HUMAN TISSUE FACTOR PATHWAY INHIBITOR FUSED TO CD4 BINDS BOTH FXA ANDTF FVIIA AT THE CELL-SURFACE/

Tissue factor pathway inhibitor (TFPI) is one of the main regulators o f the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third K unitz domain were fused with domains three and four and the carboxy-te rminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expressio n using monoclonal antibodies directed against the first two TFPI Kuni tz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition o f FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated wi th FXa, specifically bound human TF-FVIIa complexes as revealed with a n anti-human TF polyclonal antibody. No functional difference was obse rved between full length or truncated TFPI-CD4. These results demonstr ate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to th e stable expression of TFPI may inhibit the development of coronary ar tery heart disease following cardiac allotransplantation, and may inhi bit thrombosis in the context of xenotransplantation.