The long-term objective of this research proposal is to define mechanisms by which the production of coagulation factor VIII (FVIII) is regulated.
The specific aims of the studies described in this proposal are 1) to clarify the role played by the hepatocyte and by the hepatic sinusoidal endothelial cell in the production of FVIII by the liver; and 2) to determine how plasma levels of FVIII are regulated. These experiments will be conducted using a model of human hepatocytes and hepatic sinusoidal endothelial cells growing in cell culture. FVIII and von Willebrand factor (vWF) will be detected and quantitated in cell culture media using exquisitely sensitive enzyme linked immunosorbent assays for their respective antigens. The liver cells which, in cell culture, contain the FVIII antigen and which contain the messenger RNA (mRNA) for FVIII will be determined. The pattern of FVIII antigen and mRNA staining in tissue sections from human liver will be determined. The effects of vWF concentration and of cloned interleukin-6 on the synthesis of FVIII, as measured by FVIII antigen in cell culture media and by quantitation of FVIII mRNA, will be determined. A complete understanding of hemophilia A and of von Willebrand's syndrome will require knowledge of how FVIII synthesis is controlled and of how the FVIII/vWF complex is assembled. Furthermore, with a better understanding of how the production of FVIII is regulated at the cellular level, it may be possible to suggest alternate methods by which levels of FVIII can be manipulated therapeutically, either to increase its production (to control the clotting mechanism in persons with recurrent thromboembolism by down- regulating the intrinsic side of the coagulation cascade).
