Augmented intrinsic activity of Factor VIIa by replacement of residues 305, 314, 337 and 374: evidence of two unique mutational mechanisms of activity enhancement

Abstract

Coagulation Factor VIIa (FVIIa) lacks the ability to spontaneously complete the conversion to a fully active enzyme after specific cleavage of an internal peptide bond (Arg152-Ile153) in the zymogen. Recently, several variants of FVIIa with enhanced intrinsic activity have been constructed. The in vitro characterization of these variants has shed light on molecular determinants that put restrictions on FVIIa in favour of a zymogen-like conformation and warrants continued efforts. Here we describe a new FVIIa variant with high intrinsic activity containing the mutations Leu305-->Val, Ser314-->Glu, Lys337-->Ala, and Phe374-->Tyr. The variant, called FVIIa(VEAY), processes a tripeptidyl substrate very efficiently because of an unprecedented, 5.5-fold lowering of the K(m) value. Together with a 4-fold higher substrate turnover rate this gives the variant a catalytic efficiency 22 times that of wild-type FVIIa, which is reflected in a considerably enhanced susceptibility to inhibition by antithrombin and other inhibitors. For instance, the affinity of FVIIa(VEAY) for the S1 probe and inhibitor p -aminobenzamidine is represented by an 8-fold lower K(i) value compared with that of FVIIa. Activation of Factor X in solution occurs about 10 times faster with FVIIa(VEAY) than with FVIIa, due virtually exclusively to an increased kcat value. The high activity of FVIIa(VEAY) is not accompanied by an increased burial of the N-terminus of the protease domain. A comparison of the kinetic parameters and molecular properties of FVIIa(VEAY) with those of the previously described mutant V158D/E296V/M298Q-FVIIa (FVIIa(IIa)), and the locations of the substitutions in the two variants, reveals what appear to be two profoundly different structural mechanisms dictating improvements in enzymic performance.

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

“…FVIIa, however, retains zymogen-like properties after cleavage and exhibits very low enzymatic activity. There is biochemical evidence that the new N-terminal Ile-153 (16) of FVIIa (the chymotrypsin numbering is denoted in superscript with parentheses) fails to insert into the activation pocket and that binding of TF facilitates N-terminal insertion and transition to the catalytically competent form of FVIIa (5,6). Crystal structures of FVIIa in the presence of TF and active site inhibitors provide detailed information on the active form of FVIIa ( Fig.…”

“…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).…”

A reconsideration of the evidence for structural reorganization in FVII zymogen

“…We have previously built equilibrated solution models of FVII zymogen [6], FVIIa/TFs [7] and TFs/FVIIa/FXa [8] so as to extend the X-ray data into the realm of short-time dynamics (less than a few nanoseconds) to investigate local changes. Likewise, others have provided mutational studies that provide clues for the underlying nature of the allosteric effect [3,9]. Recently, it has been proposed [10] that FVII/FVIIa exists in two forms, one of which is competent to be enhanced by TF, and the other not.…”

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The presence of tissue factor (TF) enhances the activity of factor VIIa (FVIIa) toward FX by a factor of approximately 10 5 if full length [1,2] and by a factor of approximately 76 in the soluble (TFs), truncated form [3]. The activation of FVII is also greatly enhanced by the presence of TF [4].

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“…The inhibitor peptide (A183-2) bound to FVII-2 is shown in green; the residues of FVII (1) End-stage renal disease (ESRD) patients experience a high mortality rate from cardiovascular diseases [1,2]. Vitamin D analogs that activate vitamin D receptor (VDR) such as paricalcitol and calcitriol are commonly used to manage secondary hyperparathyroidism associated with ESRD [3]. Recent clinical data show that vitamin D analogs provide survival benefit for ESRD patients in the order of paricalcitol > calcitriol > no vitamin D analog therapy, independent of the PTH and calcium levels [4,5].…”