title: A role for arginine-12 in thrombin-thrombomodulin-mediated activation of thrombin-activatable fibrinolysis inhibitor.
abstract: BACKGROUND
  Thrombin-activatable fibrinolysis inhibitor (TAFI) is a proenzyme that links coagulation and fibrinolysis. TAFI can be activated by thrombin, the thrombin-thrombomodulin complex and plasmin through cleavage of the first 92 amino acids from the enzyme. In silico analysis of the TAFI sequence revealed a potential thrombin cleavage site at Arg12. The aim of this study was to determine whether TAFI can be cleaved at Arg12 and whether this cleavage plays a role in TAFI activation.
  
  
  METHODS
  A peptide based on the first 18 amino acids of TAFI was used to determine whether thrombin was able to cleave at Arg12. Mass spectrometry was performed to determine whether the Arg12-cleaved peptide was released from full-length TAFI. Furthermore, a TAFI mutant in which Arg12 was replaced by a glutamine (TAFI-R12Q) was constructed and characterized with respect to its activation kinetics.
  
  
  RESULTS
  The peptide and mass spectrometry data showed that thrombin was able to cleave TAFI at Arg12, but with low efficiency in full-length TAFI. Characterization of TAFI-R12Q showed no difference in thrombin-mediated activation from wild-type TAFI. However, there was an approximately 60-fold impairment in activation of TAFI-R12Q by the thrombin-thrombomodulin complex.
  
  
  CONCLUSIONS
  Arg12 of TAFI plays an important role in thrombomodulin-mediated TAFI activation by thrombin. Thrombin is able to cleave TAFI at Arg12, but it remains to be determined whether Arg12 is part of an exosite for thrombomodulin or whether cleavage at Arg12 accelerates thrombomodulin-mediated TAFI activation.
authors: Department of Experimental Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands.
authors: T
authors: T
authors: Plug
authors: G
authors: G
authors: Kramer
authors: J C M
authors: JC
authors: Meijers
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001120:Arginine / Q000737:chemistry*; D001777:Blood Coagulation; D025901:Carboxypeptidase B2 / Q000737:chemistry*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000737:chemistry; D005342:Fibrinolysis; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry*; D018180:Thrombomodulin / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D010455:Peptides; D018180:Thrombomodulin; D005973:Glutamine; D001120:Arginine; D025901:Carboxypeptidase B2; D013917:Thrombin; D005341:Fibrinolysin
keywords: carboxypeptidase U; fibrinolysis; thrombin; thrombin-activatable fibrinolysis inhibitor; thrombomodulin