injections of FVIII or FVIIICPI (10?IU/injection) were administered to female hemophilia A mice (test and one-way ANOVA followed by Dunnets post hoc multiple comparison test. that protects FVIII from degradation circulation time and reduced catabolism of FVIII in hemophilia A mice. FVIIICPI complex reduced inhibitor development in hemophilia A mice following intravenous and subcutaneous administration. The data suggest that PI binding reduces catabolism and immunogenicity of FVIII and has potential to be a useful therapeutic approach for hemophilia A. could not be clearly understood α-Terpineol due to rapid uptake of PS by reticuloendothelial system (RES) (29,30). Here, we have replaced PS with another anionic phospholipid, PI, which resists RES uptake (31), and investigated its effect on the immunogenicity and catabolism of FVIII. FVIIICPI reduced inhibitor development and prolonged the circulation half-life (Characterization of FVIIICPI Activity Activity of the protein associated with PI was determined using one-stage activated partial thromboplastin time (aPTT) assay and by chromogenic assay. For aPTT assay, the samples were mixed with an equal volume of FVIII-deficient plasma and incubated at 37C. Following addition of activator (platelin-L reagent) and CaCl2, the clotting time was measured using a Coag-A-Mate XM coagulometer (Organon Teknika Corporation, Durham, North Carolina, USA). Activity of FVIII samples was also determined using Coamatic FVIII kit according to manufacturer instructions. For both assays, the activities of FVIII and FVIIICPI samples were estimated from a calibration curve constructed using the clotting times or the optical densities values determined from various dilutions of a FVIII concentrate of known activity. Conformational studies The effect of PI binding on the tertiary structure of FVIII was determined by fluorescence α-Terpineol spectroscopy. The samples (5?g/mL) were either excited at 280 or at 265?nm, and the emission spectra were obtained in the α-Terpineol wavelength range α-Terpineol of 300C400?nm. Slit widths were set at 4?nm for both the excitation and emission paths. The spectra were acquired on a PTI-Quantamaster fluorescence spectrophotometer (Photon Technology International, Lawrenceville, New Jersey, USA). The contribution of PI vesicles on the emission spectra of the protein was corrected by subtracting the spectra acquired for the vesicles alone and by using a long pass filter on emission path. Circular dichroism (CD) spectra were acquired on a JASCO-715 spectropolarimeter calibrated with d-10 camphor sulfonic acid. Far-UV CD spectra of FVIII and FVIIICPI were obtained over the range of 255 to 208?nm for secondary structural analysis using a 10-mm quartz cuvette. The protein concentration used in this experiment was 20?g/mL, and the protein/lipid Rabbit Polyclonal to SUPT16H ratio was maintained at 1:2,500. Multiple scans were obtained and averaged to improve the signal quality. FVIII CD spectra were corrected by subtracting the baseline of the Tris buffer whereas FVIIICPI spectra were corrected by subtracting the baseline of PI particles. Thermal denaturation of the FVIII and FVIIICPI was determined by monitoring the ellipticity at 215?nm from 20C to 80C using a heating rate of 60C/h with a 2-min holding time at every 5C controlled by a Peltier 300 RTS unit. The cuvette was sealed with Teflon tape in order to minimize sample loss, and volume of the sample was monitored before and after each thermal stress experiment. The temperature of the sample compartment was determined using a temperature probe that was inserted in the sample cell holder adjacent to the cuvette, as recommended by the manufacturer. The transition temperatures (denotes the magnitude of the ellipticity change defined as (Tween 20, pH?7.4) and then blocked in 1% BSA (prepared in PBS) for 2?h at room temperature. One hundred microliters of 0.5?g/mL of FVIIICPI at various protein/lipid ratios (1:5,000, 10,000, and 50,000) or PI particles in blocking buffer was incubated at 37C for 1?h. Plates were washed and then incubated with 100?L of a 1:500 dilution of rat polyclonal antibody containing a 1:1,000 dilution of goat anti-rat Ig alkaline phosphatase conjugate in blocking buffer at room temperature for 1?h. Plates were washed again and 200?L of a 1-mg/mL release kinetics FVIIICPI complexes were prepared in Tris buffer as described above and incubated at 37C in the presence or absence of 10% FVIII-deficient plasma. One of each of the tubes was taken from each treatment group at.