Factor VIIa Modified in the 170 Loop Shows Enhanced Catalytic Activity but Does Not Change the Zymogen-like Property*

Kenji SoejimaDagger , Jun Mizuguchi§, Masato Yuguchi§, Tomohiro Nakagaki§, Shouichi Higashi||, and Sadaaki Iwanaga§**

From the Dagger  First Research Department and the § Blood Products Research Department, The Chemo-Sero-Therapeutic Research Institute, Kumamoto 869-1298, the || Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, and ** Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Nagoya 470-1192, Japan

Received for publication, October 9, 2000, and in revised form, January 12, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Factor VIIa (VIIa)1 is a plasma serine proteinase that is essential for the initiation of extrinsic blood coagulation (1). 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 (2). In vitro, the formation of the active complex can be evidenced by measuring the esterolytic and amidolytic activities of VIIa (3-6); this activity is also enhanced in the presence of soluble TF (sTF) and Ca2+ (4). 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) (7).

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 (8). 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 (9). 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 (9, 10). 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 (11-15). 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 (16-18). 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.

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INTRODUCTION
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Materials-- 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(Glugamma OMe)-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 alpha -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 (19). 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.

Proteins-- Recombinant human soluble TF (residues 1-218; sTF) was prepared as described (20). Relipidated human placental full-length TF and plasma-derived human clotting factors VII, VIIa, X, and Xa were prepared as described (21).

Gene Construction of VII Mutants-- 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 (22). 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 (23). 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.

Expression, Cell Culture, and Purification of VII Mutants-- 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 alpha -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 mM benzamidine-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 (21) at 4 °C. The column had been equilibrated in 50 mM Tris-HCl, pH 7.2, containing 0.1 M NaCl, 50 mM benzamidine-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.

Preparation of VIIa Mutants-- 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 Mutants-- 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 (18, 24) 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.).

Amidolysis of Synthetic Chromogenic Substrates-- 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.

TF Dependence of VIIa Mutants on Amidolysis of S-2288-- 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 (Delta mOD/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.

Measurement of Kinetic Parameters for Mutant VIIa-catalyzed Amidolysis of S-2288-- 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.

APMSF Incorporation into VIIa Mutants-- 5 µl each of a given concentration of APMSF was added to 45 µl of 1 µM VIIa 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.

Carbamylation of the alpha -Amino Group in VIIa Mutants-- 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.

Substrate Specificities of VIIa Mutants-- 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.

Zymogen X Activation by VIIa Mutants-- The mutant VIIa-mediated activation of zymogen X in the presence or absence of relipidated TF was performed as described by Komiyama et al. (2).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Purification and Activation of VII Mutants-- 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 and 2). 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] (25), 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).


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Fig. 1.   Unique amino acid sequence and location of the 170 loop in the proteinase domain of VIIa. Sequence comparisons of the 170 loop derived from serine proteinases of various species are indicated in the box. IX, factor IX; PC, protein C; IIa, thrombin; Tryp, trypsin; alpha -CT, alpha -chymotrypsin. The unique five extra amino acid (KVGDS) residues located in the 170 loop are shown in orange. Locations of various surface loops are indicated on the right. There is no direct contact between the 170 loop and the NH2-terminal domain of soluble tissue factor (left). The crystal structure is cited from Protein Data Bank code 1DAN (9). The NH2-terminal domain of sTF is colored green, and activation domains are light blue.


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Fig. 2.   Circumferential 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 in orange. 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.


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Fig. 3.   SDS-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.

TF Dependence of Amidolytic Activities of VIIa Mutants-- 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. 4A, sTF potentiated the amidolytic activities of both mutants and VIIa-W in a dose-dependent manner. The data shown in Fig. 4A were 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. 4B). 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 (Table I). In the absence of sTF, the kcat/Km value of VIIa-31 was about 7-fold higher than that of VIIa-W, whereas the kcat/Km value of VIIa-11 was about 50% of that of VIIa-W. In the presence of saturating concentration of sTF, the kcat/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. The kcat/Km values of VIIa-11-sTF and VIIa-31-sTF complexes were 9.6 and 46% of that of VIIa-W-sTF, respectively.


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Fig. 4.   TF dependence of VIIa mutants. In A, the hydrolysis of chromogenic substrate S-2288 (1 mM) by VIIa-W (circle; 2 nM), VIIa-11 (square; 2 nM), and VIIa-31 (triangle; 2 nM) in the presence of various concentrations of sTF was analyzed at 30 °C. In B, the apparent dissociation constants of VIIa-W (circle), VIIa-11 (square), and VIIa-31 (triangle) with sTF were calculated by Hanes-Woolf plot analysis. In both panels, data of plasma-derived VIIa (diamond) is equivalent to that of VIIa-W.

                              
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Table I
Kinetic parameters for VIIa mutants toward chromogenic substrate (S-2288)

Activation of the Zymogen X by VIIa Mutants-- 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.


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Fig. 5.   Zymogen X activation by VIIa mutants. In A, the activations of zymogen X (FX) by VIIa-W (open circle; 50 nM), VIIa-11 (open square; 200 nM), and VIIa-31 (open triangle; 50 nM) were analyzed at 30 °C. In B, the activation of zymogen X by the complex of VIIa-W-TF/PCPS (closed circle; 20 pM), VIIa-11-TF/PCPS (closed square; 20 pM), or VIIa-31-TF/PCPS (closed triangle; 20 pM) was also analyzed at 30 °C. In both panels, data of plasma-derived VIIa (diamond) is equivalent to that of VIIa-W.

Incorporation of APMSF into VIIa Mutants-- 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.


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Fig. 6.   Susceptibility of VIIa mutants to APMSF. In A, VIIa-W (open circle), VIIa-11 (open square), and VIIa-31 (open triangle) were incubated with various concentrations of APMSF for 5 min at 30 °C, followed by measurement of the remaining amidolytic activity with 1 mM S-2288. In B, the complexes of VIIa-W-sTF (closed circle), VIIa-11-sTF (closed square), VIIa-31-sTF (closed triangle), and human trypsin (asterisk) were incubated with various concentrations of APMSF for 5 min at 30 °C, followed by measurement of the remaining amidolytic activity with 1 mM S-2288. In both panels, data of plasma-derived VIIa (diamond) is equivalent to that of VIIa-W.

Carbamylation of VIIa Mutants-- It has been reported that treatment of VIIa with cyanate ions results in a loss of activity caused by carbamylation of the alpha -amino group of Ile153[c16] (16-18). 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 (18). 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 alpha -amino groups of Ile153[c16] of the mutants as well as that of VIIa-W are accessible to cyanate ions (Fig. 7A). After complex formation with sTF (Fig. 7B), 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.


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Fig. 7.   Effect of carbamylation on VIIa mutants. In A, VIIa-W (open circle), VIIa-11 (open square), and VIIa-31 (open triangle) were each incubated with 0.2 M KNCO at 30 °C for the periods indicated, and the remaining amidolytic activity toward S-2288 was measured. In B, VIIa-W-sTF (closed circle), VIIa-11-sTF (closed square), and VIIa-31-sTF (closed triangle) were each incubated with 0.2 M KNCO at 30 °C for the periods indicated, and the remaining amidolytic activity toward S-2288 was measured. The concentration used for the wild-type and the VIIa mutants was 1 µM, and the concentration of sTF was 5 µM. In both panels, data of plasma-derived VIIa (diamond) is equivalent to that of VIIa-W.

Substrate Specificity of VIIa Mutants-- To examine the substrate specificities of the two mutant and wild-type VIIa, we tested various chromogenic substrates that are used to measure the activities of various clotting serine proteinases. As shown in Table II, amidolytic activities of VIIa-31 toward the tested substrate were always higher than those of VIIa-W. It seems likely that VIIa-31 especially may have some favor pyroGlu structure at their P3 site.

                              
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Table II
Substrate specificities of VIIa-W, VIIa-11, and VIIa-31 toward various chromogenic substrates


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ABSTRACT
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DISCUSSION
REFERENCES

The catalytic domain of the coagulation proteinases has an active site and internal core that are similar to those of the typical serine proteinase, trypsin. Although these proteinases have a highly homologous three-dimensional structure, they display significant differences in specificity and catalytic activity. The serine proteinase domain consists of 12 beta -strands and the loops between them. The beta -strands form a scaffold of the catalytic domain, and their Calpha traces are very similar despite differences in amino acid residues. The individual proteinases may be distinguished by surface loops of varying lengths and compositions (26). These surface loops are thought to participate closely in the mechanism of the enzymatic activity. In the case of beta -tryptase, the 60 loop, 99 loop, and 170 loop have a major interface for making a tetramer, and this tetramerization is important for the expressing activity (27). The 170 loop of factor IX has some function in the interaction with factor VIIIa (28, 29). It was also reported that replacement of some residues in the 99 loop of factor IXa allows the enzyme to gain enhanced catalytic activity toward synthetic peptidyl substrates (30). The 170 loop consists of 15 amino acid residues in trypsin and many vitamin K-dependent coagulation proteinases but 20 residues in VIIa (16, 31-35). The 170 loop in VIIa is not in direct contact with TF but is located close to the region in which amino acid residues are clustered for binding with TF (Figs. 1 and 2). To clarify the role of the 170 loop in the catalytic activity and/or TF-induced conformational transition of VIIa, we prepared two mutants of VIIa in which the 170 loops were modified.

We found that both VIIa-11 and VIIa-31 had lower affinities for sTF (Fig. 4B), suggesting that not only the surface making contact with TF but also that the loop region of VIIa play important roles in the high affinity interaction between VIIa and TF. Because the 170 loop of VIIa is close to the TF-contacting surface, modification of this loop may change the conformation or the orientation of the binding site. It has been reported that reduction and alkylation of the disulfide bond between Cys310[c168] and Cys329[c182] of VIIa lead to a loss of binding ability with sTF (16). Substitution of Phe328[c181] to Ser in the 170 loop of VIIa was reported to have an adverse effect on affinity for TF (36). Alanine scanning study in the 170 loop was reported (15). These studies also suggested the importance of certain residues in the 170 loop for the interaction with TF. As shown in Table I, the magnitudes of the TF-mediated acceleration of amidolytic activity of VIIa-11 (7-fold) and of VIIa-31 (2-fold) were smaller than that of wild-type VIIa (30-fold). We speculated, therefore, that the allosteric effect of TF is not transmitted sufficiently to the active site of these mutants. After complex formation with TF, the alpha -amino group of Ile153[c16] of wild-type VIIa was protected from carbamylation. However, the Ile153[c16] of VIIa-11 was only partially protected from carbamylation, and almost no protection of the alpha -amino group was observed in the case of VIIa-31 (Fig. 7). This suggested that the allosteric effect of TF is not transmitted sufficiently to the NH2-terminal region of these mutants either. The 170 loop of VIIa would play an important role in the TF-induced conformational transition of VIIa by transmitting the effect of TF to the activation domain that contains both the catalytic center and the ion pair between Ile153[c16] and Asp343[c194].

Modification of the 170 loop also seems to affect the catalytic efficiency of VIIa. Although the 170 loop size of VIIa-11 is equal to that of VIIa-31 (Fig. 2), catalytic efficiencies are different from each other (Tables I and II). As compared with wild-type VIIa, VIIa-31 had markedly higher catalytic efficiency, whereas VIIa-11 showed reduced activity in the absence of sTF. On the other hand, because VIIa is assumed to exist in equilibrium between minor active and dominant zymogen-like inactive conformational states, the observed enhancement of the activity of VIIa after modification can be explained in two alternative ways. One explanation is that the modification leads to a shift of the unusual equilibrium into the active state, thus enhancing activity. The other explanation is that the modification converts VIIa into a trypsin-like highly efficient enzyme, without shifting the equilibrium. Based on the results shown in Fig. 7A, the latter explanation is more likely, because VIIa-31 continued to exist predominantly in a zymogen-like state after modification. Although both trypsin and VIIa-TF complex are assumed to exist in an active state, trypsin showed 40-fold higher susceptibility to APMSF inhibition as compared with the VIIa-W-sTF complex (Fig. 6B). Inactivation of trypsin by lower concentrations of APMSF may reflect higher affinity interaction between S1 subsite of trypsin and APMSF. Considering that APMSF is a transition state analog of substrate (37) and that high affinity interaction between enzyme and substrate at transition state provides an energy required for enzyme catalysis, the susceptibility to APMSF inhibition probably reflects catalytic efficiency of proteinase. Therefore, the result in Fig. 6B suggests that trypsin has much higher catalytic efficiency than the active state of VIIa. Susceptibility of VIIa-31 to APMSF inhibition was 17-fold higher than that of VIIa-W (Fig. 6A). The difference in the susceptibility between VIIa-31 and free VIIa-W was similar to that between trypsin and VIIa-W-sTF complex. Therefore, it is possible that the catalytic efficiency of VIIa-31 becomes close to that of trypsin if the zymogen-like state of the mutant VIIa is converted to the active state. However, we could not examine this possibility because TF failed to convert the zymogen-like state of VIIa-31 into its active state (Fig. 7B). Because replacement of the 170 loop of VIIa with that of trypsin conferred enhanced activity on VIIa, the loop region in trypsin may be important for its high catalytic efficiency.

Although a relationship between the flexible structure of the 170 loop of VIIa and the unusual zymogenicity of this enzyme has been suggested (16), the results of the present study showed that modification of this loop did not change the zymogenicity of VIIa. It is likely that structural elements related to the zymogenicity of VIIa exist in some regions other than the 170 loop. We are currently exploring the structural elements to clarify the mechanism responsible for TF-mediated acceleration of VIIa activity.

    ACKNOWLEDGEMENTS

We thank Noriko Mimura, Yuki Miyagawa, Mari Kawamoto, and Yasue Sakaguchi for excellent technical assistance in the preparations of recombinant proteins. We also thank Kazuhiko Tomokiyo for providing plasma-derived factor VII and VIIa. We also thank Drs. T. Matsushita and H. Saito (Nagoya University) for providing cDNA of human VII. We also thank to Dr. T. Hara (Daiichi Pharmaceutical Co., Ltd., Tokyo) for providing Synthetic Xa inhibitor (DX-9065a).

    FOOTNOTES

* 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: Blood Products Research Dept., The Chemo-Sero-Therapeutic Research Inst., Kyokushi, Kikuchi, Kumamoto 869-1298, Japan. Tel.: 81-968-37-4052; Fax: 81-968-37-3616; E-mail: mizuguchi@kaketsuken.or.jp.

Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M009206200

    ABBREVIATIONS

The abbreviations used are: VIIa, activated coagulation factor VII; VII, coagulation factor VII; VII-W, wild-type coagulation factor VII; VIIa-W, activated wild-type coagulation factor VII; VIIa-11, activated coagulation factor VII-11; VIIa-31, activated coagulation factor VII-31; X, coagulation factor X; Xa, activated coagulation factor X; TF, tissue factor (full-length; residues 1-263), sTF, soluble tissue factor (extracellular domain of TF; residues 1-218), APMSF, p-amidinophenyl methanesulfonyl fluoride-hydrochloride; PCPS, mixture of phosphatidylcholine and phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor; pNA, p-nitroanilide; PEG, polyethylene glycol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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