Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity

Abstract

A trace amount of coagulation factor VII (FVII) circulates in the blood in the activated form, FVIIa (EC 3.4.21.21), formed by internal proteolysis. To avoid disseminated thrombus formation, FVIIa remains in a conformation with zymogen-like properties. Association with tissue factor (TF), locally exposed upon vascular injury, is necessary to render FVIIa biologically active and initiate blood clotting. We have designed potent mutants of FVIIa by replacing residues believed to function as determinants for the inherent zymogenicity. The TF-independent rate of factor X activation was dramatically improved, up to about 100-fold faster than that obtained with the wild-type enzyme and close to that of the FVIIa-soluble TF complex. The mutants appear to retain the substrate specificity of the parent enzyme and can be further stimulated by TF. Insights into the mechanism behind the increased activity of the mutants, presumably also pertinent to the TFinduced, allosteric stimulation of FVIIa activity, were obtained by studying their calcium dependence and the accessibility of the N terminus of the protease domain to chemical modification. The FVIIa analogues promise to offer a more efficacious treatment of bleeding episodes especially in hemophiliacs with inhibitory antibodies precluding conventional replacement therapy.

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

“…In particular, comparative analysis of free FVIIa and of the FVIIa-TF complex showed that P303 undergoes a drastic change of solvent accessibility, from about 40% to 4%, upon TF binding. A similar process involves L305 and M298, so that a burial of apolar side chains in this domain occurs upon TF binding and FVIIa activation (Persson et al, 2001c). In this study, the difference of DG c values between the FVIIaWT and the FVIIa303T mutant form was equal to about 0AE8 kcal/mol.…”

The Endothelial Protein C Receptor Supports Tissue Factor Ternary Coagulation Initiation Complex Signaling through Protease-activated Receptors

“…Signaling of sTFFVIIa was inhibited by active site-blocked aPC, consistent with the notion that aPC and FVIIa shared the EPCR receptors that were in spatial proximity to PAR2. EPCR-dependent signaling of a cofactor-independent, superactive FVIIa DVQA mutant (29) showed that the major function of sTF was to activate FVIIa. In addition, mutation of hydrophobic residues in the ⍀ loop of the FVIIa Gla domain provided evidence that PAR2 cleavage by the sTF-FVIIa complex was dependent on Gla domain-dependent recruitment to EPCR binding sites in proximity to PAR2 (Fig.…”

The Origins of Enhanced Activity in Factor VIIa Analogs and the Interplay between Key Allosteric Sites Revealed by Hydrogen Exchange Mass Spectrometry

“…The three mutations in FVIIa DVQ are situated in the N-terminal tail of the heavy chain (V158 (21) D) and near the activation pocket (E296 (154) V and M298 (156) Q) and mimic a sequence motif from thrombin and factor IXa. The related FVIIa DVQA variant has properties similar to those of FVIIa DVQ but with a further increased activity due to the additional K337 (188) A mutation (17). In contrast to these highly active variants, FVIIa M306D has an intrinsic activity comparable with that of FVIIa.…”

“…We have discovered several such variants through site-directed mutagenesis in the protease domain of FVIIa and have selected four FVIIa analogs with unique properties for the present study. These are FVIIa M306D , which is unresponsive to activation by TF (15), FVIIa VEAY , which has optimized activity especially toward small peptidyl substrates (16), and FVIIa DVQ and FVIIa DVQA , with dramatically improved activity toward the natural macromolecular substrate factor X (FX) (17)(18)(19).…”

“…Similarly, the intrinsic activity toward FX is increased 10-fold. Similar to FVIIa VEAY , FVIIa DVQ possesses increased amidolytic activity toward small peptidyl substrates (predominantly a higher apparent k cat ), but most important, it displays a 40 -50-fold increased rate of FX activation (17). The three mutations in FVIIa DVQ are situated in the N-terminal tail of the heavy chain (V158 (21) D) and near the activation pocket (E296 (154) V and M298 (156) Q) and mimic a sequence motif from thrombin and factor IXa.…”