title: The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are required for efficient inhibition of factor Xa.
abstract: Tissue factor pathway inhibitor (TFPI) is a slow tight-binding inhibitor that inhibits factor (F)Xa in a biphasic fashion: a rapid formation of loose FXa·TFPI encounter complex is followed by slow rearrangement into a tight FXa·TFPI* complex in which the Kunitz-2 (K2) domain of TFPI binds and inhibits FXa. In the current study, full-length TFPI (TFPIfl) and various truncated TFPI constructs were used to assess the importance of TFPI domains other than K2 in the inhibition of FXa. In the absence of Ca2+ ions, FXa was more effectively inhibited by TFPIfl than Gla-domain less FXa. In turn, Ca2+ ions impaired FXa inhibition by TFPIfl but not by TFPI constructs that lack the C-terminus. This suggests that, in absence of Ca2+ ions, interactions between the C-terminus of TFPI and the Gla-domain of FXa promote FXa-inhibition. TFPIfl and K2K3 had similar efficiencies for encounter complex formation. However, K2K3 showed monophasic inhibition instead of biphasic inhibition, indicating absence of rearrangement into a tight complex. K1K2 and TFPI1-161 showed biphasic inhibition, but had less efficient encounter complex formation than TFPIfl. Finally, K2K3 was a 10-fold more efficient FXa- inhibitor than K2. These results indicate that K3-C-terminus enhances the formation of encounter complex and that K1 is required for isomerisation of the encounter- into tight complex. Since TFPIfl has a 10-fold higher Ki than K2K3-C-terminus, we propose that K1 is not only required for the transition of the loose to the tight FXa·TFPI* complex, but also inhibits FXa·TFPI encounter complex formation. This inhibitory activity is counteracted by K3 and C-terminus.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands.
authors.forename: Sameera
authors.initials: S
authors.lastname: Peraramelli
authors.forename: Dennis P L
authors.initials: DP
authors.lastname: Suylen
authors.forename: Jan
authors.initials: J
authors.lastname: Rosing
authors.forename: Tilman M
authors.initials: TM
authors.lastname: Hackeng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015951:Factor Xa / Q000737:chemistry; D065427:Factor Xa Inhibitors; D006801:Humans; D007477:Ions; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D017293:Protein S / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D007477:Ions; D008074:Lipoproteins; D010455:Peptides; D017293:Protein S; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D015951:Factor Xa; D010100:Oxygen; D002118:Calcium