Proteolytic Activation of Prothrombin: The 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 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. It 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 6000) 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.
Extinction Coefficient:E1%1cm, 280nm=19.5 (7)
Structure:Two subunits, ~Mr 6000 and 31,000
Specific Activity:~3800U/mg. Activity is measured in a thrombin specific clotting assay and compared to standardized NIH thrombin.
Storage and Stability:May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt at -20°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.
Thrombin, alpha, Bovine Products:Thrombin is also available with the active site blocked with either DFP, FPRck, or biotinlyated FPRck
Purity：≥95% by SDS-PAGE. Purified from prothrombin using a modification of the Lundblad procedure (1) as described by Nesheim, et al. (2). Purity is determined by SDS-PAGE analysis.
