecular weigh36,700 (3-6)
Extinction coefficient
E
1 %
1 c m, 280 nm
= 18.3 (human) (6)
= 19.5 (bovine) (7)
Specific Activityapproximately 3800 NIH units/mg
Isoelectric point7.0-7.6 (human) (3)
Structuretwo subunits, approximately Mr=6,000 and 31,000
Percent carbohydrateapproximately 5%
  1. Lundblad, R.L., et al., Methods Enzymol., 45, 156 (1976).
  2. Nesheim, M.E., et al., J. Biol. Chem., 258, 5386 (1983).
  3. Fenton, J.W., et al., in Chemistry and Biology of Thrombin, ed. R.L. Lundblad, J.W. Fenton, K.G. Mann, pp. 43-70. Ann Arbor, MI: Ann Arbor Science Publishers, Inc., 1977.
  4. Braughman, D.J., et al., J. Biol. Chem., 242, 5252 (1967).
  5. Winzor, D.J., et al., Arch. Biochem. Biophys., 104, 202 (1964).
  6. Fenton, J.W., et al., J. Biol. Chem., 252, 3587 (1977).
  7. Winzor, D.J., et al., J. Phys. Chem., 68, 338 (1964).
  8. Magnusson, S., in The Enzymes, ed. P.D. Boyer, vol. III, pp. 277-321. New York: Academic Press, 1971.
  9. Gaun, K.L. and Dixon, J.E., Anal. Biochem., 192, 262 (1991).
  10. Germino, J. and Bastia, D., Proc. Natl. Acad. Sci. USA, 81, 4692 (1984).
  11. Chang, J.-Y., Eur. J. Biochem., 151, 217 (1985).
  1. Rovner, A, Fagnant, P., Trybus, K., Biochemistry. 2006 April 25; 45(16): 5280–5289. (Fusion Protein Cleavage)
  2. Bai, E., et al., Biochem. J. (2006) 400, 385–392. (Fusion Protein Cleavage)
  3. Lian, L., et al., Blood. 2005 July 1; 106(1): 110–117. (Platelet Activation)
  4. Nylander, S., et al., British Journal of Pharmacology (2004) 142, 1325–1331 (Platelet Activation)
  5. Bos, M., et al., Blood. 2009 114: 686-692. (Cleavage of venom factor V from brown snake)
  6. Krisinger, M. et al., FEBS Journal 276 (2009) 6586–6602. (Activated mouse protein C)

This publication list is not all encompassing, and is only meant to provide limited examples of how Prolytix products are used. We encourage you to search the literature for other examples pertinent to your experimentation, and to contact us with any technical questions.

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