Involvement of the hydrophobic stack residues 39-44 of factor VIIa in tissue factor interactions.
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 oligopeptide 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 approximately 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 Ca(2+)-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.
Biopharmaceuticals Research, Novo Nordisk A/S, Copenhagen Gentofte, Denmark.
L C
LC
Petersen
J
J
Schiødt
U
U
Christensen
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D049449:Luminescence; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013725:Terbium / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D000577:Amides; D010446:Peptide Fragments; C075846:des(1-38) factor VIIa; C087993:des(1-44) factor VIIa; D013725:Terbium; D013925:Thromboplastin; D015942:Factor VIIa; D015032:Zinc; D002118:Calcium