Factor VII mutant V154G models a zymogen-like form of Factor VIIa

Abstract

Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.

Recombinant factor VIIa analog in the management of hemophilia with inhibitors: results from a multicenter, randomized, controlled trial of vatreptacog alfa

“…subcutaneous injection) [11,12], and the development of modified rFVIIa molecules with improved therapeutic characteristics. Strategies have focused on extending the rFVIIa plasma half-life by PEGylation or protein fusion technology [13][14][15][16][17], and/or increasing the potency and rate of onset of hemostatic action of rFVIIa through rational and targeted protein modification [6,7,[18][19][20].…”

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

“…Another conspicuous and mechanistically interesting observation is the effect (as in FVIIa IIa ) or lack of effect (as in FVIIa VEAY ) on the propensity of the N-terminal amino group of Ile 153 (16) to form a salt bridge with the side chain of Asp 343 (194). Nevertheless, the activity of both wild-type FVIIa (with a relatively exposed N-terminus) and FVIIa IIa (more buried N-terminus) suffers dramatically when an additional glycine residue is introduced [Val 154 (17) → Gly] [32] to make the tail more flexible (E. Persson, unpublished work). Thus the salient characteristic of FVIIa VEAY is a high intrinsic amidolytic activity, whereas the prominent feature of FVIIa IIa is a high intrinsic proteolytic activity.…”

Structure–Function Relationship of the Interaction between Tissue Factor and Factor VIIa

“…48 Formation of this salt bridge is critical for FVIIa activity; indeed, FVIIa with the mutation V154G is cleaved to the two-chain form normally and with wild-type macromolecular substrate affinity, 49 but with significantly reduced ability of the resultant FVIIa to activate FX. 49,50 Reduced N-terminal hydrogen-deuterium exchange upon TF binding to FVIIa supports the hypothesis that the N-terminal Ile 153 is not fully inserted into the activation pocket when free in solution. 51 Additionally, unlike most other serine proteases, oxyanion hole formation in free FVIIa does not occur upon proteolytic activation, but instead upon substrate interaction.…”