This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tissue factor pathway inhibitor (TFPI) blocks activation of factors X (FX) and IX by the tissue factorfactor VIIa (TF-FVIIa) complex. The observation that TFPI gene deletion results in embryonic lethality caused in part by a coagulopathy demonstrates the physiological importance of TFPI. In vitro, effective TFVIIa inhibition by soluble TFPI requires the product, factor Xa, to bind to TFPI, concentrating TFPI on negatively charged membrane surfaces and facilitating inhibition of TF-FVIIa. In vivo, most of the TFPI is associated with endothelial cells (EC) where it is ideally situated to regulate TF-FVIIa activity, perhaps circumventing the need for FXa concentrating effect. Cellular TFPI exists in at least two pools, one bound reversibly to unidentified """"""""receptors"""""""", and another probably mediated by a glycosyl phosphatidylinositol (GPI) anchor (i.e. sensitive to phospholipase C). The goal of this application is to determine the mechanisms by which TFPI associates with membrane surfaces. Although TFPI is released from EC by phospholipase C, TFPI lacks the classical membrane spanning sequence found in other GPI anchored proteins, suggesting either a novel GPI attachment mechanism or binding through a GPI anchored TFPI receptor. To address this, TFPI will be isolated from human placenta or cultured EC by immunoaffinity chromatography. Hydrophobic forms indicative of GPI anchorage will be separated by reversed phase HPLC, enzymatically digested and analyzed by mass spectrometry to identify the GPI-linked peptide. We will also isolate candidate TFPI receptors by affinity chromatography on immobilized TFPI. Detergent extracts from placenta or cultured EC will be used as a source for putative receptors. Bound proteins will be separated, tested for TFPI binding activity, subjected to micro sequencing and identified by searching databases. Potential GPI anchorage will be tested as described above. Finally, after classical sequences for GPI attachment or transmembrane domains are fused to the C-terminal region of TFPI, the impact of this modification on TF-FVIIa inhibition and the cellular distribution of the mutants will be studied. If successful, these studies will provide novel data about the role and mechanism of function of cell-bound TFPI in controlling the activity of TF in various pathological conditions.
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