title: TAFIa inhibiting nanobodies as profibrinolytic tools and discovery of a new TAFIa conformation.
abstract: BACKGROUND
  Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co-administration of t-PA and a TAFIa inhibitor enhances t-PA treatment.
  
  
  OBJECTIVE
  We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t-PA induced clot lysis.
  
  
  RESULTS
  Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A(147) -C(305) -I(325) -I(329) -Y(333) -Q(335) ), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16-fold molar excess of nanobody over TAFIa, IC(50) 's range between 0.38- and > 16-fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t-PA concentration. The nanobodies were found to bind in the active-site region of TAFIa and their time-dependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition.
  
  
  CONCLUSIONS
  These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa.
authors: Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
authors: M L V
authors: ML
authors: Hendrickx
authors: A
authors: A
authors: DE Winter
authors: K
authors: K
authors: Buelens
authors: G
authors: G
authors: Compernolle
authors: G
authors: G
authors: Hassanzadeh-Ghassabeh
authors: S
authors: S
authors: Muyldermans
authors: A
authors: A
authors: Gils
authors: P J
authors: PJ
authors: Declerck
mesh_terms: D000906:Antibodies / Q000494:pharmacology* / Q000627:therapeutic use; D025901:Carboxypeptidase B2 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005342:Fibrinolysis / Q000187:drug effects*; D005343:Fibrinolytic Agents; D006801:Humans; D050505:Mutant Proteins / Q000276:immunology; D019151:Peptide Library; D011487:Protein Conformation
chemical_list: D000906:Antibodies; D005343:Fibrinolytic Agents; D050505:Mutant Proteins; D019151:Peptide Library; D025901:Carboxypeptidase B2