Structural-functional relationships in the human thrombin A-chain [article]

Isis Sarah Rosemary Carter
2011
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
more » ... orders. 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 rol [...]
doi:10.14288/1.0072069 fatcat:duplk3fqxnfrhkga3lb45ghyp4