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.
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