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After tissue damage, initiation of blood coagulation takes place upon the exposure of tissue factor (TF) . TF acts as co-factor of factor (F) VIIa in the activation of the FX [2-4], a reaction that is greatly stimulated by the negatively charged phospholipids contributed by the damaged tissue. [5, 6].
Tissue factor pathway inhibitor (TFPI) is a major inhibitor of the extrinsic coagulation pathway, which inhibits both FXa and FVIIa . TFPI is a multivalent Kunitz type inhibitor that consists of an acidic N-terminus followed by three tandem Kunitz domains (KDs) and a basic C-terminus . Site-directed mutagenesis experiments showed that the Kunitz 1 domain (KD1) of TFPI inhibits FVIIa and that the Kunitz 2 domain (KD2) inhibits FXa . The physiological importance of TFPI is demonstrated by premature mortality during embryogenesis in mice expressing a mutant TFPI lacking KD1 .
FXa inhibition by TFPI is a biphasic reaction that occurs via a so-called slow-tight binding mechanism . In the first step, TFPI and FXa rapidly form a loose binary complex that slowly isomerises to a tight FXa-TFPI complex. FXa inhibition by TFPI is inhibited by Ca2+ ions and stimulated by phospholipids and protein S [12, 13]. Although KD2 of TFPI directly binds and inhibits FXa, the other domains of TFPI (KD1, KD3 and C-terminus) also contribute to FXa inhibition [14, 15]. In recent reports it was shown that KD1 is required for the transition of the loose to the tight FXa-TFPI complex and that KD3 promotes the formation of the loose FXa-TFPI complex  and is required for protein S stimulation of TFPI .
Much less is known about the inhibition of FVIIa by TFPI. Many reports showed that TF-FVIIa inhibition occurs via quaternary complex formation in which the KD2 domain of TFPI binds to the active site of FXa and the TF-FVIIa complex is subsequently inhibited by the interaction of KD1 of FXa-TFPI complex with the active site of FVIIa [17, 18]. TFPI can also inhibit TF-FVIIa in the absence of FXa [19, 20], but there is no information on the role of the different TFPI domains in FVIIa inhibition.
In the present study, we focused on the direct inhibition of FVIIa by TFPI and by various truncated TFPI constructs in order to investigate the role of the different domains of TFPI in the inhibition of TF-FVIIa. In addition, we tested the effect of relipidated TF, soluble TF, FXa and protein S on FVIIa inhibition by TFPIfl and the TFPI constructs. Our work provides new information on the contribution of the various TFPI domains, TF, phospholipid, FXa and protein S to the down-regulation of the extrinsic coagulation pathway by TFPI.
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- Materials and methods
- Disclosure of Conflict of Interests
Although direct inhibition of TF-FVIIa by TFPI was described by Pederson et al. , most of the subsequent studies on FVIIa inhibition by TFPI were performed in the presence of FXa and/or cell surfaces. It is generally accepted that the KD1 domain of TFPI inhibits FVIIa and that KD2 binds and inhibits FXa [9, 17]. For inhibition of FXa by TFPI there are several reports that show other Kunitz domains (KD1 and KD3) and the C-terminus of TFPI contribute to FXa inhibition [14, 30, 31]. As limited information is available on the role of the different Kunitz domains of TFPI in the direct inhibition of FVIIa, we have performed a detailed kinetic analysis of FVIIa inhibition by TFPIfl and TFPI constructs lacking Kunitz domains and C- and N-termini. To establish whether phospholipids and TF also contribute to FVIIa inhibition by TFPI, experiments were performed at three different conditions (i.e. in the presence of relipidated TF and sTF and in the absence of TF). Full-length TF, a membrane integral glycoprotein (46 kDa), is a 263 a.a. single-chain polypeptide that consists of a 219 a.a. extracellular N-terminus and a 23 a.a. transmembrane domain followed by an intracellular 21 a.a. C-terminus [32-34]. The extracellular region of TF contains the FVII/FVIIa binding domains. sTF lacks both the transmembrane and the intracellular domain. The transmembrane domain of TF plays a crucial role in TF-FVIIa anchorage to the cell surface [35-37], in the activation of FX by TF-FVIIa  and in the autoactivation of FVII . Hence, experiments were performed in the presence and absence of relipidated full-length TF and sTF to obtain information on the role of phospholipids and TF in the inhibition of FVIIa by TFPI and TFPI constructs.
We show that relipidated TF-FVIIa is directly inhibited by TFPIfl with a Ki value of 4.6 nm (Table 1). Progress curves of TF-FVIIa inhibition determined at increasing TFPI bend off at low TFPI concentrations (< 2 nm). This is indicative of time-dependent inhibition of FVIIa at low TFPI concentrations, which can be either due to a biphasic slow-tight binding mechanism or slow monophasic binding . As this bending-off is only observed at TFPI concentrations < 2 nm (Fig. 2A), it is likely that the time-dependent inhibition at low TFPI concentrations is due to slow monophasic inhibition (t½ ≈ 5 min at 2 nm TFPI) and not to a slow-tight binding mechanism as observed for FXa inhibition by TFPIfl or TFPI1–150 [12-15]. Inhibition of FVIIa by TFPI was much less efficient for sTF (Ki = 122 nm) and in the absence of tissue factor (Ki = 1118 nm). These differences can be either attributed to binding of TFPI to phospholipids and/or TF promoting the interaction between TFPI and FVIIa or to effects of TF and/or PL on the conformation of FVIIa, exposing the active site of FVIIa and making it more accessible to inhibition by TFPI [40, 41].
A possible role of the KD3-C-terminus in the inhibition of relipidated TF-FVIIa was studied using truncated forms of TFPI (TFPI1–150 and KD1-KD2). The KD3-C-terminus significantly contributed to the inhibition of relipidated TF-FVIIa by TFPI as the Ki values of TFPI1–150 (Ki = 44 nm) and KD1-KD2 (Ki = 36 nm) were some 7–10-fold higher than the Ki determined for TFPIfl (Ki = 4.6 nm). As the C-terminus of TFPI is known to interact with the phospholipids , the absence of the KD3-C-terminus might affect the interaction of TFPI with TF and/or FVIIa, which are both bound to the phospholipid surface. However, TFPI1–150 and KD1-KD2 also had 5–8-fold higher Ki values than TFPIfl for inhibition of sTF-FVIIa, which indicates that the KD3-C-terminus is also important for direct interaction with and inhibition of sTF-FVIIa, independent of phospholipid.
KD2 of TFPI also contributes towards the direct inhibition of FVIIa by TFPIfl or TFPI1–150 as removal of KD2 results in TFPI constructs (KD1 or N-KD1) that are very poor inhibitors of relipidated TF-FVIIa with Ki values (380–434 nm) that are more than 10–12-fold higher than the Ki of TFPI1–150 and KD1-KD2 (Table 1). In the presence of sTF the Ki values for the isolated Kunitz domains were some 3–4-fold increased and in the absence of (s)TF inhibition was not detectable. The (s)TF requirement for FVIIa inhibition by isolated Kunitz domains suggests that FVIIa inhibition by isolated Kunitz domains is a specific phenomenon. Surprisingly, FVIIa was inhibited by (N-)KD1 and by KD2 with similar Ki values. This shows that the presence of the other Kunitz domains promotes specific inhibition of TF-FVIIa by KD1, a phenomenon that is similar to that observed for FXa inhibition by KD2 [14, 15].
Protein S is a vitamin-K-dependent protein with two distinct anticoagulant functions. Protein S acts as a co-factor of APC in FVa and FVIIIa inactivation  and enhances FXa inhibition by TFPI . The current study shows that protein S also decreases the Ki value of relipidated TF-FVIIa inhibition by TFPI, which in the presence of protein S (Ki = 0.7 nm) is some 6–7-fold lower than the Ki observed in the absence of protein S (Ki = 4.6 nm). Protein S did not act as a co-factor of TFPI in FVIIa inhibition in the presence of anti-protein S antibodies, which shows that the protein S effect is specific and not due to contaminations in the protein preparations. Protein S did not stimulate relipidated TF-FVIIa inhibition by truncated forms of TFPI (TFPI1–150 and KD1-KD2) and also had no effect on the inhibition of sTF-FVIIa by TFPIfl. This indicates that both the KD3-C-terminus and phospholipids are required for protein S co-factor activity in FVIIa inhibition by TFPI.
Similar observations were made for the co-factor activity of protein S in FXa inhibition by TFPI, which is also only observed with TFPIfl in the presence of phospholipids and not with truncated forms of TFPI or in the absence of phospholipids . The KD3-C-terminus requirement for the expression of protein S co-factor activity is supported by the observations of Ndonwi et al. and Ahnstrom et al. [16, 29], who showed that the KD3 domain is essential for protein S binding to TFPI and for the co-factor activity of protein S in FXa inhibition by TFPI. The possible physiological importance of the TFPI co-factor activity of protein S is discussed below.
It has been reported that efficient inhibition of TF-FVIIa requires the presence of FXa, which promotes binding of TFPI to TF-FVIIa, and the fast formation of a tight quaternary TF-FVIIa-TFPI-FXa complex in which both FXa and FVIIa are inhibited. We performed experiments in the presence of FXa to further investigate the mechanism of quaternary complex formation. In the presence of FXa, TFPIfl rapidly formed a tight 1:1 stoichiometric quaternary complex with TF and FVIIa with a Ki value < 0.15 nm (Fig. 6). FXa was also able to form a quaternary complex with TF-FVIIa and TFPI1–150 and KD1-KD2, but not with KD1 or KD2, which indicates that the presence of both the KD1 and KD2 domains is essential for quaternary complex formation, but that the KD3-C-terminus is not an absolute requirement. Quaternary complex formation appeared to be also strictly dependent on the presence of phospholipids and on the presence of a Gla-domain in FXa because FXa did not stimulate the inhibition of sTF-FVIIa by TFPI and GD-FXa had no effect on relipidated TF-FVIIa inhibition by TFPI. Together these data indicate that actually a pentenary (quintenary) TF-FVIIa-TFPI-FXa-PL complex rather than a quaternary complex is the basis of physiological TF-FVIIa inhibition by TFPI.
The formation of the quaternary TF-FVIIa-TFPI-FXa complex is fast and efficient. We were not able to follow the quaternary complex formation in time and also did not succeed in measuring the Ki or Kd of the quaternary complex because at FXa concentrations in the range of the FVIIa concentration a tight stoichiometric complex was formed. This suggests that in the presence of FXa, inhibition of TF-FVIIa by TFPI is so efficient that it may not require protein S. Indeed Ndonwi and Broze reported that in a model system containing purified proteins, protein S does not enhance TFPI inhibition of TF-FVIIa but promotes FXa inhibition by TFPI . Experiments in more complex reaction systems (e.g. plasma) are required to appreciate the role of TFPI as an anticoagulant protein in down-regulating thrombin formation and to quantify the contribution of TF-FVIIa and FXa inhibition by TFPI and the effects of protein S.
One may question whether direct inhibition of TF-FVIIa by TFPI in the presence of phospholipids and protein S (Ki ~ 0.7 nm) is physiologically important (plasma full-length TFPI concentration ~ 0.25 nm). However, the local TFPI concentration at the site of thrombus formation may be substantially higher than 0.25 nm (e.g. through the release of TFPI from platelets at the growing thrombus). Under such conditions, local high concentrations of TFPI can directly inhibit TF-FVIIa, thereby contributing to the down-regulation of thrombin formation. An important role for TFPI released from platelets in regulating thrombus formation is indicated by a study of Maroney et al. , who demonstrated that in hemophilic mice with TFPI−/− hematopoietic cells blood loss on tail transections was significantly decreased.
In addition, it should also be emphasized that in a normal population the plasma TFPI concentration shows a large variation and individuals may have TFPI levels that are two to three times higher than the population average (0.25 nm). Levels of TFPI also considerably increase during heparin treatment  and are significantly elevated in pathological conditions such as cancer or coronary heart disease . In these situations direct inhibition of TF-FVIIa by TFPI may contribute to the inhibition of coagulation.