Abstract

Factor VIII (fVIII) participates in the intrinsic pathway of blood coagulation and is essential for normal hemostasis. The sites of hemostatically significant fVIII expression remain unknown and the control of fVIII expression is poorly understood. The problem is relevant to the basic biology of the hemostatic mechanism and to the development of better treatments for hemophilia A. Additionally, there is a clear association between elevated fVIII levels and thrombotic disease, which further underscores the need to understand the regulation of fVIII expression.This project has two specific aims.
In Aim 1, we will study the endogenous synthesis of fVIII in vivo. We will identify cellular sites of murine fVIII synthesis by immunolocalization. Additionally, we will study the tissue distribution of fVIII using eGFP-fVIII transgenic mice. Human fulminant hepatic failure is associated with increased fVIII, which is surprising because the liver is considered the dominant site of fVIII synthesis. Understanding this phenomenon may be a key to identifying mechanisms of fVIII regulation. Therefore, we will study the tissue distribution of fVIII and fVIII mRNA in a murine model of fulminant hepatic failure. Transplantation studies have suggested that the spleen synthesizes hemostatically significant amounts of fVIII, but conflicting results have been obtained. Therefore, we will determine whether transplantation of normal donor spleen corrects the hemostatic defect in hemophilia A mice.
In Aim 2, we will characterize the regulation of fVIII expression in heterologous systems. These systems are important models for the study of the regulation of fVIII synthesis. Additionally, heterologous expression is used in the commercial manufacture of fVIII. There is a worldwide shortage of fVIII that is due in part to low-level expression, in the last project period, we observed that expression of porcine B-domainless fVIII is approximately ten-fold higher than any level previously reported. We will identify sequences responsible for high-level expression of porcine fVIII. Additionally, we will identify the mechanism of differential expression of porcine versus human fVIII by using cell-free translation and co-translational processing systems.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL040921-16
Application #
6760004
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Link, Rebecca P
Project Start
1988-07-01
Project End
2006-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
16
Fiscal Year
2004
Total Cost
$304,000
Indirect Cost

  Institution

Name
Emory University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322

  Publications

Lollar, Pete; Winzor, Donald J (2014) Reconciliation of classical and reacted-site probability approaches to allowance for ligand multivalence in binding studies. J Mol Recognit 27:73-81
Grushin, K; Miller, J; Dalm, D et al. (2014) Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII. Haemophilia 20:723-31
Nguyen, Phuong-Cac T; Lewis, Kenneth B; Ettinger, Ruth A et al. (2014) High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance. Blood 123:2732-9
Markovitz, Rebecca C; Healey, John F; Parker, Ernest T et al. (2013) The diversity of the immune response to the A2 domain of human factor VIII. Blood 121:2785-95
Walter, Justin D; Werther, Rachel A; Brison, Caileen M et al. (2013) Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes. Blood 122:4270-8
Walter, Justin D; Werther, Rachel A; Polozova, Maria S et al. (2013) Characterization and solution structure of the factor VIII C2 domain in a ternary complex with classical and non-classical inhibitor antibodies. J Biol Chem 288:9905-14
Meeks, Shannon L; Cox, Courtney L; Healey, John F et al. (2012) A major determinant of the immunogenicity of factor VIII in a murine model is independent of its procoagulant function. Blood 120:2512-20
Summers, Ryan J; Meeks, Shannon L; Healey, John F et al. (2011) Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein. Blood 117:3190-8
Gershom, E S; Sutherland, M R; Lollar, P et al. (2010) Involvement of the contact phase and intrinsic pathway in herpes simplex virus-initiated plasma coagulation. J Thromb Haemost 8:1037-43
Meeks, S L; Healey, J F; Parker, E T et al. (2009) Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model. J Thromb Haemost 7:658-64

Showing the most recent 10 out of 37 publications