title: Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy.
abstract: Factor VIIa (fVIIa) is composed of four discrete domains, a gamma-carboxyglutamic acid (Gla)-containing domain, two epidermal growth factor (EGF)-like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF-like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca(2+)-dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca2+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca(2+)-induced changes in the far-UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIIa derivatives. The changes primarily appeared to reflect a Ca(2+)-induced ordering of alpha-helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K-dependent plasma proteins. The near-UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a fVIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which TF induces an enhancement of fVIIa catalytic activity.
authors.affiliation: Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Gentofte, Denmark.
authors.forename: P O
authors.initials: PO
authors.lastname: Freskgård
authors.forename: O H
authors.initials: OH
authors.lastname: Olsen
authors.forename: E
authors.initials: E
authors.lastname: Persson
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003029:Coagulants / Q000737:chemistry* / Q000378:metabolism; D015942:Factor VIIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D003029:Coagulants; D011994:Recombinant Proteins; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium