title: Inhibition of tissue factor:factor VIIa-catalyzed factor IX and factor X activation by TFPI and TFPI constructs.
abstract: BACKGROUND
  TFPI is a Kunitz-type protease inhibitor that downregulates the extrinsic coagulation pathway by inhibiting factor Xa (FXa) and FVIIa. All three Kunitz domains (KD1, KD2, and KD3) and protein S are required for optimal inhibition of FXa and FVIIa. There is limited information on Kunitz domain requirements of the inhibition of TF:FVIIa-catalyzed FIX and FX activation by TFPI.
  
  
  OBJECTIVE
  To investigate the role of the Kunitz domains of TFPI and protein S in the inhibition of FX and FIX activation.
  
  
  METHODS
  Inhibition of TF:FVIIa-catalyzed FX and FIX activation by full-length TFPI (TFPIFL ) and TFPI constructs was quantified from progress curves of FXa and FIXa generation measured with chromogenic substrates.
  
  
  CONCLUSIONS
  TFPIFL inhibited TF:FVIIa-catalyzed FIX activation with a Ki of 16.7 nmol L(-1) . Protein S reduced the Ki to 1.0 nmol L(-1) . TFPI1-150 and KD1-KD2 had 10-fold higher Ki values and were not stimulated by protein S. Single Kunitz domains were poor inhibitors of TF:FVIIa-catalyzed FIX activation (Ki >800 nm). FX activation was measured at limiting FVIIa and excess TF or vice versa. At both conditions, TFPIFL , TFPI1-150 , and KD1-KD2 showed similar inhibition of FX activation. However, at low phospholipid concentrations, TFPIFL was ~ 15-fold more active than TFPI1-150 or KD1-KD2. Apparently, excess phospholipids act as a kind of sink for TFPIFL , limiting its availability for TF:FVIIa inhibition. Preformed FXa:TFPIFL/1-150 complexes rapidly and stoichiometrically inhibited FIX and FX activation by TF:FVIIa, indicating that binary TFPI:FXa complex formation is the limiting step in TF:FVIIa inhibition. Protein S also enhanced inhibition of TF:FVIIa-catalyzed FX activation by TFPI.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, Maastricht, the Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: Peraramelli
authors.forename: S
authors.initials: S
authors.lastname: Thomassen
authors.forename: A
authors.initials: A
authors.lastname: Heinzmann
authors.forename: J
authors.initials: J
authors.lastname: Rosing
authors.forename: T M
authors.initials: TM
authors.lastname: Hackeng
authors.forename: R
authors.initials: R
authors.lastname: Hartmann
authors.forename: F
authors.initials: F
authors.lastname: Scheiflinger
authors.forename: M
authors.initials: M
authors.lastname: Dockal
mesh_terms: D001777:Blood Coagulation / Q000187:drug effects; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D015949:Factor IXa / Q000378:metabolism*; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D065427:Factor Xa Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000378:metabolism* / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017293:Protein S / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D010743:Phospholipids; D017293:Protein S; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin; D015942:Factor VIIa; D015949:Factor IXa; D015951:Factor Xa
keywords: TFPI; factor IX; factor X; phospholipids; protein S