Lipid Binding Region (2303–2332) is Involved in Aggregation of Recombinant Human FVIII (rFVIII)**Karthik Ramani and Vivek S. Purohit have contributed equally to this work.

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.

Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

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Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice

Peng

Straubinger

Balu‐Iyer

2010

“…A reduction of affinity for PS in the platelet membrane could alter clotting activity. In addition, we have shown that small conformational changes in the lipid-binding region 2303-2332 were responsible for the initiation of the aggregation process (42). Based on these data, we speculate that the N-linked glycosylation proximal to this epitope may play a critical role in the observed loss of activity and aggregation (Fig.…”

Section: Discussionmentioning

confidence: 67%

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

Kosloski

Miclea

Balu‐Iyer

2009

Self Cite

Abstract. Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein-lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.

“…35 Because DCPS binds to the phospholipid binding region, we determined the effect of DCPS on the aggregation behavior of rFVIII using CD under thermal denaturing conditions. Thermal stress was chosen to investigate the effects of DCPS, as it is frequently used to understand protein folding and stability issues.…”

Section: Effect Of Dcps On Thermal Denaturation Of Rfviiimentioning

confidence: 99%

Interaction of Dicaproyl Phosphatidylserine With Recombinant Factor VIII and Its Impact on Immunogenicity

Purohit¹,

Balasubramanian²

2006

A BSTRACTReplacement therapy with exogenous recombinant factor VIII (rFVIII) to control bleeding episodes results in the development of inhibitory antibodies in 15% to 30% of hemophilia A patients. The inhibitory antibodies are mainly directed against specifi c and universal immunodominant epitopes located in the C2 domain. Previously we have shown that complexation of O-phospho-L-serine (phosphatidylserine head group) with the phospholipid binding region of the C2 domain can lead to an overall reduction in the immunogenicity of rFVIII. Here, we have investigated the hypothesis that dicaproyl phosphatidylserine, a shortchain water-soluble phospholipid, can reduce the immunogenicity of rFVIII. Circular dichroism and fl uorescence spectroscopy studies suggest that dicaproyl phosphatidylserine interacts with rFVIII, causing subtle changes in the tertiary and secondary structure of the protein. Sandwich enzyme-linked immunosorbent assay studies indicate that dicaproyl phosphatidylserine probably interacts with the phospholipid binding region of the C2 domain. The immunogenicity of FVIII-dicaproyl phosphatidylserine complexes prepared at concentrations above and below the critical micellar concentrations of the lipid were evaluated in hemophilia A mice. Our results suggest that micellar dicaproyl phosphatidylserine may be useful to reduce the immunogenicity of rFVIII preparations.