Factor VIIa Modified in the 170 Loop Shows Enhanced Catalytic Activity but Does Not Change the Zymogen-like Property
2001; Elsevier BV; Volume: 276; Issue: 20 Linguagem: Inglês
10.1074/jbc.m009206200
ISSN1083-351X
AutoresKenji Soejima, J. Mizuguchi, Masato Yuguchi, Tomohiro Nakagaki, Shouichi Higashi, Sadaaki Iwanaga,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoFactor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appears to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond between Cys310 and Cys329, which is five residues longer than those of other trypsin types. To examine the functional role of the loop region, we prepared two mutants of VIIa. One of the mutants, named VII-11, had five extra corresponding residues 316–320 of VII deleted. The other mutant, VII-31, had all of the residues in its loop replaced with those of trypsin. Functional analysis of the two mutants showed that VIIa-11 (Kd = 41 nm) and VIIa-31 (Kd = 160 nm) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (Kd = 11 nm). The magnitude of tissue factor-mediated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced activity; the catalytic efficiencies of VIIa-31 toward various chromogenic substrates were 2–18-fold greater than those of the wild-type VIIa. Susceptibility of the α-amino group of Ile-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wild-type VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational transition, and changed the catalytic efficiency of VIIa, but they did not affect the equilibrium between active and zymogen-like conformational states. Factor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appears to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond between Cys310 and Cys329, which is five residues longer than those of other trypsin types. To examine the functional role of the loop region, we prepared two mutants of VIIa. One of the mutants, named VII-11, had five extra corresponding residues 316–320 of VII deleted. The other mutant, VII-31, had all of the residues in its loop replaced with those of trypsin. Functional analysis of the two mutants showed that VIIa-11 (Kd = 41 nm) and VIIa-31 (Kd = 160 nm) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (Kd = 11 nm). The magnitude of tissue factor-mediated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced activity; the catalytic efficiencies of VIIa-31 toward various chromogenic substrates were 2–18-fold greater than those of the wild-type VIIa. Susceptibility of the α-amino group of Ile-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wild-type VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational transition, and changed the catalytic efficiency of VIIa, but they did not affect the equilibrium between active and zymogen-like conformational states. activated coagulation factor VII coagulation factor VII wild-type coagulation factor VII activated wild-type coagulation factor VII activated coagulation factor VII-11 activated coagulation factor VII-31 coagulation factor X activated coagulation factor X tissue factor (full-length sTF, soluble tissue factor (extracellular domain of TF APMSF, p-amidinophenyl methanesulfonyl fluoride-hydrochloride mixture of phosphatidylcholine and phosphatidylserine polyacrylamide gel electrophoresis epidermal growth factor p-nitroanilide polyethylene glycol Factor VIIa (VIIa)1 is a plasma serine proteinase that is essential for the initiation of extrinsic blood coagulation (1Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1626) Google Scholar). When VIIa forms a complex with tissue factor (TF) in the presence of Ca2+ and phospholipids, the proteinase activity of VIIa toward its natural substrates, factors IX and X, is enhanced by several orders of magnitude, and the coagulation cascade is triggered (2Komiyama Y. Pedersen A.H. Kisiel W. Biochemistry. 1990; 29: 9418-9425Crossref PubMed Scopus (153) Google Scholar). In vitro, the formation of the active complex can be evidenced by measuring the esterolytic and amidolytic activities of VIIa (3Ruf W. Kalnik M.W. Lund-Hansen T. Edgington T.S. J. Biol. Chem. 1991; 266: 2158-2166Abstract Full Text PDF PubMed Google Scholar, 4Higashi S. Nishimura H. Fujii S. Takada K. Iwanaga S. J. Biol. Chem. 1992; 267: 17990-17996Abstract Full Text PDF PubMed Google Scholar, 5Lawson J.H. Butenas S. Mann K.G. J. Biol. Chem. 1992; 267: 4834-4843Abstract Full Text PDF PubMed Google Scholar, 6Neuenschwander P.F. Branam D.E. Morrissey J.H. Thromb. Haemostasis. 1993; 70: 970-977Crossref PubMed Google Scholar); this activity is also enhanced in the presence of soluble TF (sTF) and Ca2+ (4Higashi S. Nishimura H. Fujii S. Takada K. Iwanaga S. J. Biol. Chem. 1992; 267: 17990-17996Abstract Full Text PDF PubMed Google Scholar). Human zymogen VII is a single-chain enzyme precursor with an NH2-terminal Gla domain (residues 1–39), followed by two EGF-like domains, EGF 1 (residues 50–81), and EGF 2 (residues 91–127), and a COOH-terminal serine proteinase domain (residues 153–406). Through the limited proteolysis of the Arg152-Ile153 peptide bond, zymogen VII is converted to a two-chain form enzyme, activated VII (VIIa), bridged by a disulfide bond (Cys135-Cys262), which is composed of a light chain (residues 1–152) with Gla, EGF 1, and EGF 2 domains, and a heavy chain with a serine proteinase domain (residues 153–406) (7Hagen F.S. Gray C.L. O'Hara P. Grant F.J. Saari G.C. Woodbury R.G. Hart C.E. Insley M. Kisiel W. Kurachi K. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2412-2416Crossref PubMed Scopus (321) Google Scholar). On the other hand, the TF molecule consists of two immunoglobulin-like extracellular domains, a single membrane-spanning region, and a COOH-terminal cytoplasmic tail (8Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1879) Google Scholar). Solution of the crystal structure of the human VIIa-sTF complex revealed that there are several amino acid residues in the proteinase domain of VIIa that come into direct contact with TF, in addition to a number of interaction sites in the NH2-terminal portion (9Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (686) Google Scholar). Six of these, Phe275[c129F], Arg277[c134], Arg304[c162], Met306[c164], Gln308[c166], and Asp309[c167] (chymotrypsinogen numbering in brackets), are located in an almost cluster-like form (9Banner D.W. D'Arcy A. Chene C. Winkler F.K. Guha A. Konigsberg W.H. Nemerson Y. Kirchhofer D. Nature. 1996; 380: 41-46Crossref PubMed Scopus (686) Google Scholar, 10Kirchhofer D. Banner D.W. Trends Cardiovasc. Med. 1997; 7: 316-324Crossref PubMed Scopus (27) Google Scholar). The predicted sites of interaction between VIIa and TF on mutants from patients, and the results of alanine scanning analysis are consistent with the results obtained from crystallography (11O'Brien D.P. Gale K.M. Anderson J.S. McVey J.H. Miller G.J. Meade T.W. Tuddenham E.G. Blood. 1991; 78: 132-140Crossref PubMed Google Scholar, 12Marchetti G. Patracchini P. Gemmati D. DeRosa V. Pinotti M. Rodorigo G. Casonato A. Girolami A. Bernardi F. Hum. Genet. 1992; 89: 497-502Crossref PubMed Scopus (70) Google Scholar, 13Matsushita T. Kojima T. Emi N. Takahashi I. Saito H. J. Biol. Chem. 1994; 269: 7355-7363Abstract Full Text PDF PubMed Google Scholar, 14Cooper D.N. Millar D.S. Wacey A. Banner D.W. Tuddenham E.G. Thromb. Haemostasis. 1997; 78: 151-160Crossref PubMed Scopus (90) Google Scholar, 15Dickinson C.D. Kelly C.R. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14379-14384Crossref PubMed Scopus (179) Google Scholar). Furthermore, studies of the chemical modification of bovine VIIa provide a model of the mechanism of TF-mediated acceleration of VIIa activity. In this model, the proteinase domain of VIIa exists in equilibrium between the minor active and dominant zymogen-like inactive conformational states, and preferential binding of TF to the active state leads to a shift in equilibrium, thereby accelerating VIIa activity (16Higashi S. Matsumoto N. Iwanaga S. J. Biol. Chem. 1997; 272: 25724-25730Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 17Higashi S. Nishimura H. Aita K. Iwanaga S. J. Biol. Chem. 1994; 269: 18891-18898Abstract Full Text PDF PubMed Google Scholar, 18Higashi S. Matsumoto N. Iwanaga S. J. Biol. Chem. 1996; 271: 26569-26574Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). However, the structural elements of VIIa required for catalytic site formation are still not clear. The proteinase domain of VIIa contains a unique primary structure; the 170 loop (chymotrypsinogen number, 168th to 182nd) has five extra amino acid residues compared with those of other trypsin-types. In this study, we made two mutants of VIIa and studied the functional role of the 170 loop in formation of the catalytic site. The materials used were as follows: S-2288 (H-d-Ile-Pro-Arg-pNA·2HCl), S-2366 (pyroGlu-Pro-Arg-pNA·HCl), S-2238 (H-d-Phe-Pip-Arg-pNA·2HCl), S-2302 (H-d-Pro-Phe-Arg-pNA·2HCl), S-2765 (Z-d-Arg-Gly-Arg-pNA·2HCl), S-2444 (pyroGlu-Gly-Arg-pNA·HCl), S-2222 (Bz-IIe-Glu(GluγOMe)-Gly-Arg-pNA·HCl), and S-2403 (pyroGlu-Phe-Lys-pNA·HCl), from Chromogenix AB, Stockholm, Sweden; Chromozym® t-PA (MeSO2-d-Phe-Gly-Arg-pNA), and Chromozym® X (MeO-CO-d-Nle-Gly-Arg-pNA), from Roche Molecular Biochemicals; p-amidinophenyl methanesulfonyl fluoride hydrochloride (APMSF) and butyric acid, from Wako Pure Chemical Industries, Ltd., Osaka, Japan; benzamidine-HCl, from Tokyo Chemical Industry Co., Ltd.; LIPOFECTACETM Reagent, GENETICIN® (antibiotics G418), and α-minimum essential medium, from Life Technologies, Inc.; ASF-104 medium, from Ajinomoto Co., Inc., Tokyo, Japan; fetal bovine serum, from HyClone Co., Ltd.; penicillin G potassium, from Banyu Pharmaceutical Co., Ltd., Tokyo, Japan; streptomycin sulfate, from Meiji Seika Kaisha, Ltd., Tokyo, Japan; vitamin K, polyethylene glycol (PEG) 8000, bovine serum albumin (fatty acid free), phosphatidylcholine, and phosphatidylserine, from Sigma-Aldrich; human trypsin, from Athens Research & Technology, Inc.; fluorescein-Phe-Pro-Arg-chloromethyl ketone (FPR-ck), from Hematologic Technologies, Inc.; Asserachrom® VII: Ag (enzyme-linked immunosorbent assay kit), from Diagnostica Stago, Asnieres, France; Citrated control plasma (Ci-trol®), immunoabsorbed factor VII depleted plasma, and Dade® thromboplastin·C plus, from DADE International Inc., Miami, FL; and Platelin®, from Organon Teknika Corporation. Synthetic Xa inhibitor (DX-9065a) was a gift from Dr. T. Hara, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan (19Kunitada S. Nagahara T. Hara T. Uprichard A.C.G. Gallagher K.P. Antithrombotics: Handbook of Experimental Pharmacology. 132. Springer-Verlag, Berlin1999: 397-420Google Scholar). All custom oligo-DNA primers were provided by Nippon Flour Mills Co., Ltd. All other chemicals were of analytical grade or of the highest quality commercially available. Recombinant human soluble TF (residues 1–218; sTF) was prepared as described (20Shigematsu Y. Miyata T. Higashi S. Miki T. Sadler J.E. Iwanaga S. J. Biol. Chem. 1992; 267: 21329-21337Abstract Full Text PDF PubMed Google Scholar). Relipidated human placental full-length TF and plasma-derived human clotting factors VII, VIIa, X, and Xa were prepared as described (21Nakagaki T. Foster D.C. Berkner K.L. Kisiel W. Biochemistry. 1991; 30: 10819-10824Crossref PubMed Scopus (126) Google Scholar). cDNA of human VII was a gift from Drs. T. Matsushita and H. Saito, Nagoya University. Mutagenesis by polymerase chain reaction-based methods was performed for construction of mutant VII under standard conditions as described (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 14.1-15.113Google Scholar). Sequences of specific primers for cloning VII cDNA were as follows: 5′-GGGGTCGACATGGTCTCCCAGGCCCTCAGGCTCCTCTGCCTTCTG-3′ (sense primer containing a SalI site), 5′-CCCGGATCCCTAGGGAAATGGGGCTCGCAGGAGGACTCCTGGGCG-3′ (antisense primer containing a BamHI site). Sequences of primers for mutagenetic polymerase chain reaction were as follows: 5′-CAGCAGTCACGGCCAAATATCACGGAGTACATGTTCTGTGCCGGC-3′ (sense), 5′-GCCGGCACAGAACATGTACTCCGTGATATTTGGCCGTGACTGCTG-3′ (antisense) for VII-11; 5′-ATGACCCAGGACTGCGAAGCCTCCTACCCTGGAAAGATCACGGAGTACATG-3′ (sense), 5′-CATGTACTCCGTGATCTTTCCAGGGTAGGAGGCTTCGCAGTCCTGGGTCAT-3′ (antisense) for VII-31. Amplified mutant VII DNA fragments were digested with SalI and BamHI and then cloned into the mammalian expression vector pCAG-neo (23Niwa H. Yamamura K. Miyazaki J. Gene ( Amst. ). 1991; 108: 193-199Crossref PubMed Scopus (4597) Google Scholar). The vectors were confirmed to contain the entire sequences of the wild-type and mutant VII DNA fragments by sequencing on an automated DNA sequencer. Each of the mutant expression vectors was transfected into the Chinese hamster ovary cell line CHO-K1 with Lipofectin reagent (LIPOFECTACETM). Two days after transfection, the medium was changed to α-minimum essential medium containing 10% fetal bovine serum, 1 μg/ml G418, 10 units/ml penicillin G, 100 μg/ml streptomycin, and 10 μg/ml vitamin K. After 2 weeks, cells were cloned by the limiting dilution method. Expression of each clone was confirmed by enzyme-linked immunosorbent assay (Asserachrom® VII: Ag). The highly expressed clones were selected, cultured, and expanded. 48 h before harvesting, culture media were replaced with serum-free media (ASF-104) supplemented with 1 mm butyric acid and 20 μg/ml vitamin K. After harvesting, each conditioned medium was mixed with 0.1% bovine serum albumin and 50 mmbenzamidine-HCl, and centrifuged at 5000 rpm for 20 min at 4 °C. The supernatants (1.5–3 liters) were stored at −80 °C. Subsequently, the frozen media were thawed and filtrated through a 0.45 μm membrane filter (Corning Costar), mixed with 2 mm CaCl2, and subjected to the Ca2+-dependent anti-factor VII monoclonal antibody-conjugated column chromatography (21Nakagaki T. Foster D.C. Berkner K.L. Kisiel W. Biochemistry. 1991; 30: 10819-10824Crossref PubMed Scopus (126) Google Scholar) at 4 °C. The column had been equilibrated in 50 mmTris-HCl, pH 7.2, containing 0.1 m NaCl, 50 mmbenzamidine-HCl, and 2 mm CaCl2. After the medium was loaded, the column was washed with equilibration buffer. Mutant factor VII was eluted with 50 mm Tris-HCl, pH 7.2, containing 0.1 m NaCl, 50 mm benzamidine-HCl, and a 0–10 mm EDTA gradient, and eluted peak fractions were pooled. The pooled fractions were analyzed by SDS-PAGE, and clotting activity was determined in a Behring Fibrintimer® using VII-depleted plasma. The protein concentrations were determined by the Bradford method and/or absorbance at 280 nm (A280 = 13.9 for 1% VII) after dialysis to remove benzamidine-HCl. Purified VII derived from human plasma was used as a standard to estimate the protein concentration. Activation of VII mutants was achieved at 37 °C for 15–60 min by addition of a 1:100 molar ratio of plasma-derived Xa in 50 mm Tris-HCl, pH 7.45, containing 0.1 m NaCl, 0.1% PEG 8000, phospholipids (Platelin®), and 10 mm CaCl2. This reaction was terminated by the addition of 50 mm benzamidine-HCl. Mutant VIIa was purified by Ca2+-dependent anti-VII monoclonal column chromatography, as described above. The eluted VIIa fractions were pooled and dialyzed against 50 mm Tris-HCl, pH 8.0, containing 0.1 m NaCl. PEG 8000 was added to aliquots at a final concentration of 0.1%, and the samples were stored at −80 °C until use. Any potentially contaminating Xa was inactivated with a synthetic Xa-specific inhibitor (DX-9065a). Amidolytic activities of wild-type VIIa and mutants of VIIa were not affected by DX-9065a (data not shown). Active site titration of VIIa was performed by calculating the ratio of the concentrations of fluorescein and the protein in the fluorescein-labeled VIIa as described (18Higashi S. Matsumoto N. Iwanaga S. J. Biol. Chem. 1996; 271: 26569-26574Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 24Williams E.B. Mann K.G. Methods Enzymol. 1993; 222: 503-513Crossref PubMed Scopus (13) Google Scholar) with some modifications. Fluorescein-FPR-ck and VIIa were mixed (molar ratio of 50:1) and incubated for 18 h until VIIa activity was no longer detectable by clotting assay. Free fluorescein inhibitor was removed by gel filtration (Sephadex G-25) and extensive dialysis. The concentrations of fluorescein and the protein in the complex were determined by spectrofluorometry and the Bradford method, respectively. Fluorescein-labeled VIIa was excited at 485 nm, and emission was detected at 535 nm in FluoroNunc® C8-white 96-well microwell plates (NalgeNunc International) with a microplate spectrofluorometer (SPECTRAFLUOR, TECAN Austria GmbH.). Unless otherwise noted, all kinetic experiments were carried out under the following conditions: 50 mm Tris-HCl, pH 8.0, containing 0.1 m NaCl, 10 mm CaCl2, and 0.1% PEG 8000; 200 μl of total volume of sample in 96-well microplates (F96 Polysorp NuncTM-Immuno Plate; NalgeNunc International Denmark); measuring at 30 °C with a temperature controlled microplate spectrophotometer SPECTRAmax plus® (Molecular Devices Inc.). Kinetics were measured based on initial rates. All experiments were performed in at least two independent trials. A mixture of 2 nm VIIa and various concentrations (0–400 nm) of sTF was preincubated for 5 min at 30 °C. In one series of experiments, the initial rate (ΔmOD/min) was measured as amidolytic activity under the above conditions on the additional of a final concentration of 1 mm S-2288. The data were subjected to Hanes-Woolf plot analysis to determine the apparent dissociation constants. For assays in the absence of sTF, the mutant VIIa concentration was 100 nm, and substrate concentrations ranged from 0.2 to 1.2 mm. In the presence of sTF, a mixture of 1 μm enzyme and 5 μm sTF was preincubated for 5 min. 20 μl of this mixtures was diluted 10-fold, and the initial rate of hydrolysis was measured under the above conditions with substrate concentrations ranging from 0.2 to 1.2 mm. 5 μl each of a given concentration of APMSF was added to 45 μl of 1 μmVIIa with or without 5 μm sTF. The mixture was incubated for 5 min to incorporate the APMSF molecule. To measure residual VIIa amidolytic activities, 20 μl of each of the mixtures was diluted 9-fold, and kinetic analyses were carried out by adding 20 μl of 10 mm S-2288. For measurement of residual human trypsin amidolytic activity, the sample mixture was diluted 90-fold. One hundred μl of each VIIa mutant (1 μm) with or without sTF (5 μm), preincubated at 30 °C for 5 min, was mixed with 25 μl of 1 m KNCO. The mixture was incubated at 30 °C for 0, 10, 20, 40, 80, and 160 min. After each incubation, aliquots of 20 μl of each sample taken from the reaction mixture were diluted 9-fold, and kinetic analyses were carried out by addition of 20 μl of 10 mm S-2288. Amidolytic activities of VIIa toward various substrates were measured by kinetic analysis as described above. The enzyme and substrate concentrations were 100 nm and 1 mm, respectively. The mutant VIIa-mediated activation of zymogen X in the presence or absence of relipidated TF was performed as described by Komiyama et al.(2Komiyama Y. Pedersen A.H. Kisiel W. Biochemistry. 1990; 29: 9418-9425Crossref PubMed Scopus (153) Google Scholar). To investigate the function of the 170 loop, we prepared two mutants: VII-11, with deletion of the five extra residues in the 170 loop, and VII-31, in which the 170 loop was replaced with that of trypsin (Figs. 1 and2). Both mutants and wild-type VII (VII-W) were purified from 1.5–3 liters of transfected CHO-K1 cell culture conditioned medium using a monoclonal antibody column. The specific activity (clotting activity/protein concentration) of purified recombinant VII-W was equivalent to that of plasma-derived VII (about 2500 units/mg). The activation of the wild-type zymogen VII to VIIa mediated by Xa was completed within 15 min under the conditions described under "Experimental Procedures." Under these conditions, the activation of VII-31 was similar to that of VII-W, whereas that of VII-11 required a farther incubation for 15–45 min. On SDS-PAGE, the band of the heavy chain of VIIa-31 showed further mobility than those of VIIa-W and VIIa-11 (Fig. 3). This was probably because a carbohydrate attaching site in the VIIa-heavy chain, Asn322[c175] (25Klausen N.K. Bayne S. Palm L. Mol. Biotechnol. 1998; 9: 195-204Crossref PubMed Google Scholar), was lost in the creation of VIIa-31. The carbohydrate chain in the VIIa-W-heavy chain, but not in the VIIa-31-heavy chain, was detected by lectin blotting analysis (data not shown).Figure 2Circumferential structures of the 170 loop and design of VII mutants. The unique five extra amino acid (KVGDS) residues located in the 170 loop are shown inorange. A cluster of amino acid residues that are in direct contact with sTF are shown in pink. The NH2-terminal domain of sTF is colored green. Other circumferential structures of the 170 loop, which are involved in catalytic-site formation, are indicated as follows: S1 pocket,red circle; activation domains, light blue; NH2-terminal insertion of Ile153[c16],yellow dot. Sequences of the wild-type and both VII mutants are indicated in the orange box.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3SDS-PAGE of the time course of activation of each mutant VII by Xa. SDS-PAGE was performed under reducing conditions followed by Coomassie Brilliant Blue staining. Incubation time of activation is indicated above lanes. Bz indicates purified mutant VII-31 with 50 mm benzamidine-HCl. After remove benzamidine-HCl by dialysis, VII-31 showed some auto activation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Amidolytic activities of plasma-derived VIIa, VIIa-W, VIIa-11, and VIIa-31 toward S-2288, as well as the effects of TF on these activities, were examined in the absence and presence of various concentrations of sTF. In the absence of sTF, the amidolytic activity of VIIa-11 and that of VIIa-31 were about 50 and 700% of that of VIIa-W, respectively. As shown in Fig.4 A, sTF potentiated the amidolytic activities of both mutants and VIIa-W in a dose-dependent manner. The data shown in Fig. 4 Awere subjected to Hanes-Woolf plot analysis to determine the apparent dissociation constants (Kd app) for the plasma-derived VIIa-sTF, VIIa-W-sTF, VIIa-11-sTF, and VIIa-31-sTF complexes (Fig. 4 B). We found that both VIIa-11 (Kd app = 41 nm) and VIIa-31 (Kd app = 160 nm) had reduced affinities for sTF as compared with VIIa-W (Kd app = 11 nm) and plasma-derived VIIa (Kd app = 10 nm). We also determined the kinetic parameters for the hydrolysis of S-2288 catalyzed by mutants or wild-type VIIa (TableI). In the absence of sTF, thekcat/Km value of VIIa-31 was about 7-fold higher than that of VIIa-W, whereas thekcat/Km value of VIIa-11 was about 50% of that of VIIa-W. In the presence of saturating concentration of sTF, thekcat/Km value of VIIa-W was enhanced by 30-fold, whereas those of mutants VIIa-11 and VIIa-31 were enhanced by 7-fold and 2-fold, respectively. Thekcat/Km values of VIIa-11-sTF and VIIa-31-sTF complexes were 9.6 and 46% of that of VIIa-W-sTF, respectively.Table IKinetic parameters for VIIa mutants toward chromogenic substrate (S-2288)Kmkcatkcat/Kmmms−1mm−1s−1−sTF 1-aReactions were performed using plasma-derived VIIa (pd-VIIa; 100 nm), VIIa-W (100 nm), VIIa-11 (100 nm), and VIIa-31 (100 nm) in the presence of CaCl2 (5 mm).pd-VIIa2.82 ± 0.133.29 ± 0.061.17 ± 0.08VIIa-W2.22 ± 0.021.95 ± 0.010.88 ± 0.01VIIa-111.53 ± 0.170.59 ± 0.040.39 ± 0.07VIIa-313.06 ± 0.3018.53 ± 2.406.04 ± 0.20+sTF 1-bReactions were performed using the same concentrations of plasma-derived VIIa, VIIa-W, VIIa-11, and VIIa-31 as described in footnote a in the presence of sTF (500 nm) and CaCl2 (5 mm).pd-VIIa1.25 ± 0.1544.20 ± 0.2935.90 ± 4.13VIIa-W1.43 ± 0.3737.08 ± 2.8327.30 ± 5.01VIIa-112.98 ± 0.597.81 ± 1.692.61 ± 0.05VIIa-311.73 ± 0.3220.98 ± 0.5012.50 ± 2.021-a Reactions were performed using plasma-derived VIIa (pd-VIIa; 100 nm), VIIa-W (100 nm), VIIa-11 (100 nm), and VIIa-31 (100 nm) in the presence of CaCl2 (5 mm).1-b Reactions were performed using the same concentrations of plasma-derived VIIa, VIIa-W, VIIa-11, and VIIa-31 as described in footnote a in the presence of sTF (500 nm) and CaCl2 (5 mm). Open table in a new tab Plasma-derived VIIa-, VIIa-W-, VIIa-11-, and VIIa-31-mediated activation of zymogen X with phospholipids (PCPS) were examined in the presence or absence of full-length TF (Fig. 5). In the absence of TF, VIIa-31 showed the highest Xa generation as compared with VIIa-W and VIIa-11. This is consistent with data of the amidolytic activity toward S-2288. In the presence of TF, VIIa-W-mediated Xa generation was strongly enhanced and showed the highest activity as compared with VIIa-11 and VIIa-31 in this condition. APMSF is a synthetic serine proteinase inhibitor in which an amidino group was designed to interact with the Asp-189 (chymotrypsinogen numbering) in the subsite 1 (S1) pocket of trypsin-type serine proteinases. The interaction brings the sulfofluoride moiety of APMSF into proximity with the active serine residue of the enzyme, thus facilitating modification of the active site serine. Because APMSF does not interact with the S2 or S3 sites of serine proteinases, the inhibitor is a useful probe to examine the active site and S1 site of VIIa mutants without considering the contribution of extended subsite-peptidyl site interactions. We compared the susceptibilities of VIIa mutants to APMSF inhibition with those of wild-type and plasma-derived VIIa. In the absence of sTF, VIIa-31 showed the highest susceptibility to APMSF, and the concentration of APMSF required for 50% inhibition was 120 μm (Fig. 6). In contrast, 2000 μm APMSF was needed for 50% inhibition of plasma-derived VIIa, VIIa-W, and VIIa-11 activities. Therefore, it is likely that VIIa-31 but not VIIa-11 gained enhanced catalytic efficiency after modification in the 170 loop. In the presence of sTF, the concentrations of APMSF required for 50% inhibition of plasma-derived VIIa, VIIa-W, VIIa-11, and VIIa-31 were shifted to 60 μm, 40 μm, 60 μm and 20 μm, respectively. On the other hand, the susceptibility of human trypsin was much higher than those of VIIa derivatives, and the APMSF concentration required for 50% inhibition of trypsin activity was ∼1 μm. It has been reported that treatment of VIIa with cyanate ions results in a loss of activity caused by carbamylation of the α-amino group of Ile153[c16] (16Higashi S. Matsumoto N. Iwanaga S. J. Biol. Chem. 1997; 272: 25724-25730Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 17Higashi S. Nishimura H. Aita K. Iwanaga S. J. Biol. Chem. 1994; 269: 18891-18898Abstract Full Text PDF PubMed Google Scholar, 18Higashi S. Matsumoto N. Iwanaga S. J. Biol. Chem. 1996; 271: 26569-26574Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). This carbamylation reaction is inhibited by TF, hence the critical ion pair between Ile153[c16] and Asp343[c194] in VIIa appears to be formed only after complex formation with TF. This suggests that carbamylation might be useful as a tool to examine the microenvironment of the NH2-terminal Ile153[c16] and/or formation of the critical ion pair in VIIa mutants. In the presence of KNCO, the rates of inactivation of the mutants VIIa-11 and VIIa-31 and of VIIa-W were measured under the same conditions as those described previously (18Higashi S. Matsumoto N. Iwanaga S. J. Biol. Chem. 1996; 271: 26569-26574Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In the absence of sTF, the inactivation rates of both mutants VIIa-11 an VIIa-31 were almost the same as that of VIIa-W, suggesting that α-amino groups of Ile153[c16] of the mutants as well as that of VIIa-W are accessible to cyanate ions (Fig.7 A). After complex formation with sTF (Fig. 7 B), the inactivation rate of VIIa-W declined significantly. In contrast, binding of sTF partly reduced the inactivation rate of VIIa-11 but did not change the rate of VIIa-31, suggesting that the allosteric effect of TF is not transmitted sufficiently to the NH2-terminal region of these mutants. To examine the substrate specificities of the two mutant and wild-type VIIa, we tested various chromogenic substrates th
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