Involvement of the hydrophobic stack residues 39–44 of factor VIIa in tissue factor interactions

Abstract

Des(1–38) factor VIIa and des(1–44) factor VIIa were obtained by limited proteolysis. The binding of tissue factor to these factor VIIa-derivatives was assessed from its stimulation of the proteolytic activity on chromogenic obgopeptide substrates. Compared to native factor VIIa (KTF = 0.6 ± 0.1 nM), Tissue factor binds to des(1–38) factor VIIa with a lower, but still significant affinity (KTF = 4.8 ± 0.3 nM). The activity of des(1–44) factor VIIa was only slightly stimulated by TF (KTF ∼ 200 nM). Binding of TF depends critically on the presence of Ca2+ ions. Ca2+ ions stimulated the activity of factor VIIa/TF with an apparent KCa = 0.16 ± 0.02 mM. Factor VIIa in the absence of tissue factor was stimulated by Ca2+ with an apparent KCa = 0.05 ± 0.01 mM, and similar KCa values were obtained for the truncated derivatives of factor VIIa. Measurements of Ca2+-induced changes in intrinsic protein fluorescence suggest a conformational change. The Ca2+ ion concentration at which this change occurred was higher for des(1–44) factor VIIa (apparent KCa = 0.14 mM) than for des(1–38)- and native factor VIIa (apparent KCa = 0.04 mM). The Tb3+ ion luminescence technique was used to further investigate the Ca2+ binding sites. Tb3+ ions bound with a lower affinity to des(1–44) factor VIIa than to des(1–38)-and native factor VIIa. The observed drastic decrease in affinity for tissue factor as a result of truncation of the ‘hydrophobic stack’ residues 39–44, suggest that this region of factor VIIa provides a structural determinant that together with other regions participates in tissue factor binding