Factor VIII (fVIII) and von Willebrand factor (vWf) are plasma proteins that are necessary for normal hemostasis. FVIII circulates bound noncovalently to vWf, which stabilizes fVIII until it is proteolytically activated during blood coagulation. vWf also regulates fVIII expression in vivo. An understanding of how this occurs has been hindered by the fact that the cellular origin of fVIII is not known. The liver is a dominant site of fVIII synthesis, at least under some conditions. However, it is not known whether fVIII synthesis occurs in hepatocytes or non-hepatocytes (e.g., sinusoidal endothelial cells or Kupffer cells). It also is likely that spleen potentially can contribute significantly toward fVIII synthesis. However, spleen cells that synthesize fVIII have not been identified. Additionally, fVIII mRNA is found in many other tissues, including lymph node, heart, brain, lung, kidney, testes, muscle, and placenta. In this project, we will use murine models to determine the relative contribution of liver and other organs toward fVIII synthesis and/or storage. We will determine which liver and spleen cells contribute to fVIII synthesis and/or storage. We will also make these measurements in a murine model of acetaminophen-induced fulminant hepatic failure, which is characterized by an increase in fVIII synthesis and a decrease in synthesis of all other hepatic coagulation and fibrinolytic factors. To accomplish these aims, we will measure steady-state mRNA levels in murine liver, spleen, and other tissues, and in subpopulations of cells in liver and spleen under normal conditions and during acetaminophen- induced fulminant hepatic failure. Using a homologous recombination tag-and-exchange strategy, we will create a mouse that expresses a green fluorescent protein-fVIII fusion protein instead of wild-type fVIII. We will use this mouse to identify sites of fVIII synthesis and/or storage by flow cytometry and fluorescence microscopy of cell suspensions and histological tissue sections. The characterization of fVIII gene expression in vivo may lead to better approaches toward somatic cell gene therapy of hemophilia A.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL040921-13
Application #
6389066
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Link, Rebecca P
Project Start
1988-07-01
Project End
2002-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
13
Fiscal Year
2001
Total Cost
$267,058
Indirect Cost
Name
Emory University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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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
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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

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