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• W2024315929 abstract "Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal γ-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, 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 (FVIIa·TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa·TF complex. 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), FIXΔEGF1 (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 proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa·TF or FVIIa·sTF activated each mutant significantly more slowly than the FIXWT or FXWT. Importantly, in ligand blot assays, FIXWTand FXWT bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments 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 with Ki of ∼60 μm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FXWT and FXPCEGF1 normally, 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 interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain. Factors VII, IX, and X play key roles in blood coagulation. Each protein contains an N-terminal γ-carboxyglutamic acid domain, followed by EGF1 and EGF2 domains, 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 (FVIIa·TF) complex activates both factor IX (FIX) and factor X (FX). FVIIa represents the enzyme, and TF represents the membrane-bound cofactor for this reaction. The substrates FIX and FX may utilize multiple domains in binding to the FVIIa·TF complex. 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), FIXΔEGF1 (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 proteins were monitored by SDS-PAGE and by enzyme assays. FVIIa·TF or FVIIa·sTF activated each mutant significantly more slowly than the FIXWT or FXWT. Importantly, in ligand blot assays, FIXWTand FXWT bound to sTF, whereas mutants did not; however, all mutants and WT proteins bound to FVIIa. Further experiments 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 with Ki of ∼60 μm. Notably, each FIXa or FXa mutant activated FVII and bound to antithrombin, normally indicating correct folding of each protein. In additional experiments, FIXa with or without FVIIIa activated FXWT and FXPCEGF1 normally, 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 interacting with FVIIa (enzyme) interact with TF (cofactor) using, in part, the EGF1 domain. FVII, FX, and FVIII, factor FIX, FVII, FX, and FVIII, respectively FIX in which EGF1 domain has been deleted FIX or FX in which the EGF1 domain has been replaced with that of protein C normal plasma FIX normal plasma FX membrane-inserted tissue factor containing residues 1–243 membrane region deleted soluble tissue factor containing residues 1–219 epidermal growth factor antithrombin phospholipid monoclonal antibody benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide biotinylated Glu-Gly-Arg-chloromethylketone bovine serum albumin Russell's viper venom polyethylene glycol 8000 γ-carboxyglutamic acid high pressure liquid chromatography wild type N-(9-fluorenyl)methoxycarbonyl Human factor IX (FIX)1and factor X (FX) are vitamin K-dependent glycoproteins with Mr of 57,000 and 58,800, respectively (1Yoshitake S. Schach B.G. Foster D.C. Davie E.W. Kurachi K. J. Biol. Chem. 1985; 24: 3736-3750Google Scholar,2Leytus S.P. Foster D.C. Kurachi K. Davie E.W. Biochemistry. 1986; 25: 5098-5102Crossref PubMed Scopus (182) Google Scholar). Factor VIIa-tissue factor (FVIIa·TF) complex activates FIX to FIXa and FX to FXa by cleaving Arg145–Ala146and Arg180–Val181 peptide bonds in FIX (3Bajaj S.P. Birktoft J.J. Methods Enzymol. 1993; 222: 96-128Crossref PubMed Scopus (41) Google Scholar) and the Arg194–Ile195 peptide bond in FX (2Leytus S.P. Foster D.C. Kurachi K. Davie E.W. Biochemistry. 1986; 25: 5098-5102Crossref PubMed Scopus (182) Google Scholar). 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 γ-carboxyglutamic acid (Gla) domain and two epidermal 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 activates prothrombin to thrombin in a reaction that requires factor Va, PL, and calcium (4Bajaj S.P. Joist J.H. Semin. Thromb. Hemost. 1999; 25: 407-418Crossref PubMed Scopus (88) Google Scholar). In addition, both FIXa and FXa activate FVII to FVIIa (5Masys D.R. Bajaj S.P. Rapaport S.I. Blood. 1982; 60: 1143-1150Crossref PubMed Google Scholar, 6Bajaj S.P. Rapaport S.I. Brown S.F. J. Biol. Chem. 1981; 256: 253-259Abstract Full Text PDF PubMed Google Scholar, 7Butenas S. Mann K.G. Biochemistry. 1996; 35: 1904-1910Crossref PubMed Scopus (94) Google Scholar) and are inhibited by antithrombin (AT) (8Di Scipio R.G. Hermodson M.A. Yates S.G. Davie E.W. Biochemistry. 1977; 16: 698-706Crossref PubMed Scopus (415) Google Scholar, 9Rosenberg R.D. Rosenberg J.S. J. Clin. Invest. 1984; 74: 1-6Crossref PubMed Scopus (190) Google Scholar). The conversion of single chain zymogen FVII to enzyme FVIIa involves the cleavage of a single peptide bond between Arg152 and 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 (10Hagen 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). 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 (10Hagen 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). TF, the cellular cofactor for FVIIa, is composed of two fibronectin type III β-sandwich domains (11Harlos K. Martin D.M. O'Brien D.P. Jones E.Y. Stuart D.I. Polikarpov I. Miller A. Tuddenham E.G. Boys C.W. Nature. 1994; 370: 662-666Crossref PubMed Scopus (214) Google Scholar, 12Muller Y.A. Ultsch M.H. Kelley R.F. de Vos A.M. Biochemistry. 1994; 36: 10864-10870Crossref Scopus (133) Google Scholar). Recently, high resolution x-ray structure of the complex of soluble tissue factor (sTF) and FVIIa has been reported (13Banner 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). 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 (13Banner 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). Thus, FVIIa uses all of its four domains in binding to the N- and C-terminal domains of TF (13Banner 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). Efforts have been directed to understanding the regions in FVIIa·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 (14Kirchhofer D. Lipari M.T. Moran P. Eigenbrot C. Kelley R.F. Biochemistry. 2000; 39: 7380-7387Crossref PubMed Scopus (68) Google Scholar). Similarly, by mutagenesis and docking experiments, it has been proposed that the Gla domain of FVIIa interacts with the Gla domain of FX (15Ruf W. Shobe J. Rao S.M. Dickinson C.D. Olson A. Edgington T.S. Biochemistry. 1999; 38: 1957-1966Crossref PubMed Scopus (49) Google Scholar). Further, we reported earlier that the EGF1 domain of FIX is required for its activation by the FVIIa·TF complex (16Zhong D. Smith K.J. Birktoft J.J. Bajaj S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3574-3578Crossref PubMed Scopus (58) Google Scholar). However, 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·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 FVIIa·TF 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 (17Esmon C.T. J. Biol. Chem. 1989; 264: 4743-4746Abstract Full Text PDF PubMed Google Scholar, 18Mather T. Oganessyan V. Hof P. Huber R. Foundling S. Esmon C. Bode W. EMBO J. 1996; 15: 6822-6831Crossref PubMed Scopus (193) Google Scholar). Further, activated protein C is not involved in the TF-induced coagulation, and its EGF1 domain near the N terminus has an eight-residue insertion (18Mather T. Oganessyan V. Hof P. Huber R. Foundling S. Esmon C. Bode W. EMBO J. 1996; 15: 6822-6831Crossref PubMed Scopus (193) Google Scholar). Therefore, substituting 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·TF complex. In this report, in addition to the above two replacement mutants (FIXPCEGF1 and FXPCEGF1), we used a point mutant (FIXQ50P) and an EGF1 deletion mutant (FIXΔEGF1) 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 domain in FIX and FX. Our findings represent the first report that assigns a specific function to the EGF1 domain in these proteins. 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. Nitrocellulose membrane, polyethylene glycol 8000 (PEG), p-nitrophenyl phosphate, bovine serum albumin (BSA), bovine brain phosphatidylcholine, and phosphatidylserine were purchased from Sigma. Horseradish peroxidase-goat anti-mouse IgG and enhanced chemiluminescence (ECL) detection reagents were purchased from Amersham Biosciences. FVII-depleted plasma and Neoplastin were obtained from Amersham Biosciences. Normal plasma FIX (FIXNP), plasma FX (FXNP), FXIa, Russell's viper venom (RVV), and AT were obtained from Enzyme Research Laboratory. Low molecular weight heparin was purchased from Rhône-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 nm thrombin in the presence of 0.1% BSA and 5 mm CaCl2 in Tris/NaCl at 37 °C for 2 min as described earlier (20Mathur A Zhong D Sabharwal A.K. Smith K.J. Bajaj S.P. J. Biol. Chem. 1997; 272: 23418-23426Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The formed FVIIIa was diluted and used immediately in the activation of FX by FIXa·FVIIIa·PL. For ligand blot experiments, a Ca2+-dependent FIX monoclonal antibody (mAb) cell line was provided by Dr. Shirly Miekka of the American Red Cross, and the IgG was purified as described (21Tharakan J. Strickland D. Burgess W. Drohan W.N. Clark D.B. Vox. Sang. 1990; 58: 21-29Crossref PubMed Scopus (25) Google Scholar). A Ca2+-dependent mAb to the heavy chain of FX used for the ligand blot experiments was purchased from American Diagnostics, Inc. PL vesicles (75% phosphatidylcholine, 25% phosphatidylserine) were prepared by the method of Husten et al. (22Husten E.J. Esmon C.T. Johnson A.E. J. Biol. Chem. 1987; 262: 12953-12961Abstract Full Text PDF PubMed Google Scholar). TF containing the transmembrane region (residues 1–243) was a gift from Genetech, Inc. The relipidation of the TF was performed as described (16Zhong D. Smith K.J. Birktoft J.J. Bajaj S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3574-3578Crossref PubMed Scopus (58) Google Scholar). sTF that lacks the transmembrane 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 (16Zhong D. Smith K.J. Birktoft J.J. Bajaj S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3574-3578Crossref PubMed Scopus (58) Google Scholar) and activating FX with RVV (23Fujikawa K. Legaz M.E. Davie E.W. Biochemistry. 1972; 11: 4892-4899Crossref PubMed Scopus (138) Google Scholar) in 50 mm Tris, 0.15 m NaCl (Tris/NaCl), pH 7.4, containing 5 mm CaCl2 and 0.1% PEG at 37 °C for 2 h. Complete activation of FIX or FX was confirmed by SDS-PAGE (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). SDS-gel electrophoresis was performed using the Laemmli buffer system (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). 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. Gla and amino acid sequence 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 sequencer. Gla analysis of each protein was performed by alkaline hydrolysis followed by HPLC analysis. The amount of Gla was quantitated based upon Asp and Asn present per mol of each protein. Recombinant FIXWT, FIXΔEGF1, FIXPCEGF1, and FIXQ50P were expressed in human embryonic kidney 293 cells and purified by using the IX A-7 mAb column as described (16Zhong D. Smith K.J. Birktoft J.J. Bajaj S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3574-3578Crossref PubMed Scopus (58) Google Scholar, 25Zhong D. Bajaj S.P. BioTechniques. 1993; 15: 874-878PubMed Google Scholar). Each FIX protein had ∼12 Gla residues/mol (16Zhong D. Smith K.J. Birktoft J.J. Bajaj S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3574-3578Crossref PubMed Scopus (58) Google Scholar). 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 (26Hippenmeyer P. Highkin M. Bio/Technology. 1993; 11: 1037-1041PubMed Google Scholar) that was modified to contain AflII andXhoI sites. A stable cell line that expressed FVIIWT was established as described in detail by Hippenmeyer and Highkin (26Hippenmeyer P. Highkin M. Bio/Technology. 1993; 11: 1037-1041PubMed Google Scholar). Medium was collected in the presence of vitamin K as outlined earlier for FIX (16Zhong D. Smith K.J. Birktoft J.J. Bajaj S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3574-3578Crossref PubMed Scopus (58) Google Scholar, 25Zhong D. Bajaj S.P. BioTechniques. 1993; 15: 874-878PubMed Google Scholar). FVIIWT was purified by using a Ca2+-dependent mAb as described (27Kazama Y. Pastuszyn A. Wildgoose P. Hamamoto T. Kisiel W. J. Biol. Chem. 1993; 268: 16231-16240Abstract Full Text PDF PubMed Google Scholar). 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 (6Bajaj S.P. Rapaport S.I. Brown S.F. J. Biol. Chem. 1981; 256: 253-259Abstract Full Text PDF PubMed Google Scholar). The resin was removed by centrifugation, and the supernatant was passed over a small Chelex-100 column to remove Ca2+. Aliquots were kept frozen at −80 °C until used. An expression vector for FXWT was constructed in which the prepro-leader sequence of FXWT was replaced with that of prothrombin as described by Camire et al. (28Camire R.M. Larson P.J. Stafford D.W. High K.A. Biochemistry. 2000; 39: 14322-14329Crossref PubMed Scopus (60) Google Scholar). The prepro-leader sequence of prothrombin was amplified by PCR using primers A and B (TableI) 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 (25Zhong D. Bajaj S.P. BioTechniques. 1993; 15: 874-878PubMed Google Scholar). The resulting chimeric DNA, containing the prepro-leader sequence of prothrombin followed by the FX sequence, was digested withAflII and XhoI and ligated into pMon3360b expression vector. A stable cell line that expressed FXWTwas established as described (26Hippenmeyer P. Highkin M. Bio/Technology. 1993; 11: 1037-1041PubMed Google Scholar). Medium was collected in the presence of vitamin K as outlined earlier for FIX (16Zhong D. Smith K.J. Birktoft J.J. Bajaj S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3574-3578Crossref PubMed Scopus (58) Google Scholar, 25Zhong D. Bajaj S.P. BioTechniques. 1993; 15: 874-878PubMed Google Scholar). FXWT was purified using a Ca2+-dependent mAb to the Gla domain of FX (25Zhong D. Bajaj S.P. BioTechniques. 1993; 15: 874-878PubMed Google Scholar) followed by FPLC Mono Q column. The conditions for the FPLC Mono Q column were the same as described previously for FIX purification (16Zhong D. Smith K.J. Birktoft J.J. Bajaj S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3574-3578Crossref PubMed Scopus (58) Google Scholar, 25Zhong D. Bajaj S.P. BioTechniques. 1993; 15: 874-878PubMed Google Scholar). Construction of FXPCEGF1 was performed by the overlap extension method as described (25Zhong D. Bajaj S.P. BioTechniques. 1993; 15: 874-878PubMed Google Scholar), and primers D and E (Table I) were used to amplify the protein C EGF1 domain. The establishment of a stable cell line and the purification of FXPCEGF1 were the same as for FXWT.Table ISequence of synthetic oligonucleotide primers for construction of FXWT and FXPCEGF1PrimerSequence1-aPrimer A contains aAflII site, and primer C contains an XhoI 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 containing FX and protein C DNA sequences. The sequences in parenthesis correspond to the human protein C sequences.A5′-CTAGACTTAAGCTTCCACC(ATGGCCACGTCCGAGGCTTG)B5′-CTTCATCTCTTCAAGAAAGGAATTGGC(TCGCCGGACCCGCTGGAG)C5′-TCTGACTCGAG(TCACTTTAATGGAGAGGA)D5′-AAAGATGGCGACCAGTGT(TTGGTCTTGCCGTTGGAG)E5′-GCTGCAGAGCTTCCGTGT(CTCCCGCTGGCAGAAGCG)1-a Primer A contains aAflII site, and primer C contains an XhoI 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 containing FX and protein C DNA sequences. The sequences in parenthesis correspond to the human protein C sequences. Open table in a new tab For activation of FIX by FVIIa·TF·PL, 2 μm FIX was activated with 8 nm VIIa and 0.5 nm TF in the presence of 1 mm PL, 5 mm CaCl2, 0.1% PEG in Tris/NaCl buffer. At different times, 20 μl 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 μm, 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 μl (10 μg/ml in 0.1 m of NaHCO3) of the Ca2+-dependent FIX mAb at 4 °C overnight. The wells were blocked with 200 μl of 1% BSA and 0.1% Tween 20 in Tris/NaCl for 2 h at 37 °C. At this point, each biotin-EGR-IXa sample was further diluted 50-fold in Tris/NaCl containing 1% BSA and 0.1% Tween 20. 100 μl 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 μl of alkaline phosphatase-streptavidin in Tris/NaCl containing 1% BSA, 0.1% Tween 20, and 5 mm CaCl2. The plate was incubated at 37 °C for 1 h. After three washings, each well received 100 μl of substrate p-nitrophenyl phosphate (4 mg/ml) in the alkaline phosphatase buffer (100 mm NaCl, 5 mmMgCl2, 100 mm Tris, 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·sTF, 4 μm FIX was activated with 0.16 μm FVIIa·sTF in the absence of PL. All other assay conditions were the same as those for the activation of FIX with FVIIa·TF·PL outlined above. For SDS-PAGE, 12 μl of the reaction mixture was removed at different times and added to 2 μl of 0.5 m EDTA and 5 μl of 5-fold concentrated SDS-reducing buffer. Samples were placed in boiling water for 5 min and analyzed by SDS-PAGE (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). For activation of FX by FVIIa·TF·PL, 2 μm FX was activated with 8 nm FVIIa and 0.5 nm TF in the presence of 1 mm PL, 5 mm CaCl2, 0.1% PEG in Tris/NaCl. These conditions are the same as used for the activation of FIX. At different times, 20 μl of the reaction mixture was removed and diluted 10-fold with 20 mm EDTA 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 μm S-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·sTF, 4 μm FX was activated with 0.16 μm FVIIa·sTF in the absence of PL. All other assay conditions were the same as above for the activation of FX with FVIIa·TF·PL. For SDS-PAGE, 12 μl of the reaction mixture was removed at different times and added to 2 μl of 0.5 mEDTA and 5 μl of 5-fold SDS-reducing buffer for analysis by SDS-PAGE. For activation of FXWT and FXPCEGF1 by FVIIa·PL, 50 nm FVIIa was incubated at 37 °C with 1 μm FXWT or FXPCEGF1 in the presence of 35 μm PL and 5 mm CaCl2 in Tris/NaCl containing 0.1% BSA. 10-μl aliquots were removed at 0, 5, 10, and 15 min and added to 40 μl of 20 mm EDTA in Tris/NaCl. The reaction mixture was further diluted as needed, and the concentration of FXa was determined by the hydrolysis of 250 μm S-2222. These experiments were performed exactly as described in detail earlier (30Mathur A. Bajaj S.P. J. Biol. Chem. 1999; 274: 18477-18486Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The concentration of each reagent used is given in the legend to Fig. 6. A 50 nm concentration of each FIXa or FXa was incubated at 37 °C with 1 μm FVII in the presence of 35 μm PL and 5 mm CaCl2 in Tris/NaCl. 2-μl aliquots were removed at different times and added to 100 μl of 0.1% BSA in Tris/NaCl containing 10 mm EDTA. 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 (31Rao L.V. Bajaj S.P. Rapaport S.I. Blood. 1985; 65: 218-226Crossref PubMed Google Scholar). For this assay, 50 μl of FVII-depleted plasma was incubated with 50 μl of Neoplastin for 3 min at 37 °C. Then 25 μl of test sample and 50 μl of prewarmed (37 °C) 35 mmCaCl2 were 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. For these experiments, the final reaction mixtures contained the following: 2 μmFIXa or FXa, 2 μm AT, 5 mm CaCl2, and 10 units/ml low molecular weight heparin in Tris/NaCl, pH 7.4. The total reaction mixture in each case was 150 μl, and 20-μl aliquots were removed at 0.15, 0.5, 1.5, 4, 12, and 30 min and added to 5 μl 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 formation 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. First sTF and FVIIa were electrophoresed on SDS-PAGE. The proteins were then transferred to a 0.2-μm nitrocellulose membrane. The protocol used was that outlined by Sambrook et al. (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), 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 μg/ml in Tris/NaCl, 1% milk, 0.05% Tween 20, and 5 mm CaCl2 at 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 μg/ml) at room temperature for 2 h. A second antibody (horseradish peroxidase-goat anti-mouse IgG) and the ECL Western blotting detection kit were used to detect the primary mAb. 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 α-amino group of the amino acid was Fmoc-protected, and the side chain groups were protected by t-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 (33Gorka J. McCourt D.W. Schwartz B.D. Pept. Res. 1989; 2: 376-380PubMed Google Scholar). The purified reduced peptide gave a molecular 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 (34Jaenicke R. Rudolph R. Creighton T.E. Protein Structure: A Practical Approach. IRL Press, Oxford1989: 208-209Google Scholar, 35Whiteman P. Downing A.K. Smallridge R. Winship P.R. Handford P.A. J. Biol. Chem. 1998; 273: 7807-7813Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The lyophilized peptide was dissolved at a concentration of ∼0.4 mg/ml in a solution containing 0.1m Tris-HCl, pH 8.3, 50 mm CaCl2, 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 0.1 m NaOH (36Goodwin T.W. Morton R.A. Biochem. J. 1946; 40: 628-632Crossref PubMed Scopus (1098) Google Scholar). Two tyrosines and one tryptophan contained in our peptide were taken into account in calculating its concentration. 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 μm in 4 ml of Tris/NaCl, pH 7.4, were performed by" @default.
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• W2024315929 title "The N-terminal Epidermal Growth Factor-like Domain in Factor IX and Factor X Represents an Important Recognition Motif for Binding to Tissue Factor" @default.
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