Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor

Brit Binow Sørensen,Egon Persson,Per-Ola Freskgård,Marianne Kjalke,Mirella Ezban,Todd Williams,L Vijaya Mohan Rao

doi:10.1074/jbc.272.18.11863

Abstract

Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active site-inhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.

Highlights

Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems
A 5-fold higher concentration of factor VIIa (FVIIa) (0.28 Ϯ 0.05 nM) was required to restore 50% of the maximal activity in the presence of 0.1 nM FFR-FVIIa. Since these data suggested that FFRFVIIa bound to tissue factor (TF) with a higher affinity than FVIIa, we carried out further experiments to investigate the binding of FFR-FVIIa to cell surface TF
The direct binding of FVIIa and FFR-FVIIa to TF was characterized by two different methods: (i) by measuring the binding parameters of radiolabeled FVIIa and FFR-FVIIa to monolayers of J82 cells that constitutively express cell surface TF and (ii) by monitoring the binding of FVIIa and FFR-FVIIa to soluble TF (sTF) using a BIAcore instrument

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—A human bladder carcinoma cell line (J82) was obtained from American Type Culture Collection (ATCC: HTB-1). In experiments to measure binding at equilibrium, the monolayers were incubated for 2 h at 4 °C with varying concentrations of radiolabeled FVIIa or FFR-FVIIa in Buffer B (Buffer A supplemented with 1 mg/ml bovine serum albumin and 5 mM Ca2ϩ) in a final volume of 300 ␮l. FX Activation Assay on J82 Cells—Confluent cell monolayers in a 24-well plate were incubated with FVIIa or a combination of FVIIa and FFR-FVIIa in Buffer B (final volume, 300 ␮l) at 37 °C for 30 min to allow for the saturation of ligand binding to the cell surface TF. Binding analyses were performed on the same sTF-coated surface (regenerated with 50 mM EDTA between runs) at a protein concentration of 10 or 30 nM FVIIa and FFR-FVIIa in 20 mM Tris, pH 7.5, containing 0.1 M NaCl, 2 mM CaCl2, and 0.02% Tween 80 at 25 °C and at a flow rate of 5 ␮l/min. In the far-UV region, a 0.1-mm path length was used, whereas a 5-mm path length was used in the near-UV region

RESULTS

DISCUSSION

Additions and Corrections