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

FEBS Letters

Petersen, Lars C.¹  Schiødt, Jakob²  Christensen, Ulla³ 

[1] Biopharmaceuticals Research, Novo Nordisk A/S Hagedornsvej 1, DK-2820 Copenhagen Gentofte, Denmark;Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark;Department of Chemistry, University of Copenhagen, Universitetsparken, DK-2100 Copenhagen, Denmark

关键词: Factor VII;    Tissue factor;    Des(1–38) factor VIIa;    Des(1–44) factor VIIa;    Ca2+-ion binding;    Gla-;    γ-carboxy-glutamic acid;    EGF domain;    epidermal growth factor-like domain;    TF;    tissue factor;    (H-d-Ile-Pro-Arg-pNA);    H-d-isoleycyl-l-prolyl-l-arginine-p-nitroanilide;    tPA;    tissue type plasminogen activator;    uPA;    urokinase-type plasminogen activator;    PC;    phosphatidyl choline;    PS;    phosphatidyl serine;   

DOI  :  10.1016/0014-5793(94)00513-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Des(1–38) factor VII_a and des(1–44) factor VII_a were obtained by limited proteolysis. The binding of tissue factor to these factor VII_a-derivatives was assessed from its stimulation of the proteolytic activity on chromogenic obgopeptide substrates. Compared to native factor VII_a (K _TF = 0.6 ± 0.1 nM), Tissue factor binds to des(1–38) factor VII_a with a lower, but still significant affinity (K _TF = 4.8 ± 0.3 nM). The activity of des(1–44) factor VII_a was only slightly stimulated by TF (K _TF ∼ 200 nM). Binding of TF depends critically on the presence of Ca²⁺ ions. Ca²⁺ ions stimulated the activity of factor VII_a/TF with an apparent K _Ca = 0.16 ± 0.02 mM. Factor VII_a in the absence of tissue factor was stimulated by Ca²⁺ with an apparent K _Ca = 0.05 ± 0.01 mM, and similar K _Ca values were obtained for the truncated derivatives of factor VII_a. 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 VII_a (apparent K _Ca = 0.14 mM) than for des(1–38)- and native factor VII_a (apparent K _Ca = 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 VII_a than to des(1–38)-and native factor VII_a. 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 VII_a provides a structural determinant that together with other regions participates in tissue factor binding.

【 授权许可】

Unknown