title: Identification of a thrombomodulin interaction site on thrombin-activatable fibrinolysis inhibitor that mediates accelerated activation by thrombin.
abstract: BACKGROUND
  Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM) or plasmin. Evidence from several studies suggests that TM and TAFI make direct contact at sites remote from the activating cleavage site to facilitate acceleration of thrombin-mediated TAFI activation. The elements of TAFI structure that allow accelerated activation of thrombin by TM are incompletely defined.
  
  
  OBJECTIVE
  To identify TM interaction regions on TAFI that mediate acceleration of activation by thrombin and therefore indicate TM binding sites on TAFI.
  
  
  METHODS
  We mutated selected surface-exposed charged residues on TAFI to alanine in order to identify sites that mediate acceleration of activation by TM. The kinetics of activation of the mutants by thrombin in the presence or absence of TM, as well as their thermal stabilities and antifibrinolytic potentials, were determined.
  
  
  RESULTS
  TAFI variants R15A, E28A, K59A, D75A/E77A/D78A, E99A and E106A all exhibited moderately reduced catalytic efficiencies of activation by thrombin-TM. TAFI variants R377A and, particularly, R12A and R12A/R15A exhibited severely reduced activation by thrombin-TM that was not explained by differences in activation by thrombin alone.
  
  
  CONCLUSIONS
  We have identified R12 as a critical residue for the activation of TAFI by thrombin-TM, extending a previous report that identified a role for this residue. R12 is likely to directly bind to TM while another key residue, R377, may affect the thrombin-TAFI interaction specifically in the presence of TM.
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada.
authors.forename: T T
authors.initials: TT
authors.lastname: Marar
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada.
authors.forename: M B
authors.initials: MB
authors.lastname: Boffa
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D025901:Carboxypeptidase B2 / Q000737:chemistry* / Q000235:genetics; D006224:Cricetinae; D005341:Fibrinolysin / Q000737:chemistry; D005342:Fibrinolysis; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013499:Surface Properties; D013917:Thrombin / Q000737:chemistry*; D018180:Thrombomodulin / Q000737:chemistry* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C546931:THBD protein, human; D018180:Thrombomodulin; D001120:Arginine; D025901:Carboxypeptidase B2; D013917:Thrombin; D005341:Fibrinolysin
keywords: carboxypeptidase B2; enzyme kinetics; fibrinolysis; thrombin; thrombin-activatable fibrinolysis inhibitor; thrombomodulin