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The N-terminal Epidermal Growth Factor-like Domain in Factor IX and Factor X Represents an Important Recognition Motif for Binding to Tissue Factor*

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The N-terminal Epidermal Growth Factor-like Domain in Factor IX

and Factor X Represents an Important Recognition Motif for

Binding to Tissue Factor*

Received for publication, November 23, 2001 Published, JBC Papers in Press, November 26, 2001, DOI 10.1074/jbc.M111202200

Degang Zhong, Madhu S. Bajaj, Amy E. Schmidt, and S. Paul Bajaj‡

From the Department of Internal Medicine and Department of Pharmacological and Physiological Sciences, Saint Louis University Health Sciences Center, St. Louis, Missouri 63110

Factors VII, IX, and X play key roles in blood coagula-tion. Each protein contains an N-terminal -carboxyglu-tamic acid domain, followed by EGF1 and EGF2 do-mains, and the C-terminal serine protease domain. Protein C has similar domain structure and functions as an anticoagulant. During physiologic clotting, the factor VIIa-tissue factor (FVIIaTF) complex activates both fac-tor IX (FIX) and facfac-tor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofac-tor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIaTF com-plex. To investigate the role of the EGF1 domain in this context, we expressed wild type FIX (FIXWT), FIXQ50P,

FIXPCEGF1(EGF1 domain replaced with that of protein

C), FIXEGF1 (EGF1 domain deleted), FXWT, and

FXPCEGF1. Complexes of FVIIa with TF as well as with

soluble TF (sTF) lacking the transmembrane region were prepared, and activations of WT and mutant pro-teins were monitored by SDS-PAGE and by enzyme as-says. FVIIaTF or FVIIasTF activated each mutant sig-nificantly more slowly than the FIXWT or FXWT.

Importantly, in ligand blot assays, FIXWT and FXWT

bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further ex-periments revealed that the affinity of the mutants for sTF was reduced 3–10-fold and that the synthetic EGF1 domain (of FIX) inhibited FIX binding to sTF withKiof

60M. Notably, each FIXa or FXa mutant activated

FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experi-ments, FIXa with or without FVIIIa activated FXWTand

FXPCEGF1normally, which is interpreted to mean that

the EGF1 domain of FX does not play a significant role in its interaction with FVIIIa. Cumulatively, our data reveal that substrates FIX and FX in addition to inter-acting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain.

Human factor IX (FIX)1and factor X (FX) are vitamin

K-de-pendent glycoproteins with Mrof 57,000 and 58,800, respec-tively (1, 2). Factor VIIa-tissue factor (FVIIa䡠TF) complex acti-vates FIX to FIXa and FX to FXa by cleaving Arg145–Ala146and Arg180–Val181peptide bonds in FIX (3) and the Arg194–Ile195 peptide bond in FX (2). The resulting FIXa or FXa molecule consists of an N-terminal light chain and a C-terminal heavy chain linked by a disulfide bond. The light chain in each case contains a␥-carboxyglutamic acid (Gla) domain and two epi-dermal growth factor-like domains (EGF1 and EGF2), whereas the heavy chain contains the serine protease domain. In the blood coagulation cascade, FIXa also activates FX to FXa in a reaction that requires factor VIIIa (FVIIIa), phospholipid (PL), and calcium. FXa formed by either pathway then acti-vates prothrombin to thrombin in a reaction that requires factor Va, PL, and calcium (4). In addition, both FIXa and FXa activate FVII to FVIIa (5–7) and are inhibited by anti-thrombin (AT) (8, 9).

The conversion of single chain zymogen FVII to enzyme FVIIa involves the cleavage of a single peptide bond between Arg152and Ile153. The FVIIa formed consists of a light chain of 152 amino acids and a heavy chain of 254 amino acids held together by a disulfide bond (10). Like FIXa and FXa, the N-terminal light chain of FVIIa contains the Gla domain and two EGF-like domains, whereas the heavy chain contains the serine protease domain (10). TF, the cellular cofactor for FVIIa, is composed of two fibronectin type III␤-sandwich domains (11, 12). Recently, high resolution x-ray structure of the complex of soluble tissue factor (sTF) and FVIIa has been reported (13). In this structure, the Gla and EGF1 domains make contact with the C-terminal domain of TF and the EGF2 and the protease domains make contact with the N-terminal domain of TF (13). Thus, FVIIa uses all of its four domains in binding to the N-and C-terminal domains of TF (13).

Efforts have been directed to understanding the regions in FVIIa䡠TF that interact with the substrates FIX and FX. By studying the effect of mutations in the C-terminal domain of TF, it has been proposed that this domain may interact with the Gla domains of FIX and FX (14). Similarly, by mutagenesis and docking experiments, it has been proposed that the Gla * This work was supported by National Institutes of Health Grant

HL36365 and American Heart Association Grant 9950228N. The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked “

advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this

fact.

A preliminary account of this work has been presented in abstract form (19).

‡ To whom correspondence should be addressed: Division of Hema-tology and Oncology, Saint Louis University Health Sciences Center, 3635 Vista Ave., P. O. Box 15250, St. Louis, MO 63110-0250. Tel.: 314-577-8499; Fax: 314-773-1167; E-mail: Bajajps@slu.edu.

1The abbreviations used are: FIX, FVII, FX, and FVIII, factor FIX,

FVII, FX, and FVIII, respectively; FIXEGF1, FIX in which EGF1

do-main has been deleted; FIXPCEGF1or FXPCEGF1, FIX or FX in which the

EGF1 domain has been replaced with that of protein C; FIXNP, normal

plasma FIX; FXNP, normal plasma FX; TF, membrane-inserted tissue

factor containing residues 1–243; sTF, membrane region deleted soluble tissue factor containing residues 1–219; EGF, epidermal growth factor; AT, antithrombin; PL, phospholipid; mAb, monoclonal antibody;

S-2222, benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide; biotin-EGR-CK,

bio-tinylated Glu-Gly-Arg-chloromethylketone; BSA, bovine serum albu-min; RVV, Russell’s viper venom; PEG, polyethylene glycol 8000; Gla, ␥-carboxyglutamic acid; HPLC, high pressure liquid chromatography;

WT, wild type; Fmoc,N-(9-fluorenyl)methoxycarbonyl.

© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org

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domain of FVIIa interacts with the Gla domain of FX (15). Further, we reported earlier that the EGF1 domain of FIX is required for its activation by the FVIIa䡠TF complex (16). How-ever, the role of the EGF1 domain of FX in this context has not been investigated. Moreover, it is not known whether FVIIa or TF in the FVIIa䡠TF complex interacts with the EGF1 domains of FIX and FX. Thus, the precise function of EGF1 domain of FIX or FX in its interaction with the FVIIaTF complex is not known.

Protein C is a serine protease with an anticoagulant function whose domain organization is similar to that of FVIIa, FIXa, or FXa (17, 18). Further, activated protein C is not involved in the TF-induced coagulation, and its EGF1 domain near the N ter-minus has an eight-residue insertion (18). Therefore, substitut-ing the EGF1 domain of FIX (or FX) with the EGF1 domain of protein C should replace the unique determinants present in the EGF1 domain of FIX (or FX) that provides specificity for its interaction with the FVIIa䡠TF complex. In this report, in addi-tion to the above two replacement mutants (FIXPCEGF1 and FXPCEGF1), we used a point mutant (FIXQ50P) and an EGF1 deletion mutant (FIXEGF1) of FIX to understand the function of this domain in TF-induced coagulation. Data are provided, which strongly indicate that TF interacts with the EGF1 do-main in FIX and FX. Our findings represent the first report that assigns a specific function to the EGF1 domain in these proteins.

EXPERIMENTAL PROCEDURES

Reagents—Carrier-free Na125I was obtained from ICN Biomedicals,

Inc. Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222) was obtained from

Diapharma Inc. Biotinylated Glu-Gly-Arg-chloromethylketone (biotin-EGR-CK) was purchased from Hematologic Technologies, Inc.

Nitrocel-lulose membrane, polyethylene glycol 8000 (PEG),p-nitrophenyl

phos-phate, bovine serum albumin (BSA), bovine brain phosphatidylcholine, and phosphatidylserine were purchased from Sigma. Horseradish per-oxidase-goat anti-mouse IgG and enhanced chemiluminescence (ECL) detection reagents were purchased from Amersham Biosciences. FVII-depleted plasma and Neoplastin were obtained from Amersham

Bio-sciences. Normal plasma FIX (FIXNP), plasma FX (FXNP), FXIa,

Rus-sell’s viper venom (RVV), and AT were obtained from Enzyme Research Laboratory. Low molecular weight heparin was purchased from Rhoˆne-Poulenc Rorer Pharmaceuticals Inc. A monoclonal antibody-purified human FVIII concentrate was obtained from Dr. Leon Hoyer (American

Red Cross, Rockville, MD). It was activated with 1 nMthrombin in the

presence of 0.1% BSA and 5 mMCaCl2in Tris/NaCl at 37 °C for 2 min

as described earlier (20). The formed FVIIIa was diluted and used

immediately in the activation of FX by FIXa䡠FVIIIa䡠PL.

For ligand blot experiments, a Ca2⫹-dependent FIX monoclonal

an-tibody (mAb) cell line was provided by Dr. Shirly Miekka of the

Amer-ican Red Cross, and the IgG was purified as described (21). A Ca2⫹

-de-pendent mAb to the heavy chain of FX used for the ligand blot experiments was purchased from American Diagnostics, Inc. PL vesi-cles (75% phosphatidylcholine, 25% phosphatidylserine) were prepared

by the method of Hustenet al. (22). TF containing the transmembrane

region (residues 1–243) was a gift from Genetech, Inc. The relipidation of the TF was performed as described (16). sTF that lacks the trans-membrane region (residues 1–219) was a gift from Tom Gerard of Pharmacia Corp., St. Louis, MO.

For studies of AT binding and FVII activation, FIXa and FXa were

prepared by activating FIX with FXIa (16) and activating FX with RVV

(23) in 50 mMTris, 0.15MNaCl (Tris/NaCl), pH 7.4, containing 5 mM

CaCl2and 0.1% PEG at 37 °C for 2 h. Complete activation of FIX or FX

was confirmed by SDS-PAGE (24).

SDS-Gel Electrophoresis—SDS-gel electrophoresis was performed using the Laemmli buffer system (24). The acrylamide concentration was 12%, and the gels were stained with Coomassie Brilliant Blue dye.

All proteins used in the present study were⬃98% pure.

Amino Acid Sequencing and Gla Analysis—Gla and amino acid se-quence analysis were performed by Commonwealth Biotechnologies,

Inc. (Richmond, VA). Automated degradation of each protein (⬃0.5

nmol) was performed using an Applied Biosystems gas phase se-quencer. Gla analysis of each protein was performed by alkaline hy-drolysis followed by HPLC analysis. The amount of Gla was quantitated based upon Asp and Asn present per mol of each protein.

Expression and Purification of Recombinant Factors IX and VII—

Recombinant FIXWT, FIX⌬EGF1, FIXPCEGF1, and FIXQ50Pwere expressed

in human embryonic kidney 293 cells and purified by using the IX A-7

mAb column as described (16, 25). Each FIX protein had ⬃12 Gla

residues/mol (16). To express FVIIWT, the restriction sitesAflII and

XhoI were introduced at the 5⬘- and 3⬘-ends of VII cDNA for ligation into

the pMon3360b expression vector (26) that was modified to contain

AflII andXhoI sites. A stable cell line that expressed FVIIWTwas

established as described in detail by Hippenmeyer and Highkin (26). Medium was collected in the presence of vitamin K as outlined earlier

for FIX (16, 25). FVIIWTwas purified by using a Ca

2⫹-dependent mAb

as described (27). It contained 9 –10 Gla residues/mol and had ANAFL as the N-terminal sequence. FVIIa was obtained as earlier, except insoluble FXa (Sepharose-FXa) was used instead of the soluble FXa as the activator (6). The resin was removed by centrifugation, and the supernatant was passed over a small Chelex-100 column to remove

Ca2⫹. Aliquots were kept frozen at80 °C until used.

Expression and Purification of Recombinant FX—An expression

vec-tor for FXWTwas constructed in which the prepro-leader sequence of

FXWTwas replaced with that of prothrombin as described by Camireet

al. (28). The prepro-leader sequence of prothrombin was amplified by

PCR using primers A and B (Table I) and a human liver cDNA library. The prepro-leader sequence of prothrombin was then linked to the FX cDNA sequence by the overlap extension method using primers A and C (25). The resulting chimeric DNA, containing the prepro-leader se-quence of prothrombin followed by the FX sese-quence, was digested with

AflII andXhoI and ligated into pMon3360b expression vector. A stable

cell line that expressed FXWTwas established as described (26).

Me-dium was collected in the presence of vitamin K as outlined earlier for

FIX (16, 25). FXWTwas purified using a Ca

2⫹-dependent mAb to the Gla

domain of FX (25) followed by FPLC Mono Q column. The conditions for the FPLC Mono Q column were the same as described previously for

FIX purification (16, 25). Construction of FXPCEGF1was performed by

the overlap extension method as described (25), and primers D and E (Table I) were used to amplify the protein C EGF1 domain. The

estab-lishment of a stable cell line and the purification of FXPCEGF1were the

same as for FXWT.

Measurements of Rates of Activation of FIX by FVIIa—For activation

of FIX by FVIIa䡠TF䡠PL, 2␮MFIX was activated with 8 nMVIIa and 0.5

nMTF in the presence of 1 mMPL, 5 mMCaCl2, 0.1% PEG in Tris/NaCl

buffer. At different times, 20␮l of the reaction mixture was removed

and diluted 10-fold with 20 mMEDTA, pH 7.4. Biotin-EGR-CK was

added to the diluted mixture to a final concentration of 20␮M, and the

sample was incubated at 37 °C for 2 h and then at 4 °C overnight. To measure the amount of biotin-EGR-IXa, a 96-well microtiter plate was

coated with 100␮l (10␮g/ml in 0.1Mof NaHCO3) of the Ca

2⫹-dependent

FIX mAb at 4 °C overnight. The wells were blocked with 200␮l of 1%

BSA and 0.1% Tween 20 in Tris/NaCl for 2 h at 37 °C. At this point,

TABLE I

Sequence of synthetic oligonucleotide primers for construction of FXWTand FXPCEGF1

Primer Sequencea A 5⬘-CTAGACTTAAGCTTCCACC(ATGGCCACGTCCGAGGCTTG) B 5⬘-CTTCATCTCTTCAAGAAAGGAATTGGC(TCGCCGGACCCGCTGGAG) C 5⬘-TCTGACTCGAG(TCACTTTAATGGAGAGGA) D 5⬘-AAAGATGGCGACCAGTGT(TTGGTCTTGCCGTTGGAG) E 5⬘-GCTGCAGAGCTTCCGTGT(CTCCCGCTGGCAGAAGCG)

aPrimer A contains aAflII site, and primer C contains anXhoI site. The restriction site sequences are underlined. The sequence in parenthesis

for primers A and B correspond to the prepro-leader sequence of human prothrombin. The sequences in parenthesis is primer C correspond to the

C-terminal six amino acid residues of FX as well as the stop codon. Primers D and E were used to construct FXPCEGF1. These are hybrid primers

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each biotin-EGR-IXa sample was further diluted 50-fold in Tris/NaCl

containing 1% BSA and 0.1% Tween 20. 100␮l of the diluted sample

was added to each well, and the plate was incubated at 37 °C for 2 h for capture of the biotin-EGR-IXa by the FIX mAb. The plate was washed

three times with Tris/NaCl containing 0.1% Tween 20 and 5 mMCaCl2.

Each well then received 100␮l of alkaline phosphatase-streptavidin in

Tris/NaCl containing 1% BSA, 0.1% Tween 20, and 5 mMCaCl2. The

plate was incubated at 37 °C for 1 h. After three washings, each well

received 100␮l of substratep-nitrophenyl phosphate (4 mg/ml) in the

alkaline phosphatase buffer (100 mMNaCl, 5 mMMgCl2, 100 mMTris,

pH 9.5). The amount ofp-nitrophenol generated was measured in a

microtiter plate reader at 405 nm (Bio-Rad). FIXa concentration was then calculated from a standard curve generated starting with known amounts of preformed FIXa and formation of the biotin-EGR-IXa using the above protocol.

For activation of FIX with FVIIa䡠sTF, 4␮MFIX was activated with

0.16␮MFVIIa䡠sTF in the absence of PL. All other assay conditions were

the same as those for the activation of FIX with FVIIa䡠TF䡠PL outlined

above. For SDS-PAGE, 12␮l of the reaction mixture was removed at

different times and added to 2 ␮l of 0.5MEDTA and 5␮l of 5-fold

concentrated SDS-reducing buffer. Samples were placed in boiling wa-ter for 5 min and analyzed by SDS-PAGE (24).

Measurements of Rates of Activation of FX by FVIIa—For activation

of FX by FVIIa䡠TF䡠PL, 2␮MFX was activated with 8 nMFVIIa and 0.5

nMTF in the presence of 1 mMPL, 5 mMCaCl2, 0.1% PEG in Tris/NaCl.

These conditions are the same as used for the activation of FIX. At

different times, 20␮l of the reaction mixture was removed and diluted

10-fold with 20 mMEDTA in Tris/NaCl, pH 7.4 to stop the reaction. The

reaction mixture was further diluted as needed, and the concentration

of FXa was determined by the hydrolysis of 250␮MS-2222. The amount

of FXa generated was calculated from a standard curve constructed using known amounts of fully activated FXa.

For activation of FX with FVIIa䡠sTF, 4␮MFX was activated with 0.16

␮MFVIIa䡠sTF in the absence of PL. All other assay conditions were the

same as above for the activation of FX with FVIIa䡠TF䡠PL. For

SDS-PAGE, 12␮l of the reaction mixture was removed at different times and

added to 2␮l of 0.5MEDTA and 5␮l of 5-fold SDS-reducing buffer for

analysis by SDS-PAGE.

Measurements of Rates of Activation of FIX and FX by VIIaPL in the Absence of TF—For activation of FXWTand FXPCEGF1by FVIIa䡠PL, 50

nMFVIIa was incubated at 37 °C with 1␮MFXWTor FXPCEGF1in the

presence of 35␮MPL and 5 mMCaCl2in Tris/NaCl containing 0.1%

BSA. 10-␮l aliquots were removed at 0, 5, 10, and 15 min and added to

40␮l of 20 mMEDTA in Tris/NaCl. The reaction mixture was further diluted as needed, and the concentration of FXa was determined by the

hydrolysis of 250␮MS-2222.

Measurements of Rates of Activation of FX by FIXa with or without FVIIIa—These experiments were performed exactly as described in detail earlier (30). The concentration of each reagent used is given in the legend to Fig. 6.

Measurements of Rates of FVII Activation by FIXa or FXa—A 50 nM

concentration of each FIXa or FXa was incubated at 37 °C with 1␮M

FVII in the presence of 35␮MPL and 5 mMCaCl2in Tris/NaCl. 2-␮l

aliquots were removed at different times and added to 100␮l of 0.1%

BSA in Tris/NaCl containing 10 mMEDTA. The aliquots were further

diluted in 0.1% BSA in Tris/NaCl without EDTA and analyzed for FVII/FVIIa clotting activity in a one-stage assay (31). For this assay, 50 ␮l of FVII-depleted plasma was incubated with 50␮l of Neoplastin for

3 min at 37 °C. Then 25␮l of test sample and 50␮l of prewarmed

(37 °C) 35 mM CaCl2were added and the clotting time was noted.

Citrated pooled normal human plasma was used as a standard (1 unit/ml FVII). For SDS-PAGE analysis, samples were removed after a 2-h incubation period.

Binding of FIXa and FXa to AT—For these experiments, the final

reaction mixtures contained the following: 2␮MFIXa or FXa, 2␮MAT,

5 mMCaCl2, and 10 units/ml low molecular weight heparin in Tris/

NaCl, pH 7.4. The total reaction mixture in each case was 150␮l, and

20-␮l aliquots were removed at 0.15, 0.5, 1.5, 4, 12, and 30 min and

added to 5␮l of 5-fold concentrated SDS-reducing buffer and analyzed

by SDS-PAGE. The protein bands were visualized by Coomassie Blue staining and quantitated by densitometry. The rate of complex forma-tion of heavy chain of FIXa or FXa with AT was then calculated from the decrease in the intensity of band corresponding to the heavy chain of each enzyme and increase in intensity of the AT-heavy chain complex. Ligand Blotting for Binding of sTF or FVIIa to FIX and FX—First sTF and FVIIa were electrophoresed on SDS-PAGE. The proteins were

then transferred to a 0.2-␮m nitrocellulose membrane. The protocol

used was that outlined by Sambrook et al. (32), using the Bio-Rad

Mini-transfer apparatus. The membrane was blocked with 5% fat-free milk, 0.05% Tween 20 in Tris/NaCl at room temperature for 1 h. Each membrane was then incubated with various FIX or FX proteins at 5

␮g/ml in Tris/NaCl, 1% milk, 0.05% Tween 20, and 5 mMCaCl2at 4 °C

overnight. After three washes with 0.05% Tween 20 in Tris/NaCl and 5

mMCaCl2, the membrane was incubated with FIX mAb or FX mAb (1

␮g/ml) at room temperature for 2 h. A second antibody (horseradish peroxidase-goat anti-mouse IgG) and the ECL Western blotting detec-tion kit were used to detect the primary mAb.

Synthesis and Folding of the EGF1 Domain of FIX—The EGF1

domain of FIX corresponding to amino acid residues 45– 87 (NH2

-

YVDGQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVT-CONH2) was synthesized on an ABI model 433A peptide synthesizer by

Biomolecules Midwest, Inc. (Waterloo, IL). The C-terminal amino acid was coupled to Fmoc-Rink Amide MBHA resin using standard ABI

protocols. Amino acid activation was performed using HBTU. The␣

-amino group of the -amino acid was Fmoc-protected, and the side chain

groups were protected byt-butyl (Tyr and Ser), t-butyl ester (Asp and

Glu),t-butoxycarbonyl (Lys),

2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Arg), and trityl (His, Cys, Gln, and Asn). Fmoc deprotection was performed using 20% piperidine in 1-methyl-2-pyrrolidinone, and the peptide was simultaneously deprotected and cleaved from the resin using trifluoroacetic acid/phenol/anisole/water/methyl sulfide (85/5/4/ 4/2; v/v/v/v/v) for 2 h. The crude peptide was purified by reverse phase HPLC on a Vydac C-18 column using standard trifluoroacetic acid/ acetonitrile conditions (33). The purified reduced peptide gave a molec-ular mass of 4755 Da (expected mass 4755.2 Da) as determined by mass spectrometry analysis using a Finnigan LCQ Iontrap Electrospray mass spectrometer.

The synthetic FIX-EGF1 domain peptide was folded using an oxido-shuffling system (34, 35). The lyophilized peptide was dissolved at a

concentration of⬃0.4 mg/ml in a solution containing 0.1MTris-HCl, pH

8.3, 50 mMCaCl2, 3 mM L-cysteine, and 0.3 mM L-cystine. The mixture

was allowed to sit for 40 h at room temperature, at which time 10% trifluoroacetic acid was added to adjust the pH to 2.5. The refolded peptide was then purified by reverse phase HPLC and lyophilized, and its concentration was determined using the molar extinction coefficient

of 2748 at 294.4 nm for a single tyrosine or tryptophan residue in 0.1M

NaOH (36). Two tyrosines and one tryptophan contained in our peptide were taken into account in calculating its concentration.

Ca2Binding to the Folded IX-EGF1 Domain—Calcium ion activity

was determined by using a Ca2⫹-specific electrode and a model 601A

digital Ionlyzer (Orion Research). Titrations of the folded synthetic

EGF1 domain at 400␮Min 4 ml of Tris/NaCl, pH 7.4, were performed

by adding 1-␮l increments of 400 mMCaCl2at room temperature. In

these titrations, bound Ca2⫹was taken as the difference between the

measured free Ca2⫹concentration and the total added.

Iodination of FIXWTand FXWT—FIX and FX were labeled with

125I using IODO-GEN-precoated iodination tubes obtained from Pierce. The standard Tris buffer was used, and the procedure followed was that outlined by the manufacturer. The iodinated proteins were precipitated by the slow addition of solid ammonium sulfate to 80% saturation. Each suspension was centrifuged at 10,000 rpm in Eppendorf tubes for 10 min at 4 °C. The pellets were dissolved in Tris/NaCl, pH 7.4, and dialyzed against the same buffer until the cpm in the dialysate were

close to background. The radiospecific activity of125I-FIX was 8.5106

cpm/␮g, and that of125I-FX was 3.6106cpm/g. SDS-PAGE

radio-activity profiles of the proteins revealed a single molecular species characteristic of each protein (37). The radiolabeled proteins retained

⬃90% clotting activity as compared with the nonlabeled controls.

Determination of the Apparent Kdof Binding of sTF to FIX and FX

Proteins and to FIX-EGF1 Domain—ApparentKdvalues of

125I-FIX and

125I-FX binding to sTF were determined by direct binding assays using

Costar enzyme immunoassay/radioimmunoassay, high binding, type I

strip plates. Each well was coated with 100␮l of either 10␮g/ml sTF or

BSA in 0.1 M NaHCO3, pH 8.5, overnight at 4 °C. The wells were

washed three times with Tris/NaCl, pH 7.4, containing 0.01% Tween 20. The wells were then blocked with 3% BSA in Tris/NaCl, pH 7.4, containing 0.05% Tween 20 for 4 h at room temperature. The wells were

washed with Tris/NaCl, pH 7.4, containing 1 mMbenzamidine, 1 mg/ml

BSA, and 5 mMCaCl2(binding buffer). Each well (sTF or BSA control)

then received 100␮l of125I-FIX (3.7–366 n

M) or of125I-FX (25– 800 n

M) in binding buffer. The wells were then allowed to sit at room tempera-ture for 4 h, after which time they were washed three times with the

binding buffer. The wells were separated and counted for125I

radioac-tivity in a Packard Cobra II ␥-counter. Each BSA control cpm was

subtracted from the corresponding experimental cpm to obtain the specific cpm due to binding of FIX or FX to sTF. The nonspecific cpm

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ranged from 22 to 33% of the experimental cpm. The specific cpm data

were plotted against 125I-FIX or 125I-FX to obtain approximate K

d

values for the binding of FIX or FX to sTF. The data were fitted to a hyperbolic curve using the program GraFit from Erithacus Software.

In further experiments, the affinity of each protein (WT, mutants, FIX-EGF1 domain) was determined by equilibrium competition assays.

In these experiments, a fixed final concentration of125I-FIX (10 n

M) or

125I-FX (30 n

M) was used with varying concentrations of the competitor.

A 100-␮l aliquot of each mixture was added to the sTF or BSA wells to generate the specific binding data as outlined above. The data were analyzed using the nonlinear regression analysis program (GraFit) to

obtain the IC50values (concentration of the competitor yielding 50%

inhibition) using the IC50four-parameter logistic equation of Halfman

(38). To obtain the Kd(app) values for the interaction of competitor

proteins with sTF, the following equation described by Craig was used (39).

Kd共app兲⫽ IC50

1⫹共A/Kd共WT兲兲 (Eq. 1)

whereAis the concentration of125I-FIX or125I-FX, andK

d(WT)

repre-sents the dissociation constant of WT protein for sTF.

RESULTS

Expression of Recombinant FVII and FX—Purified recombi-nant FVII had normal Gla content and the expected N-terminal sequence corresponding to zymogen FVII. It had 2000⫾200 units/mg as measured in a one-stage clotting assay (31). SDS-PAGE analysis is shown in subsequent figures.

To obtain fully␥-carboxylated FX, we replaced the prepro-leader sequence of FX with that of prothrombin as described by Camireet al. (28). FXWTand FXPCEGF1were expressed in BHK cells and purified using a Ca2⫹-dependent mAb whose epitope

is in the Gla domain of FX (29). The purified proteins had normal Gla content (11 ⫾ 1) and revealed two N-terminal sequences in equimolar amounts: one corresponding to the heavy chain (SVAQA) and a second corresponding to the light chain (ANSFL). This indicates that the prepro-leader se-quences of FXWTand FXPCEGF1were completely removed. The specific activity was 92 ⫾ 10 units/mg for FXWT, which is similar to that of plasma FX (8510 units/mg), and it was 12⫾2 units/mg for FXPCEGF1in the two-stage clotting assay (31). SDS-PAGE analysis is presented in subsequent figures.

Activation of FIXWT, FIXQ50P, FIXPCEGF1, and FIXEGF1by FVIIaTFPL—The activation rates of FIX mutants by FVIIa䡠TF䡠PL obtained by measuring the amount of biotin-EGR-IXa formed are presented in Fig. 1A. Examination of the data in Fig. 1Areveals that the initial rates of activation of FIX-PCEGF1and FIXQ50Pare⬃7 and⬃30% of FIXWT, respectively. Under these conditions, FIXEGF1 was activated only mini-mally, and FIXWTwas activated at a rate similar to that ob-tained with FIXNP(data not shown).

Activation of FXWT and FXPCEGF1 with FVIIaTFPL—The rates of activation of FXWTand FXPCEGF1by FVIIa䡠TF䡠PL are presented in Fig. 1B. The initial rate of activation of FXPCEGF1 was⬃5% of FXWT. It should be noted that the rate of activation of FXWTwas similar to the rate obtained with FXNP(data not shown).

Activation of FIX and FX Proteins by FVIIasTF Complex— Studies presented above reveal that the activation rates of FIX and FX EGF1 domain mutants with FVIIaTFPL are impaired. Gla domains of FIX and FX bind to the PL vesicles, and the membrane surface provides a platform for their assembly for maximal activation. Thus, it is possible that the mutations in the EGF1 domain alter the distance between the cleavage site of FIX or FX and the PL surface, which results in the misalign-ment of the substrate and the enzyme FVIIa. This possibility was tested by using sTF (transmembrane region deleted TF) under reaction conditions that do not require assembly of FVIIa䡠TF and substrates on the PL surface.

The activation of each FIX mutant by FVIIa䡠sTF in this

system where PL is absent is presented in Fig. 2A. Similar to the PL-containing system (Fig. 1A), activations of FIXPCEGF1 and FIXQ50Pwere impaired. However, in contrast to the PL-containing system, the rate of activation of FIXEGF1 in the absence of PL was similar to that of FIXPCEGF1. The initial activation rate was ⬃33% for FIXQ50P,⬃12% for FIXPCEGF1, and⬃9% for FIXEGF1when compared with the rate obtained with FIXWT (Fig. 2A). Again, it should be noted that FIXWT activated at a rate similar to FIXNP in this system (data not shown). Activation rates of FXWT and FXPCEGF1 with FVIIa䡠sTF were also determined. As is the case with the FIXPCEGF1 mutant, the FXPCEGF1 with FVIIa䡠sTF was acti-vated at an initial rate that is10% of FXWT(Fig. 2B).

SDS-PAGE Analysis of FIX and FX Activation Products— Next, we performed SDS-PAGE of various FIX and FX proteins activated by FVIIa䡠sTF. The data obtained for FIXWTare pre-sented in Fig. 3A; data for FIXQ50P are in Fig. 3B; data for FIXPCEGF1are in Fig. 3C; data for FIX⌬EGF1are in Fig. 3D; data for FXWTare in Fig. 3E; and data for FXPCEGF1are in Fig. 3F. As is the case with the enzyme assays, the rate of activation in decreasing order for each FIX protein was FIXNP (not shown)⬇FIXWT⬎FIXQ50P⬎FIXPCEGF1⬇FIX⌬EGF1. Further, the rate of activation of FXPCEGF1as analyzed by SDS-PAGE was also considerable slower than the FXWT.

Rates of Activation of FX Proteins by FVIIaPL in the Absence of TF—The activation of FIX or FX mutants by FVIIa䡠TF or FVIIa䡠sTF with or without PL is impaired (Figs. 1–3). Next we measured the activation of FX mutants by FVIIa䡠PL without TF (or sTF) as outlined earlier (57, 58). These results are depicted in Fig. 4. The activation rate was 0.19⫾0.02 nM/min

for FXWT and 0.18 ⫾ 0.02 nM/min for FXPCEGF1. Thus, the interaction between FXPCEGF1and FVIIa䡠PL is normal in the absence of TF (or sTF). We were unable to measure the rates of activation of FIX under these conditions.

Rate of FVII Activation by FIXa and FXa—FIXa and FXa are the enzymes that convert FVII to FVIIa (5–7). This activation requires only Ca2⫹and PL without the need for protein

cofac-tors. Thus, this analysis should provide important data regard-ing whether or not FIXa and FXa mutants can function as FIG. 1.Activation of FIX and FX proteins with FVIIaTFPL.A

2␮Mconcentration of each protein was mixed with 8 nMFVIIa and 0.5

nMTF in Tris/NaCl containing 0.1% PEG, 1 mMPL, and 5 mMCaCl2.

The biotin-EGR-FIXa or FXa formed at each time point was measured

as described under “Experimental Procedures.” A, activation of FIX.

The proteins used were FIXWT(open circles), FIXQ50P(closed circles),

FIXPCEGF1(open squares), and FIX⌬EGF1(closed squares).B, activation

of FX. The proteins used were FXWT(open circles) and FXPCEGF1(closed

(5)

enzymes in activating FVII to FVIIa. Such experiments are presented in Fig. 5. An examination of the results reveals that the activation rate of FVII by each FIXa or FXa protein is similar. The activation of FVII was also confirmed by SDS-PAGE (Fig. 5, inset). These data provide evidence that the mutant proteins are folded correctly and that the interaction of mutant enzymes (FIXa mutants and FXaPCEGF1) with their substrate FVII is normal. It also indicates that the EGF1 domain in FIXa or FXa does not play an important role in the activation of FVII.

Binding of FIXa and FXa Proteins to AT—AT binds to the heavy chain of FIXa or FXa. It forms a covalent bond with the active site serine of FIXa as well as that of FXa (8, 9). Whether or not the mutations in the EGF1 domain in FIXa or FXa influence AT binding was examined by studying the tight com-plex formation of FIXa or FXa with AT. The rate of tight complex formation between AT and FIXa or FXa mutants ap-peared to be similar to that of FIXaWTand FXaWTas analyzed by SDS-PAGE. In each case, the FIXaAT or FXaAT tight complex formed at 0.15 min was⬃20%, at 0.5 min it was⬃50%, at 1.5 min it was ⬃80%, and at 4 min it was essentially complete. These initial data indicate that the EGF1 domains of FIXa and FXa are most likely not involved in AT binding and imply that the protease domains of the mutant FIXa and FXa are correctly folded to bind to their natural inhibitor. These data are consistent with the previous observation that the protease domain in these enzymes plays an important role in binding to AT (8, 9).

Activation of FXWT and FXPCEGF1 by FIXaPL and FIXaPLFVIIIa—In the coagulation cascade, FX can be acti-vated either by the extrinsic pathway or by the intrinsic path-way. Studies presented above reveal that the activation of FXPCEGF1by the extrinsic pathway is impaired. Here, we in-vestigated whether or not the activation of FXPCEGF1 by the intrinsic pathway is normal. When the activation was carried out in absence of FVIIIa, the initial rate of activation for FXWT was 0.096 nM/min, and for FXPCEGF1 it was 0.073 nM/min.

These data are presented in Fig. 6A. When the activation was carried out in the presence of FVIIIa, the rates of activation for FXWTand FXPCEGF1were the same (2.2 nM/min). These data are presented in Fig. 6B. It should be noted that FXNP was activated by FIXa䡠PL with or without FVIIIa at the same rate as obtained with FXWT. These results indicate that the EGF1 domain of FX plays virtually no role or a very minor role in the activation of FX by the intrinsic pathway.

Ligand Blotting of FIX or FX Proteins to sTF or FVIIa on Nitrocellulose Membrane—FIXa and FXa mutants function normally as enzymes in activating FVII and in binding to the serpin inhibitor AT. Thus, our results indicate that the defect in FIX and FX mutants is in their binding either to FVIIa or to TF (or both) in the FVIIaTF complex. To distinguish between these possibilities, we performed SDS-PAGE and transferred the FVIIa and sTF to the nitrocellulose membrane. Each FIX mutant was then used as a ligand to probe FVIIa or sTF on the membrane. When we probed the membrane with FIXWT, it bound to FVIIa as well as to sTF (Fig. 7A). In these experi-ments, FIXQ50P, FIXPCEGF1, and FIXEGF1also bound to FVIIa but not to sTF (Fig. 7, B–D). These results indicate that the EGF1 domain of FIX is important for its binding to TF. We also performed similar ligand blotting experiments using FX as a ligand. Both FXWTand FXPCEGF1bound to FVIIa, whereas only FXWTbound to the sTF (Fig. 7,FandG). These results indicate that the EGF1 domain of FX, like that of FIX, is important in its binding to TF.

Affinity of sTF for Various Proteins—The Western ligand blot assays described above provide qualitative data for binding of sTF to various FIX and FX mutants. To obtain quantitative information, we studied binding of 125I-FIX and 125I-FX to immobilized sTF. A direct binding plot for FIX is presented in Fig. 8Aand theKd(app) obtained from this plot for binding of 125I-FIX to sTF was 13020 n

M. The affinity of WT and of each

mutant FIX was then obtained by its ability to compete with 125I-FIX in binding to sTF. These data are presented in Fig. 8B. Analysis of these data reveal that sTF interacts with FIXWT withKd(app) of⬃150 nM, FIXQ50PwithKd(app) of⬃500 nM, FIXPCEGF1 with Kd(app) of ⬃1500 nM, and FIX⌬EGF1 with Kd(app) of⬃1600 nM. Thus, as compared with FIXWT, the point mutant (FIXQ50P) has ⬃3-fold reduced affinity, whereas the replacement (FIXPCEGF1) or the deletion (FIX⌬EGF1) mutant has⬃10-fold reduced affinity for binding to sTF.

A direct binding plot for binding of 125I-FX to sTF is pre-sented in Fig. 9Aand theKd(app) obtained from this plot for binding of FX to sTF was 500⫾100 nM. The affinity of WT and PCEGF1 mutant was then determined by competition with 125I-FX for binding to sTF. These data are shown in Fig. 9B. The Kd(app) for WT was 900 nM, and the Kd(app) for

FXPCEGF1was6Min binding to sTF. Thus, compared with

WT, the FXPCEGF1has significantly reduced affinity for sTF. Studies with FIX-EGF1 Synthetic Domain—Data presented thus far indicate that mutations in the EGF1 domain of FIX and FX impair their abilities to be activated by the FVIIasTF complex, and this property may be related to diminished bind-ing of the mutants to sTF in the FVIIasTF. To examine whether isolated EGF1 domain binds to sTF, we synthesized the 45– 87-residue segment of FIX representing its EGF1 do-main. The fully reduced peptide had the correct mass (4755 Da) indicating no error in synthetic steps. The peptide was then oxidized and purified as outlined under “Experimental Proce-dures.” The mass spectrometric analysis of the oxidized puri-fied peptide is shown in Fig. 10. A peak corresponding to the correct mass of 4749 Da represented⬎90% of the total molec-ular species. This analysis also indicated that six cysteines had been oxidized to yield three disulfide pairs. Since only properly FIG. 2.Activation of FIX and FX proteins with FVIIasTF.Four

␮Mof each protein was mixed with 160 nMFVIIa䡠sTF in Tris/NaCl

containing 0.1% PEG and 5 mMCaCl2. The biotin-EGR-IXa or FXa

formed at each time point was measured as described under

“Experi-mental Procedures.”A, activation of FIX. The proteins used were FIXWT

(open circles), FIXQ50P(closed circles), FIXPCEGF1(open squares), and FIXEGF1(closed squares).B, activation of FX. The proteins used were FXWT(open circles) and FXPCEGF1(closed circles).

(6)

disulfide-paired and correctly folded EGF1 domain binds Ca2⫹

(35), we studied the binding of Ca2⫹ to the folded peptide.

These data are presented in Fig. 10 (inset) using a Ca2⫹-specific

electrode. This peptide contained a single Ca2⫹ binding site

(0.95/mol) with a Kd of ⬃0.6 mM, which is consistent with correct folding of the domain. Importantly, the folded peptide inhibited the binding of FIX to sTF with aKiof60M(Fig.

8B). Moreover, reduced and carboxymethylated peptide pre-pared essentially by the method of Sodetz and Castellino (40) neither bound Ca2⫹(data not shown) nor inhibited FIX binding

to sTF (Fig. 8B).

DISCUSSION

The FVIIa䡠TF complex has high specificity and affinity for its substrates FIX and FX, which quite possibly involve regions that are remote from the cleavage sites. The peptides that do not bind to the active site of FVIIa specifically inhibit the activation of FX by FVIIa䡠TF as well as the TF-initiated clot-ting (41). Further, when the active site of FVIIa is blocked with peptidyl substrates, the resulting FVIIa䡠TF complex has nearly the same affinity toward its substrate FX as the uninhibited FVIIa䡠TF (42). Thus, exosites exist in FX that are responsible for this specific recognition of FX by the FVIIaTF complex. Moreover, a FVIIa molecule with a point mutation in the Gla domain (Arg363Ala) activates FX at a reduced rate in the presence but not in the absence of TF (15). These results indicate that TF facilitates an optimal interaction between the Gla domains of FVIIa and FX (15). Further, TF residues Lys165 and Lys166 that are exposed in the crystal structure of the FVIIa䡠TF complex (13) are thought to interact with the Gla domain of FX or FIX (43, 44). These TF mutants were deficient in supporting FVIIa activation of normal FX (or FIX) but not of the Gla domainless FX (43). In support of this, when Kirchhofer

et al. (14) made a panel of additional TF mutants involving surface-exposed residues 157–185, they found that the mutant TF molecules had reduced affinity for FIX and FX. Based upon FIG. 3.Analysis of activation of FIX and FX proteins with FVIIasTF complex by SDS-PAGE.Proteins were analyzed by reducing

SDS-PAGE using 12% acrylamide gels (for details, see “Experimental Procedures”).A–D, analysis of FIX activation.H, the heavy chain of FIX;

H, the heavy chain of FIXa;L, the light chain of FIXor FIXa.EandF, analysis of FX activation.FX-H, the heavy chain of FX;H, the heavy

chain of FXa␤;L, the light chain of FX␤or FXa␤. The nomenclature used for FIX is that of Di Scipioet al. (8); that used for FX is from Fujikawa

et al. (23). The minor band migrating slightly slower than theH␤in each set may represent the heavy chain of FVIIa.

FIG. 4.Activation of FX proteins with FVIIaPL without TF.A

1␮Mconcentration of each protein was mixed with 50 nMFVIIa䡠PL in

Tris/NaCl containing 0.1% BSA and 5 mMCaCl2. The FXa formed at

each time point was measured as described under “Experimental

Pro-cedures.” The proteins used were FXWT(open circles) and FXPCEGF1

(closed circles).

FIG. 5.Activation of FVII by FIXa or FXa.A 50 nMconcentration

of each FIXa or FXa protein was incubated at 37 °C with 1 ␮M(50

␮g/ml) FVII in the presence of 35␮MPL vesicles and 5 mMCaCl2in

Tris/NaCl containing 0.1% PEG. Aliquots were removed at different times, diluted, and assayed for clotting activity as described under

“Experimental Procedures.”A, FVII activation by FIXa. The proteins

used were FIXaWT(open circles), FIXaQ50P(closed squares), FIXaPCEGF1

(open squares), and FIXaEGF1(closed circles).Inset, Analysis of FVII activation by SDS-PAGE. The incubation time was 2 h, and the

activa-tors are identified at thetopof eachlane. Lane 1, standard protein

markers;lane 2, FVII starting material;lane 3, FVII incubated with

buffer alone;lane 4, FVII incubated with FXIa (0.6␮g/ml);lane 5, FVII

incubated with FIXaWT;lane 6, FVII incubated with FIXa⌬EGF1;lane 7,

FVII incubated with FIXaPCEGF1;lane 8, FVII incubated with FIXaQ50P.

B, FVII activation by FXa. The proteins used were FXaWT(open circles)

and FIXaPCEGF1(closed circles).Inset, analysis of FVII activation by

SDS-PAGE. The incubation time was 2 h, and the activators used were

identified at thetopof eachlane.Lane 1, standard protein markers;

lane 2, FVII starting material;lane 3, FVII incubated with buffer alone; lane 4, FVII incubated with RVV (1.2␮g/ml);lane 5, FVII incubated

with FXaWT; lane 6, FVII incubated with FXaPCEGF1; lane 7, FVII

incubated with FXaNP. In bothAandB,FVIIrepresents zymogen,HC

represents heavy chain of FVIIa, andLCrepresents the light chain of

(7)

the topology of FVIIasTF complex (13), these residues in the C-terminal domain of TF would appear to interact with the Gla domain of substrates FIX and FX.

The EGF1 domain of FIX (or FX) also appears to play an important role in its activation by FVIIaTF. In an earlier study when FVIIa䡠TF䡠PL was used as the activator, it appeared that in this system FIXEGF1 could not be activated. Further,

FIXPCEGF1 was activated at a slower rate, whereas FIXQ50P was activated at a nearly normal rate in this system (16). The data in these studies were analyzed by SDS-PAGE of the reac-tion mixtures drawn at different times and thus were only qualitative. Further, the failure of activation of FIXEGF1 in this system could stem from the altered spatial alignment of domains in this mutant. To circumvent this problem, we have now developed an assay to quantitatively measure the forma-tion of FIXa and have conducted studies using the FVIIa䡠sTF system, where anchoring of the Gla domain on the PL surface is not required. Additionally, we have performed studies using FXPCEGF1and compared its activation properties with FXWT. Our data indicate that the activation rates of FIXQ50P, FIXPCEGF1, FIX⌬EGF1, and FXPCEGF1are impaired both by the FVIIaTF (Fig. 1) and by the FVIIasTF (Figs. 2 and 3). A recent report revealed that mutations at residue 48 in FIX result in delayed activation by FVIIa䡠TF (45). This observation is con-sistent with the extensive kinetic data presented in this paper. In ligand blot assays, all EGF1 FIX and FX mutants bound to FVIIa but not to TF (Fig. 7). However, results obtained by Western ligand blot assays are at best qualitative in nature. Further, such data do not allow estimation of the binding energy involved in complex formation. For these reasons, we measured the affinity of WT and mutant FIX and FX proteins for binding to immobilized sTF. These data indicate that FIXWT binds to sTF withKd(app) of⬃150 nM. The affinity of the point mutant (FIXQ50P) for sTF was reduced 3-fold, whereas the affinity of the replacement (FIXPCEGF1) or the deletion mutant (FIXEGF1) was reduced⬃10-fold (Fig. 8). Similarly, the affin-FIG. 6.Activation of FXWTand FXPCEGF1by FIXaPL with and

without FVIIIa.A, without FVIIIa. A 100 nMconcentration of each FX

protein was mixed with 20 nMFIXa in Tris/NaCl containing 25␮MPL

and 5 mMCaCl2.B, with FVIIIa. A 50 nMconcentration of each FX

protein was mixed with 0.5 nMFIXa and 0.1 unit/ml FVIIIa in Tris/

NaCl containing 10␮MPL and 5 mMCaCl2. The FXa formed at each

time point was measured as described under “Experimental

Proce-dures.” The proteins used were FXWT(closed circles) and FXPCEGF1

(open squares).

FIG. 7.Ligand blotting of FIX or FX proteins to FVIIa and sTF.

FVIIa (0.4␮g), sTF (0.2␮g), FIX (20 ng), or FX (20 ng) was

electro-phoresed on a 12% SDS-PAGE gel. The proteins were then transferred to nitrocellulose membranes. Each protein that was electrophoresed

and blotted onto the nitrocellulose membrane is labeled at thetopof

eachpanel. The FIX proteins that were used as ligands to probe the

membranes were FIXWT(A), FIXQ50P(B), FIXPCEGF1(C), and FIX⌬EGF1

(D). The FX proteins that were used as ligands to probe the membranes

were FXWT(F) and FXPCEGF1(G). Inpanels EandH, each labeled as

buffer control, 1% milk solution was used to probe the membranes instead of the ligand FIX or FX, respectively. The FIX (or FX) bound to sTF and FVIIa was detected by FIX mAb (or FX mAb) as outlined under “Experimental Procedures.”

FIG. 8.Binding of FIXWTand EGF1 domain mutants to sTF.The

data inArepresent direct specific binding of125I-FIX to immobilized

sTF. An approximateKd(app) value for FIXWTand sTF interaction was

calculated to be⬃120 nM. The data inBdepict the abilities of various

EGF1 domain mutants and synthetic FIX-EGF1 domain to inhibit the

binding of 125I-FIX to sTF. Competitors were FIX

WT(solid circles), FIXQ50P(open circles), FIXPCEGF1(open triangles), FIX⌬EGF1(solid tri-angles), folded EGF1 domain (open squares), and reduced and

car-boxymethylated EGF1 domain peptide (closed squares). The

concentra-tion of 125I-FIX used was 10 n

M with increasing amounts of the

competitors. Thecurvesrepresent best fit to the IC50four-parameter

(8)

ity for sTF of FXPCEGF1 mutant was reduced ⬃6 –10-fold as compared with FXWT (Fig. 9). Of interest is the observation that FIXWT bound to sTF with 3-fold higher affinity than FXWT. The reason(s) for this observation is not known.

Exten-sive additional data are needed to understand whether there are real differences in the affinities of FIXWT and FXWT in binding to sTF or if they simply reflect experimental difficulties inherent to the technique employed. However, the conclusions drawn from this paper do not depend upon resolution of this issue.

The studies conducted with the EGF1 domain mutants yield data pertaining to loss of function. Direct evidence that the EGF1 domain is involved in binding to sTF comes from the ability of synthetic FIX-EGF1 domain to inhibit the interaction of FIXWT with sTF with a Ki 60 ␮M. An interesting point

emerges from such studies. FIX protein lacking the EGF1 domain or having the EGF1 domain of protein C only has a 10-fold reduced affinity for sTF (Fig. 8B). This information coupled with the basic thermodynamic principles involving equilibrium constants predicts that the isolated FIX-EGF1 do-main should bind to sTF with aKdin the mMrange. However,

such is not the case, and the isolated properly folded FIX-EGF1 domain binds sTF with 60 ␮M Kd. Two explanations may be forwarded to explain this phenomenon. First, the binding of FIX to sTF involves two regions (Gla and EGF1 domains) and conformational strain occurs upon binding to the second site either in FIX or sTF. This would yield lowerKdvalues for the isolated two fragments (FIX lacking the EGF1 domain and the EGF1 domain alone) due to the absence of steric constraints inherent in the full-length molecule. An alternative explana-tion might be that the synthetic EGF1 domain we have used is devoid of glycosylation, which might interfere with binding to sTF. Further studies are needed to address this issue. How-ever, we currently prefer the first explanation.

These impaired interactions of mutants with FVIIa䡠TF (or sTF) are not due to defective folding of the proteins. All acti-vated mutants (FIX by FXIa, and FX by RVV) bound AT and activated FVII to FVIIa normally. Furthermore, FXPCEGF1 could be activated by the FIXa䡠FVIIIa䡠PL complex at the same rate as FXWT (Fig. 6). These data are consistent with the observation of Lapan and Fay (46), who found that the protease domain of FX interacts with FVIIIa in the FIXa䡠FVIIIa com-plex. Importantly, our data indicate that the EGF1 domain of FIX or FX contains an exosite(s) that appears to directly inter-act with TF.

The EGF1 domain in FIXa or FXa also contributes to the assembly of FIXa䡠FVIIIa or FXa䡠FVa on the PL surface. Data exist that suggest that the EGF1 domain in FIXa or FXa may FIG. 9.Binding of FXWTand FXPCEGF1to sTF. The data inA

represent direct specific binding of 125

I-FX to immobilized sTF. An

approximateKd(app) value for FXWTand sTF interaction was

calcu-lated to be⬃500 nM. The data inBdepict the abilities of FXWTand

FXPCEGF1to inhibit the binding of

125I-FX to sTF. Competitors were

FXWT(open circles) and FXPCEGF1(closed circles). The concentration of

125I-FX used was 30 n

Mwith increasing amounts of the competitors.

Thecurvesrepresent best fit to the IC50four-parameter logistic equa-tion of Halfman (38).

FIG. 10. Mass spectrometry and

Ca2binding analyses of the folded

FIX-EGF1 domain. The deconvoluted

spectrum of the peptide obtained using a Finnigan LCQ Iontrap Electrospray mass

spectrometer is depicted. Theinsetshows

binding of Ca2⫹to the folded peptide as

determined by a Ca2⫹-specific electrode.

The concentration of the peptide used was

400␮M, and the free Ca2⫹concentration

is plotted against r(mol of Ca2⫹bound/

(9)

directly interact with its cofactor FVIIIa or FVa, respectively (47, 48). However, data also exist that indicate that the EGF1 domain primarily serves as a spacer to properly align the protease domain in FIXa or FXa for optimal interaction with the cofactor (30, 49). In support of this, a recombinant FIX molecule in which the EGF1 domain was replaced with that of FX had normal clotting activity (50), and another recombinant FIX molecule in which the EGF1 domain was replaced with that of FVII had 2-fold increased clotting activity (51). These data suggest that there are no unique determinants in the EGF1 domain of FIX that cannot be replaced by that of FX or FVII. Moreover, to study the functions of EGF domains in protein C, a replacement mutant in which both EGF domains

were from FIX was constructed. This mutant was activated by thrombomodulin-thrombin complex at ⬃70% of the rate ob-tained with the wild type protein C (52). The activated protein C mutant was also defective in inactivating FVa and FVIII/ FVIIIa in a PL-containing system (52). The decreased activity of activated protein C mutant was attributed to direct protein-protein interaction and/or to misalignment of domains/recogni-tion sites with its physiological substrates. Thus, the EGF1 domain in each vitamin K-dependent protease may be involved in direct binding as well as in specific alignments of recognition motifs with other proteins involved in the assembly. We are currently examining the role of EGF1 domain of FXa in the activation of prothrombin in the presence and absence of FVa and PL using our FXPCEGF1mutant.

Last, we have made attempts to define an exosite in the EGF1 domain of FIX or FX that may interact with TF. Three important considerations were given in search for such an exosite. First, since FVIIa䡠TF can activate FVII䡠TF efficiently (53), we opted to select regions that are not involved in binding of FVIIa EGF1 domain to TF. This excluded the interface region involving residues Gln64, Ile69, Phe71, Glu77, and Arg79 of FVIIa (54) and by inference of FIX and FX. Further, this interface is not structurally conserved in FIXa and FXa. Sec-ond, we excluded side chains of those residues (Asn47, Gln50, and Asp64) that are involved in binding to Ca2⫹(54). Third, our

data indicate that FIXQ50Pmutant in which the Ca2⫹-binding site is impaired is defective in binding to TF. Thus, we exam-ined the region surrounding the Ca2⫹site that might be

per-turbed and therefore a likely candidate for binding to TF. Using these three criteria, we postulate an extended exosite region in the EGF1 domain of FIX as well as in FX that could be involved in binding to TF. This is shown in Fig. 11. Residues Asp49, Glu52, Ser53, Asn58, Phe77, Asn81, and Glu83are located on the surface and may be the key determinants for binding to TF. Numerous point mutations in these residues cause hemophilia B (55). Studies are in progress to mutate these residues to examine if they are indeed involved in TF binding.

Acknowledgments—We thank Dr. Shirley Miekka (American Red Cross) for FIX mAb cell line, Dr. Leon Hoyer (American Red Cross) for human factor FVIII concentrate, Dr. Harold L. James (University of Texas at Tyler) for FX mAb cell line, Dr. W. Kisiel (University of Mexico) for FVII mAb cell line, Dr. Bob Kelly (Genetech Inc.) for TF, and Dr. Tom Girard (Monsanto Co.) for sTF.

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References

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