Isolation of the tissue factor inhibitor produced by HepG2 hepatoma cells

Progressive inhibition of tissue factor activity occurs upon its addition to human plasma (serum). This process requires the presence of factor VII(a), factor X(a), Ca²⁺, and another component in plasma that we have called the tissue factor inhibitor (TFI). A TFI secreted by HepG2 cells (human hepatoma cell line) was isolated from serum-free conditioned medium in a four-step procedure including CdCl₂ precipitation, diisopropylphosphoryl-factor X(a) affinity chromatography, Sephadex G-75 superfine gel filtration, and Mono Q ion-exchange chromatography. The purified TFI contained a predominant band at M(r) 38,000 on NaDodSO₄/polyacrylamide gel electrophoresis that comigrates with inhibitory activity. Like the activity present in plasma, this TFI requires the presence of factor VII(a), factor X(a), and Ca²⁺ to express inhibitory activity. Its specific activity (assuming an extinction coefficient of 10 at 280 nM, for a 1-cm path length through a 1% solution) was 9800 units/mg of protein, where 1 unit of TFI activity was defined as that present in 1 ml of normal pooled serum.