Dynamic character of the complex of human blood coagulation factor VIIa with the extracellular domain of human tissue factor: a normal mode analysis

As an attempt to investigate the dynamic interactions between plasma serine protease, coagulation factor VIIa (VIIa) and its cofactor, tissue factor (TF), we performed normal mode analysis (NMA) of the complex of VIIa with soluble TF (the extracellular part of TF; sTF). We compared fluctuations of Calpha atoms of VIIa or sTF derived from NMA in the VIIa-sTF complex with those of VIIa or sTF in an uncomplexed condition. The atomic fluctuations of the Calpha atoms of sTF complexed with VIIa did not significantly differ from those of sTF without VIIa. In contrast, the atomic fluctuations of VIIa complexed with sTF were much smaller than those of VIIa without sTF. These results suggest that domain motions of VIIa molecule alone are markedly dampened in the VIIa-sTF complex and that the sTF molecule is relatively more rigid than the VIIa molecule. This may indicate functions of TF as a cofactor.     

Crystallization and preliminary X-ray analysis of active site-inhibited human coagulation factor VIIa (des-Gla)

Human coagulation factor VIIai that lacks the Gla domain (residues 1-44) has been prepared, purified, and crystallised. First, recombinant factor VII was activated to form factor VIIa, the active site was then inhibited with 1,5-dansyl-Glu-Gly-Arg-chloromethyl ketone, and finally the Gla domain was removed by chymotryptic digestion, yielding factor VIIai (des-Gla). After further purification single crystals suitable for x-ray analysis were obtained by vapour diffusion. Crystals of factor VIIai (des-Gla) belong to the tetragonal space group P41212 or P43212 with unit cell dimensions a = b = 94.85 A, c = 114.30 A, contain one molecule per asymmetric unit, and diffract to 2.3-A resolution when exposed to synchrotron radiation.     

Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The gamma-carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF.PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF.PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF.PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.     

Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor.

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.     

Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.     

Transition state analysis of the complex between coagulation factor VIIa and tissue factor: suggesting a sequential domain-binding pathway

Injury of a blood vessel exposes membrane-bound tissue factor (TF) to blood, which allows binding of coagulation factor VIIa (FVIIa). This initiation of the coagulation cascade is dictated by a specific multi-domain interaction between FVIIa and TF. To examine the energies involved in the transition state of the FVIIa:TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with a smaller cysteine residue. Determination of Phi values in each of the positions using surface plasmon resonance measurements enabled us to characterize the transition state complex between the resulting sTF variants and FVIIa. We found that the interactions in the transition state seemed to be most pronounced between the protease domain of FVIIa and sTF while detailed specific interactions between the Gla-domain and sTF were missing. Thus, the transition state energy data indicate a sequential binding event between these two macromolecules.     

Similar molecular interactions of factor VII and factor VIIa with the tissue factor region that allosterically regulates enzyme activity

Tissue factor (TF) binds the zymogen (VII) and activated (VIIa) forms of coagulation factor VII with high affinity. The structure determined for the sTF-VIIa complex [Banner, D. W., et al. (1996) Nature 380, 41-46] shows that all four domains of VIIa (Gla, EGF-1, EGF-2, and protease) are in contact with TF. Although a structure is not available for the TF-VII complex, the structure determined for free VII [Eigenbrot, C., et al. (2001) Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K(D)) measured for binding to wild-type sTF are 7.5 +/- 2.4 nM for VII and 5.1 +/- 2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.     

Sequential coagulation factor VIIa domain binding to tissue factor

Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the γ-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced.     

Deletion of the membrane anchoring region of tissue factor abolishes autoactivation of factor VII but not cofactor function. Analysis of a mutant with a selective deficiency in activity.

The activation of human blood coagulation factor VII can occur by the feedback activity of either factor VIIa (autoactivation) or factor Xa. Both of these reactions are known to be enhanced by the presence of tissue factor, an integral membrane protein and the cofactor for factor VIIa. We examine here the activation of 125I-factor VII by both factor VIIa and factor Xa employing a mutant soluble form of tissue factor which has had its transmembrane and cytoplasmic domains deleted (sTF1-219). This mutant soluble tissue factor retains cofactor activity toward factor VIIa in a single-stage clotting assay but shows a strong dependence on initial plasma levels of factor VIIa (from 1 to 10,000 ng/ml) when compared to wild-type tissue factor. We show that this dependence is due to a deficiency of sTF1-219 in ability to both promote autoactivation and enhance the factor Xa-catalyzed activation of 125I-factor VII. sTF1-219 does not, however, inhibit the tissue factor-independent activation of 125I-factor VII by factor Xa. The results strongly suggest that the phospholipid anchoring region of tissue factor is essential for autoactivation and beneficial for factor Xa-catalyzed activation of 125I-factor VII. In addition, when taken together with the dependence of clotting times on initial factor VIIa levels observed with sTF1-219, these results indicate that factor VII autoactivation may be of greater importance in the initiation of blood coagulation via tissue factor than has been previously realized.     

The tissue factor region that interacts with substrates factor IX and Factor X

The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 A(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding. The calculated surface-exposed area of this substrate interaction region is about 1100 A(2), which is approximately half the size of the TF area that is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 A(2)) is engaged in protein-protein interactions during substrate catalysis.     

Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains

We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.     

The 99 and 170 loop-modified factor VIIa mutants show enhanced catalytic activity without tissue factor

To elucidate the functions of the surface loops of VIIa, we prepared two mutants, VII-30 and VII-39. The VII-30 mutant had all of the residues in the 99 loop replaced with those of trypsin. In the VII-39 mutant, both the 99 and 170 loops were replaced with those of trypsin. The k(cat)/K(m) value for hydrolysis of the chromogenic peptidyl substrate S-2288 by VIIa-30 (103 mm(-)1s(-)1) was 3-fold higher than that of wild-type VIIa (30.3 mm(-)1 s(-)1) in the presence of soluble tissue factor (sTF). This enhancement was due to a decrease in the K(m) value but not to an increase in the k(cat) value. On the other hand, the k(cat)/K(m) value for S-2288 hydrolysis by VIIa-39 (17.9 mm(-)1 s(-)1) was 18-fold higher than that of wild-type (1.0 mm(-)1 s(-)1) in the absence of sTF, and the value was almost the same as that of wild-type measured in the presence of sTF. This enhancement was due to not only a decrease in the K(m) value but also to an increase in the k(cat) value. These results were in good agreement with their susceptibilities to a subsite 1-directed serine protease inhibitor. In our previous paper (Soejima, K., Mizuguchi, J., Yuguchi, M., Nakagaki, T., Higashi, S., and Iwanaga, S. (2001) J. Biol. Chem. 276, 17229-17235), the replacement of the 170 loop of VIIa with that of trypsin induced a 10-fold enhancement of the k(cat) value for S-2288 hydrolysis as compared with that of wild-type VIIa in the absence of sTF. These results suggested that the 99 and the 170 loop structures of VIIa independently affect the K(m) and k(cat) values, respectively. Furthermore, we studied the effect of mutations on proteolytic activity toward S-alkylated lysozyme as a macromolecular substrate and the activation of natural macromolecular substrate factor X.     

Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF--FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.     

Spectroscopic probing of the influence of calcium and the gla domain on the interaction between the first EGF domain in factor VIIa and tissue factor.

The binding of factor VIIa (FVIIa) to tissue factor (TF) initiates blood coagulation. The binary complex is dependent on Ca2+ binding to several sites in FVIIa and is maintained by multiple contacts distributed throughout the various domains. Although the contributions from various residues and domains, including the Ca2+ coordination, to the global binding energy have been characterized, their importance for specific local interactions is virtually unknown. To address this aspect, we have attached four spectroscopic probes to an engineered Cys residue replacing Phe140 in soluble TF (sTF). This allows the monitoring of local changes in hydrophobicity and rigidity upon complex formation at the interface between the first epidermal growth factor-like (EGF1) domain of FVIIa and sTF. The fluorescent labels used sense a more hydrophobic environment and the spin labels are dramatically immobilized when FVIIa binds sTF. The results obtained with a 4-carboxyglutamic acid (Gla)-domainless derivative of FVIIa indicate that the Gla domain has no or minimal influence on the interaction between EGF1 and sTF. However, there is a difference in local Ca2+ dependence between Gla-domainless and full-length FVIIa.     

Loop Dynamics of the Extracellular Domain of Human Tissue Factor and Activation of Factor VIIa

In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro⁷⁹-Pro⁹² loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu⁸⁴ resides in the Pro⁷⁹-Pro⁹² loop, and Thr¹²¹ resides in the turn between the first and second antiparallel β-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu⁸⁴ nor Thr¹²¹ makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro⁷⁹-Pro⁹² loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro⁷⁹-Pro⁹² loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation.     

Properties of spin and fluorescent labels at a receptor-ligand interface.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.     

Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W

Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gaveriaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTF(R200W)) compared to that of the wild-type protein (sTF(wt)). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTF(R200W) mutant and sTF(wt) were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTF(R200W) indicated a population of partially folded intermediates during denaturation. sTF(R200W) showed a dramatically increased propensity for aggregate formation compared to sTF(wt) at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTF(R200W) in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TF(R200W) carriers, which could protect from thrombotic disease.     

Tissue factor-dependent autoactivation of human blood coagulation factor VII.

We recently showed that single-chain zymogen factor VII is converted to two-chain factor VIIa in an autocatalytic manner following complex formation with either cell-surface or solution-phase relipidated tissue factor apoprotein (Nakagaki, T., Foster, D. C., Berkner, K. L., and Kisiel, W. (1991) Biochemistry 30, 10819-10824). We have now performed a detailed kinetic analysis of the autoactivation of human plasma factor VII in the presence of relipidated recombinant tissue factor apoprotein and calcium. Incubation of factor VII with equimolar amounts of relipidated tissue factor apoprotein resulted in the formation of factor VIIa amidolytic activity coincident with the conversion of factor VII to factor VIIa. The time course for the generation of factor VIIa amidolytic activity in this system was sigmoidal, characterized by an initial lag phase followed by a rapid linear phase until activation was complete. The duration of the lag phase was decreased by the addition of exogenous recombinant factor VIIa. Relipidated tissue factor apoprotein was essential for factor VII autoactivation. No factor VII activation was observed following complex formation between factor VII and a recombinant soluble tissue factor apoprotein construct consisting of the aminoterminal extracellular domain in the presence or absence of phospholipids. Kinetic analyses revealed that factor VII activation in the presence of relipidated tissue factor apoprotein can be defined by a second-order reaction mechanism in which factor VII is activated by factor VIIa with an apparent second-order rate constant of 7.2 x 10(3) M-1 S-1. Benzamidine inhibited factor VII autoactivation with an apparent Ki of 1.8 mM, which is identical to the apparent Ki for the inhibition of factor VIIa amidolytic activity by this active site competitive inhibitor. Our data are consistent with a factor VII autoactivation mechanism in which trace amounts of factor VIIa rapidly activate tissue factor-bound factor VII by limited proteolysis.     

Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa.

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF alpha (high molecular weight form) and sTF beta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF alpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTF beta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF alpha and sTF beta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTF beta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTF beta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.