Thrombin, alpha, Human

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. \n\nThrombin 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. \n\nThrombin 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.\n\nProteolytic Activation of Prothrombin:\nThe serine protease a-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, a-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. \n\nLocalization: Plasma \n\nMode of Action: \nSerine 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.\n\nExtinction Coefficient: E1%1cm, 280nm=18.3 (6)\n\nSpecific Activity: ~3800U/mg, Relative to human NIH standard thrombin.\n\nIsoelectric Point: \n7.0-7.6 (3)\n\nStructure: \nTwo subunits, ~Mr=6,000 and 31,000\n\nPercent Carbohydrate: ~5%\n\nStorage and Stability: \nMay be stored at 4 degrees C for short-term only. For long-term storage, store at -20 degrees C. Aliquots are stable for 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

T4040

Size

100ug

Form

Supplied as a liquid in 50% glycerol/H2O. Thrombin is also available with the active site blocked with either DFP, FPRck, or biotinlyated FPRck.

Purity

(same/more than) 95% by SDS-PAGE. Human thrombin is prepared from purified prothrombin using a modification of the Lundblad procedure (1) as described by Nesheim et al. (2). Purity is determined by SDS-PAGE analysis and activity is measured in a thrombin specific clotting assay, and compared to standardized NIH thrombin.

References

US Biological application reference: Osborne, S.A. et al., (2008) Glycobiology 18:225-234. 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).