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

Abstract

The structural and functional effects of Ca'+ 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. Ca'+ 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 Caz+ 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 y-carboxyglutamicacid-dependent Ca2+ binding. Mg" appeared to bind to the site(s) of relatively higher affinity since, although it was less efficient than Ca", it stimulated the amidolytic activity and induced quenching of the intrinsic fluorescence. In contrast, Mg'+ did not induce expression of the phospholipid-interactive structure. The binding properties of two monoclonal antibodies that recognized epitopes in the y-carboxyglutamic-acid-rich domain of factor VIIa corroborated the occurrence of two Ca' ' -induced, sequential structural changes and only one of the antibodies recognized the Mg2' -induced structure. Thus Ca2+ binding to the y-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 Ca'+ 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 Ca'+ enhancement was observed at 0.25 mM. This coincided with a Ca'+-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 Ca" binding to a site located in the serine protease part.

Site‐directed mutagenesis but not γ‐carboxylation of Glu‐35 in factor VIIa affects the association with tissue factor

“…A lower apparent binding of F6A4 was observed for 735V-fVIIa. Residue 35 is either part of or in close proximity of the epitope [14] and the lower response supports the assumption that the T35V mutation introduces structural changes that also may cause the decreased TF binding, The Ca 2+ dependencies of all fVIIa mutants for binding to F6A4 were nevertheless similar to that of the wild-type protein (Fig. 2).…”

“…The two forms were identified by mass spectrometry and N-terminal amino acid sequencing (with positive identification of Gla) of peptic and tryptic peptides (manuscript in preparation). The ELISA employing the monoclonal antibodies F1A2 and F6A4 was carried out as described [14]. SDS-PAGE was run according to Laemmli [15].…”

Mechanism of the Ca2+-induced Enhancement of the Intrinsic Factor VIIa Activity

“…Removal of Ca 2ϩ reduces the activity of FVIIa to less than 10% (14), indicating the potentially essential charge-neutraliz- (43,44). FIXa is also modestly stimulated (3-fold), and this has, like for FVIIa, been shown to be accompanied by insertion of the N terminus into the activation pocket (45) , and Met 298 play significant roles in, but are not fully responsible for, maintaining this physiological control mechanism.…”

“…Upon binding of the cofactors, an activity increase of several 1,000-fold is observed, in part by shifting the equilibrium from the zymogen-like form toward the catalytically active conformation of FVIIa (12,13). Ca 2ϩ binding alone leads to a more than 10-fold increase in activity (14).…”

Activation and Active Site Occupation Alter Conformation in the Region of the First Epidermal Growth Factor-like Domain of Human Factor VII

“…It is the light chain of FVII that contains the first epidermal growth factor-like domain (EGF-1), a stretch of 37 amino acids with characteristic structure that has been implicated as the principal site of FVII interaction with TF (5)(6)(7)(8). The FVII molecule also requires calcium for the expression of optimum activity, one molecule of which is bound in each of the protease domains (9,10) and the EGF-1 domain at a high affinity site (11), with seven more Ca 2ϩ molecules bound with variable affinity by the Gla domain (12).…”