Intrinsic pathway activation of factor X (fX) is catalyzed by an enzymatic complex consisting of activated factor IX (fIXa) and activated factor VIII (fVIIIa) on a phospholipid membrane surface. The regulated assembly of the intrinsic pathway fX activator is necessary for normal hemostasis. Hemophilia A and hemophilia B, which result from fVIII and fIX deficiency respectively, are the two most common inherited severe bleeding disorders. Many aspects of the biology of fVIII and fIX are not understood, particularly the structure-function relationships that govern assembly of the intrinsic pathway fX activator. Progress in this area has been hampered by difficulties associated with the isolation of FVIII. Recent progress in several laboratories including ours has resulted in the isolation of well-characterized FVIII. Accordingly, it is now feasible to directly study the macromolecular interactions in which these proteins are involved. Porcine proteins will be used as a model system because procine FVIIIA but not human FVIIIA has been isolated in stable form. However, human fVIIIa and fVIIIa will used in selected circumstances. The assembly of the intrinsic pathway fX activator on synthetic phospholipid vesicles will be studied by direct binding measurements using fluorescence polarization and fluorescence energy transfer spectroscopy and compared to the kinetics of fX activation to develop an overall model of assembly of the complex. The structure of fVIIIa ad the fIXa/FvIIIa/phospholipid vesicle complex will be studied by scanning transmission electron microscopy. Structural components of fIXa that participate in intrinsic pathway fX activation will be identified by using synthetic peptides that interfere with binding and function. The mechanism of non- proteolytic loss of fVIIIa activity will be approached by preparing plasma-derived and recombinant procine/human hybrid fVIII molecules to attempt to determine the structural basis for the increased stability of porcine VIIIa relative to the human homolog. Additionally, stabilization of fVIIIa by covalent modification of histidine and cysteine groups will be attempted. Finally, the proteolytic control of fVIII by fXa will be evaluated to determine the identity of fXa-activated species and mechanisms of their loss of activity. The new knowledge that will result from this project will provide information that will be basic to the understanding of bleeding and thrombotic disorders.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Emory University
Schools of Medicine
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Parker, Ernest T; Healey, John F; Barrow, Rachel T et al. (2004) Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope. Blood 104:704-10
van den Brink, Edward N; Bril, Wendy S; Turenhout, Ellen A M et al. (2002) Two classes of germline genes both derived from the V(H)1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII. Blood 99:2828-34
Wu, H; Reding, M; Qian, J et al. (2001) Mechanism of the immune response to human factor VIII in murine hemophilia A. Thromb Haemost 85:125-33
Barrow, R T; Healey, J F; Jacquemin, M G et al. (2001) Antigenicity of putative phospholipid membrane-binding residues in factor VIII. Blood 97:169-74
Lian, F; He, L; Colwell, N S et al. (2001) Anticoagulant activities of a monoclonal antibody that binds to exosite II of thrombin. Biochemistry 40:8508-13
Barrow, R T; Healey, J F; Gailani, D et al. (2000) Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules. Blood 95:564-8
Lollar, P (1999) Characterization of factor VIII B-cell inhibitory epitopes. Thromb Haemost 82:505-8
Healey, J F; Barrow, R T; Tamim, H M et al. (1998) Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII. Blood 92:3701-9
Lubin, I M; Healey, J F; Barrow, R T et al. (1997) Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis. J Biol Chem 272:30191-5
Lollar, P (1997) Analysis of factor VIII inhibitors using hybrid human/porcine factor VIII. Thromb Haemost 78:647-51

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