Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Abstract

The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.

Engineering of Substrate Selectivity for Tissue Factor·Factor VIIa Complex Signaling through Protease-activated Receptor 2

“…1C). Exceptions were T99A and K192A variants with 30 -50% reduced activity as previously reported (16,29,37). Similarly, FVIIa-catalyzed FIX activation was markedly unaffected by modifications in the FVIIa protease domain (Fig.…”

“…We focused on the P2-S2 interaction known from FVIIa T99A and T99Y variants to modulate the efficacy of PAR2 cleavage while still preserving PAR2-tethered ligand interactions following activation. The P2 preference of TF⅐FVIIa has previously been determined using combinatorial fluorogenic tetrapeptide substrate libraries (29). To allow for direct comparison we determined the P2 preference of FXa under identical conditions (Fig.…”

Antibody-induced Enhancement of Factor VIIa Activity through Distinct Allosteric Pathways

“…The interaction between the enzyme and the serpin requires a well defined active site, and especially interactions in the substrate binding pockets S1-S4 seem to be crucial (30). We found that FVIIa alone is relatively slowly inhibited by AT with a second-order rate constant of 310 M Ϫ1 s…”

Activation mechanism and substrate specificity of the Drosophila initiator caspase DRONC

“…Such 'exosite' interactions are important in enhancing the catalysis of inherently poor enzymes, including metalloproteases 28 and coagulation proteases. 29,30 Since DRONC has no intrinsic preference for Glu over Asp, why is the primary autolytic cleavage site at position 352 conserved as Glu in both D. melanogaster and Drosophila pseudoobscura? To answer this will require some sophisticated experiments, but it could simply be to prevent other caspases, such as DCP-1 and DRICE with the canonical Asp specificity, 16 from adventitiously cleaving DRONC.…”