Abstract. Factor VIII (FVIII) is an important cofactor in blood coagulation cascade. It is a multidomain protein that consists of six domains, NH 2 -A1-A2-B-A3-C1-C2-COOH. The deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. Replacement therapy using recombinant FVIII (rFVIII) is the first line of therapy, but a major clinical complication is the development of inhibitory antibodies that abrogate the pharmacological activity of the administered protein. FVIII binds to anionic phospholipids (PL), such as phosphatidylinositol (PI), via lipid binding region within the C2 domain of FVIII. This lipid binding site not only consists of immunodominant epitopes but is also involved in von Willebrand factor binding that protects FVIII from degradation in vivo. Thus, we hypothesize that FVIII-PL complex will influence immunogenicity and catabolism of FVIII. The biophysical studies showed that PI binding did not alter conformation of the protein but improved intrinsic stability as measured by thermal denaturation studies. ELISA studies confirmed the involvement of the C2 domain in binding to PI containing lipid particles. PI binding prolonged the in vivo circulation time and reduced catabolism of FVIII in hemophilia A mice. FVIII-PI 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.

Hemophilia A, a life-threatening bleeding disorder, is caused by deficiency of factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII-containing cochleate cylinders, utilizing PS-Ca(2+) interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze-thawing and rate-limiting diffusion studies revealed that small cochleate cylinders with a particle size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications.

Liposomes consisted of phosphatidylinositol (PI) and phosphatidylcholine (PC) have been utilized as delivery vehicle for drugs and proteins. In the present work, we studied the effect of soy PI on physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes such as phase state of lipid bilayer, lipid packing and phase properties using multiple orthogonal biophysical techniques. The 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan) fluorescence studies showed that presence of PI induces the formation of fluid phases in DMPC. Differential scanning calorimetry (DSC), temperature dependent fluorescence anisotropy measurements, and generalized polarization values for Laurdan showed that the presence of as low as 10 mol% of PI induces substantial broadening and shift to lower temperature of phase transition of DMPC. The fluorescence emission intensity of DPH labeled, PI containing DMPC lipid bilayer decreased possibly due to deeper penetration of water molecules in lipid bilayer. In order to further delineate the effect of PI on the physico chemical properties of DMPC is due to either significant hydrophobic mismatch between the acyl chains of the DMPC and that of soy PI or due to the inositol head group, we systematically replaced soy PI with PC species of similar acyl chain composition (DPPC and 18:2 (Cis) PC) or with diacylglycerol (DAG) respectively. The anisotropy of PC membrane containing soy PI showed largest fluidity change compared to other compositions. The data suggests that addition of PI alters structure and dynamics of DMPC bilayer in that it promotes deeper water penetration in the bilayer, induces fluid phase characteristics and causes lipid packing defects that involve its inositol head group.

A major clinical complication in the treatment of Hemophilia A using exogenously administered recombinant Factor VIII (FVIII) is the development of neutralizing antibodies. It has been shown previously that FVIII complexed with phosphatidylserine (PS) reduces the development of total and neutralizing antibody titers in hemophilic mice. The effect of complexation of FVIII with PS upon dendritic cell (DC) uptake, maturation and processing, T-cell proliferation and cytokine secretion profiles was investigated. Flow cytometric studies of DC showed that PS inhibited the up-regulation of cell surface co-stimulatory markers (CD86 and CD40). PS reduced T-cell proliferation and increased significantly levels of TGF-β and IL-10 but reduced secretion of IL-6 and IL-17 compared to controls. The data suggest that PS reduces immunogenicity of FVIII by regulating dendritic cell maturation and subsequent T-lymphocyte activity through modulation of cytokine secretion. A possible mechanism for PS-mediated induction of FVIII tolerance is discussed.

Factor VIII is a multi-domain glycoprotein and is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes Hemophilia A, a bleeding disorder. Replacement using exogenous recombinant Factor VIII (FVIII) is the first line of therapy for Hemophilia A. Immunogenicity, the development of binding (total) and neutralizing (inhibitory) antibody against administered protein is a clinical complication of the therapy. There are several product related factors such as presence of aggregates, route and frequency of administration and glycosylation have been shown to contribute to immunogenicity. The effect of route of administration of Factor VIII on antibody development in Hemophilia A is not completely understood. Here we investigated the effect of route of administration (sc or iv) on immunogenicity in Hemophilia A mice. The total and inhibitory titers were determined using ELISA and modified Bethesda Assay respectively. The results indicated that sc is more immunogenic compared to iv route in terms of total antibody titer development (binding antibodies) but no significant differences in inhibitory titer levels could be established.