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Biophysical characterization of the activity state of thrombin

  • Author(s): Koeppe, Julia Rachel
  • et al.
Abstract

The final step of the blood coagulation cascade is the activation of thrombin. Active thrombin cleaves fibrinogen to create fibrin which polymerizes into clots. Regulation of thrombin is imperative to maintaining normal hemostasis. The blood contains a high concentration of prothrombin, which must be proteolytically cleaved at two sites to generate active alpha-thrombin. Very little alpha -thrombin is ever generated, and this is rapidly captured by either thrombomodulin (TM) and/or antithrombin III. This work investigates the dynamics of thrombin in several activity states by H/2H exchange and MALDI-TOF mass spectrometry. Chapter II investigates the activation mechanism of conversion of prothrombin to active thrombin. This process involves cleavage at Arg320-Ile321 producing meizothrombin or cleavage at Arg271-Thr272 producing prethrombin-2. Full activation of thrombin requires both cleavage events. The H/2H results show that upon activation half of thrombin becomes more dynamic and the other half becomes less dynamic. In Chapter III solvent accessibility changes which occur upon active site occupation of thrombin are investigated. The dynamics of thrombin are studied in the presence of two different active site inhibitors of thrombin. Development of a flow- quench protocol allowed for the capture of early time points in H/2H exchange and aided our ability to observe conformational changes in key surface loops. The results show a path of communication between the active site and remote co-factor binding regions of thrombin. Chapter IV presents a study of the dynamics of thrombin in the presence of its cofactor TM. When an active TM cofactor (TMEGF45) binds at ABE1 far from the thrombin active site, dynamics changes are observed in the thrombin active site. When an inactive TM cofactor (TMEGF56) binds, there are no changes observed in the thrombin active site. In Chapter V several mutants of TMEGF45 are studied for their cofactor activity in accelerating the cleavage of protein C by thrombin. Several TM residues that appear far from thrombin in the thrombin-TMEGF456 crystal structure are known to have large effects on protein C activation. Both kinetic data and H/2H exchange data for the TMEGF45 mutants show TM interacts with both thrombin and protein C

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