Principal Investigator: Andrey Sarafanov, PhD
Office / Division / Lab: OTAT / DPPT / HB
Hemophilia A is a bleeding disorder caused by congenital deficiency of a protein called factor VIII (FVIII) found in blood. The condition occurs in about one in 5,000-10,000 males but is rare in women. The treatment for Hemophilia A involves infusions of an FVIII concentrate, which compensates for the deficiency of this protein in blood. The therapeutic FVIII is either obtained (purified) from human blood or manufactured using recombinant DNA technology. FVIII obtained from blood carries a risk of transmitting blood borne infections to the patient; therefore, most physicians prefer to use recombinant FVIII. At the same time, a number of other problems are still associated with the treatment. These are complexity of the treatment because it includes up to three-four infusions of FVIII each week and its high cost that can be more than $100,000 per year. Another complication of the treatment is that about 30% of the patients develop FVIII inhibitors (anti-FVIII antibodies) that makes the treatment ineffective. In part, this can be related to presence of FVIII forms compromised in structure (product-related impurities) that can appear during the manufacturing process. The treatment would be more efficient if the therapeutic FVIII lasts longer in the circulation, thus is infused less often, and has better purity.
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 that occurs through the liver. In our project, we study how FVIII interacts with liver receptors (proteins) that bind FVIII and remove it from the blood flow. Another direction of our study is characterization of FVIII products purity as this factor contributes to their safety for 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 mechanisms of FVIII removal from the blood and also improve purity of FVIII concentrates. This will facilitate review and approval of FVIII products, especially, those designed to have prolonged therapeutic effect in the circulation. Altogether, this will improve safety and efficacy of FVIII products for better treatment of Hemophilia A.
Congenital deficiency in blood coagulation factor VIII (FVIII) results in a blood coagulation disorder (Hemophilia A), which is treated with infusions of plasma-derived (pdFVIII) or recombinant (rFVIII) FVIII concentrates. Due to potential of transmitting the blood borne infections to the patient by using pdFVIII, using rFVIII could be preferable. The hemophilia community hopes for improvements in FVIII products, in particular, for those with the prolonged therapeutic effect. This would reduce the infusions frequency, and thus, the complexity and, possibly, cost of the treatment. Therefore, the manufacturers modify the structure of the rFVIII to make it lasting longer in the circulation. 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 life-time of FVIII in the circulation depends on a number of factors that include its interactions von Willebrand Factor (vWF), to which FVIII is bound in plasma, and a several hepatic clearance receptors. Among these receptors are the low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP), which act in concert to catabolize FVIII and FVIII/vWF, from the circulation. We plan to map the LRP- and LDLR-binding sites on both FVIII and vWF by using site-directed mutagenesis combined with hydrogen-deuterium exchange mass-spectrometry, testing interactions of the mutated FVIII and vWF with the receptors in purified system, cell culture and in vivo. The FVIII variant(s) and vWF with reduced binding to LRP (LDLR) will be characterized for functional properties and tested whether their half-lives are prolonged as compared to the respective wild-type proteins. An FVIII variant(s), which retains the functional properties of FVIII, could be a model of a longer-lasting therapeutic FVIII. In turn, a vWF variant with increased half-life could be a model of longer-lasting vWF that would be a resource of further increase of the half-life of FVIII, and improving of vWF products used for treatment of von Willebrand Disease (deficiency in vWF).
Another part of the project is assessment of FVIII products for product-related impurities. Upon the treatment with FVIII products, about 30% of the patients develop FVIII inhibitors (anti-FVIII antibodies, which neutralize the FVIII function), which makes the treatment ineffective. Recently, it was shown that the risk of inhibitors formation upon the use of rFVIII is higher than of pdFVIII. Most likely, this is due to difference in the structure between these forms of FVIII; in particular, rFVIII has higher content of FVIII fraction which is (FVIII*) unable to bind vWF. The FVIII* consists of structurally compromised forms of the protein proposed to contribute to immunogenicity of rFVIII products. We develop a new methodology to control such impurities in FVIII products, which is expected to result in their better purity, and thus safety and efficacy.
J Thromb Haemost 2017 Apr;15(4):709-20
Expression and characterization of a codon-optimized blood coagulation factor VIII.
Shestopal SA, Hao JJ, Karnaukhova E, Liang Y, Ovanesov MV, Lin M, Kurasawa JH, Lee TK, McVey JH, Sarafanov AG
Haemophilia 2016 Sep;22(5):780-9
Optimization of the thrombin generation test components to measure potency of factor VIII concentrates.
Jha NK, Shestopal SA, Gourley MJ, Woodle SA, Liang Y, Sarafanov AG, Weinstein M, Ovanesov MV
Biochim Biophys Acta 2016 Jun;1858(6):1216-27
Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.
Podoplelova NA, Sveshnikova AN, Kurasawa JH, Sarafanov AG, Chambost H, Vasil'ev SA, Demina IA, Ataullakhanov FI, Alessi MC, Panteleev MA
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