The structure of the FVIIa-TF complex . The backbone traces of FVIIa and TF are shown as gray ribbon and a black wire, respectively. The nine calcium ions bound to FVIIa are shown as spheres. The Gla and EGF-like domains of FVIIa form a stalk along TF and the protease domain rests on top of TF. The catalytic triad and residue M306 in FVIIa are shown in ball-and-stick representation (marked with a solid arrow and a dotted arrow, respectively, for clarity).
In the complex with membrane-bound TF, FVIIa generates FXa (and FIXa) at a dramatically higher rate than it does in the free form . As the same peptide bond in FX is to be cleaved during activation, free and TF-bound FVIIa presumably position themselves identically relative to FX, but the presence of TF helps to bring enzyme and substrate together. More importantly, TF association also stimulates the enzymatic activity of FVIIa. The mechanism behind the TF-induced increase in activity remains elusive. The differences between free and TF-bound FVIIa are very subtle, both when their available crystal structures [14,15] are compared and when the structural changes induced by TF are monitored spectroscopically . Thus, no conspicuous structural rearrangements appear to occur. Recent work has identified residue M306 in FVIIa as a key mediator of the TF-induced allosteric effect [17,20]. However, the precise route of the allosteric signal from the TF-inter-active surface to the active site and the conformational switches are unknown. But it is justifiable to state that interactions between the protease domain of FVIIa and TF unleash the full enzymatic potential, and that the three other domains tether FVIIa to its cofactor and to the membrane surface. In the free form not bound to TF, the form in which we assume that the therapeutic rFVIIa molecule exerts its function, FVIIa has low biological activity and poor membrane affinity. Therefore, high doses of rFVIIa are needed for it to be efficacious in the treatment of bleeding episodes.
By activation, the single-chain rFVII is converted to the two-chain form (rFVIIa), consisting of light and heavy chains linked by a disulfide bridge (Figure 10.3). The structural characterization of rFVIIa includes determination of the amino acid sequence as well as determination of the posttranslational modifications. These are y-carboxylation of glutamic acid in the N-terminal Gla domain, N-linked glycosylation of N145 and N322, and O-linked glycosylation of S52 and S60.
The amino acid sequence of rFVIIa has been determined by sequence analysis and compared to the sequence of human plasma-derived (pd) FVIIa . After reduction and
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