From the Haemostasis Research Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom, the § School of Allied Medical Science, Shinsu University, Asahi 3-1-1, Matsumoto, Japan, and ¶ Pharma Division, Hoffmann-La Roche, Grenzacherstrasse 124, 4002 Basel, Switzerland
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ABSTRACT |
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We have used recombinant mammalian expression and
purification of the factor VII (FVII) variant Gln100
Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue
factor (sTF); however, unlike WTFVIIa no typical increase in activity
was seen after addition of sTF. In a factor X activation assay using
relipidated transmembrane truncated tissue factor (residues 1-243),
Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate
factor X.
We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant.
In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.
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INTRODUCTION |
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Plasma FVII1 is essential for the initiation of blood coagulation via the tissue factor (TF: CD142) pathway; plasma FVII binds to exposed subendothelial TF on tissue injury and is rapidly activated to FVIIa, the TF-bound serine protease which then activates plasma factors IX and X, leading ultimately to thrombin generation (1). Severe reduction in plasma FVII activity usually leads to bleeding problems in human patients although the phenotype is heterogeneous (2). Complete absence of circulating FVII appears to be lethal due to traumatic postnatal bleeding2; in addition, a recent report on FVII knock-out mice indicates poor survival of neonates, with the majority dying within 24 h from severe bleeding and virtually no survival beyond 21 days (3).
Many subjects listed in the FVII mutation data
base,3 when characterized at
the molecular genetic level, are revealed to be heterozygous for one or
more mutations in the FVII gene, making it difficult to assess the
functional deficit resulting from a particular amino acid substitution
in the molecule. FVII Gln100 Arg (Q100RFVII) is a
naturally occurring EGF2 variant found in compound heterozygotes with
other FVII mutations; it has been suggested that its circulating plasma
antigen level is low (4), although its functional characteristics have
not been determined. Previously we identified a subject heterozygous
for Q100RFVII with greatly reduced plasma FVII activity (5), but we
were unable to determine whether this was due to reduced plasma levels of a functionally normal factor or a dysfunctional protein. In the
latter case, reduced or abolished function might result from one of the
following different reasons: the inability to be activated to FVIIa;
absence of active site peptide bond cleavage; defective binding to
macromolecular substrate (factor X); or altered binding to TF. In the
present study, we undertook the expression, purification, and
characterization of Q100RFVII to determine the nature of the functional
deficiency in subjects with this mutation.
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EXPERIMENTAL PROCEDURES |
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Buffer Solutions and Reagents-- Unless stated all buffers were based on 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 (TBS). TBS-A was TBS containing 1 mg/ml human albumin (Bio Products Laboratory, Elstree, UK). All chemicals were of analytical grade or better and were obtained from Sigma (Poole, UK) unless stated otherwise. Protein grade Tween 20 was supplied by Calbiochem (Nottingham, UK). Chromatography media were purchased from Pharmacia (St. Albans, UK). SDS-PAGE was performed on 10% Nupage precast gels using SeeBlue markers (Novex GmBH, Frankfurt, Germany). Non-activated human FX was obtained from ERL (Swansea, UK). Recombinant human transmembrane tissue factor (TF1-243, residues 1-243) was a kind gift of Dr. D. L. Eaton (Genentech Inc., San Francisco) and was relipidated before use as follows: 5 µl of 10 µM TF1-243 were mixed with 30 µl of TBS-A, 30 µl of 9:1 (v/v) platelet substitute mixed phospholipid (Diagnostic Reagents, Thame, UK), 25 mg/ml sodium deoxycholate, and 3 µl of 100 mM CdCl2 and incubated at 37 °C for 30 min. The mixture was then diluted in TBS-A to 16 nM final TF1-243 concentration before use in FX activation assays. Chromogenic substrates used to detect amidolytic activity of FXa (S-2765) and FVIIa (S-2288) were from Chromogenix/Quadratech (Epsom, UK). Immobilized FXa beads were produced by incubation of 270 µg of human FXa (ERL), predialyzed into 50:50 (v/v) glycerol, H20, with 150 µl of AminoLink Plus beads (Pierce, Chester, UK) according to the instructions supplied with the beads. FXa beads were extensively washed before use, and no FXa activity was detected in the washings after prolonged incubation with the amidolytic substrate S-2765, whereas inclusion of a few beads led to rapid color generation.
Construction of FVII Expression Vectors-- Vectors for stable transfection of wild-type and variant FVII were constructed by site-directed mutagenesis essentially as described previously (6), and the desired variant nucleotide sequence was confirmed by direct sequencing (7).
Isolation of Recombinant Proteins-- Recombinant human soluble tissue factor (sTF, residues 1-219) was produced in Escherichia coli essentially as described previously (8), apart from omission of the FLAG sequence. sTF was purified directly from clarified culture broth by sequential cation and anion exchange chromatography. First, broth was buffer-exchanged into 50 mM sodium acetate, pH 4.6, clarified, and loaded onto an SP-Sepharose high performance column and eluted with a linear gradient of the identical buffer containing 0.25 M NaCl. Second, sTF-containing fractions were pooled and buffer-exchanged into 10 mM Tris-HCl, pH 7.4, and then loaded onto a 6-ml Resource Q column; sTF was eluted with a linear gradient of start buffer to 10 mM Tris-HCl, 0.25 M NaCl. The protein was homogeneous as assessed by SDS-PAGE. Recombinant WTFVII and Q100RFVII were produced by mammalian roller bottle tissue culture in CHO-K1 cells using protein-free expression medium as described previously (9). Single chain FVII preparations were isolated at 4 °C from clarified media by sequential barium citrate precipitation (9:1:1 conditioned medium, 3.8% w/v trisodium citrate, 1 M BaCl2 added dropwise), resolution of the precipitate in 200 mM EDTA, 50 mM Tris-HCl, 150 mM NaCl, 20 mM benzamidine HCl, pH 7.4, and extensive buffer exchange at 4 °C into 50 mM Tris-HCl, 5 mM benzamidine HCl, pH 7.4. Following clarification the crude FVII was loaded onto a 6-ml Resource Q column and eluted by a linear gradient to the same buffer containing 1 M NaCl. The resulting FVII was further purified by affinity chromatography on a 1-ml Hi-Trap heparin-Sepharose column using a linear gradient from 50 mM Tris-HCl, 0.05% Tween 20, pH 7.4, to the same buffer containing 1 M NaCl.
Post-translational Modification of Glu Residues to Gla in Recombinant Q100RFVII-- To rule out incomplete gamma-carboxylation of candidate Glu residues (residue numbers 6, 7, 14, 16, 19, 20, 25, 26, 29, and 35) (10), purified single chain Q100RFVII was subjected to direct Edman sequencing on an Applied Biosystems 473A instrument (Warrington, UK).
Specific Activity of Q100RFVII-- FVII:C and FVII:Ag values of conditioned media and purified single chain Q100RFVII were measured to estimate specific activity in comparison with normal plasma FVII; the 6th British Plasma Standard for Blood Coagulation Factors (code 93/622, NIBSC, South Mimms, UK) was used as reference. One-stage coagulant assays were performed with human thromboplastin (Thromborel, Hoechst/Behring, Milton Keynes) and FVII-deficient plasma (Sigma) using a Coag-a-Mate XC instrument (Organon Teknika, Cambridge, UK), whereas antigen assays were performed with the Asserachrom FVII:Ag kit (Shield, Dundee, UK). A normal plasma level of 1.0 IU/ml is equivalent to a plasma concentration of approximately 0.5 µg/ml (11).
Activation of FVII-- Recombinant WTFVII and Q100RFVII (0.8 µM) were incubated overnight at room temperature in the presence and absence of an equal volume of immobilized FXa beads to produce activated FVII species. After removal of the beads the activated and non-activated samples were analyzed by SDS-PAGE to confirm complete FXa cleavage and then compared in functional and ligand-binding systems. In addition, several milligrams of Resource-Q-purified wild-type FVII (see above) were concentrated to ~4 mg/ml and autoactivated by overnight incubation at room temperature in TBS, 5 mM CaCl2, followed by repurification using anion exchange chromatography on Mono Q to remove small amounts of des-Gla FVIIa and residual single chain FVII; this material was homogeneous as judged by SDS-PAGE and was used as an additional reference preparation of highly purified wild-type FVIIa to ensure that the FXa bead treatment produced fully activated FVIIa.
Functional Tests on Q100RFVII-- In this section all reagent levels given are final concentrations. FVIIa direct amidolytic activity was assessed in 96-well plates by incubation of wild-type and variant FVII/FVIIa samples (10 nM) with varying concentrations of purified recombinant human sTF (0-8.8 µM) in the presence of 5 mM CaCl2 followed by addition of S-2288 (1 mM). Generation of color was followed at 405 nM using a Thermomax plate reader (Molecular Devices, Crawley, UK). The ability of FVII/FVIIa preparations to activate FX was studied by coincubating relipidated TF1-243 (3.2 nM), wild-type, or variant FVII/FVIIa (2 pM), S-2765 (0.68 mM), and CaCl2 (5 mM) in a 96-well plate. The reaction was initiated by addition of FX to a final concentration of 20 nM and exponential color generation followed in the Thermomax reader.
Surface Plasmon Resonance Analysis of FVII/FVIIa Binding to Tissue Factor-- All experiments were performed using a Biacore X instrument and reagents unless otherwise stated below (Biacore, St. Albans, UK). Recombinant sTF (4 µM) was injected and immobilized onto flow cell 2 of a CM5 sensor chip by amine coupling according to the manufacturer's instructions to a total of approximately 500 resonance units (RU). An irrelevant protein (an anti-FVIII monoclonal antibody) was immobilized to a similar RU on flow cell 1 to act as a control surface. 80-µl injections of wild-type or variant FVII and FVIIa (40 and 200 nM) were made at a flow rate of 40 µl/min, using running buffer of TBS, 2.5 mM CaCl2, 0.01% protein grade Tween 20 (Calbiochem, Nottingham, UK), and HBS buffer (Biacore) containing 3 mM EDTA for regeneration. TF-specific association was detected as the net signal when nonspecific RU changes in flow cell 1 were subtracted from flow cell 2. Data from both injections were prepared for analysis using BiaEvaluation 2.2 software and exported to the global analysis program CLAMP 3.0 (12), which was also used for kinetic simulations.
Molecular Environment of FVIIa Q100R-- To examine the environment of residue Gln100 in FVIIa, the coordinates of FVIIa were extracted from the FVIIa-sTF complex crystal structure 1DAN.PDB (13); visualizations were performed using MOLSCRIPT (14). All residue numbers correspond to mature human FVII.
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RESULTS |
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Expression of Wild-type and Variant Q100RFVII in Mammalian Tissue Culture-- As previously reported (9), WTFVII accumulated into medium consistently reaching levels between 2.5 and 5 µg/ml (approximately 5-10 IU/ml FVII:Ag): however, in more than 10 independent expression experiments, Q100RFVII:Ag levels were found to be extremely low, varying between 0.05 and 0.4 µg/ml (0.1-0.8 IU/ml), suggesting an expression defect in comparison with wild-type FVII. Increasing the levels of methotrexate used for amplification of the FVII cDNA in the Q100RFVII-expressing cells stepwise from 12.8 µM (9) to as high as 100 µM did not result in increased accumulation of FVII:Ag in the medium (data not shown). In addition, while FVII:C values for WTFVII were similar to those of FVII:Ag, FVII:C was essentially undetectable in Q100RFVII medium. Typical FVII:C and FVII:Ag levels obtained after expression from CHO-K1 cells into protein-free medium are given in Table I.
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Purification of Variant Q100RFVII-- Due to the very low level of expressed Q100RFVII from CHO-K1 cells, a large volume (9 liters) of conditioned medium (0.4 µg/ml Q100RFVII as assessed by FVII:Ag assay, total 3.6 mg) was generated to allow purification of the variant for further characterization. Q100RFVII was quantitatively precipitated from conditioned medium by addition of BaCl2 and eluted similarly to WTFVII from the Resource Q and heparin-Sepharose columns but with low yields. A total of approximately 140 µg of variant protein was isolated with an overall yield of approximately 4%. After purification SDS-PAGE showed full-length single chain FVII with a minor component presumed to be des-Gla FVII. Due to the low yields of variant protein and the requirements for characterization by several different methods, further repurification to remove this minor contaminant was not attempted. Table I shows that purified Q100RFVII possessed very low FVII:C activity (~0.15 IU/ml) but with a FVII:Ag level of 90 IU/ml; thus Q100RFVII possessed a specific coagulant activity less than 0.2% of WTFVII, approximately 3.3 versus 2180 IU/mg, assuming a plasma unit to be equivalent to 0.5 µg (11).
Gla Content of Recombinant Q100RFVII-- Following Edman degradation and direct sequencing, signals were absent in all the residue positions given above up to 29, despite clear and unambiguous responses for other residues as far as position 33. We were also unable to detect a signal at position 35 (the 10th and last Gla position), but the surrounding residue signals were too attenuated to be certain as to the significance of the absence of signal at Glu35; however, it has been stated that a Gla rather than Glu at position 35 does not increase the affinity of FVIIa for TF (15). Thus the level of gamma-carboxylation in Q100RFVII is comparable to that in WTFVII (10).
Activation of FVII Samples with Immobilized FXa-- SDS-PAGE of wild-type and variant FVII incubated together with immobilized FXa beads showed complete activation of both FVII samples, as seen by quantitative loss of the single chain form and generation of heavy and light chains for both proteins following silver staining (Fig. 1). Purified Q100RFVII contained a minor des-Gla component that was apparently cleaved normally to give a small amount of des-Gla light chain (just visible, marked by gdLC).
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sTF-enhanced Direct Amidolytic Activity of Wild-type and Q100RFVIIa-- The FVIIa amidolytic activity found in the presence of a wide range of sTF concentrations was then studied using the oligopeptide substrate S-2288. Without prior FXa bead treatment, both wild-type and Q100R samples were virtually inactive (not shown). As shown in Fig. 2A, after FXa activation, wild-type FVIIa (10 nM) demonstrated a typical TF-promoted increase in amidolytic activity (16), with approximately 50% of maximum stimulation at 8.8 nM sTF and essentially 100% at 88 nM sTF. Purified FVIIa at the same concentration produced by bulk autoactivation and repurification (see "Experimental Procedures") gave a very similar response. In comparison, Q100RFVIIa showed no stimulation in amidolytic activity at an sTF concentration of 88 nM; a small augmentation was seen only at very high sTF concentrations (approximately 2-fold at 8.8 µM, Fig. 2B). Interestingly, in the absence of sTF the amidolytic activities of WTFVIIa and Q100RFVIIa were similar (1.24 milliabsorbance units/min and 0.98 milliabsorbance units/min), indicating that the underlying functionality of the Q100RFVIIa active site toward the oligopeptide substrate was essentially normal but was not greatly augmented in the presence of sTF. Results shown are representative of duplicate experiments; data obtained from two separate purified preparations of Q100RFVIIa were very similar.
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TF1-243-enhanced Activation of FX by Wild-type and Q100RFVIIa-- To assess the ability of wild-type and variant FVII and FVIIa to activate FX in the presence of TF1-243, FVII and FVIIa samples (10 pM) produced by FXa bead treatment were incubated with equal volumes of a large molar excess of relipidated TF1-243, CaCl2, and S-2765, and the reaction was started with addition of purified FX to a final concentration of 20 nM. Cleavage of the FXa chromogenic substrate was monitored at 405 nM, and FXa generation curves representative of two duplicate experiments are shown in Fig. 3. WTFVIIa (activated by FXa beads) generated FXa rapidly, and bulk autoactivated/purified WTFVIIa gave very similar results (not shown); wild-type non-activated FVII gave similar FXa generation following a lag phase resulting from the necessity for back activation of FVII by FXa. In contrast, Q100RFVIIa gave a very low level of FXa generation (less than 5% that of WTFVIIa), and no color was generated in the presence of non-activated Q100RFVII suggesting absence of back activation (not shown). Thus, the results of both direct amidolytic and FX activation systems indicate a profound defect in TF-modulated FVIIa function.
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Analysis of FVII/FVIIa Binding to sTF by Surface Plasmon Resonance-- The lack of TF-promoted activity in the Q100R variant might result from a defect in TF binding or the lack of allosteric activation following normal binding. The binding of Q100RFVII/FVIIa to immobilized sTF was therefore compared with that of wild-type protein by surface plasmon resonance. As seen in Fig. 4, on injection of 200 nM wild-type FVII and FVIIa prepared by FXa bead treatment onto the sTF chip both gave a significant response although the shapes of the curves were qualitatively different. Two separate purified preparations of Q100RFVII and Q100RFVIIa gave similar results. Careful analysis of the sensorgrams by global fitting indicated that the interactions of both analytes were heterogeneous, as has been found previously using randomly immobilized ligand (17); however, estimation of the apparent dissociation constant KD for the highest affinity interaction taking place gave values of approximately 20 nM for wild-type FVII and 8-10 nM for FVIIa, in broad agreement with previously published values (6, 15, 18). In contrast, the variant Q100R samples (± FXa bead activation) showed very weak interactions with the sTF surface, giving response curves that were difficult to resolve from instrument noise even at this high injection concentration; a lower limit for KD values in these cases was estimated using kinetic simulation as >500 nM. Thus, a severe binding defect has been demonstrated for variant Q100R, found in both the non-activated and amidolytically functional activated form.
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Molecular Environment of FVIIa Residue
Gln100--
Fig.
5A shows a ribbon diagram (14)
of the WTFVIIa/sTF complex; the structure was taken from 1DAN.PDB (13).
It can be seen that the Gln100 side chain is positioned
between FVIIa EGF2 and the protease domain, at a considerable distance
from the N-terminal TF domain. Fig. 5B shows a stereo
diagram of the Gln100 side chain and its immediate
environment. Gln100 lies in the interface between the EGF2
domain of the light chain (to left) and the heavy chain catalytic
domain (to right); this residue has unusual main chain torsion angles
( =
123° and
=
99°) and falls on the edge of the allowed
region of the Ramachandran plot. The homologous residues in FXa (19)
and activated protein C (20) have also been reported to have strained
bond angles. In the crystal structure, the Gln100 side
chain is held in place by a hydrogen bond to its amide oxygen OE-1 from
the peptide ---NH of His115. This leaves the
Gln100 amide amino group (upper right atom as shown)
apparently with no hydrogen bonding partners. However, to the lower
right is a water molecule (HOH-1:O-216) at a distance of 3.6 Å in the
correct direction to accept a hydrogen bond; in addition, above the
amino group is the phenyl ring of Tyr118, in the correct
position and orientation to accept a hydrogen bond to its
electrons. Mutation of Gln100 to arginine as in Q100RFVIIa
would seriously disrupt this region of the molecule. Furthermore, any
displacement of Tyr118 would also affect the loop
Pro134-Cys135-Gly136-Lys137;
in WTFVII the Cys135 residue makes the disulfide bridge to
Cys262 in the heavy chain as shown, enabling the formation
of disulfide-linked FVIIa after cleavage at Arg152.
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DISCUSSION |
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There are few reports in the literature describing recombinant expression and functional characterization of specific FVII missense mutations originally identified in human subjects. Two mutations in EGF1 have been studied, N57D (21) and R79Q (6, 22-24), both of which showed absent or reduced affinity toward sTF as might be predicted from the extensive interaction of EGF1 with sTF in the crystal structure (13). The FVII activation site variant R152Q was found to be severely dysfunctional as expected (23), and in the protease domain R304Q was also shown to have decreased affinity for sTF (25). Recently the characterization of the variant F328S in the protease domain was reported (26); although its binding affinity to sTF was not greatly reduced, this interesting variant showed undetectable coagulant activity. Both amidolytic and proteolytic activity in the presence of sTF were abolished, indicating a severe dysfunction in the interaction of substrate within the FVIIa active site. To date there has been no recombinant expression and detailed characterization of an EGF2 variant found in a human subject.
Our studies indicate that the Q100RFVII EGF2 variant is unusual in two distinct ways. First, the purified protein lacks, in the presence of TF, both coagulant activity and (following activation) the ability to activate factor X, although it appears to possess basal amidolytic function in the absence of TF; a gross deficiency in TF binding is responsible for this defect. Second, it apparently circulates at a very low or absent level in subjects with the mutation. We wanted to account for both these characteristics in terms of its single molecular defect.
Amidolytic assays with FXa-activated Q100RFVIIa demonstrated that no functional defect is evident in the absence of sTF, suggesting that dysfunctional interaction with TF is responsible for its lack of coagulant activity. Although this could result from simple loss of binding, defective allosteric activation at the active site, or defective interaction with macromolecular substrate, the surface plasmon resonance results presented here indicate that the recombinant purified variant Q100RFVII(a) shows no detectable TF binding. In this situation it is to be expected that the variant would show little or no activity in FX activation assays; in a tissue thromboplastin-initiated clotting system, Q100RFVII procoagulant activity would be further compromised by the inability of non-TF-bound zymogen to be activated.
A possible defect in post-translational modifications of Glu residues in the Gla domain is very unlikely to be responsible for affecting TF binding to the extent seen, since under-carboxylated FVIIa binds TF similarly to wild-type FVIIa (27) and des-Gla FVIIa shows only a 7-fold increase in KD of sTF binding (28); in any case gamma-carboxylation in Q100RFVII appeared normal as assessed by amino acid sequencing.
Thus, we can make a hypothesis at the level of molecular structure as to how the single amino acid substitution in this variant can result in this dramatic loss of affinity for sTF. Several years ago, the solution of the crystal structure of the TF extracellular portion (sTF) by two groups (29, 30) allowed a structure-based reinterpretation (31) of previous TF-FVIIa studies using a variety of techniques including antibody mapping (32), alanine-scanning mutagenesis (8, 33, 34), proteolytic fragmentation (35), and chemical cross-linking (36). A large interfacial area of binding was predicted, likely to involve multiple domains in the two proteins. More recently, the solution of the crystal structure of FVIIa/sTF (13) demonstrated the extended nature of this interface, with intermolecular chain contact contributions from all four FVIIa domains (Gla, EGF1, EGF2, and protease) and both fibronectin type III-like domains of sTF. The most important interactions are contributed by the EGF1 and protease domains as evidenced both by the contact chain areas in the crystal structure and the consequences of FVIIa alanine scanning mutagenesis (37). However, the contact areas of EGF2 are relatively small, and the contribution of the residues involved in stability of the complex has been found to be minor. In addition, the Gln100 residue does not form part of the TF binding face of EGF2 (Fig. 5) but rather is placed at the interface of EGF2 and the protease domain. These two domains are often described as forming a single rigid structural unit with no interdomain flexibility.
It has been reported that the WTFVIIa molecule free in solution has an
extended conformation (38) and possesses interdomain flexibility
(resulting from Gla-EGF1 and EGF1-EGF2 flexibility) which is reduced on
binding to sTF (39). It is likely that both the high affinity of the
sTF-WTFVIIa interaction and part of the allosteric promotion of FVIIa
activity which results derive from the stabilization by TF of FVIIa
inter- and intradomain conformation, requiring some movement of FVIIa
domains to orient the large surface involved. The FVIIa EGF2-protease
interface is rigid, as seen also in the crystal structures of the
homologous serine proteases factor Xa (19), factor IXa (40), and
activated protein C (20); however, it is possible that a disruption of
the main chain fold in this region could have a dramatic effect on the
mutual orientation of the protease domain and EGF1, both of whose
surfaces are closely involved in TF interaction. Abnormal main chain
dihedral angles and
(resulting from a very short three-residue
bridge between two disulfide links) are seen for Gln100 in
FVIIa and also the homologous residues in both factor Xa (19) and
activated protein C (20). The Gln100 side chain position is
stabilized by a hydrogen bond between its amide oxygen and the peptide
---NH of His115, and by hydrogen bonds from the amino group
to a water molecule and to the
electrons of Tyr118.
Replacement of the glutamine side chain by the larger, positively charged arginine side chain in Q100RFVII will disrupt this hydrogen bonding network and destabilize this interdomain area. In addition movement of the Tyr118 side chain will affect the short
loop C-terminal to EGF2 which bears Cys135; since this
cysteine forms one-half of the light chain-heavy chain disulfide link
(with Cys262), proper interchain disulfide formation in
variant Q100RFVII may be significantly disturbed. In this respect it is
of interest that mutation of the FIX homologous residue
Gln97 to Glu in a case of severe hemophilia B results in
absence of circulating FIX protein (41).
We suggest that folding and/or disulfide linkage disturbances in the EGF2 protease region of Q100RFVII are responsible both for the absence of measurable affinity for TF (and thus, coagulant activity) and also for the apparent defect in secretion or stability in human subjects with this variant. This variant may define a new class of mutation affecting function via substitutions of amino acid side chains positioned at interdomain interfaces, distorting global conformation and indirectly preventing extended receptor-ligand interactions.
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ACKNOWLEDGEMENT |
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We are indebted to Dr. Peter Byfield of the Haemostasis Research Group for carrying out the protein sequencing of Q100FVII and WTFVII.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-181-383-8253;
Fax: 44-181-383-8273.
1
The abbreviations used are: FVII, factor VII;
FVIIa, activated factor VII; FVII Gln100 Arg/Q100RFVII,
FVII with residue glutamine 100 replaced by arginine; Gla,
-carboxyglutamic acid-rich domain; des-Gla, lacking the Gla domain;
EGF1, epidermal growth factor-like domain 1; EGF2, epidermal growth
factor-like domain 2; FIX, factor IX; FX, factor X; FXa, activated FX;
sTF, soluble truncated tissue factor (residues 1-219);
TF1-243, transmembrane truncated tissue factor; CHO-K1,
Chinese hamster ovary cell line; RU, resonance units; PAGE,
polyacrylamide gel electrophoresis; Ag, antigen; WT, wild type.
2 J. H. McVey, personal communication.
3 Available on the World Wide Web at the following address: http://europium.mrc.rpms.ac.uk.
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REFERENCES |
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