title: Binding of human factors VII and VIIa to a human bladder carcinoma cell line (J82). Implications for the initiation of the extrinsic pathway of blood coagulation.
abstract: We have studied the binding of radioiodinated human factor VII and its activated form, factor VIIa, to monolayers of a human bladder carcinoma cell line (J82) that expresses functional cell surface tissue factor. The binding of factors VII and VIIa to these cells was found to be time-, temperature-, and calcium-dependent. In addition, the binding of each protein to J82 cells was specific, dose-dependent, and saturable. The binding isotherms for factors VII and VIIa were hyperbolic, and Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for each protein with Kd values of 3.20 +/- 0.51 and 3.25 +/- 0.31 nM, respectively. Factors VII and VIIa, respectively, interacted with 256,000 +/- 39,000 and 320,000 +/- 31,000 binding sites/cell. Competition experiments suggested a common receptor for factors VII and VIIa. Binding of factor VIIa to the cells was completely blocked by preincubation of the cells with polyclonal anti-tissue factor IgG, whereas binding of factor VII was inhibited approximately 90%, suggesting the presence of a small number of tissue factor-independent binding sites specific for factor VII on this cell. Functional studies revealed that factor X activation by increasing amounts of cell-bound factor VII or VIIa was hyperbolic in nature. Half-maximal rates of factor Xa formation occurred at factor VII and VIIa concentrations of 3.7 +/- 0.47 and 3.2 +/- 0.31 nM, respectively. No factor VII- or VIIa-mediated activation of factor X was observed when cells were preincubated with anti-tissue factor IgG. Two-chain 125I-factor VIIa recovered from the cells was identical to the offered ligand as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In contrast, the offered single-chain 125I-factor VII was progressively converted to two-chain 125I-factor VIIa upon binding to the cells. When the J82 cells were pretreated with anti-tissue factor IgG, both factor VII recovered from the cells and factor VII in the supernatant were in the single-chain form, indicating that cell-surface tissue factor was essential for the activation of factor VII on these cells. These data indicate that binding of factor VII to tissue factor appears to be a prerequisite for its conversion to factor VIIa and the initiation of the extrinsic pathway of coagulation on these cells.
authors: Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131.
authors: T
authors: T
authors: Sakai
authors: T
authors: T
authors: Lund-Hansen
authors: L
authors: L
authors: Paborsky
authors: A H
authors: AH
authors: Pedersen
authors: W
authors: W
authors: Kisiel
mesh_terms: D001667:Binding, Competitive; D001777:Blood Coagulation; D002460:Cell Line; D005167:Factor VII / Q000378:metabolism*; D015942:Factor VIIa; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000378:metabolism; D001749:Urinary Bladder Neoplasms / Q000378:metabolism
chemical_list: D005167:Factor VII; D005170:Factor X; D012697:Serine Endopeptidases; D015942:Factor VIIa; D015951:Factor Xa