Structurally and Functionally Distinct Ca2+ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

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Abstract
The structural and functional effects of Ca2+ binding to vitamin-K-dependent coagulation factor VIIa were investigated. Conformational changes with a midpoint around 0.7 mM Ca2+ quenched the intrinsic protein fluorescence of a fragment of factor VIIa comprising only the light chain and this coincided with an increase in factor VIIa amidolytic activity in the absence of tissue factor. Ca2+ binding to sites in factor VIIa and in the fragment with an apparent dissociation constant of 1.3-1.4 mM induced binding to phospholipids. A similar Ca2+ dependency was not observed with factor VIIa lacking the N-terminal 38 or 44 residues of the light chain and the observed effects could thus be attributed to gamma-carboxyglutamic-acid-dependent Ca2+ binding. Mg2+ appeared to bind to the site(s) of relatively higher affinity since, although it was less efficient than Ca2+, it stimulated the amidolytic activity and induced quenching of the intrinsic fluorescence. In contrast, Mg2+ did not induce expression of the phospholipid-interactive structure. The binding properties of two monoclonal antibodies that recognized epitopes in the gamma-carboxy-glutamic-acid-rich domain of factor VIIa corroborated the occurrence of two Ca(2+)-induced, sequential structural changes and only one of the antibodies recognized the Mg(2+)-induced structure. Thus Ca2+ binding to the gamma-carboxyglutamic-acid-containing domain appeared to result in at least two distinct structural transitions with different functional consequences. The two (sets of) sites responsible for the observed effects could be distinguished based upon differences in Ca2+ affinity and metal ion selectivity. The interaction between factor VIIa and tissue factor was monitored by means of a direct binding assay and an amidolytic assay. In both systems, half-maximal Ca2+ enhancement was observed at 0.25 mM. This coincided with a Ca(2+)-induced conformational change in factor VIIa associated with fluorescence quenching. The same effect on amidolytic activity was observed with the two N-terminally truncated forms of factor VIIa and it is presumably mediated by Ca2+ binding to a site located in the serine protease part.