文摘
Activated Factor XIII is a 83 kDa transglutaminase crucial in the final steps of blood coagulation. FXIII can be activated both via a proteolytic pathway and a non-proteolytic pathway. Activation in plasma takes place by a thrombin catalyzed cleavage after residue Arg37 (FXIIIa′) and a Ca2+ dependent conformational change (FXIIIa*). The non-proteolytic activation is the result of a conformation change only in the absence of thrombin or other proteolytic cleavage (FXIIIa°). Hydrogen/deuterium exchange (HX) detected by mass spectrometry (MS) has proven a powerful technique for analyzing the conformational properties of proteins in solution. In this study, we apply HX-MS analyses on the entire span of conformational states of recombinant FXIII, i.e., rFXIII, rFXIIIa′, rFXIIIa* and rFXIIIa°. 79 peptic peptides were used for analysis providing 90 % coverage of the 83 kDa non-redundant sequence of FXIII. The HX-MS data show increased deuterium exchange along the dimer interface suggesting weakened dimer interaction in all rFXIIIa′, rFXIIIa* and rFXIIIa°. Apart from this, rFXIIIa′ resembles zymogen rFXIII and no conformational changes seem to have taken place. In contrast, extensive changes occur upon full activation to either rFXIIIa* or rFXIIIa°. All domains of rFXIII are involved, but the major changes occur in the catalytic core and β-barrel 1 domains. Furthermore, these experiments show highly similar HX-MS data for rFXIIIa* and rFXIIIa° clearly demonstrating that both the thrombin dependent and the non-proteolytic activation pathways of rFXIII produce similar activated conformations.
