Factor VIII (fVIII) is an important plasma component required for haemostasis, since genetic defects in this molecule cause a life-threatening coagulation disorder known as Hemophilia A. This genetic disease is treated by repeated infusions of expensive fVIII products. A more effective therapy can be provided if the molecular basis of fVIII clearance is understood and a novel recombinant fVIII protein with a prolonged lifetime in circulation is developed. We have previously found that the low density lipoprotein receptor-related protein (LRP), the main endocytic liver receptor, and cell surface heparan sulfate proteoglycans (HSPGs) cooperate in the clearance of fVIII, since simultaneous blocking of these two receptor systems dramatically prolonged the lifetime of fVIII in mice. While in purified system both LRP and HSPGs were shown to interact with fVIII via the sites located within the A2 domain, the precise molecular events responsible for fVIII catabolism are currently not well characterized. We propose to identify the amino acid residues critical for fVIII interaction with LRP and HSPGs by mutational analysis of the regions previously identified as LRP and HSPGs binding sites of fVIII. The mutations will be introduced into B-domain depleted recombinant fVIII, which is functionally identical to plasma-derived fVIII and is presently used for Hemophilia A therapy. We will express these fVIII mutants in mammalian cells and test them for binding to LRP and heparin, used as model of HSPGs, in purified systems. The catabolism of the mutants will be examined in vitro using LRP-expressing cells and in vivo in a murine model of Hemophilia A. These experiments will identify fVIII mutants with reduced binding to LRP and HSPGs and will clarify the role of these two receptor systems in fVIIII clearance. The proposed studies should develop an insight into the mechanism of fVIII regulation in circulation and will provide a basis for generation of a novel type of recombinant fVIII products, having a prolonged lifetime in circulation. Development of such fVIII derivatives, which may be prospective for less expensive Hemophilia A therapy, is the long-term goal of our studies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL066101-04
Application #
6702276
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Link, Rebecca P
Project Start
2001-02-01
Project End
2004-06-30
Budget Start
2004-02-01
Budget End
2004-06-30
Support Year
4
Fiscal Year
2004
Total Cost
$4,797
Indirect Cost
Name
American National Red Cross
Department
Type
DUNS #
003255213
City
Washington
State
DC
Country
United States
Zip Code
20006
Ananyeva, Natalya M; Makogonenko, Yevgen M; Kouiavskaia, Diana V et al. (2008) The binding sites for the very low density lipoprotein receptor and low-density lipoprotein receptor-related protein are shared within coagulation factor VIII. Blood Coagul Fibrinolysis 19:166-77
Sarafanov, Andrey G; Makogonenko, Evgeny M; Pechik, Igor V et al. (2006) Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein. Biochemistry 45:1829-40
Panteleev, Mikhail A; Ananyeva, Natalya M; Greco, Nicholas J et al. (2006) Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex. FEBS J 273:374-87
Panteleev, Mikhail A; Ovanesov, Mikhail V; Kireev, Dmitrii A et al. (2006) Spatial propagation and localization of blood coagulation are regulated by intrinsic and protein C pathways, respectively. Biophys J 90:1489-500
Ovanesov, M V; Ananyeva, N M; Panteleev, M A et al. (2005) Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate. J Thromb Haemost 3:321-31
Panteleev, M A; Ananyeva, N M; Greco, N J et al. (2005) Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex. J Thromb Haemost 3:2545-53
Sarafanov, Andrey; Saenko, Evgueni (2004) High-throughput optimization of protein expression in the baculovirus system based on determination of relative expression efficiency of viral stocks. Anal Biochem 328:98-100
Saenko, Evgueni L; Ananyeva, Natalya M; Moayeri, Morvarid et al. (2003) Development of improved factor VIII molecules and new gene transfer approaches for hemophilia A. Curr Gene Ther 3:27-41
Ananyeva, Natalya; Tjurmin, Alexey; Saenko, Evgueni et al. (2003) Low density lipoproteins interact with acidic fibroblast growth factor and modify its function. Arterioscler Thromb Vasc Biol 23:601-7
Ananyeva, Natalya M; Kouiavskaia, Diana V; Shima, Midori et al. (2002) Intrinsic pathway of blood coagulation contributes to thrombogenicity of atherosclerotic plaque. Blood 99:4475-85