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Thrombin

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.

PURCHASING AND PRODUCT INFORMATION

Catalog Number HCT-0020 HCT-DFP HCT-FPRCK HCT-BFPRCK BCT-1020 BCT-DFP BCT-FPRCK BCT-BFPRCK MCT-5020**	Description Human α-Thrombin   Human α-Thrombin-DFP Human α-Thrombin-FPRck (FPR-CMK*) Human α-Thrombin-BFPRck (Biotin) Bovine α-Thrombin Bovine α-Thrombin-DFP Bovine α-Thrombin-FPRck (FPR-CMK*) Bovine α-Thrombin-BFPRck (Biotin)  Mouse Thrombin	Size 100 µg or 1 mg 100 µg 100 µg 100 µg 200 µg or 1 mg 100 µg 100 µg 100 µg 50 µg	Formulation 50% glycerol/water (v/v) 20 mM Hepes, 150 mM NaCl, pH 7.4 20 mM Hepes, 150 mM NaCl, pH 7.4 20 mM Hepes, 150 mM NaCl, pH 7.4 50% glycerol/water (v/v) 20 mM Hepes, 150 mM NaCl, pH 7.4 20 mM Hepes, 150 mM NaCl, pH 7.4 20 mM Hepes, 150 mM NaCl, pH 7.4 50% glycerol/water (v/v)
Storage Glyercol formulation: -20oC Hepes formulation: -80oC	Purity >95% by SDS-PAGE	Activity Determination Fibrinogen clotting or chromogenic assay	Shelf Life (properly stored) 12 months
Compliment fluorogenic substrate(s): HTI Catalog # SN-17a, SN-20, and SN-59		DOWNLOAD THROMBIN PDF
		Sample Gel Information: Gel: Novex 4-12% Bis-Tris Load: Human Thrombin, 1 µg per lane Buffer: MES Standard: SeeBluePlus 2; Myosin (191 kDa), Phosphorylase B (97 kDa), BSA (64 kDa), Glutamic Dehydrogenase (51 kDa), Alcohol Dehydrogenase (39 kDa), Carbonic Anhydrase (28 kDa), Myoglobin Red (19 kDa), Lysozyme (14 kDa)
Sample publications referencing our Thrombin: Rovner, A, Fagnant, P., Trybus, K., Biochemistry. 2006 April 25; 45(16): 5280–5289. (Fusion Protein Cleavage) Bai, E., et al., Biochem. J. (2006) 400, 385–392. (Fusion Protein Cleavage) Lian, L., et al., Blood. 2005 July 1; 106(1): 110–117. (Platelet Activation) Nylander, S., et al., British Journal of Pharmacology (2004) 142, 1325–1331 (Platelet Activation) This publication list is not all encompassing, and is only meant to provide limited examples of how Haematologic Technologies' 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.
U.S. Pricing	Product inquiry	SAMPLE DATA SHEET	ORDER NOW!

* Chloromethylketone

**Due to mouse plasma quality issues our mouse proteins are not being sold until further notice.  At the present time we cannot give a completion date.  Thank you..

Overview of Thrombin

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 -20^oC. 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.

Properties of Thrombin

PURCHASING AND PRODUCT INFORMATION

* Chloromethylketone

References 

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).