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

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

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