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.
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.
Sakai
T
T
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131.
Lund-Hansen
T
T
Paborsky
L
L
Pedersen
A H
AH
Kisiel
W
W
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
D005167:Factor VII; D005170:Factor X; D012697:Serine Endopeptidases; D015942:Factor VIIa; D015951:Factor Xa