Disulfide locked variants of factor VIIa with a restricted beta-strand conformation have enhanced enzymatic activity

Abstract

Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three-residue shift in the position of beta-strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF*FVIIa-like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Notably, FVIIa disulfide-locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their V(max)/K(m) values for FX activation but no significant improvement in TF-dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro-molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.

Figure 1.

FVIIa disulfide lock strategy. The registration of strands A2 and B2 in the TF•FVIIa-like active enzyme state is shown. The link between cysteine pairs is depicted in bold lines that were introduced at residue pairs that could form a disulfide only in the strand registration of the TF•FVIIa-like active state. The distances between C_α atoms in Å for the TF•FVIIa-like registration and (zymogen FVII) registrations are noted in the table with arrows pointing toward the engineered disulfide residue pair. The Leu-X-Val-Leu-X-Val residues important for reregistration in the zymogen and TF•FVIIa-like conformations (Eigenbrot et al. 2001) are depicted in bold ovals for both registrations. Hydrogen bonds are depicted as dashed lines.

Figure 2.

Representative SDS-PAGE gels of FVII and FVIIa mutants. Purified FVII mutant 139:157 was run on SDS-PAGE gels under both nonreduced (A) and reduced (B) conditions. FVIIa mutant 139:157 activated by FXa as described in Materials and Methods was run on a reduced SDS-PAGE gel (C) where heavy and light chains are indicated. All mutants were expressed as zymogen and remained intact. Molecular mass markers are shown in kDa. All other mutants basically gave the same results from SDS-PAGE analysis under these conditions.

Figure 3.

Kinetics of FVIIa mutants with S-2765 amidolytic activity. Representative individual kinetic analysis for amidolytic activity of S-2765 with 30 nM wild-type FVIIa (•) and 30 nM FVIIa mutants (all normalized by active site titration) 136:160 (▴), 137:159 (♦), 138:160 (▾), and 139: 157 (▪). Data for the Michaelis-Menten plot were fit to a hyperbolic equation using Kaleidagraph, from which values for K_m and V_max were derived; independent determinations were performed in triplicate.

Figure 4.

Amidolytic activity of FVIIa mutants. The fold increases in amidolytic activity (V_max/K_m) for FVIIa disulfide-locked variants in the absence of sTF relative to wild type are shown. Data for mutants with S-2765 is shown in black and with Spectrozyme fXa in gray; the fold increase is shown above the respective column.

Figure 5.

Relative TF-dependent clotting of FVIIa mutants in FVII-deficient plasma. Relative clotting times were normalized to the clotting time in FVII-deficient plasma. Data is shown for wild-type FVIIa (•) and FVIIa mutants 136:160 (▴), 137:159 (♦), 138:160 (▾), and 139:157 (▪). The average data from three independent determinations were fit by a four-parameter fit using Kaleidagraph; the error as standard deviation is shown.