Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor

• Published in: The Journal of biological chemistry 1987-07, Vol.262 (20), p.9503-9508
• Main Authors: Ploplis, V A, Edgington, T S, Fair, D S
• Format: Article
• Language: English
• Subjects: Binding, Competitive; Biological and medical sciences; Blood Coagulation; Blood coagulation. Blood cells; Cell Line; Factor VII - metabolism; Factor VIIa; Factor X - metabolism; Factor Xa; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Humans; Kinetics; Molecular and cellular biology; Thromboplastin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)47961-6

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.