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 coagulation cascade. TF plays an important role in hemostasis, but may also have noncoagulation functions in vascular development, angiogenesis, and tumor cell metastasis. In tumor cells, analysis of the role of TF has been hampered by the lack of purified TF. In this study, TF antigen was identified on human A375 malignant melanoma cells using flow cytometry. We further purified TF apoprotein 2,000-fold to homogeneity from A375 melanoma cells using immunoaffinity chromatography. On SDS-polyacrylamide gel electrophoresis under reduction, 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 human 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 and contains about 22% total carbohydrates. The coagulant activity of the purified apoprotein was reconstituted by the addition of phospholipids. The effects of varying concentrations (0 to 8 microg) of polyclonal antibodies to TF and FVII on TF procoagulant activity were studied. Both antibodies inhibited more than 70% of the procoagulant activity of TF in an FX activation assay. The complex formation between purified TF apoprotein and FVIIa was demonstrated 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 forms and appears to have similar properties as reported for TF from other sources.
Siddiqui
F A
FA
Center for Hemostasis & Thrombosis, Clinical and Research Laboratories, Florida Hospital Cancer Institute, Orlando 32804, USA. Farooq_Siddiqui_Ph.D.@mail.fhmis.net
Amirkhosravi
A
A
Amaya
M
M
Desai
H
H
Meyer
T
T
Francis
J L
JL
D000906:Antibodies / Q000494:pharmacology; D002846:Chromatography, Affinity; D019281:Dimerization; D005167:Factor VII / Q000276:immunology / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000737:chemistry*; D009363:Neoplasm Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D013925:Thromboplastin / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000906:Antibodies; D009363:Neoplasm Proteins; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa