Purification and properties of human melanoma cell tissue factor

Tissue factor (TF) is a transmembrane glycoprotein that acts as a receptor for nonactivated and activated factor VII (FVII) and triggers the coagulati on cascade. TF plays an important role in hemostasis, but may also have non coagulation functions in vascular development, angiogenesis, and tumor cell metastasis. In tumor cells, analysis of the role of TF has been hampered b y the lack of purified TF. In this study, TF antigen was identified on huma n A375 malignant melanoma cells using flow cytometry. We further purified T F apoprotein 2,000-fold to homogeneity from A375 melanoma cells using immun oaffinity chromatography. On SDS-polyacrylamide gel electrophoresis under r eduction, purified TF apoprotein gave two major protein bands corresponding to molecular weights of 53 and 34 to 36 KD. The identity of these forms of TF was confirmed by Western blotting using a polyclonal antibody against h uman brain TF. Under reduction, the TF antibody bound with a monomeric form of TF (53 KD), and without reduction, to several forms of TF (34 to 128 KD ). Preliminary carbohydrate analysis suggested that TF is a glycoprotein an d contains about 22% total carbohydrates. The coagulant activity of the pur ified apoprotein was reconstituted by the addition of phospholipids. The ef fects of varying concentrations (0 to 8 mug) of polyclonal antibodies to TF and FVII on TF procoagulant activity were studied. Both antibodies inhibit ed more than 70% of the procoagulant activity of TF in an FX activation ass ay. The complex formation between purified TF apoprotein and FVIIa was demo nstrated by using an enzyme-linked immunosorbent assay. TF formed a complex with FVIIa in a concentration-dependent and saturable manner. We conclude that in human melanoma cells, TF occurs in monomeric and heterodimeric form s and appears to have similar properties as reported for TF from other sour ces.