Journal of Thrombosis and Haemostasis, 3: 1250–1256
ORIGINAL ARTICLE
Interspecies exchange mutagenesis of the first epidermal growth factor-like domain of human factor VII V . W I L L I A M S O N , * A . P Y K E , * S . S R I D H A R A , * R . F . K E L L E Y , M . A . B L A J C H M A N * à and B . J . C L A R K E * *Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; Genentech Inc., San Francisco, CA, USA; and àThe Canadian Blood Services, Hamilton, Ontario, Canada
To cite this article: Williamson V, Pyke A, Sridhara S, Kelley RF, Blajchman MA, Clarke BJ. Interspecies exchange mutagenesis of the first epidermal growth factor-like domain of human factor VII. J Thromb Haemost 2005; 3: 1250–6.
Introduction Summary. The first epidermal growth factor-like (EGF1) domain of human factor VII (FVII) is essential for binding to tissue factor (TF). We hypothesized that the previously observed increased coagulant activity of rabbit plasma (i.e. FVII) with human TF might be explained by the five nonconserved amino acids in the rabbit vs. the human FVII EGF1 domain. Accordingly, we ÔrabbitizedÕ the human FVII EGF1 domain either by exchanging the entire EGF1 domain creating human FVII(rabEGF1) or by the single amino acid substitutions S53N, K62E, P74A, A75D and T83K. After transient expression in HEK293 cells, the recombinant FVII (rFVII) mutant proteins were analyzed for biological activity and binding affinity to human TF by competitive enzyme-linked immunosorbent assay (ELISA). Biological activity of the unpurified rFVII mutant proteins was either depressed or statistically unchanged vs. rFVII(WT). However, three of six rFVII mutant proteins had increased affinity for human TF in the rank order rFVII(rabEGF1) (3.3-fold) > rFVII(K62E) (2.9-fold) > rFVII(A75D) (1.7-fold). The mutant protein rFVII(K62E) was then permanently expressed and purified. Fully activated, purified rFVIIa(K62E) had a twofold greater clotting activity and 2.8-fold greater direct FVIIa amidolytic activity when compared with rFVIIa(WT). Quantitation of the affinity of TF binding by surface plasmon resonance indicated that the KD of purified rFVII(K62E) for human soluble TF (sTF) was 1.5 nM compared with 7.5 nM for rFVII(WT), i.e. fivefold greater affinity. We conclude that substitution of selected amino acid residues of the FVII EGF1 domain facilitated the creation of human rFVII chimeric proteins with both enhanced biological activity and increased affinity for TF. Keywords: coagulation, EGF, FVII, rabbit, tissue factor. Correspondence: Dr Bryan J. Clarke, Department of Pathology & Molecular Medicine, HSC 4N65, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5, Canada. Tel.: (905)-525-9140 ext 22372; fax: (905)-777-7856; e-mail:
[email protected] Received 29 July 2004, accepted 10 February 2005
Factor VII (FVII), a 50-kDa glycoprotein of human plasma [1] is essential for the initiation of the clotting cascade in man [2,3]. Recent evidence has supported a role for activated FVII (FVIIa) in tissue factor (TF)-mediated signal transduction [4,5], tumor angiogenesis and metastasis [6,7] and the inflammatory response during disseminated intravascular coagulation [8]. In the quest for new anticoagulant and anti-inflammatory drugs, a number of strategies have been explored in attempts to inhibit the FVII–TF interaction. This is a formidible biological task as both zymogen FVII and FVIIa bind with high affinity to human sTF with a KD of 7.5 and 5.1 nM, respectively [9]. New, potentially important anticoagulants include humanized monoclonal antibodies to TF [10] and variants of human sTF [11]. A different approach resulted in the description of inhibitory peptides to exosites in the heavy chain of human FVII [12,13]. To date, studies of both natural [14–16] and sitedirected mutants of FVII [17] have generally described FVII mutant proteins with decreased affinity for TF with one exception being the M156Q mutation [18]. The impetus for this work was an early observation of Janson et al. [19] who described a fourfold increased clotting activity of rabbitplasma FVII vs. human-plasma FVII, on incubation with human TF. As 43% of the contact area of FVII with TF lies within the FVII epidermal growth factor-like (EGF1) domain [20], we postulated that the discrepancy in FVII clotting activities noted might reside in the five amino acid residue differences between the rabbit [21] and human FVII EGF1 domains. Accordingly, we synthesized human FVII chimeras in which either the entire EGF1 domain was substituted with the rabbit EGF1 domain or each of the five rabbit amino acids was individually substituted for its human counterpart. This resulted in the synthesis of human rFVII mutant proteins with enhanced affinity for human TF and increased enzymatic activity. A preliminary report of this work was presented at the XIX Congress of the International Society on Thrombosis and Haemostasis [22]. 2005 International Society on Thrombosis and Haemostasis
Mutations of the FVII EGF1 domain 1251
Experimental procedures Reagents
Human FVII cDNA was subcloned in the EcoRI–HindIII site of the expression vector pCMV5 as described [15]. Cloning vector pUC19 DNA was obtained from New England Biolabs (Beverly, MA, USA). Plasmid DNA was amplified in Escherichia coli XL-1 Blue (Stratagene, La Jolla, CA, USA). Oligonucleotide synthesis and automated DNA sequence analysis were performed in the molecular biology facility MOBIX (McMaster University). Dulbecco’s modified Eagles medium (DMEM)-Ham’s F12 media, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT) and paranitrophenyl phosphate (PNPP) were obtained from SigmaAldrich Co. (St. Louis, MO, USA). Lipofectin reagent was from InVitrogen Corp. (San Diego, CA, USA). The tissue culture medium supplement bovine albumin–insulin–transferrin (BIT9500) was from Stem Cell Technologies (Vancouver, Canada). Vitamin K1 was purchased from EMD Biosciences (La Jolla, CA, USA). Purified plasma-derived human factor FVII (pdFVII) and pdFVIIa was from Enzyme Research Labs (South Bend, IN, USA). Purified, recombinant wildtype human factor VII [rFVII(WT)] was kindly donated by Dr R. Kelley, Genentech, Inc. (South San Francisco, CA, USA). Purified rFVIIa(WT) was also purchased from Novo Nordisk Inc. (Bagsvaerd, Denmark) or purified in our laboratory from rFVII(WT)HEK293 cell-line culture medium as required. The chromogenic peptidyl substrates Pefachrome FVIIa (CH3SO2-D-CHA-But-Arg-pNA.AcOH) and S-2222 were purchased from Pentapharm Ltd. (Basel, Switzerland) and Chromogenix (Westchester, OH, USA), respectively. Human thromboplastin (Thromborel S) was a product of Dade Behring (Marburg, Germany). Streptavidin conjugated to alkaline phosphatase was from Jackson Immune Research Laboratories (West Grove, PA, USA). The total-protein bicinchoninic acid (BCA) and Bradford Coomassie blue assay reagents were from Pierce Scientific Co. (Rockford, IL, USA). Mutagenesis of the FVII EGF1 domain
Oligonucleotide site-directed mutagenesis (Clontech, Palo Alto, CA, USA) was performed on the FVII EGF1 domain in the vector pUC19 as previously described [15]. The mutagenic primers employed were S53(5¢-AGT-3¢) fi N K62 (5¢-GTGTGCCTCAAACCCATGCCAGAATG-3¢), (5¢-AAG-3¢) fi E (5¢-GGGCTCCTGCGAGGACCAGCTC-3¢), P74(5¢-CCT-3¢) fi A (5¢-GCTTCTGCCTCGCTGCCTTC GAG-3¢), A75(5¢-GCC-3¢) fi D (5¢-CTGCCTCCCTGACT TCGAGGGC-3¢), and T83(5¢-ACG-3¢) fi K (5¢-GCCGGAA GTGTGAGAAACACAAGGATGACC-3¢). Human rFVII with a rabbit EGF1 domain (Fig. 1), i.e. rFVII(rabEGF1) was created by site-directed mutagenesis using the unique restriction sites BstEII and NsiI at the 5¢ and 3¢ ends of the human FVII EGF1 domain, respectively. The BstEII site was generated using the primer 5¢-CTTACAGTGATGGTGACC 2005 International Society on Thrombosis and Haemostasis
Fig. 1. Alignment of the EGF1 domains of human and rabbit FVII. The N-terminal amino acid residues 46–83 of FVII were aligned using the software program GENEPRO. The five non-conserved amino acid residues at positions 53, 62, 74, 75 and 83 are highlighted in bold.
AGTGTGCCTC-3¢. This base substitution did not change the amino acid sequence of human FVII. The NsiI site was generated using the primer 5¢-CGGAACTGTGAGATGCAT AAGGATGACCAGC-3¢. Creation of the new NsiI restriction site also altered the amino acid sequence of human FVII changing residue T83 fi M. After excision of the human FVII EGF1 domain DNA by BstEII–NsiI restriction endonuclease digestion and subcloning of the rabbit FVII EGF1 domain DNA in its place, the codon at position 83 was corrected from M83 fi K by site-directed mutagenesis using the primer 5¢-GGTCGCAACTGTGAGAAACACAAGGATGACCA GC-3¢. Rabbit FVII EGF1 domain DNA was prepared using rabbit FVII template cDNA [21] by a standard polymerase chain reaction utilizing the forward primer 5¢-TACAATGATGGTGACCAGTGTGCCTCC-3¢ and the reverse primer 5¢-TCTTATGCATCTCACAGTTGCGACCC TCG-3¢. The fidelity of all FVII mutant DNAs were confirmed by automated DNA sequence analysis. Mammalian cell culture and transient expression of rFVII mutant proteins
Wild-type and mutant FVII cDNAs in the vector pCMV5 were transfected into HEK293 cells using Lipofectin reagent as previously described [23]. HEK293 cells were routinely maintained in DMEM-F12 medium supplemented with 10% fetal calf serum, 100 U mL)1 penicillin–streptomycin and 100 ng mL)1 vitamin K1. HEK293 cell-conditioned media (3 mL medium, approximately 1 · 106 cells/35 mm well, 6 · 35 mm wells per FVII cDNA) containing the serum-free supplement BIT9500 were collected for analysis 72 h post-transfection and concentrated sixfold using an Amicon centrifugal filtration device, 10 kDa molecular weight cut-off (Millipore Corporation, Billerica, MA, USA) prior to quantitation by FVII-specific ELISA. Permanent expression of rFVII mutant proteins
A HEK293 cell line permanently expressing rFVII(K62E) was established essentially as described [21]. Briefly, the rFVII(K62E) mutant cDNA was subcloned into the EcoRI– HindIII site of the expression vector pCMV5 and cotransfected into HEK293 cells with the selection plasmid pSV2neo. After 2–3 weeks post-transfection, G418-resistant clones were assayed for synthesis of human rFVII by ELISA. Optimal FVII-synthesizing cell clones were expanded into NUNC triple-flask cell factories and the supernatant medium
1252 V. Williamson et al
was collected weekly. Purification of rFVII from HEK293 cell-conditioned medium was greatly facilitated by the use of phenol red-free DMEM-F12 supplemented with BIT9500. Confluent HEK293 cells remained adherent to the plastic substratum and continued to synthesize rFVII normally for 3–4 weeks in the above medium. Purification of rFVII mutant proteins
Both rFVII(WT) and rFVII(K62E) mutant protein were purified using a modification of the Q-Sepharose pseudoaffinity chromatography technique [24]. Briefly, HEK293 cell serumfree conditioned medium was collected and benzamidine and Na2 ethylenediaminetetraacetic acid (EDTA) were added to final concentrations of 10 and 5 mM, respectively. The medium was stored frozen at )40 C. One liter of HEK293 cellconditioned medium was concentrated to 250 mL using a Millipore Masterflex pump and prep-scale tangential flow filter (TFF) cartridge (30 kDa molecular weight cut-off). The 250-mL concentrate was dialyzed overnight against 20 mM Tris, pH 8.0, 50 mM NaCl, 0.05% azide, 1 mM benzamidine, 1 mM EDTA at 4 C. The dialyzed sample was readjusted to 10 mM benzamidine and 5 mM EDTA. Conductivity of the dialyzed sample was routinely 95% pure rFVII/rFVIIa. Quantitation of FVII antigen and total protein
Total rFVII antigen levels were determined by solid-phase ÔtrapÕ ELISA in 96-well microtiter plates. Briefly, a single sheep was hyperimmunized with 300–400 lg of purified human
rFVII and total sheep IgG purified as previously described [21]. Routinely, 500 ng of the above polyclonal sheep anti-human rFVII IgG was coated per microtiter well using carbonate antigen coating buffer (0.15 M Na2CO3, 0.35 M NaHCO3, pH 9.6) overnight at 4 C. All proteins and unknown samples were diluted in phosphate-buffered saline (PBS)-Tween buffer (25 mM Na phosphate, pH 8.0, 0.15 M NaCl, 0.05% Tween 20) containing 0.1% bovine serum albumin as a carrier protein; 100 ng per well of biotinylated polyclonal sheep anti-human rFVII IgG was employed as the detecting antibody. Biotinylated antibody binding to human rFVII was quantitated using streptavidin–alkaline phosphatase and the enzyme substrate PNPP (1 mg mL)1 in diethanolamine buffer, 5 mM MgCl2, pH 9.8). Either purified plasma-derived human FVII or purified human rFVII were used to generate a standard curve. After subtraction of background (routinely 95% pure as judged by SDS-PAGE with Coomassie blue staining and Western blot analysis (data not shown). Purification of rFVII(K62E) resulted in 10–20% activation of rFVII(K62E) to rFVIIa(K62E). rFVII(WT) from Genentech was 50% activated to rFVIIa whereas pdFVII was purely in the zymogen form (data not shown). The relative affinity of purified rFVII(K62E) for full-length relipidated human TF calculated by competitive ELISA (Fig. 2) was substantially greater than either human plasma FVII (IC50 is 19-fold lower) or human rFVII(WT) (IC50 is 18-fold lower). On average, using three different preparations, rFVII(K62E) exhibited 5-fold tighter binding than rFVII(WT) in competitive ELISA experiments. These results were confirmed independently by surface plasmon resonance experimentation on two of the above FVII preparations. In this representative experiment (Table 2), testing the identical FVII preparations used in Fig. 2, the ka of rFVII(K62E) for sTF was 5.6-fold higher than that of rFVII(WT) resulting in a KD of rFVII(K62E) for human sTF of 1.5 nM, i.e. 5-fold lower than rFVII(WT) and more than 10-fold lower than plasma FVII. In addition to its enhanced affinity for TF, rFVIIa(K62E) exhibited a 2.0- and 5.5-fold increase in
1254 V. Williamson et al
in Table 3 were purified in our laboratory and 100% activated to the rFVIIa form as judged by SDS-PAGE followed by Coomassie blue staining and densitometry.
FVII bound (A405nm)
0.9 Plasma FVII FVII(WT) FVII(K62E)
0.8 0.7 0.6
Discussion
0.5 0.4 0.3 0.2 0.1 0
1 5 2.5 Competitor:probe (molar ratio)
10
0.5
Fig. 2. Relative affinity of purified rFVII(K62E) mutant protein for TF. rFVII(WT) was from Genentech Inc. The data illustrate the inhibition of binding of biotinylated, plasma-derived zymogen FVII to full-length, relipidated human TF via competitive ELISA. Data are the mean ± SEM of triplicate determinations.
Table 2 Absolute affinity of purified FVII for human sTF determined by surface plasmon resonance FVII sample
ka · 105 (M)1 s)1)
kd · 10)3 (s)1)
KD (nM)
rFVII(WT) pdFVII rFVII(K62E)
3.9 ± 0.5 1.4 ± 0.1 22.1 ± 3.5
2.9 ± 0.1 3.7 ± 0.1 3.2 ± 0.03
7.5 ± 1.1 26.0 ± 0.9 1.5 ± 0.2**
Purified rFVII(WT) was from Genentech Inc. Data are mean ± SEM. **A statistically significant difference between rFVII(K62E) and rFVII(WT), P £ 0.01 using a two-tailed t-test, Welch-corrected. For rFVII(WT), n ¼ 6; pdFVII and rFVII(K62E), n ¼ 3.
Table 3 Comparative enzyme activity of purified rFVIIa
FVII sample
Clotting activity (U mg)1)
Indirect amidolytic assay (DA405nm min)1 lg)1)
rFVIIa 20740 ± 4680 35.3 ± 2.2 (WT) pdFVIIa 7540 ± 785 36.5 ± 2.1 rFVIIa 41635 ± 385** 39.3 ± 2.4 (K62E)
Direct amidolytic assay (DA405nm h)1 lg)1) 2.09 ± 0.02 2.88 ± 0.06 5.89 ± 0.62**
Both rFVIIa(WT) and rFVIIa(K62E) were purified in our laboratory. Data are mean ± SEM. n ‡ 3. **A statistically significant difference between rFVIIa(K62E) and rFVIIa(WT) in column 1 and plasma FVIIa in column 3, P £ 0.01, using the Tukey–Kramer multiple comparisons t-test.
coagulant activity (Table 3) when compared with rFVIIa(WT) and pdFVIIa, respectively. It is of interest to note that the amidolytic activity of rFVIIa(K62E) for the FVIIa-specific substrate Pefachrome VIIa was increased 2.8-fold vs. rFVIIa(WT), whereas there was no statistically significant difference in activity of the FVIIa preparations in the classical indirect FXa-dependent amidolytic assay. Both preparations of rFVIIa(WT) and rFVIIa(K62E) compared
The first epidermal growth factor-like domain of human coagulation FVII is essential for high-affinity binding to its cell-surface receptor TF [14,20,27,28]. Initially, replacement of the entire human FVII EGF1 domain with the homologous rabbit FVII EGF1 region resulted in the formation of a chimeric human rFVII(rabEGF1) molecule which exhibited greater than a threefold increase in affinity for human TF (Table 1) and an approximate twofold increase in specific clotting activity with human TF relative to pdFVII (data not shown). Subsequently, we examined the effects of the five individual amino acid differences between the human and rabbit FVII EGF1 domains (Fig. 1). Transient expression of the rFVII mutants in HEK293 cells revealed that only rFVII(K62E) demonstrated a statistically significant increase in affinity for human TF (Table 1). The purified rFVII(K62E) mutant protein exhibited a fivefold increased affinity for human TF (Fig. 2, Table 2). The biological activity of rFVIIa(K62E) was elevated twofold in coagulant activity and 2.8-fold in direct amidolytic activity (Table 3) relative to human rFVIIa(WT). These data confirm and extend previous observations [19,29,30] all of which described significant differences in the affinity and/or activity of FVII for homologous vs. non-homologous TF. We previously described two naturally occurring mutations of the FVII EGF1 domain which affect binding to TF. Both mutants rFVII(N57D) [15] and rFVII(R79Q) [14,31] exhibited a five- to ten-fold decrease in TF binding but the mechanisms of the two defects differed. Amino acid residue R79 of FVII was shown to form both hydrophobic and hydrogen bonds with TF [20], whereas the mutation FVII(N57D) did not directly alter FVII contact with TF but caused a misfolding of the FVII EGF1 domain [15]. rFVII(R79Q) mutant protein bound normally to the EGF1 conformation-sensitive monoclonal antibody 231-7 but rFVII(N57D) mutant protein did not [15]. Notably, the increased affinity of rFVII(K62E) protein for TF is associated with enhanced binding of rFVII(K62E) protein to monoclonal antibody 231-7 (data not shown), suggesting that the K fi E substitution at position 62 has resulted in a conformational change in the rFVII(K62E) EGF1 domain. Although K62 is far removed from the FVII–TF interface [20], the K fi E substitution potentially affects its neighboring amino acid residues D63, Q64, I69, C70 and F71 all of which either directly contact TF and/or act as ligands for the single Ca++ bound within the FVII EGF1 domain. Recently, reverse-charge mutations of surface-exposed amino acid residues have been correlated with faster association and tighter binding protein complexes in other biological systems [32]. Thus the increased affinity of rFVII(K62E) for TF is likely because of either electrostatic or conformational changes (or a combination of the two) in the EGF1 domain of FVII. 2005 International Society on Thrombosis and Haemostasis
Mutations of the FVII EGF1 domain 1255
Naturally occurring mutants of the EGF1 domain have not been reported for either FVII or FIX at the K62/K63 amino acid residue, respectively. Furthermore, the C–K–D tripeptide sequence is absolutely conserved in the FIX EGF1 and FX EGF1 domains across species but highly variable in protein C and FVII. The FVII EGF1 amino acid residue 62 is variously K (man), D (chicken), E (rabbit and bovine), Q (mouse and rat) and T (zebrafish) [21,33,34]. We are currently analyzing the effect of each of these interspecies EGF1 domain amino acid substitutions, i.e. K62 fi X on the biological activity of FVII. A number of studies provide evidence for reciprocal allosteric interaction(s) between the EGF1 and protease domains of FVII [35–38]. Perturbations of the FVII EGF1 domain including monoclonal antibody 231-7 binding [34] and site-directed mutagenesis affecting the high-affinity Ca++ binding site [36] have been shown to either enhance or decrease rFVIIa catalytic activity. We therefore suggest that the K62 fi E amino acid substitution enhances rFVIIa activity via an allosteric effect on the protease domain. Our results are an interesting contrast to the recent data of Persson [39] and Harvey et al. [40], who have demonstrated that replacement of selected amino acid residues in the protease domain and Gla domain of rFVII, respectively, can substantially increase the enzymatic activity of rFVIIa without a concomittent change in the affinity of these rFVII mutants for TF. Recombinant FVIIa is currently approved for use in hemophilia A patients with acquired inhibitors to FVIII [41]. In addition, chemically inactivated rFVIIa, termed rFVIIai, which exhibits increased affinity for TF in vitro [42], has been shown to be an effective inhibitor of thrombosis in vivo in a baboon model [8] and to enhance apoptosis of tumor cells in vitro [43–45]. The clinical use of human rFVIIa/rFVIIai could be greatly facilitated by the advent of mutants of rFVIIa with either increased enzymatic activity (in hemophilia) or increased affinity for TF (a better competitive inhibitor) in patients with thrombosis and cancer. Acknowledgements This work was supported by project grants from the R & D Fund of the Canadian Blood Services (MAB and BJC) and the Heart and Stroke Foundation of Ontario (no. NA5229 to BJC). References 1 Hagen FS, Gray CL, O’Hara PJ, Grant FJ, Saari GC, Woodbury RG, Hart CE, Insley m, Kisiel W, Kurachi K, Davie EW. Characterization of a cDNA coding for human factor VII. Proc Natl Acad Sci USA 1986; 83: 2412–6. 2 Broze GJ, Jr. Tissue factor pathway inhibitor and the revised theory of coagulation. Annu Rev Med 1995; 46: 103–12. 3 Hockin MF, Jones KC, Everse SJ, Mann KG. A model for the stoichiometric regulation of blood coagulation. J Biol Chem 2002; 277: 18322–33.
2005 International Society on Thrombosis and Haemostasis
4 Versteeg HH. Tissue factor as an evolutionary conserved cytokine receptor: implications for inflammation and signal transduction. Semin Hematol 2004; 41: 168–72. 5 Ruf W, Dorfleutner A, Riewald M. Specificity of coagulation factor signalling. J Thromb Haemost 2003; 1: 1495–503. 6 Chen J, Bierhaus A, Schiekofer S, Andrassy M, Chen B, Stern DM, Nawroth PP. Tissue factor – a receptor involved in the control of cellular properties including angiogenesis. Thromb Haemost 2001; 86: 334–45. 7 Konigsberg W, Kirchhofer D, Riederer MA, Nemerson Y. The TF:VIIa complex: clinical significance, structure–function relationships and its role in signalling and metastasis. Thromb Haemost 2001; 86: 757–71. 8 Taylor FB, Jr, Chang A, Peer G, Li A, Ezban m, Hedner U. Active site inhibited factor VIIa(DEGR VIIa) attenuates the coagulant and interleukin-6 and -8 but not tumor necrosis factor responses of the baboon to LD100 Escherichia coli. Blood 1998; 91: 1609–15. 9 Kelley RF, Yang J, Eigenbrot C, Moran P, Peek M, Lipari M, Kirchhofer D. Similar molecular interactions of factor VII and factor VIIa with the tissue factor region that allosterically regulates enzyme activity. Biochemistry 2004; 43: 1223–9. 10 Presta L, Sims P, Meng YG, Moran P, Bullens S, Bunting S, Schoenfeld J, Lowe D, Lai J, Rancatore P, Iverson M, Lim A, Chisholm V, Kelley RF, Riederer M, Kirchhofer D. Generation of a humanized, high-affinity anti-tissue factor antibody for use as a novel therapeutic. Thromb Haemost 2001; 85: 379–89. 11 Yang J, Lee GF, Riederer MA, Kelley RF. Enhancing the anticoagulant potency of soluble tissue factor mutants by increasing their affinity to factor VIIa. Thromb Haemost 2002; 87: 450–8. 12 Dennis MS, Eigenbrot C, Skeleton NJ, Ultsch MH, Santell L, Dwyer MA, O’Connell MP, Lazarus RA. Peptide exosite inhibitors of factor VIIa as anticoagulants. Nature 2000; 404: 465–70. 13 Maun HR, Eigenbrot C, Lazarus RA. Engineering exosite peptides for complete inhibition of factor VIIa using a protease switch with substrate phage. J Biol Chem 2003; 278: 21823–30. 14 Chaing S, Clarke BJ, Sridhara S, Chu K, Friedman P, Van Dusen W, Roberts HR, Blajchman M, Monroe DM, High KA. Severe factor VII deficiency caused by mutations abolishing the cleavage site for activation and altering binding to tissue factor. Blood 1994; 83: 3524–35. 15 Leonard BL, Chen Q, Blajchman MA, Ofosu FA, Sridhara S, Yang D, Clarke BJ. Factor VII deficiency caused by a structural variant N57D of the first epidermal growth factor domain. Blood 1998; 91: 142–8. 16 McVey JH, Boswell E, Mumford AD, Kemball-Cook G, Tuddenham EGD. Factor VII deficiency and the factor VII mutation database. Hum Mut 2001; 17: 3–17. 17 Dickinson CD, Kelly CR, Ruf W. Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa. Proc Natl Acad Sci USA 1996; 93: 14379–84. 18 Petrovan RJ, Ruf W. Residue Met156 contributes to the labile enzyme conformation of coagulation factor VIIa. J Biol Chem 2001; 276: 6616– 20. 19 Janson TL, Stormorken H, Prydz H. Species specificity of tissue thromboplastin. Haemostasis 1984; 14: 440–4. 20 Banner DW, D’Arcy A, Che`ne C, Winkler FK, Guha A, Konigsberg WH, Nemerson Y, Kirchhofer D. The crystal structure of the complex of factor VIIa with soluble tissue factor. Nature 1996; 380: 41–6. 21 Ruiz S, Sridhara S, Blajchman MA, Clarke BJ. Expression and purification of recombinant rabbit factor VII. Thromb Res 2000; 98: 203– 11. 22 Williamson V, Pyke A, Sridhara, S, Blajchman MA, Clarke BJ. Interspecies exchange mutagenesis of the first epidermal growth factorlike domain of human factor VII. J Thromb Haemost 2003; 1: abstract PO559. 23 Clarke BJ, Sridhara S. Incomplete gamma carboxylation of human coagulation factor VII: differential effects on tissue factor binding and enzymatic activity. Br J Haematol 1996; 93: 445–50
1256 V. Williamson et al 24 Yan SC, Razzano P, Chao B, Walls J, Berg D, McClure D, Grinnell B. Characterization and novel purification of recombinant human protein C from three mammalian cell lines. Biotechnology 1990; 8: 655–61. 25 Sridhara S, Chaing S, High KA, Blajchman MA, Clarke BJ. Activation of a recombinant human factor VII structural analogue alters its affinity of binding to tissue factor. Am J Hematol 1996; 53: 66–71. 26 Kelley RF, Costas KE, OÔConnell MP, Lazarus RA. Analysis of the factor VIIa binding site on human tissue factor: effects of tissue factor mutations on the kinetics and thermodynamics of binding. Biochemistry 1995; 34: 10383–92. 27 Clarke BJ, Ofosu FA, Sridhara S, Bona RD, Rickles FR, Blajchman MA. The first epidermal growth factor domain of human coagulation factor VII is essential for binding to tissue factor. FEBS Lett 1992; 298: 206–10. 28 Chang JY, Stafford DW, Straight DL. The roles of factor VII’s structural domains in tissue factor binding. Biochemistry 1995; 34: 12227–32. 29 Clarke BJ, Sridhara S, Blajchman MA, Hatton MWC. Inhibition of the rabbit extrinsic pathway of coagulation: A comparison of rabbit and human FVII structural analogues. Can J Cardiol 2003; 18: abstract 331. 30 Kelley RF, Refino CJ, O’Connell MP, Modi N, Sehl P, Lowe D, Pater C, Bunting S. A soluble tissue factor mutant is a selective anticoagulant and antithrombotic agent. Blood 1997; 89: 3219–27. 31 O’Brien DP, Kemball-Cook G, Hutchinson AM, Martin DMA, Johnson DJD, Byfield PGH, Takamiya O, Tuddenham EGD, McVey JH. Surface plasmon resonance studies of the interaction between factor VII and tissue factor. Biochemistry 1994; 33: 14162–9. 32 Selzer T, Albeck S, Schreiber G. Rational design of faster associating and tighter binding protein complexes. Nat Struct Biol 2000; 7: 537–41. 33 Davidson CJ, Tuddenham EG, McVey JH. 450 million years of hemostasis. J Thromb Haemost 2003; 1: 1487–94. 34 Seetharam S, Murphy K, Atkins C, Feuerstein G. Cloning and expresssion of rat coagulation factor VII. Thromb Res 2003; 109: 225– 31. 35 Leonard BJN, Clarke B J, Sridhara S, Kelley R, Ofosu FA, Blajchman MA. Activation and active site occupation alter conformation in the
36
37
38
39 40
41 42
43
44
45
region of the first epidermal growth factor-like domain of human factor VII. J Biol Chem 2000; 275: 34894–900. Persson E, Olsen OH, Ostergaard A, Nielsen LS. Ca2+ binding to the first epidermal growth factor-like domain of factor VIIa increases amidolytic activity and tissue factor affinity. J Biol Chem 1997; 272: 19919–24. Jin J, Perera L, Stafford D, Pedersen L. Four loops of the catalytic domain of factor VIIa mediate the effect of the first EGF-like domain substitution on factor VIIa catalytic activity. J Mol Biol 2001; 307: 1503–17. Dickinson CD, Shobe J, Ruf W. Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa. J Mol Biol 1998; 277: 959–71 Persson E. Variants of recombinant factor VIIa with increased intrinsic activity. Semin Hematol 2004; 41: 89–92. Harvey SB, Stone MD, Martinez MB, Nelsestuen G. Mutagenesis of the c-carboxyglutamic acid domain of human factor VII to generate maximum enhancement of the membrane contact site. J Biol Chem 2003; 278: 8363–9. Hedner U. Dosing with recombinant factor VIIa based on current evidence. Semin Hematol 2004; 41: 35–9. Sorensen BB, Persson E, Freskga˚rd PO, Kjalke M, Ezban M, Williams T, Rao LVM. Incorporation of an active site inhibitor in factor VIIa alters the affinity for TF. J Biol Chem 1997; 272: 11863–8. Sorensen BB, Rao LV, Tornehave D, Gammeltoft S, Petersen LC. Antiapoptotic effect of coagulation factor VIIa. Blood 2003; 102: 1708– 15. Jiang X, Bailly MA, Panetti TS, Cappello M, Konigsberg WH, Bromberg ME. Formation of tissue factor -factor Xa complex promotes cellular signalling and migration of human breast cancer cells. J Thromb Haemost 2003; 2: 93–101. Versteeg HH, Spek CA, Richel DJ, Peppelenbosch MP. Coagulation factors VIIa and Xa inhibit apoptosis and anoikis. Oncogene 2004; 23: 410–7.
2005 International Society on Thrombosis and Haemostasis
