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.

Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

“…Although the K2 domain of TFPI is considered to be responsible for the inhibition of FXa (4), there are several reports that indicate that other domains of TFPI also contribute to factor Xa inhibition because inhibition of FXa by full-length TFPI is ϳ1000-fold more effective than inhibition of FXa by the isolated K2 domain (9). Apparently, the K1 domain of TFPI significantly contributes to the differences in FXa inhibition by the K2 domain and TFPI by promoting transition of the weak encounter to the tight FXa-TFPI complex, which is in line with recent observations of Peraramelli et al (12). Because the K i of TFPI for the weak encounter complex formation is some 100-fold higher than the K i for tight complex formation (1,2), the N terminus and/or K1 domain of TFPI have an important contribution to the inhibition of FXa.…”

Aptamer BAX 499 mediates inhibition of tissue factor pathway inhibitor via interaction with multiple domains of the protein

“…The Kunitz 3 (K3) domain, in conjunction with the C‐terminal domain, mediates the interaction of TFPI with cell surfaces . Additionally, the K3 domain enhances TFPI inhibition of FXa in vitro , and is the putative primary interaction site for protein S . Protein S enhances TFPI interaction with and inhibition of FXa , but does not appear to impact inhibition of the TF–FVIIa complex .…”

Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz‐1 domain

“…Progress curves of FXa inhibition by TFPI in the absence of mAb2F22 were fitted to the integrated rate equation for slow‐tight binding inhibition . Progress curves of FXa inhibition by TFPI in the presence of mAb2F22 were fitted to a straight line.…”

“…K1 interacts with the active site of FVIIa, and TFPI at high concentrations can inhibit TF/FVIIa, although in the presence of protein S or FXa the K i of TF/FVIIa inhibition is reduced . K2 interacts with the active site of FXa, and TFPI at low concentrations can inhibit FXa in a process described by the formation of a loose encounter FXa/TFPI complex, which subsequently slowly isomerizes to a tight FXa/TFPI complex .…”