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Structural-functional relationships in the human thrombin A-chain

University of British Columbia

Thrombin is the terminal protease in the coagulation cascade and plays a pivotal role in haemostasis, affecting both amplification and down-regulation of coagulation. Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain region has been neglected in comparison to the other domains. While originally considered to be simply an activation remnant with little physiological function, mutations in the prothrombin A-chain region lead to bleeding disorders. There is evidence that the thrombin A-chain may play a role as an allosteric effector in enzymatic reactions and may also represent a structural scaffold to stabilize the protease domain; however, the exact role(s) of the A-chain remain to be elucidated. In this thesis, the roles of the A-chain region in prothrombin folding and activation, thrombin Ca²⁺⁺ binding, enzyme stability and function were investigated. The results from this study suggest that the A-chain region is not required for prothrombin folding and secretion out of the cells; however, the A-chain is required for prothrombin activation. In an independent study using x-ray crystallographic techniques, NMR and activity assays, no evidence of a Ca²⁺⁺ binding site was found in the thrombin A-chain or elsewhere in the thrombin molecule. During prothrombin activation, nascent thrombin undergoes autolysis of a 13-residue N-terminal peptide of the A-chain to produce α-thrombin. Nascent thrombin and α-thrombin were compared to assess the effects of the A13 peptide. Contrary to expectation, autolysis of the A13 peptide at the N-terminus of the thrombin A-chain was very slow, with a half life of 46 minutes. Investigation of whether retention of this peptide affected thrombin structure and activity revealed that nascent thrombin was significantly different than α-thrombin in terms of 1) chromogenic activity and fibrinogen clotting activity, 2) thermal stability, 3) heparin binding and 4) inhibition by antithrombin. These studies further our knowledge of the roles the A-chain plays in the zymogen prothrombin and protease thrombin, and demonstrate that the A-chain A13 peptide of nascent thrombin may be a procoagulant stabilizer of thrombin in coagulation.

Text

2011-08-18

Attribution 3.0 Unported

http://hdl.handle.net/2429/36768

Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by/3.0/