title: Hemoglobin binds melanoma cell tissue factor and enhances its procoagulant activity.
abstract: Tissue factor (TF), the membrane-bound glycoprotein that normally initiates the coagulation pathway, is expressed on the surface of various cells including endothelial cells, fibroblasts, monocytes and tumor cells. We recently reported that hemoglobin (Hb) enhances TF expression and procoagulant activity on TF-bearing human A375 malignant melanoma cells. To elucidate the mechanism of Hb-induced TF expression, we studied the interaction between purified TF from human A375 malignant melanoma cells and Hb. Selective binding of highly purified melanoma cell TF-apoprotein to Hb was demonstrated under native conditions using a dot-immunobinding assay and under denaturing conditions by Western blotting. The complex formation between purified melanoma cell TF-apoprotein and Hb was also demonstrated by the binding of fluid-phase Hb to immobilized TF-apoprotein (0-2.0 microg/ml) in an enzyme-linked immunosorbent assay. The binding was specific, concentration-dependent, saturable and inhibited significantly (60%) by Concanavalin-A. Hb enhanced the factor X-activating procoagulant activity of melanoma cell TF in a concentration-dependent manner, but had no effect on recombinant human TF. Concanavalin-A and wheat germ agglutinin significantly (60%) inhibited the Hb-induced procoagulant activity of malignant cell TF. We conclude that TF-apoprotein selectively binds Hb, most probably via the carbohydrate moieties (alpha-d-glucosyl; alpha-d-mannosyl and N-acetyl-beta-d-glucosaminyl residues) of TF, and enhances its procoagulant activity. The physiological significance of this interaction remains to be established.
authors.affiliation: Center for Hemostasis and Thrombosis, Clinical and Research Laboratories, Florida Hospital Cancer Institute, Orlando, Florida 32804, USA. farooq.siddiqui@flhosp.org
authors.forename: F A
authors.initials: FA
authors.lastname: Siddiqui
authors.forename: J L
authors.initials: JL
authors.lastname: Francis
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005170:Factor X / Q000187:drug effects; D006031:Glycosylation; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000494:pharmacology; D008545:Melanoma / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000494:pharmacology; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013925:Thromboplastin / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014909:Wheat Germ Agglutinins / Q000494:pharmacology
chemical_list: D001059:Apoproteins; C038095:Helix lectin; D006454:Hemoglobins; D037102:Lectins; D009363:Neoplasm Proteins; D010835:Phytohemagglutinins; D011993:Recombinant Fusion Proteins; D014909:Wheat Germ Agglutinins; C100156:leukoagglutinins, plants; D003208:Concanavalin A; D005170:Factor X; D013925:Thromboplastin