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

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 Our 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

E1%(280nm) = 18.3 (human)15
E1%(280nm) = 19.5 (bovine)16

Prothrombin

Measurement of Thrombin Activity

Our 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.18-20

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

Thrombin (human and bovine) will catalyze the hydrolysis of several peptide p-nitroanilides, tosyl-arg-nitrobenzyl ester, and a thiobenzyl ester synthetic substrates.21

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 8.0.22

Thrombin cleavage of fusion proteins can be carried out at a thrombin to fusion protein ratio of 1:500.23
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.24

Products

Human Recombinant Thrombin
Loading
Human Thrombin
Loading
Bovine Thrombin
Loading
Rabbit Thrombin
Loading
Rat Thrombin
Loading

Substrates

Natural Substrates Fibrinogen, Factor V,25 Factor VIII,25 Factor XIII,25 Thrombospondin,26 Prothrombin,12 and Protein C.27

Synthetic Substrates 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,28 phenylmethylsulfonylfluoride, 28 AEBSF, hirudin,28 tetranitromethane,28 proflavine,28 antithrombin III,28 a1-antitrypsin28 , a1-antiplasmin,28 gabexate mesylate,29 antipain,30 and tosyl-L-lysinechloromethylketone.31

References

1.
EC 3.4.21.5. [Internet]. International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature.[updated 05 Jun 2020; cited 18 Jul 2020]. Available from: https://www.qmul.ac.uk/sbcs/iubmb/enzyme/EC3/4/21/5
2.
CHANG J. 1985. Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur J Biochem. 151(2):217-224. http://dx.doi.org/10.1111/j.1432-1033.1985.tb09091.x
3.
1975. The Plasma Proteins. http://dx.doi.org/10.1016/c2013-0-11337-2
4.
Machovich R. 1984. The Thrombin. 1. Boca Raton (FL): CRC Press.
5.
Machovich R. 1984. The Thrombin. 1. Boca Raton (FL): CRC Press.
6.
Prasad S, Cantwell AM, Bush LA, Shih P, Xu H, Di Cera E. 2004. Residue Asp-189 Controls both Substrate Binding and the Monovalent Cation Specificity of Thrombin. J. Biol. Chem.. 279(11):10103-10108. http://dx.doi.org/10.1074/jbc.m312614200
7.
Kisiel W. 1979. Human Plasma Protein C. J. Clin. Invest.. 64(3):761-769. http://dx.doi.org/10.1172/jci109521
8.
1975. The Plasma Proteins. http://dx.doi.org/10.1016/c2013-0-11337-2
9.
Liu T, Qian W, Gritsenko MA, Camp DG, Monroe ME, Moore RJ, Smith RD. 2005. Human PlasmaN-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry. J. Proteome Res.. 4(6):2070-2080. http://dx.doi.org/10.1021/pr0502065
10.
Nilsson B, Horne MK, Gralnick HR. 1983. The carbohydrate of human thrombin: Structural analysis of glycoprotein oligosaccharides by mass spectrometry. Archives of Biochemistry and Biophysics. 224(1):127-133. http://dx.doi.org/10.1016/0003-9861(83)90196-0
11.
Boyer PD. 1971. The Enzymes. 3. New York: Academic Press.
12.
Prothrombin precursor - Homo sapiens (Human) - F2 gene & protein. [Internet]. Universal Protein Resource (UniProt).[updated 16 Jun 2020; cited 18 Jul 2020]. Available from: https://www.uniprot.org/uniprot/P00734
13.
Boissel JP, Bonniec BL, Rabiet MJ, Labie D, Elion J. 1984. Covalent structures of beta and gamma autolytic derivatives of human alpha-thrombin. J. Biol. Chem.. 2595691-7.
14.
Righetti PG, Tudor G, Ek K. 1981. Isoelectric points and molecular weights of proteins. Journal of Chromatography A. 220(2):115-194. http://dx.doi.org/10.1016/s0021-9673(00)88456-3
15.
Butkowski RJ, Elion J, Downing MR, Mann KG. 1977. Primary structure of human prethrombin 2 and alpha-thrombin. J. Biol. Chem. 252(14):4942-57.
16.
Winzor D, Scheraga H. 1964. Titration behavior of bovine thrombin. Archives of Biochemistry and Biophysics. 104(2):202-207. http://dx.doi.org/10.1016/s0003-9861(64)80004-7
17.
Biggs R. 1976. Human Blood Coagulation, Haemostasis and Thrombosis. 2. Blackwell Scientific Publications.
18.
Hemker HC. 1983. Handbook of Synthetic Substrates. Dordrecht: Springer Netherlands.
19.
Gaffney PJ, Edgell TA. 1995. The International and ?NIH? Units for Thrombin - How Do They Compare?. Thromb Haemost. 74(03):900-903. http://dx.doi.org/10.1055/s-0038-1649844
20.
Whitton C, Sands D, Lee T, Chang A, Longstaff C. 2005. A reunification of the US (?NIH?) and International Unit into a single standard for Thrombin. Thromb Haemost. 93(02):261-266. http://dx.doi.org/10.1160/th04-10-0677
21.
Lottenberg R, Christensen U, Jackson CM, Coleman PL. 1981. [28] Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates.341-361. http://dx.doi.org/10.1016/s0076-6879(81)80030-4
22.
CHANG J. 1985. Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur J Biochem. 151(2):217-224. http://dx.doi.org/10.1111/j.1432-1033.1985.tb09091.x
23.
Hakes DJ, Dixon JE. 1992. New vectors for high level expression of recombinant proteins in bacteria. Analytical Biochemistry. 202(2):293-298. http://dx.doi.org/10.1016/0003-2697(92)90108-j
24.
Guan K, Dixon JE. 1991. Eukaryotic proteins expressed in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Analytical Biochemistry. 192(2):262-267. http://dx.doi.org/10.1016/0003-2697(91)90534-z
25.
De Cristofaro R, De Candia E. 2003. Thrombin Domains: Structure, Function and Interaction with Platelet Receptors. J Thromb Thrombolysis. 15(3):151-163. http://dx.doi.org/10.1023/b:thro.0000011370.80989.7b
26.
Sherwood JA, Roberts DD, Spitalnik SL, Lawler JW, Miller LH, Howard RJ. 1990. Falciparum malaria parasitized erythrocytes bind to a carboxy-terminal thrombospondin fragment and not the amino-terminal heparin-binding region. Molecular and Biochemical Parasitology. 40(2):173-181. http://dx.doi.org/10.1016/0166-6851(90)90039-o
27.
Berg DT, Wiley MR, Grinnell BW. 1996. Enhanced Protein C Activation and Inhibition of Fibrinogen Cleavage by a Thrombin Modulator. Science. 273(5280):1389-1391. http://dx.doi.org/10.1126/science.273.5280.1389
28.
Lundblad RL, Kingdon HS, Mann KG. 1976. [14] Thrombin.156-176. http://dx.doi.org/10.1016/s0076-6879(76)45017-6
29.
MATSUOKA S, FUTAGAMI M, OHNO H, IMAKI K, OKEGAWA T, KAWASAKI A. 1989. Inhibitory effects of ONO-3307 on various proteases and tissue thromboplastin in vitro and on experimental thrombosis in vivo.. Jpn.J.Pharmacol. 51(4):455-463. http://dx.doi.org/10.1254/jjp.51.455
30.
Wiman B. 1981. [32] Human ?2-antiplasmin.395-408. http://dx.doi.org/10.1016/s0076-6879(81)80034-1
31.
Magnusson S. 1971. 9 Thrombin and Prothrombin.277-321. http://dx.doi.org/10.1016/s1874-6047(08)60400-x