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• W2068033906 abstract "Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15–30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations. Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15–30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations. Factor VIII (FVIII) is a multidomain protein that functions as a cofactor in the coagulation cascade. FVIII is composed of six domains, NH2-A1-A2-B-A3-C1-C2-COOH. It is synthesized as a 2351-residue single chain precursor protein, which is subsequently cleaved at residue 1680 to form the heavy chain (A1-A2-B) and the light chain (A3-C1-C2) (1Toole J.J. Knopf J.L. Wozney J.M. Sultzman L.A. Buecker J.L. Pittman D.D. Kaufman R.J. Brown E. Shoemaker C. Orr E.C. Amphlett G.W. Foster B.W. Coe M.L. Knutson G.J. Fass D.N. Hewick R.N. Nature. 1984; 312: 342-347Crossref PubMed Scopus (660) Google Scholar, 2Kaufman R.J. Wasley L.C. Dorner A.J. J. Biol. Chem. 1988; 263: 6352-6362Abstract Full Text PDF PubMed Google Scholar, 3Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.D. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (658) Google Scholar). The light chain has a molecular mass of 80 kDa. The heavy chain undergoes further processing and is cleaved at several sites between the A2 and B domains, generating polypeptides with molecular masses ranging from 90 to 180 kDa (2Kaufman R.J. Wasley L.C. Dorner A.J. J. Biol. Chem. 1988; 263: 6352-6362Abstract Full Text PDF PubMed Google Scholar). The heavy chain and the light chain are held together by a divalent metal ion such as calcium (4Fay P.J. Arch. Biochem. Biophys. 1988; 262: 525-531Crossref PubMed Scopus (61) Google Scholar). The deficiency or dysfunction of FVIII activity causes hemophilia A, a life-threatening bleeding disorder. Replacement therapy with preparations of recombinant FVIII (rFVIII) 1The abbreviations used are: rFVIII, recombinant human factor VIII; ELISA, enzyme-linked immunosorbent assay; FVIII, factor VIII; OPLS, O-phospho-l-serine; PA, phosphatidic acid; PBT, phosphate buffer containing Tween; PC, phosphocholine; PHR, peak height ratio; PSP, plate-specific parameter; SEC, size exclusion chromatography; ITT, immune tolerance therapy. 1The abbreviations used are: rFVIII, recombinant human factor VIII; ELISA, enzyme-linked immunosorbent assay; FVIII, factor VIII; OPLS, O-phospho-l-serine; PA, phosphatidic acid; PBT, phosphate buffer containing Tween; PC, phosphocholine; PHR, peak height ratio; PSP, plate-specific parameter; SEC, size exclusion chromatography; ITT, immune tolerance therapy. is the treatment of choice (5VanAken W.G. Transfus. Med. Rev. 1997; 11: 6-14Crossref PubMed Scopus (10) Google Scholar). The administration of exogenous FVIII, however, leads to the development of inhibitory antibodies in 15–30% of hemophilia A patients, complicating the replacement therapy (6Jacquemin M.G. Saint-Remy J.M. Haemophilia. 1998; 4: 552-557Crossref PubMed Scopus (53) Google Scholar, 7Lollar P. Healey J.F. Barrow R.T. Parker E.T. Adv. Exp. Med. Biol. 2001; 489: 65-73Crossref PubMed Scopus (28) Google Scholar). Numerous treatment strategies, such as factor VIIa (a bypass agent), porcine FVIII (8Ingerslev J. Haematologica. 2000; 85: 15-20PubMed Google Scholar), immune tolerance therapy (ITT) with high doses of rFVIII (9Ho A.Y. Height S.E. Smith M.P. Drugs. 2000; 60: 547-554Crossref PubMed Scopus (43) Google Scholar), etc., are currently employed clinically to manage patients with inhibitors. However, rFVIII preparations that prevent the formation of inhibitors present alternate clinical approaches. The epitope regions and the mechanisms by which FVIII antibodies inhibit clotting activity are well understood. Most immunodominant epitopes of rFVIII are located in the C2, A3, and A2 domains of FVIII (7Lollar P. Healey J.F. Barrow R.T. Parker E.T. Adv. Exp. Med. Biol. 2001; 489: 65-73Crossref PubMed Scopus (28) Google Scholar, 10Gensana M. Altisent C. Aznar J.A. Casana P. Hernandez F. Jorquera J.I. Magallon M. Massot M. Puig L. Haemophilia. 2001; 7: 369-374Crossref PubMed Scopus (63) Google Scholar, 11Ananyeva N.M. Lacroix-Desmazes S. Hauser C.A. Shima M. Ovanesov M.V. Khrenov A.V. Saenko E.L. Blood Coagul. Fibrinolysis. 2004; 15: 109-124Crossref PubMed Scopus (72) Google Scholar). The immunoprecipitation and inhibitor neutralization assays of the inhibitor plasma from hemophilia A patients clearly indicated that the anti-light chain antibody titer was the highest (12Scandella D.H. Nakai H. Felch M. Mondorf W. Scharrer I. Hoyer L.W. Saenko E.L. Thromb. Res. 2001; 101: 377-385Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). More recently, Reding et al. (13Reding M.T. Okita D.K. Diethelm-Okita B.M. Anderson T.A. Conti-Fine B.M. J. Thromb. Haemost. 2003; 1: 1777-1784Crossref PubMed Scopus (86) Google Scholar) and Pratt et al. (14Pratt K.P. Qian J. Ellaban E. Okita D.K. Diethelm-Okita B.M. Conti-Fine B. Scott D.W. Thromb. Haemost. 2004; 92: 522-528Crossref PubMed Scopus (48) Google Scholar) have identified several universal epitopes for CD4+ T-cells in the 2291–2330 region of the C2 domain using proliferation assays with CD4+ cells from normal humans, hemophilia A patients (13Reding M.T. Okita D.K. Diethelm-Okita B.M. Anderson T.A. Conti-Fine B.M. J. Thromb. Haemost. 2003; 1: 1777-1784Crossref PubMed Scopus (86) Google Scholar), and mice (14Pratt K.P. Qian J. Ellaban E. Okita D.K. Diethelm-Okita B.M. Conti-Fine B. Scott D.W. Thromb. Haemost. 2004; 92: 522-528Crossref PubMed Scopus (48) Google Scholar). Three-dimensional models proposed based on crystallographic studies and mutational analysis show that the C2 domain also contains 2–4 hydrophobic loops and other charged residues that promote lipid binding (Fig. 1) (15Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.L. Nature. 1999; 402: 439-442Crossref PubMed Scopus (285) Google Scholar, 16Gilbert G.E. Kaufman R.J. Arena A.A. Miao H. Pipe S.W. J. Biol. Chem. 2002; 277: 6374-6381Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 17Stoilova-McPhie S. Villoutreix B.O. Mertens K. Kemball-Cook G. Holzenburg A. Blood. 2002; 99: 1215-1223Crossref PubMed Scopus (130) Google Scholar). Further, the C2 domain has structural features characteristic of universal immunodominant CD4+ epitopes (shown as sticks in Fig. 1) (18Raju R. Navaneetham D. Okita D. Diethelm-Okita B. McCormick D. Conti-Fine B.M. Eur. J. Immunol. 1995; 25: 3207-3214Crossref PubMed Scopus (39) Google Scholar). In the blood coagulation cascade, FVIII binds to the membrane surface of activated platelets via specific interaction between phosphatidylserine and the C2 domain (19Kemball-Cook G. Barrowcliffe T.W. Thromb. Res. 1992; 67: 57-71Abstract Full Text PDF PubMed Scopus (14) Google Scholar, 20Gilbert G.E. Furie B.C. Furie B. J. Biol. Chem. 1990; 265: 815-822Abstract Full Text PDF PubMed Google Scholar). The specificity of the interaction is mediated by O-phospho-l-serine (OPLS) the head group of phosphatidylserine (21Gilbert G.E. Drinkwater D. Biochemistry. 1993; 32: 9577-9585Crossref PubMed Scopus (88) Google Scholar). Since OPLS is known to interact with C2 domain that contains immunodominant epitopes, it would be of significance to know the effect of OPLS on the immunogenicity of rFVIII. Here we have investigated the effects of OPLS on the conformation, folding, and stability of rFVIII using physical and biochemical methods. We have also evaluated the impact of OPLS on the activity and immunogenicity of rFVIII in vivo in FVIII knock-out (hemophilia A) mice. The results suggest that binding of OPLS to immunodominant epitopes of rFVIII results in improved stability and reduction in immunogenicity of rFVIII in hemophilia A mice. We used recombinant human factor VIII (Baxter, Glendale, CA) for in vivo experiments. Monoclonal antibodies ESH4 and ESH8 were obtained from American Diagnostica Inc. (Greenwich, CT). Normal coagulation control plasma and FVIII deficient plasma for the activity assays were purchased from Trinity Biotech (County Wicklow, Ireland). Platelin L reagent used in activated partial thromboplastin time and Bethesda assays was purchased (BioMerieux, Durham, NC). The activated partial thromboplastin time and Bethesda assays were performed using a COAG-A-MATE coagulation analyzer (Organon Teknika Corp., Durham, NC). Diethanolamine, OPLS, phosphocholine calcium salt (PC), and glycerol-1-phosphate (PA) were obtained from Sigma. p-Nitrophenyl phosphate was purchased from Pierce. All buffer salts were purchased from Fisher and used without further purification. The l-form of O-phosphoserine (OPLS) was selected for our studies, since this form has been shown to interact with rFVIII to a greater extent than the d-form (21Gilbert G.E. Drinkwater D. Biochemistry. 1993; 32: 9577-9585Crossref PubMed Scopus (88) Google Scholar). The rFVIII·OPLS complex was prepared by diluting rFVIII stock solution in buffer composed of 5, 10, or 20 mm OPLS, 25 mm Tris, 5 mm CaCl2, and 300 mm NaCl, pH 7.0. The solution was incubated at room temperature for 30 min. rFVIII complexes with PA or PC were prepared as described for rFVIII·OPLS complex by diluting with Tris buffer containing 10 mm PA or PC. For immunogenicity studies, all solutions were prepared using pyrogen-free water and were sterile filtered prior to use. Complexation of rFVIII with OPLS can also be accomplished during purification of rFVIII. Briefly, rFVIII was expressed transiently by transfecting COS-7 cells with the plasmid containing the cDNA of human FVIII. The transfected cells were cultured in serum free media. The medium was subjected to a two-step ion exchange chromatography (22Doering C. Parker E.T. Healey J.F. Craddock H.N. Barrow R.T. Lollar P. Thromb. Haemost. 2002; 88: 450-458Crossref PubMed Scopus (57) Google Scholar). The factor VIII-containing medium was loaded on a HiPrep 16/10 SP FF column (Amersham Biosciences) and eluted using a linear gradient from 100–650 mm NaCl in HEPES-CaCl2 buffer containing 10 mm OPLS. Fractions containing rFVIII were loaded on a Resource Q column (Amersham Biosciences) and eluted with a 200 mm to 1 m NaCl linear gradient in the HEPES-CaCl2 buffer. The purity of the isolated protein was assessed by silver-stained SDS-PAGE and size exclusion chromatography (SEC) using a Biosep SEC-S-4000 (Phenomenex, Torrance, CA) (details to be published elsewhere). It was also found that inclusion of OPLS in the elution buffer enhanced the recovery of rFVIII. rFVIII was used at a concentration of 5 μg/ml (0.017 μm) in Tris buffer (Tris (25 mm), sodium chloride (300 mm), and calcium chloride (5 mm), pH 7). OPLS was dissolved in Tris buffer, and pH was adjusted to 7.0. Stock solution of OPLS at 1 mm concentration was prepared in Tris buffer containing 5 μg/ml rFVIII. Required volumes (as calculated by the alligation method) of the above solution were mixed with a 5 μg/ml solution of rFVIII in Tris buffer to obtain various concentrations of OPLS. Following each addition, the sample was allowed to equilibrate for 5 min before the fluorescence intensity was measured. Changes in tertiary structure of the protein in the presence of various concentrations of OPLS were monitored using a PTI-Quantamaster fluorescence spectrophotometer (Photon Technology International, Lawrenceville, NJ). Excitation wavelength was set at 285 nm, and emission was monitored at 335 nm (23Purohit V.S. Ramani K. Kashi R.S. Durrani M.J. Kreiger T.J. Balasubramanian S.V. Biochim. Biophys. Acta. 2003; 1617: 31-38Crossref PubMed Scopus (23) Google Scholar). An I-shaped cuvette was used to minimize any inner filter effect (24Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic/Plenum Publishers, New York1999: 291-318Crossref Google Scholar). The fluorescence intensity (F) at 335 nm in the presence of a given concentration of OPLS was normalized to the fluorescence intensity of rFVIII (Fo), to obtain the F/Fo ratio. The F/Fo is related to rFVIII and lipid concentrations by the following set of equations, FFo=1−(Fmax∗PLPT)(Eq. 1) PL=nPT∗[L]KD+[L](Eq. 2) where P represents protein, L is OPLS, PL is protein-OPLS complex, [L] is free OPLS concentration, PT is total protein concentration, n is stoichiometry, and Fmax is maximal change in F/Fo. The data were plotted as F/Fo versus OPLS concentration (μm) and fitted using WinNonlin (Pharsight, Mountainview, CA) with the following expression derived from the above equations to obtain estimates for KD. FFo=1−FmaxPT∗2∗[[KD+PT+LT]−[KD+PT+LT]2−4∗PT∗LT](Eq. 3) The above equation assumes a stoichiometry (n) of 1 and a single binding site. Binding of OPLS to the phospholipid-binding region of rFVIII was confirmed by evaluating the ability of OPLS to compete with ESH4, an antibody against the phospholipid-binding region of the C2 domain (25Saenko E.L. Scandella D. J. Biol. Chem. 1997; 272: 18007-18014Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) in a sandwich ELISA. Nunc-Maxisorb 96-well plates were coated with ESH4 antibody by incubating 50 μl/well solution of the antibody at a concentration of 5 μg/ml in carbonate buffer (0.2 m, pH 9.4) overnight at 4 °C. The plate was then washed with phosphate buffer containing 0.05% Tween 20 (PBT consisting of 10 mm Na2HPO4, 1.8 mm KH2PO4, 0.14 mm NaCl, 2.7 mm KCl, and 0.02% NaN3). The remaining nonspecific protein binding sites on the plastic's adsorptive surface were blocked by incubating 200 μl of blocking buffer consisting of 1% bovine serum albumin in phosphate buffer (consisting of 10 mm Na2HPO4, 1.8 mm KH2PO4, 0.14 mm NaCl, and 2.7 mm KCl) for 2 h at room temperature. The plates were washed with PBT, and 50 μl of 100 ng/ml rFVIII in the presence or absence of OPLS in blocking buffer was added and incubated at 37 °C for 1 h. The plates were washed with PBT and incubated with 50 μl of biotinylated ESH8 at a 1 μg/ml concentration and 50 μl of a 1:1000 dilution of avidin-alkaline phosphatase conjugate, both in blocking buffer at room temperature for 1 h. The plates were washed with PBT and 100 μl of 1 mg/ml p-nitrophenyl phosphate solution in diethanolamine buffer (consisting of 1 m diethanolamine and 0.5 mm MgCl2). The plates were incubated at room temperature for 30 min, and the reaction was quenched by adding 100 μl of 3 n NaOH. Optical density at 405 nm was monitored using a plate reader. Circular Dichroism Spectroscopy—CD spectroscopy was used to monitor the unfolding of rFVIII upon thermal stress. CD spectra were acquired on a JASCO-715 spectropolarimeter calibrated with d-10-camphor sulfonic acid. Thermal denaturation was performed in the presence and in the absence of 5 mm OPLS at a controlled heating rate of 60 °C/h from 20 to 80 °C. Protein concentrations typically used were 25 μg/100 μl in Tris buffer. Ellipticity was monitored at 215 nm, and the spectrum was obtained from 250 to 208 nm in a 0.1-cm quartz cuvette. The shorter path length cuvette was used to minimize the contribution of OPLS on rFVIII signal. Further the CD spectrum of rFVIII was corrected by subtracting the base-line spectrum of 5 mm OPLS buffer. Higher concentrations of OPLS were not investigated as it contributed significantly to the buffer base line. It is appropriate to mention here that thermal denaturation studies were carried out in Tris buffer because of its low metal ion binding capacity (26Derrick T.S. Kashi R.S. Durrani M. Jhingan A. Middaugh C.R. J. Pharm. Sci. 2004; 93: 2549-2557Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). However, Tris buffer has a high temperature coefficient, which causes pH changes at elevated temperatures. Hence the observed changes at elevated temperatures could be due to combination of temperature and pH changes. Steady State Fluorescence Anisotropy Studies—Changes in the steady-state emission anisotropy (r) of rFVIII upon thermal denaturation in the presence and in the absence of OPLS were measured by a PTI-Quantamaster fluorescence spectrophotometer, equipped with a Peltier unit and motorized Glan-Thompson polarizing prisms. The concentration of the protein used was typically 5 μg/ml, and a variable path length cuvette was used to minimize the inner filter effect. The G-factor was determined at 20 °C and was applied to data collected at higher temperatures. Thermal denaturation was conducted at a heating rate of 60 °C/h. Studies were carried out over the temperature range of 20–80 °C with a holding time of 1–2 min every 5 °C. Samples were excited at 280 nm, and the emission was monitored at 335 nm. Excitation and emission slit widths were set at 4 nm. Anisotropy was calculated using the following equation (24Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic/Plenum Publishers, New York1999: 291-318Crossref Google Scholar), r=III−G∗I⊥III+2∗G∗I⊥(Eq. 4) where I∥ represents emission intensity with the emission polarizer oriented parallel to the excitation polarizer, I∥ is emission intensity with the emission polarizer oriented perpendicular to the excitation polarizer, and G is instrument-specific G-factor (G = I∥/I⊥). Data were represented as percentage change in anisotropy (r) as a function of temperature. The percentage change in anisotropy (r) was computed as follows, % change in r=riro∗100(Eq. 5) where ro represents the anisotropy of the protein at 20 °C, and rt is the anisotropy at a given temperature. Size Exclusion Chromatography—SEC was performed to confirm the onset of aggregation observed in the CD experiments. SEC was performed using a Biosep-SEC4000S 300 × 4.6-mm (Phenomenex, Torrance, CA) column with an exclusion limit of 2,000,000 daltons. The column was calibrated using a standard protein mixture of known molecular weight from Bio-Rad (Hercules, CA). The chromatograph consisted of a Waters 510 isocratic pump (Waters, Milford, MA), equipped with a Shimadzu (Shimadzu, Braintree, MA) autoinjector, a column oven, fluorescence detector, and an integrator. Tris buffer was used as the mobile phase, and eluent was monitored at an excitation of 285 nm and emission of 335 nm. Typical sample injection volumes were 50 μl. SEC profiles of rFVIII (20 μg/ml) in the presence and in the absence of OPLS (5 mm) at different temperatures of thermal denaturation were obtained by heating the protein at controlled heating rates of 60 °C/h. Samples were withdrawn at 25, 35, 45, 50, 55, 60, 65, 70, and 75 °C and stored at 4 °C prior to injection onto the column. A colony of hemophilia A mice (C57BL/6J with a target deletion in exon 16 of the FVIII gene) was established with breeding pairs from the original colony (27Bi L. Lawler A.M. Antonarakis S.E. High K.A. Gearhart J.D. Kazazian Jr., H.H. Nat. Genet. 1995; 10: 119-121Crossref PubMed Scopus (513) Google Scholar). Equal numbers of adult male and female mice, aged 8–12 weeks, were used for the studies. The sex of the animal has no impact on the immune response (28Qian J. Borovok M. Bi L. Kazazian Jr., H.H. Hoyer L.W. Thromb. Haemost. 1999; 81: 240-244Crossref PubMed Scopus (117) Google Scholar). The in vivo activity of rFVIII·OPLS complex was confirmed by the tail clip method in the hemophilia A mice (29Sarkar R. Xiao W. Kazazian Jr., H.H. J. Thromb. Haemost. 2003; 1: 220-226Crossref PubMed Scopus (49) Google Scholar). Groups of animals (n = 3) were administered subcutaneously either with Tris buffer or with rFVIII alone or rFVIII·OPLS complex (10 mm). The dose of rFVIII used was ∼2 μg of protein (9.85 IU) per animal. The tip (1 cm) of the tail of each animal was cut off with a sharp scalpel about 2 h after administration. The animals were then monitored for the next 18 h and the survival was noted at the end of the period. Immunization and Sampling—Immunization of hemophilia A mice consisted of two intravenous injections that were 2 weeks apart or four subcutaneous injections 1 week apart of rFVIII or rFVIII·OPLS complex (2 μg in 100 μl of Tris buffer). The subcutaneous route of administration was investigated mainly to amplify the immune response. Further, Reipert et al. (30Reipert B.M. Ahmad R.U. Turecek P.L. Schwarz H.P. Thromb. Haemost. 2000; 84: 826-832Crossref PubMed Scopus (66) Google Scholar) have reported that the IgG subtype levels were identical after subcutaneous and intravenous administration in hemophilia A mice, suggesting an identical mechanism of immune response for subcutaneous and intravenous routes. Blood samples were obtained in acid citrate dextrose buffer at various time points up to 6 weeks by cardiac puncture. Immunogenicity of rFVIII with PA or PC was also evaluated after subcutaneous administration. All studies were performed in accordance with the guidelines of the Institutional Animal Care and Use Committees at the University at Buffalo. Measurement of Total and Inhibitory Anti-rFVIII Antibody Titers— Antibody titers were determined by standard antibody capture ELISA (31Crowther J.R. Methods in Molecular Biology. 149. Humana Press, Totowa, NJ2000Google Scholar). Briefly, Nunc-Maxisorb 96-well plates were coated overnight at 4 °C with 50 μl/well of 5 μg/ml rFVIII and subsequently blocked with 1% bovine serum albumin (blocking buffer). 50 μl/well of various dilutions of the sample and standard concentrations (12.5–150 μg/ml) of ESH8 antibody in blocking buffer were incubated at 37 °C for 1 h. The plates were washed and incubated with 50 μl of goat anti-mouse Ig (IgG + IgM + H + L)-alkaline phosphatase conjugate at a 1:1000 dilution in blocking buffer at room temperature for 1 h. Color was developed for 30 min with 100 μl of 1 mg/ml p-nitrophenyl phosphate solution in diethanolamine buffer (consisting of 1 m diethanolamine and 0.5 mm MgCl2). Reaction was quenched by the addition of 100 μl of 3 n NaOH. Optical density at 405 nm was monitored using a plate reader. The plate-specific parameter (PSP) was used to normalize the plate to plate variability. The PSP for each plate was obtained as follows. A linear standard curve of known ESH8 antibody concentrations was obtained for each plate and used to calculate the maximum and minimum predicted absorbance. Half of the difference between the maximum and minimum predicted absorbance was calculated as the PSP. A linear regression of the plot of absorbance of various dilutions (1:100 to 1:40,000) versus log of dilution was used to calculate the dilution that gave an optical density equal to the PSP. The dilution so obtained was the antibody titer for the sample. Inhibitory antibody titers were determined using the Nijmegen modification of the Bethesda assay as described previously (32Verbruggen B. Novakova I. Wessels H. Boezeman J. van den Berg M. Mauser-Bunschoten E. Thromb. Haemost. 1995; 73: 247-251Crossref PubMed Scopus (547) Google Scholar). Briefly, 100 μl of various dilutions of mouse plasma diluted in human FVIII-deficient plasma were mixed with an equal volume of normal human plasma and incubated at 37 °C for 2 h. Residual activity at the end of the incubation was determined using an activated partial thromboplastin time assay. A linear regression of the plot of residual activity of various dilutions (1:2 to 1:32,000) versus log of dilution was performed to calculate the dilution with 50% reduction in activity and was expressed in Bethesda units. Statistical analysis was conducted using Minitab Statistical Software, Minitab Release 14 (Minitab Inc., State College, PA). Effect of OPLS and Phosphatidylserine Analogs on the Tertiary Structure of rFVIII—In order to determine the effect of OPLS on the tertiary structure of the protein, the steady state fluorescence spectrum of the protein was monitored in the presence of various concentrations of OPLS. A concentration-dependent decrease in F/Fo of rFVIII at 335 nm was observed in the presence of OPLS (Fig. 2a) without any change in emission λmax. Similar spectral changes have been interpreted as conformational fluctuations for factor Va upon interaction with short chain phospholipids (33Zhai X. Srivastava A. Drummond D.C. Daleke D. Lentz B.R. Biochemistry. 2002; 41: 5675-5684Crossref PubMed Scopus (33) Google Scholar). The data were fit using Equation 3 (described under “Experimental Procedures”) to estimate the binding parameters. A high apparent KD value (5.71 μm) indicates that OPLS interaction with rFVIII is weak. Phosphatidylserine analogues with a hydrophobic acyl chain such as dihexyl phosphatidylserine showed a lower apparent KD value (3.31 μm), and the observed higher affinity is probably due to both hydrophobic and electrostatic interactions (data not shown). It is appropriate to mention that the apparent KD is a phenomenological parameter and may not represent the actual binding affinity of OPLS to rFVIII. Sandwich ELISA: Interaction of OPLS with rFVIII—Sandwich ELISA studies were carried out to determine whether OPLS binds to the lipid-binding region (2303–2332) of the C2 domain. The monoclonal antibody, ESH4, that binds to the lipid-binding region was used as a stationary antibody, whereas biotinylated ESH8 that recognizes a nonoverlapping region in the C2 domain was used as probe antibody. If OPLS binds to the lipid-binding region of the protein, it would compete with ESH4, leading to a reduction in rFVIII binding to ESH4 (Fig. 2b). As can be seen from the figure, the binding of rFVIII to ESH4 antibody reduces with increasing concentrations of OPLS, suggesting that the binding site of OPLS and ESH4 overlap. In order to evaluate the possible interference from OPLS (a charged molecule) on the amounts of ESH4 coated on the plates, a control experiment was conducted where OPLS alone was incubated with ESH4-coated wells prior to the addition of rFVIII. As can be seen from the inset of Fig. 2b, the optical densities were unaltered and are independent of the concentration of OPLS. The result indicates that the reduction of rFVIII binding to ESH4 observed with OPLS is mainly due to competition between ESH4 and OPLS for binding to rFVIII. Effect of OPLS on Thermal Denaturation of rFVIII—Thermal stress has been used to evaluate the folding and stability relationship of several proteins (34Pace C.N. Hebert E.J. Shaw K.L. Schell D. Both V. Krajcikova D. Sevcik J. Wilson K.S. Dauter Z. Hartley R.W. Grimsley G.R. J. Mol. Biol. 1998; 279: 271-286Crossref PubMed Scopus (141) Google Scholar, 35Vermeer A.W. Norde W. Biophys. J. 2000; 78: 394-404Abstract Full Text Full Text PDF PubMed Scopus (52" @default.
• W2068033906 created "2016-06-24" @default.
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• W2068033906 date "2005-05-01" @default.
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• W2068033906 title "Lower Inhibitor Development in Hemophilia A Mice following Administration of Recombinant Factor VIII-O-Phospho-L-serine Complex" @default.
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