Abstract

Assembly of the extrinsic pathway on cell surfaces was investigated by studying the binding and activity of factor VII on the bladder carcinoma cell line J82 which expressed 18,800 milliunits of tissue factor activity/10(6) cells. In binding studies, the association of factor VII to monolayers of cells was time-, temperature-, and calcium-dependent. The ligand binding was specific, reversible, and saturable. This interaction was inhibited by a monoclonal antibody to human brain tissue factor. Factor VII added to the cells was recovered as factor VII rather than factor VIIa when incubated in the presence of factor X neutralizing antibodies, suggesting that these cells produced factor X. Specific factor VII binding to the cell revealed a sigmoidal binding isotherm with half-maximal binding occurring at 314 +/- 145 pM to 38,300 +/- 14,300 sites/cell. Hill plots of the binding data indicated an average slope of 2.1. Binding parameters were also determined kinetically. At maximal factor VII-tissue factor complex formation the apparent Km for factor X was 274 nM, the Vmax was 4.15 nM/min, and the kcat was estimated to be 14 s-1. In the presence of excess tissue factor and factor X, increasing amounts of factor VII added to the J82 cells demonstrated a sigmoidal relationship with the rate of factor Xa formation. Hill plots indicated a slope of 2.0 at the lower factor VII concentrations which changed to 1.0 at the higher input amounts of factor VII. Hanes plots were used to determine the apparent dissociation constant of the interaction (222 +/- 85 pM). The Vmax was 5.54 +/- 1.04 nM/min for the cleavage of factor X. These data are consistent with factor VII binding to at least two sites on tissue factor (receptor) with positive cooperativity. Because at saturation the stoichiometry of the factor VII-tissue factor complex is 1:1, tissue factor must be expressed as a dimer on the surface of the J82 cells.

Highlights

  • Of the extrinsic pathway on cell surfaces was investigatedby studying thebinding and activity of factor VI1 on the bladder carcinoma cell line 582 which expressed 18,800 milliunits of tissue factor activity/lO’ cells

  • Factor VI1 can be synthesized by induced monocytes (24) and macrophages (25) indicating that the components required for the initiation of coagulation by the extrinsic pathway can be produced locally bythese effector cells

  • We describe the binding of factor VI1 to cell surface-expressed

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Summary

RESULTS

Factor was a gift from Dr James H. The specificity of binding was studied by incubating the cells with 1nM 1251-factoVr I1 and a 100-fold molar excess of incubated with 1 ml of buffer A+ or buffer A+ containing either 0.1 unlabeled factor VII, other coagulation proteins, or unrelated mg of normal mouse IgG or specific antibody for 2 h at 37 "C. The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; BSA, bovine serum albumin; SDS, sodium about 50%.Other vitaminK-dependent or unrelated proteins did not interfere with '251-factorVI1 binding to the cells.

Cooperative Interaction of Factor VII a n d Tissue Factor r
Calcium In"
Factor B
CellslWellx lo"
DISCUSSION
Findings
Cooperative Interaction of Factor V I I and Tissue Factor
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