TY - JOUR
T1 - The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are required for efficient inhibition of factor Xa
AU - Peraramelli, Sameera
AU - Suylen, Dennis P. L.
AU - Rosing, Jan
AU - Hackeng, Tilman M.
PY - 2012/8
Y1 - 2012/8
N2 - 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.
AB - 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.
KW - Anticoagulant
KW - TFPI
KW - protein S
KW - factor Xa
U2 - 10.1160/TH11-12-0902
DO - 10.1160/TH11-12-0902
M3 - Article
C2 - 22627666
SN - 0340-6245
VL - 108
SP - 266
EP - 276
JO - Thrombosis and Haemostasis
JF - Thrombosis and Haemostasis
IS - 2
ER -