FUNCTIONS OF ENDOTHELIAL CELL FACTOR IX BINDING PROTEIN

Recent studies have indicated that stimulation of endothelium by physiologic mediators results in suppression of anticoagulant and enhancement of procoagulant properties. One endothelial cell procoagulant cofactor whose expression is enhanced by tumor necrosis factor (TNF) is a cell surface binding site for Factors IX and IXa, which in the presence of Factors VIII and X is relatively selective for the enzyme (Factors IXa) and promotes it procoagulant activity. Previous work has indicated that this binding site is comprised, at least in part, of a trypsin- sensitive, cell surface protein Mr 140,000 and has established a protocol for solubilization and partial purification in a form which retains activity for several weeks. This proposal concerns completion of the isolation of this protein and characterization of its role in regulating the coagulant activity of Factor IX/IXa on quiescent and tumor necrosis factor (TNF)-stimulated endothelium. Isolation of the binding site will be followed by production of antisera, both monoclonal and polyclonal, to assess its role in kinetic studies of Factor IX and Factor X activation, and the binding of radiolabelled Factor IX/IXa to endothelium. Factor X activation on endothelium, using confluent and pre- confluent cultures, will be compared with that observed on other vascular and vessel wall-derived cells, phospholipid vesicles of varying composition, and in the presence of purified binding site. Mechanisms through which TNF up-regulates and excess Factor IX suppresses binding site expression will be examined. Regulation of endocytosis and degradation of Factor IX/IXa on quiescent and stimulated endothelium will be compared and the role of the Factor IX/IXa binding protein and of Factors VIII and X assessed. Complementary studies will examine domains of the Factor IX/IXa molecule involved in interaction with cellular binding site. Preliminary studies using a modified form of Factor IX with the delta-carboxyglutamic acid residues removed, a mutant Factor IX molecule with a defect at the start of the growth factor region, and purified activation peptide have implicated each of these domains in recognition of the cellular binding site. These studies will be extended using recombinant Factor IX molecules, naturally occurring mutants and peptides prepared from the growth factor and activation peptide domains. Using the approach one peptide, comprising residues 41-50 from Factor IX, which blocks Factor IX/IXa-binding site interaction has already been identified. Although Factor IX binding to the vessel wall has been demonstrated in vivo, a role of the Factor IX/IXa binding site in thrombosis has not been demonstrated. To address this issue, two experimental thrombosis models in which Factor IXa makes a major contribution, Wessler, stasis and TNF-induced thrombosis, will be studied. Protection from thrombosis by agents which antagonize Factor IX/IXa-cell surface interaction in vitro, such as the peptide comprising residues 41-50 of Factor IX, blocking antibodies to the binding site and active site-blocked Factor IXa, will be examined.