Vaccines, Blood & Biologics

Studies of Clearance from the Circulation of the Blood Clotting Factor VIII, Aimed at Improving the Safety and Efficacy of FVIII Products

Principal Investigator: Andrey Sarafanov, PhD
Office / Division / Lab: OBRR / DH / LH

General Overview

Hemophilia A is a bleeding disorder caused by a deficiency of a protein called factor VIII (FVIII) found in the blood. The condition occurs in about one in 5,000-10,000 males but is very rare in women. The treatment for hemophilia A involves infusions of FVIII, which compensates for the deficiency of this protein in the blood. The therapeutic FVIII is either obtained (purified) from human blood or manufactured using recombinant DNA technology. FVIII obtained from the blood carries a risk of transmitting the blood borne infections to the patient. Therefore, most physicians prefer to use FVIII that is made through the recombinant DNA technology. At the same time, a number of other problems are still associated with the treatment. These are the complexity of the treatment since it includes as many as three to four infusions of FVIII each week and a high cost as more than $100,000 per year. Upon the treatment, about 30% of the patients develop FVIII inhibitors (immune response) that makes the treatment ineffective. Thus, the treatment would be more efficient if the therapeutic FVIII lasts longer in the circulation, thus infused less often, and is less immunogenic.

The Division of Hematology Research and Review is responsible for the review of FVIII products used for the treatment of Hemophilia A patients. In order to better characterize these products, we study how the structure of FVIII affects the rate with which it is removed from the circulation through the liver. In our project, we study how FVIII interacts with liver receptors (proteins) that bind FVIII from the blood flow. Another direction of our study is characterization of FVIII products purity as this factor contributes to formation of FVIII inhibitors in the patients. Our experimental approach includes development of relevant analytical methods, use of recombinant DNA technology, expression of proteins in cell culture and testing their interactions in purified system; also we use cell culture and animal (mice) models. The results of our study are expected to improve understanding the mechanisms of FVIII removal from the blood to facilitate review and approval of FVIII products, especially of those designed to have prolonged lifetime in the circulation. This will improve safety and efficacy of FVIII products.

Scientific Overview

Deficiency in factor VIII (FVIII) results in the blood coagulation disorder (Hemophilia A), which is treated with infusions of either plasma-derived (pd) or recombinant (r) FVIII. Due to potential of transmitting the blood borne infections to the patient by the using of pd-FVIII, the using of r-FVIII is preferable. The hemophilia community hopes for improvements in r-FVIII products, in particular, for those prolonging the lifetime of FVIII in the circulation. This would reduce the infusions frequency, and thus, the complexity and cost of the treatment. The manufacturers modify the structure of the FVIII to make it lasting longer in the circulation. Therefore, it is important for FDA regulatory scientists to understand the structure of FVIII and mechanisms of its clearance from the circulation in order to effectively assess the efficacy and safety of licensed and emerging FVIII products being developed to improve the management of Hemophilia A.

The half-life of FVIII in the circulation depends on a number of factors, among which is FVIII interaction with hepatic clearance receptors. These are low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP), acting in concert to catabolize FVIII from the circulation. We plan to map the LDLR and LRP binding sites on FVIII using site-directed mutagenesis and testing the interactions with the receptors in purified system, in cell culture and in vivo. The selected FVIII variant(s) with reduced binding to LDLR and/or LRP will be characterized for FVIII functional properties (in particular, the activity and ability to bind von Willebrand factor) and whether its half-life is increased as compared to wild-type FVIII. An FVIII variant(s), which retains the functional properties of FVIII, could be a model of a longer-lasting therapeutic FVIII.

Another part of our project is assessment of FVIII products for product-related impurities. Upon the treatment with FVIII products, about 30% of the patients develop anti-FVIII immune response, i.e. anti-FVIII antibodies neutralizing its function (inhibitors), which makes the treatment ineffective. From the literature, the risk of inhibitors formation upon the use of r-FVIII may be higher than of pd-FVIII. Most likely, this is due to difference in the structure between these forms of FVIII; in particular, r-FVIII has relatively higher content of a fraction unable to bind von Willebrand factor (FVIII*). This form of FVIII, is most likely structurally compromised and contributes to the immunogenicity of r-FVIII products. Therefore, we plan to characterize FVIII* in various in FVIII products. It is expected to define FVIII* as a product-related impurity to be controlled (regulated) in FVIII products.


Biochemistry 2015 Jan;54(2):481-9
Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.
Kurasawa JH, Shestopal SA, Woodle SA, Ovanesov MV, Lee TK, Sarafanov AG

J Biol Chem 2013 Jul 26;288(30):22033-41
Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.
Kurasawa JH, Shestopal SA, Karnaukhova E, Struble EB, Lee TK, Sarafanov AG

Protein Expr Purif 2013 Jan 7;88(2):201-6
Insect cell-based expression and characterization of a single-chain variable antibody fragment directed against blood coagulation factor VIII.
Kurasawa JH, Shestopal SA, Jha NK, Ovanesov MV, Lee TK, Sarafanov AG

Prostate 2011 Aug 1;71(11):1231-8
Prostate cancer outcome and tissue levels of metal ions.
Sarafanov AG, Todorov TI, Centeno JA, Macias V, Gao W, Liang WM, Beam C, Gray MA, Kajdacsy-Balla AA

J Virol Methods 2010 Nov;169(2):322-31
Repertoire of antibodies against type 1 poliovirus in human sera.
Rezapkin G, Neverov A, Cherkasova E, Vidor E, Sarafanov A, Kouiavskaia D, Dragunsky E, Chumakov K

J Infect Dis 2010 Jan 15;201(2):214-22
Preterm Infants' T Cell Responses to Inactivated Poliovirus Vaccine.
Klein NP, Gans HA, Sung P, Yasukawa LL, Johnson J, Sarafanov A, Chumakov K, Hansen J, Black S, Dekker CL

Biol Trace Elem Res 2008 Oct;125(1):1-12
Clinical and analytical toxicology of dietary supplements: a case study and a review of the literature.
van der Voet GB, Sarafanov A, Todorov TI, Centeno JA, Jonas WB, Ives JA, Mullick FG

Blood Coagul Fibrinolysis 2008 Sep;19(6):543-55
Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484-509.
Ananyeva NM, Makogonenko YM, Sarafanov AG, Pechik IV, Gorlatova N, Radtke KP, Shima M, Saenko EL

J Trace Elem Med Biol 2008;22(4):305-14
Analysis of iron, zinc, selenium and cadmium in paraffin-embedded prostate tissue specimens using inductively coupled plasma mass-spectrometry.
Sarafanov AG, Todorov TI, Kajdacsy-Balla A, Gray MA, Macias V, Centeno JA

Thromb Haemost 2007 Dec;98(6):1170-81
Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII.
Sarafanov AG, Makogonenko EM, Andersen OM, Mikhailenko IA, Ananyeva NM, Khrenov AV, Shima M, Strickland DK, Saenko EL

Biochemistry 2006 Feb 14;45(6):1829-40
Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.
Sarafanov AG, Makogonenko EM, Pechik IV, Radtke KP, Khrenov AV, Ananyeva NM, Strickland DK, Saenko EL

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Page Last Updated: 03/08/2016
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