Thrombin Factor IIa

Specificity
Thrombin is an endolytic serine protease that selectively cleaves the Arg--Gly bonds of fibrinogen to form fibrin and release fibrinopeptides A and B.^1,2

The optimal cleavage sites for thrombin have been determined to be 1) A-B-Pro-Arg-||-X-Y where A and B are hydrophobic amino acids and X and Y are nonacidic amino acids and 2) Gly-Arg-||-Gly.²

Thrombin from any mammalian species will clot the fibrinogen of any other mammalian species.³

Thrombin cleaves fibrinogen in 2 ways, but only at arginine sites. The primary cleavage product, fibrinopeptide A is cleaved from fibrinogen after amino acid 16 and sometimes after amino acid 19, while a secondary cleavage product, fibrinopeptide B is produced by cleavage at amino acid 14.⁴

Thrombin is active in the pH range of 5-10.⁵
Catalytic optimum is pH 8.3.⁵
Thrombin precipitates at pH 5 or less.⁵

Thrombin does not require divalent metal ions or cofactors for activity. However, Na+-dependent allosteric activation of thrombin has been shown to play a role in defining the primary specificity of thrombin to cleave after Arg residues.⁶ Thromobmodulin serves as a cofactor to thrombin during the activation of protein C.⁷

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In vivo Processing and Physical Properties
The predominant form of thrombin in vivo is it's zymogen, prothrombin (factor II), which is produced in the liver. The concentration of prothrombin in normal human plasma is ~5-10 mg/dL.⁸ Prothrombin is a glycoprotein with a glycan content of ~12%.⁸

Prothrombin is cleaved in vivo by activated factor X releasing the activation peptide and cleaving thrombin into light and heavy chains yielding catalytically active α-thrombin. α-Thrombin is composed of a light chain (A chain)(MW ~ 6,000) and a heavy chain (B chain)(~31,000). These two chains are joined by one disulfide bond.¹² The B chain of human thrombin consists of a peptide portion (MW 29,485) and a carbohydrate portion (2334) with N-linked glycosylation at three Asn sites.^9,10 Bovine thrombin contains 1.7% glucosamine, 1.8% sialic acid, 0.61% galactose, and 0.95% mannose.¹¹ Thrombin also contains γ-carboxyglutamyl residues. These modified glutamyl residues are the result of carboxylation by a microsomal enzyme, vitamin K-dependent carboxylase. γ-carboxyglutamyl residues are necessary for the calcium-dependent interaction with a negatively charged phospholipid surface, which is essential for the conversion of prothrombin to thrombin.¹² In vivo, prothrombin is activated on the surface of a phospholipid membrane that binds the amino terminus of prothrombin along with factors Va and Xa. The activation process starts slowly because factor V itself has to be activated by the initial small amounts of thrombin.

Under certain storage conditions, autolytic digestion of α-thrombin results in the formation of β- and γ-thrombins that lack fibrinolytic activity, but retain some activity against synthetic peptide substrates and protein substrates other than fibrinogen.¹³ Sigma's thrombin preparations are predominantly the α-thrombin form.

Human thrombin consists of several isozymes with isoelectric points in the range of 6.35-7.6.
For bovine the pI range is 7.05 - 7.114

E^1%(280nm) = 18.3 (human)¹⁵
E^1%(280nm) = 19.5 (bovine)¹⁶

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Measurement of Thrombin Activity
Sigma's thrombin assay procedure is expressed in NIH units obtained by direct comparison to a NIH Thrombin Reference Standard.

The NIH assay procedure uses 0.2 mL of diluted plasma (1:1 with saline) as a substrate and 0.1 mL of thrombin sample (stabilized in a 1% buffered albumin solution at pH 7.35) based on a modification of the method of Biggs.¹⁷ Only clotting times in the range of 15-25 seconds are used for determining thrombin concentrations.

Thrombin concentrations in the literature are typically reported in terms of different units of activity.¹⁸

Several conventions are used in thrombin literature:
1 IOWA unit= 0.83 NIH unit
1 WHO unit = 0.56 NIH unit
1 NIH unit = 0.324 +/- 0.073 µg
1 NIH unit = 1 USP unit

Thrombin (human and bovine) will catalyze the hydrolysis of several peptide p-nitroanilides, tosyl-arg-nitrobenzyl ester, and a thiobenzyl ester synthetic substrates.¹⁹

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Applications
Production of fibrin clot in plasma:
Typicallty one to two units of thrombin will clot one mL of plasma.

Cleavage of Fusion Proteins:
Thrombin can be used for the cleavage of many peptides at the thrombin recognition site using concentrations of 0.5 NIH units thrombin per one nanomole polypeptide in 20 microliters of 50 mM ammonium bicarbonate, pH 8.0.²⁰

Thrombin cleavage of fusion proteins can be carried out at a thrombin to fusion protein ratio of 1:500.²¹
Fusion proteins may be cleaved in thrombin cleavage buffer consisting of 50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl₂ and 0.1% 2-mercaptoethanol. 2 mg of fusion protein was incubated with 4 µg of thrombin for 20 minutes at RT in the cleavage buffer.²²

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Products

Human Recombinant Thrombin
Product No.	Product Name	Add to Cart
T9326	Thrombin from human, recombinant, expressed in HEK 293 cells, ≥2,000 units/mg protein

Human Thrombin
Product No.	Product Name	Add to Cart
T1063	Thrombin from human plasma lyophilized powder, >2800 NIH units/mg protein (E1%/280, 18.3)
T6884	Thrombin from human plasma lyophilized powder, ≥2,000 NIH units/mg protein (E1%/280, 18.3)
T9010	Thrombin from human plasma lyophilized powder, Suitable for routine use in the thrombin time test
T7009	Thrombin from human plasma lyophilized powder, ~1,000 NIH units/mg protein (E1%/280, 18.3)
T9135	Thrombin from human plasma lyophilized powder, Suitable for routine use in the thrombin time test
T4393	Thrombin from human plasma lyophilized powder, ~1,000 NIH units/mg protein (E1%/280, 18.3), cell culture tested
T7572	Thrombin from human plasma 50-300 NIH units/mg protein

Bovine Thrombin
Product No.	Product Name	Add to Cart
T7513	Thrombin from bovine plasma lyophilized powder, ≥2,000 NIH units/mg protein (E1%/280 = 19.5)
T9549	Thrombin from bovine plasma powder, cell culture tested, 1,500-2,500 NIH units/mg protein (E1%/280 = 19.5)
T9681	Thrombin from bovine plasma lyophilized powder, ≥700 NIH units/mg protein (Bradford)
T6634	Thrombin from bovine plasma lyophilized powder, 600-1,500 NIH units/mg protein (biuret)
T4265	Thrombin from bovine plasma lyophilized powder, 175-350 NIH units/mg protein (biuret)
T9000	Thrombin from bovine plasma buffered aqueous solution, ≥125 NIH units/mg protein (biuret)
T6200	Thrombin from bovine plasma lyophilized powder, 40-300 NIH units/mg protein (biuret)
T3399	Thrombin from bovine plasma lyophilized powder, 50-150 NIH units/mg protein (biuret)

Murine Thrombin
Product No.	Product Name	Add to Cart
T8397	Thrombin from murine plasma lyophilized powder, ~1,000 NIH units/mg protein (biuret)

Rabbit Thrombin
Product No.	Product Name	Add to Cart
T4897	Thrombin from rabbit plasma

Rat Thrombin
Product No.	Product Name	Add to Cart
T5772	Thrombin from rat plasma lyophilized powder, ≥1,000 units/mg protein (biuret)

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Substrates
Natural Substrates
Fibrinogen, Factor V,²³ Factor VIII,²³ Factor XIII,²³ Thrombospondin,²⁴ Prothrombin,¹² and Protein C.²⁵

Synthetic Substrates
N-Benzoyl-Phe-Val-Arg-p-nitroanilide, N-Benzoyl-Phe-Val-Arg 4-methoxy-b-naphthylamide, N-Benzoyl-Phe-Val-Arg-p-nitroanilide, Boc-β-benzyl-Asp-Pro-Arg-7-amido-4-methylcoumarin, Boc-Val-Pro-Arg-7-amido-4-methylcoumarin, Sar-Pro-Arg p-nitroanilide, Thrombin generation chromogenic, N-p-Tosyl-Gly-Pro-Arg 7-amido-4-methylcoumarin, N-(p-Tosyl)-Gly-Pro-Arg p-nitroanilide.

Inhibitors
Diisopropylfluorophosphate,²⁶ phenylmethylsulfonylfluoride, ²⁶ AEBSF, hirudin,²⁶ tetranitromethane,²⁶ proflavine,²⁶ antithrombin III,²⁶ a1-antitrypsin²⁶ , a1-antiplasmin,²⁶ gabexate mesylate,²⁷ antipain,²⁸ and tosyl-L-lysinechloromethylketone.²⁹

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References

1. Enzyme Nomenclature: EC 3.4.21.5
2. Chang, J.Y., Eur. J. Biochem., 151, 217?224 (1985).
3. The Plasma Proteins, 2nd ed., 2, Putnam, F. W., ed, p. 148.
4. Machovich, R., The Thrombin, 1, 63-66 (1984)
5. Machovich, R., The Thrombin, 1, 111 (1984)
6. Prasad, S., J. Biol. Chem. 279, 10103-10108 (2004)
7. Kisiel, W., Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. J. Clin. Invest. 64, 761-769, (1979)
8. The Plasma Proteins, 2nd ed., 2, Putnam, F. W., ed: Table 2. See also: The Enzyme Explorer: Plasma and Blood Protein Resource
9. Qian, W.J., et al., J. Proteome Res., 4, 2070-2080 (2005).
10. Nilsson, B., et al., Arch. Biochem. Biophys., 224, 127-133 (1983)
11. Boyer, P.D., The Enzymes, Academic Press (New York), 3rd ed., Vol. III, p. 277-321 (1971).
12. Expasy/SwissProt: P00743
13. Boissel, J.P., et al., J. Biol. Chem., 259, 5691-5697 1984).
14. Righetti, P.G., and Tudor, G., Isoelectric points and molecular weights of proteins, a new table. Journal of Chromatography, 220, 115-194 (1981).
15. Butkowski, R.J. et al., J. Biol. Chem., 252, 4942 (1977).
16. Winzor, D. J. and Scheraga, H. A., Arch. Biochem. Biophys. 104, 202-207 (1964)
17. Human Blood Coagulation, Haemostasis and Thrombosis, 2nd ed., R. Biggs, ed., p. 722 (1976).
18. The Handbook of Synthetic Substrates, Hemker, H. C., Martinus Nijhoff publisher (1983).
19. Lottenberg, R., et al., Assay of Coagulation Proteases Using Peptide Chromogenic and Fluorogenic Substrates. Meth. Enzymol., 80-C, 341-361 (1981).
20. Chang, Y., Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur. J. Biochem., 151(2), 217-224 (1985).
21. Hakes, D.J. and Dixon, J.E., Anal. Biochem., 202, 293 (1992).
22. Gaun, KL and Dixon, JE,, Anal. Biochem., 192, 262, 1991
23. De Cristofaro, R. and De Candia, E., J. Thromb. Thrombolysis, 15, 151-163 (2003)
24. Sherwood, J.A., Mol. Biochem. Parisitol., 40, 173-181 (1990)
25. Berg, D.T., et al., Science, 273, 1389-1391 (1996)
26. Lundblad, R.L. et al., Methods Enzymol., 45, 156 (1976)
27. Matsuoka, S., et al., JP. J. Pharmacol., 51, 455-463 (1989)
28. Wimen, B., Meth. Enzymol., 80, 395-408 (1981)
29. Magnusson, S. The Enzymes, 3^rd ed., III, pp. 277-321, Boyer, P.D., ed., Academic Press (1971)

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