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• W2167633411 abstract "Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIIIa. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at positions 467, 511, 652, or 1683 displayed attenuated FXa binding, whereas the prothrombin affinity was unaffected. The affinity toward FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicates that the enzyme:cofactor assembly of the prothrombinase and the tenase complexes are homologous and provide a useful platform for further investigation of specific structural elements involved in the FVa·FXa complex assembly. Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIIIa. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at positions 467, 511, 652, or 1683 displayed attenuated FXa binding, whereas the prothrombin affinity was unaffected. The affinity toward FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicates that the enzyme:cofactor assembly of the prothrombinase and the tenase complexes are homologous and provide a useful platform for further investigation of specific structural elements involved in the FVa·FXa complex assembly. The formation of thrombin from prothrombin is a key event in the coagulation process. In this reaction, the activated form of coagulation Factor V (FVa) 1The abbreviations used are: FVa, activated Factor V; FV, Factor V; FXa, activated Factor X; FVIII, Factor VIII; FVIIIa, activated Factor VIII; FIXa, activated Factor IXa; APC, activated protein C; bFX, bovine Factor X; bFXa, bovine Factor Xa; WT, wild-type; PPACK, Phe-Pro-Arg-chloromethyl ketone, BSA, bovine serum albumin; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; ELISA, enzyme-linked immunosorbent assay functions as a cofactor to the enzyme Factor Xa (FXa). The two proteins are assembled on the surface of negatively charged phospholipid membranes to the highly efficient prothrombinase complex (1Nesheim M.E. Mann K.G. J. Biol. Chem. 1979; 254: 1326-1334Abstract Full Text PDF PubMed Google Scholar, 2Kane W.H. Davie E.W. Blood. 1988; 71: 539-555Crossref PubMed Google Scholar, 3Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar, 4Rosing J. Tans G. Thromb. Haemost. 1997; 78: 427-433Crossref PubMed Scopus (73) Google Scholar). Factor V (FV) is a single-chain procofactor (330 kDa), having the domain organization A1-A2-B-A3-C1-C2 in common with the homologous Factor VIII (FVIII) (5Jenny R.J. Pittman D.D. Toole J.J. Kriz R.W. Aldape R.A. Hewick R.M. Kaufman R.J. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4846-4850Crossref PubMed Scopus (343) Google Scholar, 6Kane W.H. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6800-6804Crossref PubMed Scopus (152) Google Scholar, 7Jenny R.J. Tracy P.B. Mann K.G. Bloom A.L. Forbes C.D. Thomas D.P. Tuddenham E.G.D. Haemostasis and Thrombosis. Churchill Livingstone Inc., New York1994: 456-476Google Scholar). FV circulates in human plasma at a concentration of 7 μg/ml but is also present in platelets from where it is released and activated during hemostasis (8Tracy P.B. Eide L.L. Bowie E.J. Mann K.G. Blood. 1982; 60: 59-63Crossref PubMed Google Scholar). FV is converted to its activated form FVa upon proteolytic cleavage mediated by either thrombin or FXa. During this reaction, the B domain of FV is released from the active FVa molecule (9Monkovic D.D. Tracy P.B. Biochemistry. 1990; 29: 1118-1128Crossref PubMed Scopus (211) Google Scholar, 10Thorelli E. Kaufman R.J. Dahlback B. Eur. J. Biochem. 1997; 247: 12-20Crossref PubMed Scopus (64) Google Scholar, 11Esmon C.T. J. Biol. Chem. 1979; 254: 964-973Abstract Full Text PDF PubMed Google Scholar, 12Suzuki K. Dahlback B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar). FVa is composed of the 105-kDa heavy chain (A1-A2 domains) and the 71/74-kDa light chain (A3-C1-C2 domains), the two chains being held together by non-covalent, calcium-ion dependent forces (1Nesheim M.E. Mann K.G. J. Biol. Chem. 1979; 254: 1326-1334Abstract Full Text PDF PubMed Google Scholar, 11Esmon C.T. J. Biol. Chem. 1979; 254: 964-973Abstract Full Text PDF PubMed Google Scholar, 12Suzuki K. Dahlback B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar). The activity of the prothrombinase complex is regulated by activated protein C (APC), which inhibits FVa by proteolytic cleavage at Arg-306, Arg-506, and Arg-679 (13Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: 31869-31880Abstract Full Text PDF PubMed Google Scholar). Factor X (FX) is a zymogen to a serine protease. It is composed of a Gla domain, two epidermal growth factor-like domains, an activation peptide, and a serine-protease domain. During blood coagulation, FX is activated by either the extrinsic pathway Factor VIIa·tissue factor complex or the intrinsic pathway tenase complex that comprises the phospholipid-bound Factor IXa (FIXa) and activated FVIII (FVIIIa) (14DiScipio R.G. Hermadson M.A. Yates S.G. Davie E.W. Biochemistry. 1977; 16: 698-706Crossref PubMed Scopus (415) Google Scholar). FVIIIa is a cofactor to FIXa in the activation of FX, a reaction that in many respects is very similar to the prothrombin activation (2Kane W.H. Davie E.W. Blood. 1988; 71: 539-555Crossref PubMed Google Scholar). After activation of FX, the light chain (18 kDa) containing the Gla and two epidermal growth factor-like domains remains associated via a disulfide bridge to the heavy chain (27 kDa) composed of the serine-protease domain (15Steinberg M. Nemerson Y. Hemostasis and Thrombosis. 2nd Ed. Lippincott, Philadelphia1987: 112-119Google Scholar, 16McMullen B.A. Fujikawa K. Kisiel W. Sasagawa T. Howald W.N. Kwa E.Y. Weinstein B. Biochemistry. 1983; 22: 2875-2884Crossref PubMed Scopus (96) Google Scholar). Even though the kinetics of the prothrombinase complex have been well characterized, surprisingly little is known about structure-function relationships of the complex and the sites of protein-protein interaction between FVa and FXa. Two regions in the A2 domain of FVa, 493–506 (17Heeb M.J. Kojima Y. Hackeng T.M. Griffin J.H. Protein Sci. 1996; 5: 1883-1889Crossref PubMed Scopus (50) Google Scholar) and 311–325 (18Kojima Y. Heeb M.J. Gale A.J. Hackeng T.M. Griffin J.H. J. Biol. Chem. 1998; 273: 14900-14905Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), have, based on peptide studies, been suggested to be important for binding of FXa. In addition, removal of the C-terminal region of the A2 domain, 684–709, has been shown to result in reduced FXa affinity (19Bakker H.M. Tans G. Thomassen M.C. Yukelson L.Y. Ebberink R. Hemker H.C. Rosing J. J. Biol. Chem. 1994; 269: 20662-20667Abstract Full Text PDF PubMed Google Scholar). In contrast to the FVa·FXa complex, extensive interaction studies have been carried out on the structurally and functionally homologous FVIIIa·FIXa complex. Various interaction sites for FIXa have been identified on FVIIIa. Three of these binding sites, comprising residues 511–530, 558–565, and 692–710, are located in the A2 domain (20Celie P.H. Van Stempvoort G. Jorieux S. Mazurier C. Van Mourik J.A. Mertens K. Br. J. Haematol. 1999; 106: 792-800Crossref PubMed Scopus (21) Google Scholar, 21O'Brien L.M. Medved L.V. Fay P.J. J. Biol. Chem. 1995; 270: 27087-27092Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22Jorquera J.I. McClintock R.A. Roberts J.R. MacDonald M.J. Circulation. 1992; 86: 685aGoogle Scholar). Furthermore, a FIXa binding sequence, including residues 1811–1818 in the A3 domain of the FVIIIa light chain, has also been identified previously (23Lenting P.J. van de Loo J.W. Donath M.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1996; 271: 1935-1940Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). To map the regions on FVa that are important for binding of FXa, targeted glycosylation created by site-directed mutagenesis was used as the experimental strategy. It has been shown that mutations aiming at introduction of N-linked glycosylation at selected residues can be a useful way to probe molecular regions involved in protein-protein interaction (24Ueda T. Iwashita H. Hashimoto Y. Imoto T. J Biochem. (Tokyo). 1996; 119: 157-161Crossref PubMed Scopus (19) Google Scholar, 25Cook B.C. Rudolph A.E. Kurumbail R.G. Porsche-Sorbet R. Miletich J.P. J. Biol. Chem. 2000; 275: 38774-38779Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 26Amano K. Sarkar R. Pemberton S. Kemball-Cook G. Kazazian Jr., H.H. Kaufman R.J. Blood. 1998; 91: 538-548Crossref PubMed Google Scholar). The size of a carbohydrate chain is convenient for searching binding site regions, because it probes larger areas than can be achieved via amino acid scanning, while it is more directed than peptide or antibody inhibition studies. The targeted glycosylation was achieved by introduction of the sequence signaling for N-linked glycosylation, Asn-X-Thr. Threonine rather than serine was chosen at the third position of the consensus sequence of glycosylation, because it has been shown to result in a higher degree of glycosylation than when serine occupies the third position (27Kasturi L. Eshleman J.R. Wunner W.H. Shakin-Eshleman S.H. J. Biol. Chem. 1995; 270: 14756-14761Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 28Nicolaes G.A. Villoutreix B.O. Dahlback B. Biochemistry. 1999; 38: 13584-13591Crossref PubMed Scopus (59) Google Scholar). A recently created three-dimensional model of FVa was very helpful for the selection of adequate sites for the novel carbohydrate side chains (29Villoutreix B.O. Dahlback B. Protein Sci. 1998; 7: 1317-1325Crossref PubMed Scopus (79) Google Scholar, 30Pellequer J.L. Gale A.J. Getzoff E.D. Griffin J.H. Thromb. Haemost. 2000; 84: 849-857Crossref PubMed Scopus (70) Google Scholar). We focused on introducing the glycosylation sites at residues that are surface-exposed in the three-dimensional model of FVa. Unfortunately, in the model, the end of the A2 domain (after residue 656) is missing. In addition to the computer model of the A domains of FVa, we designed our mutations taking into account documented binding sequences of FVIIIa and corresponding homologous sequences in FVa (20Celie P.H. Van Stempvoort G. Jorieux S. Mazurier C. Van Mourik J.A. Mertens K. Br. J. Haematol. 1999; 106: 792-800Crossref PubMed Scopus (21) Google Scholar, 21O'Brien L.M. Medved L.V. Fay P.J. J. Biol. Chem. 1995; 270: 27087-27092Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22Jorquera J.I. McClintock R.A. Roberts J.R. MacDonald M.J. Circulation. 1992; 86: 685aGoogle Scholar, 23Lenting P.J. van de Loo J.W. Donath M.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1996; 271: 1935-1940Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The results of this study show that regions surrounding residues 467, 511, 652, and 1683 of FVa are important for binding of FXa and thus for the assembly of the prothrombinase complex. Bln1 was from Roche Molecular Biochemicals (Germany), and Bsu36I andBspEI were from New England BioLabs (Beverly, MA).Pwo polymerase and T4 DNA ligase were purchased from Roche Molecular Biochemicals (Penzberg, Germany). A double-stranded DNA sequencing kit was obtained from PerkinElmer Life Sciences. Cell culture media (Optimem Glutamax) were from Invitrogen. LipofectAMINE 2000 was from Invitrogen. Tunicamycin was from Sigma Chemical Co. (St. Louis, MO). Benzamidine was purchased from Acros Organics. Phe-Pro-Arg-chloromethylketone (PPACK) was obtained from Calbiochem (La Jolla, CA). A monoclonal antibody (Mk1) directed to the B domain of Factor V has been described before (31Dahlback B. Hildebrand B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1396-1400Crossref PubMed Scopus (341) Google Scholar). Monoclonal HV-1 reacting with the C2 domain of Factor V was from Sigma. Monoclonal antibody AHV-5101 was from Hematologic Technologies Inc. A polyclonal antibody (A299) against FV was from Dako (Copenhagen, Denmark). Polyclonal anti-human FV antibody (8806) was raised by our group and showed epitopes to both the heavy and light chain as well as to the B domain. Egg extracts of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and brain extracts of phosphatidylserine (PS) were purchased from Avanti Polar Lipids (Birmingham, AL). The chromogenic substrated-Phe-(pipelocyl)-Arg-p-nitroaniline (S-2238) was a kind gift of Chromogenix (Milano, Italy). Streptavidin-coated magnetic beads (Dynabeads, Streptavidin) were purchased from Dynal (Oslo, Norway). Biotinylated PE was purchased from Molecular Probes (Eugene, OR) and [14C]PC from Amersham Biosciences (Uppsala, Sweden). The buffers used were as follows: Buffer A: 25 mm Hepes (pH 7.5), 150 mm NaCl, 5 mm CaCl2; buffer B: same as buffer A but with 2m NaCl; cell lysis buffer: 10 mm Tris (pH 7.5), 150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, 0.1% SDS, 2 μm PPACK, 10 mm benzamidine, 50 μg/ml soybean trypsin inhibitor; immune precipitate washing buffer: 10 mm Tris (pH 7.5), 150 mm NaCl, 2 mm EDTA, 0.5% Nonidet P-40, 0.1% SDS; activation buffer: 66.6 mm Tris (pH 7.5), 200 mm NaCl, 3.33 mm CaCl2, 6.66% glycerol; endoglycosidase H buffer: 50 mm sodium acetate buffer (pH 5.2), 2 μm PPACK, 10 mm benzamidine, 50 μg/ml soybean trypsin inhibitor,10 μg/ml aprotinin, 1 μg/ml leupeptin. Human prothrombin and human FXa were purchased from Kordia (Leiden, Netherlands). Human α-thrombin was obtained from Hematologic Technologies Inc. Bovine serum albumin (BSA), ovalbumin, soybean trypsin inhibitor, and leupeptin were purchased from Sigma. Aprotinin was from Bayer (Leverkusen, Germany). Bovine FX (bFX) was purified as described (32Stenflo J. J. Biol. Chem. 1976; 251: 355-363Abstract Full Text PDF PubMed Google Scholar) and activated using Russel's viper venom (33Furie B.C. Furie B. Methods Enzymol. 1976; 45: 191-205Crossref PubMed Scopus (44) Google Scholar). Bovine- Factor Xa (bFXa) was labeled with 125I using the chloramine T method. The specific activity was 20,000 cpm/ng, equal to 0.4–0.5 MBq/μg of protein. The labeled bFXa was characterized on SDS-PAGE and functionally analyzed using a prothrombinase assay and appeared unaffected by the labeling procedure. Mutations were introduced into the expression vector pMT2 containing the full-length cDNA of human FV using the QuikChange site-directed mutagenesis kit (Stratagene). For each mutant two complementary oligonucleotides containing the appropriate mutations were used. The mutated fragments were then isolated by restriction enzymes and used to replace corresponding fragments in the template. For each mutant, the sense primers used for mutagenesis and the restriction enzymes used for digestion are shown in Table I. The sequences of the fragments were confirmed by DNA sequencing.Table ISense primers used for mutagenesis and restriction enzymes employed for isolating the fragments containing the mutations, which were then used to replace the corresponding fragment in WT FV cDNAMutantPrimerEnzymesEGG172NGT5′-GAC CCT AAC TAA CGG TAC GAC ACA GAA GAC G-3′Bln1/Bsu36I (2 kb)MKR319NKT5′-AGG CGG CAC AACAAG ACG TGG GAA TAC TTC ATT GC-3′Bln1/Bsu36I (2 kb)KKY345NKT5′-GAA TAT GGA CAA CAA AAC CAG GTC TCA GCA TTT GG-3′Bln1/Bsu36I (2 kb)DES373NET5′-CAG TAC GAA AAT GAG ACC TTC ACC AAA CAT ACA GTG-3′Bln1/Bsu36I (2 kb)FTS435NTT5′-GTC AAC TCT TCT AAC ACC ACA GGC AGG AAC-3′Bln1/Bsu36I (2 kb)GET450NET5′-GCA GTT CAA CCA AAC GAA ACC TAT ACT TAT AAG TGG-3′Bsu36I/BspEI (2 kb)EFD461NFT5′-CAT CTT AAA CTT TAC TGA ACC CAC AGA AAA TGA TGC CC-3′Bsu36I/BspEI (2 kb)END467NNT5′-GAT GAA CCC ACA AAC AAT ACT GCC CAG TGC-3′Bsu36I/BspEI (2 kb)AAD511NAT5′-CAG AGG AAC GCA ACC ATC GAA CAG CAG G-3′Bsu36I/BspEI (2 kb)RDV652NDT5′-GAG GCT GAA ATT CAA CGA TAC TAA ATG TAT CCC AG-3′Bsu36I/BspEI (2 kb)KCI655NCT5′-GGG ATG TTA ACT GTA CCC CAG ATG ATG ATG-3′Bsu36I/BspEI (2 kb)SYT1677NTT5′-CAA ATA GCA ATT ATA CCT ACG TAT GGC ATG CC-3′Bsu36I/BspEI (2 kb)HAT1683NAT5′-TAC GTA TGG AAT GCC ACT GAG CGA TCA GGG-3′Bsu36I/BspEI (2 kb)Changes introduced are in boldface; antisense counterpart primers are not shown. The size of each cleaved fragment, which was isolated and ligated into the WT FV cDNA lacking the corresponding fragment, is shown in parentheses. The three amino acids given before the number represent WT FV sequence; the number, referring to the amino acid residue after mutagenesis, represents the carbohydrate attachment site. Open table in a new tab Changes introduced are in boldface; antisense counterpart primers are not shown. The size of each cleaved fragment, which was isolated and ligated into the WT FV cDNA lacking the corresponding fragment, is shown in parentheses. The three amino acids given before the number represent WT FV sequence; the number, referring to the amino acid residue after mutagenesis, represents the carbohydrate attachment site. Expression plasmids containing the various FV cDNA constructs were transfected into COS 1 cells using both the DEAE-dextran method as described (34Kaufman R.J. Methods Enzymol. 1990; 185: 487-511Crossref PubMed Scopus (150) Google Scholar) and the LipofectAMINE 2000 (Invitrogen) method as described by the manufacturer. Proteins were collected 72 h after transfection in serum-free media (Optimem Glutamax). For the expression in the presence of tunicamycin, cells were pretreated with 10 μg/ml tunicamycin prior to protein collection in serum-free media in the presence of 10 μg/ml tunicamycin. Media containing the recombinant proteins were concentrated ∼40-fold using Vivaspin 100,000 molecular weigh cut off, on the day of harvest, and the FV concentration was measured using an ELISA, after minor modifications (35Thorelli E. Kaufman R.J. Dahlback B. Blood. 1999; 93: 2552-2558Crossref PubMed Google Scholar). In brief, microtiter plates were coated overnight with 10 μg/ml monoclonal Mk1 and then quenched for 30 min. Samples were diluted in ELISA buffer supplemented with 10 mm benzamidine and 2 mm CaCl2 and incubated in 4 °C overnight. Standard curves were created using pooled normal citrated plasma, assuming the FV concentration to be 7 μg/ml (8Tracy P.B. Eide L.L. Bowie E.J. Mann K.G. Blood. 1982; 60: 59-63Crossref PubMed Google Scholar). Biotinylated monoclonal HV-1 (0.1 μg/ml) was used as secondary antibody, and after 2 h streptavidin-peroxidase was added. After 30 min, the plates were developed for 3 min, and the absorbance was measured at 490 nm. To rule out that the cell culture medium interfered with the ELISA, plasma-purified FV was diluted in mock medium or in buffer. No difference was detectable, indicating that the medium does not interfere. A pulse Labeling experiment was performed 24 h after transfection by radioactive labeling with [35S]methionine and [35S]cysteine as previously described with some modifications (36Katsumi A. Senda T. Yamashita Y. Yamazaki T. Hamaguchi M. Kojima T. Kobayashi S. Saito H. Blood. 1996; 87: 4164-4175Crossref PubMed Google Scholar). In brief, cell extracts were prepared by lysis in a cell lysis buffer supplemented with inhibitors. Cell lysate was precleared with protein A-Sepharose (Amersham Biosciences) for 2 h at 4 °C and immune precipitated overnight at 4 °C using a polyclonal anti-FV antibody, 8806 (20 μg). The immune complexes were precipitated and washed with immune precipitate washing buffer. After elution by boiling, samples were subjected to 2 units/ml thrombin for 30 min in an activation buffer. Proteins were deglycosylated by endoglycosidase H digestion (1 unit/ml) overnight at 37 °C in an endoglycosidase H buffer, supplemented with inhibitors. Boiling the sample terminated the digestion. Proteins were separated by electrophoresis on a 7.5% SDS-PAGE (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207165) Google Scholar). The gels were then exposed in a cassette and finally scanned using a PhosphorImager (Amersham Biosciences). The purification was performed as described by Heeb et al. (38Heeb M.J. Rehemtulla A. Moussalli M. Kojima Y. Kaufman R.J. Eur. J. Biochem. 1999; 260: 64-75Crossref PubMed Scopus (20) Google Scholar) after some modifications. The supernatant of conditioned medium of rFV (100 ml) was concentrated to 3 ml using the Vivaspin 100,000 molecular weight cut off. Concentrated medium was treated with 10 mm benzamidine and 2 μm PPACK and incubated on ice for 30 min. The supernatant was loaded onto a 5-ml Hi-trap column (AmershamBiosciences) coupled with 2 mg of anti-(FV light chain) monoclonal antibody AHV 5101 at a flow rate of 0.18 ml/min. The column was then washed with 20 ml of buffer at a flow rate of 0.18 ml/min. Finally, the column was then eluted with buffer B at a flow rate of 0.5 ml/min and collected in 1-ml fractions. Fractions were analyzed for FVa activity and the SDS-PAGE profile. Pooled fractions were desalted using PD-10 (Amersham Biosciences) and stored at −80 °C. Phospholipids dissolved in chloroform/methanol (9:1, v/v) were dried in a glass tube under a mild flow of nitrogen. The phospholipids were suspended in 25 mmHepes, 150 mm NaCl (pH 7.5), vigorously vortexed for 2 min, and then sonicated for 10 min at room temperature at amplitude 3, using an XL 2020 sonicator (Misonix). For the binding experiments, the phospholipid composition of the vesicles was PE/PS/PC at molar ratio of 10/20/70. A trace amount of 14C-labeled PC was included, and 10% of the PE was biotinylated. For the prothrombinase assay, the phospholipid composition was PS/PC, at molar ratio of 10/90. Recombinant FV proteins were incubated with 0.5 unit/ml of α-thrombin (Hematologic Technologies Inc.) for 10 min at 37 °C prior to analysis in the prothrombinase assay. Typically, if not specifically noted, the conditions for the prothrombinase assay were 50 pm FVa, 5 nm FXa, 0.5 μm prothrombin, and 50 μm phospholipid vesicles (10/90 of PS/PC). Thrombin generation was allowed to continue for 1 min before the reaction was stopped by dilution with ice-cold EDTA buffer. The amount of generated thrombin was quantified using the chromogenic substrate S-2238. In control experiments, plasma-purified FVa and recombinant wild-type (WT) FVa in conditioned medium behaved similarly with respect to FXa cofactor activity and FXa affinity. Moreover, in the absence of added FV, there was no appreciable thrombin generation. The prothrombinase assay was used to determine the apparent K d for the binding of FXa to the thrombin-activated FV variants. The formation of membrane-bound FXa·FVa complexes was measured by determining the rates of prothrombin activation in the presence of phospholipid vesicles at increasing concentrations of FXa and a fixed concentration of FVa. FVa (50 pm) was preincubated for 4 min with FXa (0.1–50 000 pm) and phospholipid vesicles (50 μm of 10/90, PS/PC). Thrombin generation was started by addition of 0.5 μm preheated prothrombin and allowed to continue for 1 min. The apparent K d for the membrane-bound FXa·FVa complex was obtained from plots of the rate of thrombin generation as a function of the FXa concentration. TheK d was obtained by fitting the data to the following equation for a single-site binding isotherm using non-linear least squares regression analysis, B=Bmax/(1+(Kd/[FXa]))Equation 1 The free concentration of FXa [FXa] was assumed to be equal to the added concentration of FXa. The binding maximum,B max, set as the value where the binding of FXa to WT FVa, was saturated. The K mfor prothrombin activation by the different prothrombinase complexes was determined by varying the prothrombin concentrations. FVa (200 pm) was preincubated for 4 min with increasing concentrations of prothrombin (5–2500 nm) and 25 μm phospholipid vesicles (5/95, PS/PC). Thrombin generation was started by addition of preheated FXa (5 nmfinal concentration) and allowed to proceed for 1 min before being stopped. A magnetic bead-based assay for FXa binding to membrane-bound FVa was performed as described by Rudolph, after some modifications (39Rudolph A.E. Porche-Sorbet R. Miletich J.P. Biochemistry. 2000; 39: 2861-2867Crossref PubMed Scopus (30) Google Scholar). In this assay, biotinylated phospholipid vesicles were immobilized on the surface of streptavidin-coated magnetic beads. The beads were washed twice with the assay buffer and then quenched during constant shaking with BSA-containing buffer for 4 h at 37 °C. The beads (2.5 mg/ml) were mixed with 2 mm small unilamellar phospholipid vesicles (biotinylated-PE/PE/PS/PC/[14C]PC, with the molar ratio 1/9/20/70/trace amounts) and incubated for 2 h at 37 °C during constant shaking. The beads were then washed twice and resuspended in assay buffer. The concentration of phospholipid was determined by counting [14C]PC and typically found to be around 50 μm. To measure the binding of FXa to membrane-bound FVa, FVa (0.5 nm) was incubated for 10 min with the phospholipid-coated beads. The final concentration of the phospholipid was 625 nm (this concentration was experimentally found to give the best signal-to-noise ratio). Increasing concentrations of125I-bFXa (200–6000 pm) were incubated with the beads for 30 min. The binding reaction was stopped by isolation of the magnetic beads using an MPC-96 plate (Dynal). The collected beads were washed with ice-cold assay buffer to reduce nonspecific binding. The amount of FXa that was associated with the beads and the FXa remaining in the supernatants were measured. Nonspecific binding determined from reactions containing a 100-fold excess of unlabeled bFXa was subtracted from the total binding. In addition, binding that was not dependent on FVa was determined in parallel from reactions lacking added FVa. To estimate the K d of the FXa binding to FVa, the amount of bound FXa was plotted as a function of added FXa concentration. The data were fitted to the above equation for a single site-binding isotherm via non-linear least squares regression analysis. In control experiments, plasma-purified FVa and recombinant WT FVa in conditioned medium behaved in an identical manner. All results are presented as mean values ± S.E. of three independent experiments performed in duplicate. To identify potential interaction sites on FVa for FXa and/or prothrombin, the consensus' sequence Asn-X-Thr for N-linked glycosylation was introduced at thirteen different positions in FV. We took advantage of previously identified interaction sites for FIXa in FVIIIa when choosing probing sites, assuming the FIXa·FVIIIa and FXa·FVa complexes to have similar molecular architectures. Four different FIXa interaction sites in FVIIIa have been identified; three in the A2 domain comprising regions 511–530, 558–565, and 692–710 and one in the A3 domain composed of residues 1811–1818 (20Celie P.H. Van Stempvoort G. Jorieux S. Mazurier C. Van Mourik J.A. Mertens K. Br. J. Haematol. 1999; 106: 792-800Crossref PubMed Scopus (21) Google Scholar, 21O'Brien L.M. Medved L.V. Fay P.J. J. Biol. Chem. 1995; 270: 27087-27092Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22Jorquera J.I. McClintock R.A. Roberts J.R. MacDonald M.J. Circulation. 1992; 86: 685aGoogle Scholar, 23Lenting P.J. van de Loo J.W. Donath M.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1996; 271: 1935-1940Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Corresponding regions in FVa include residues 455–474, 502–509, and 638–655 in the A2 domain and 1676–1683 in the A3 domain. Therefore, carbohydrate side chains were introduced at FV residues 461, 467, 511, 652, 655, 1677, and 1683 to probe the different regions for FXa interaction sites. In addition, six other carbohydrate side chains were introduced in areas surrounding these segments. Thes" @default.
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• W2167633411 title "Defining the Factor Xa-binding Site on Factor Va by Site-directed Glycosylation" @default.
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