
Assignment of molecular properties of a superactive coagulation
factor VIIa variant to individual amino acid changes
Egon Persson
1
and Ole H. Olsen
2
1
Haemostasis Biology and
2
Medicinal Chemistry Research IV, Novo Nordisk A/S, Ma
˚løv, Denmark
The most active factor VIIa (FVIIa) variants identified to
date carry concurrent substitutions at positions 158, 296 and
298 with the intention of generating a thrombin-mimicking
motif, optionally combined with additional replacements
within the protease domain [Persson, E., Kjalke, M. &
Olsen, O. H. (2001) Proc. Natl Acad. Sci. USA 98, 13583–
13588]. Here we have characterized variants of FVIIa
mutated at one or two of these positions to assess the relative
importance of the individual replacements. The E296V and
M298Q mutations gave an increased intrinsic amidolytic
activity (about two- and 3.5-fold, respectively) compared
with wild-type FVIIa. An additive effect was observed upon
their combination, resulting in the amidolytic activity of
E296V/M298Q-FVIIa being close to that of the triple
mutant. The level of amidolytic activity of a variant was
correlated with the rate of inhibition by antithrombin (AT).
Compared with wild-type FVIIa, the Ca
2+
dependence of
the intrinsic amidolytic activity was significantly attenuated
upon introduction of the E296V mutation, but the effect was
most pronounced in the triple mutant. Enhancement of the
proteolytic activity requires substitution of Gln for Met298.
The simultaneous presence of the V158D, E296V and
M298Q mutations gives the highest intrinsic activity and is
essential to achieve a dramatically higher relative increase in
the proteolytic activity than that in the amidolytic activity.
The N-terminal Ile153 is most efficiently buried in V158D/
E296V/M298Q-FVIIa, but is less available for chemical
modification also in the presence of the E296V or M298Q
mutation alone. In summary, E296V and M298Q enhance
the amidolytic activity and facilitate salt bridge formation
between the N-terminus and Asp343, E296V reduces the
Ca
2+
dependence, M298Q is required for increased factor X
(FX) activation, and the simultaneous presence of the
V158D, E296V and M298Q mutations gives the most pro-
found effect on all these parameters.
Keywords: factor VIIa variant; factor X activation; intrinsic
activity; superactivity; zymogenicity.
Coagulation factor VIIa (FVIIa), in contrast to other,
homologous serine proteases, possesses an active confor-
mation that is energetically unfavourable. The consequence
is a far from optimal enzymatic activity of free FVIIa, which
is dramatically enhanced upon binding to the cognate,
membrane-bound cofactor tissue factor (TF) or to its
extracellular, soluble portion (sTF) [1]. In the natural
environment, the zymogenicity of free FVIIa ensures timely
triggering and appropriate location of FVIIa haemostatic
activity upon vascular lesion and concomitant TF exposure.
The three-dimensional structure of the protease domain
of free FVIIa is, apart from certain loop regions, virtually
identical to that of thrombin and other constitutively active
and homologous serine proteases [2,3]. In addition, the
structural differences between free [3–5] and TF-bound
FVIIa [6,7] are subtle; thus the details in molecular
architecture that restrict the activity of free FVIIa remain
elusive. However, the high degree of similarity may be due
to the presence of an active site inhibitor in the structure of
the free FVIIa. The crystal (or solution) structure of
noninhibited FVIIa is presumably needed to reveal the
structural differences between latent(zymogen-like) and
activeFVIIa. However, information possibly pertaining to
the latent conformation of free FVIIa has been obtained
from the crystal structure of zymogen FVII [8]. This
structure suggests that relative bstrand movements and a
hydrogen bond involving Glu296{154} (chymotrypsinogen
numbering is given in curly brackets to facilitate compar-
isons with homologous enzymes) regulate the activity state
of FVIIa.
Recent advances in our understanding of the mechanisms
regulating the activity of FVIIa have pinpointed side chains
that function as zymogenicity determinants in the free
enzyme. Replacements of these amino acid residues have
resulted in FVIIa molecules with improved intrinsic
(TF-independent) catalytic efficiency [9–11]. The relatively
high intrinsic activity of some of these FVIIa variants
suggests that the zymogen-like conformation of free factor
VIIa is dictated by a limited number of key amino acid
residues. We have previously shown that one of these
superactive FVIIa variants, containing the mutations
V158{21}D, E296{154}V and M298{156}Q, exhibits several
properties resembling TF-bound rather than free FVIIa [9].
Apart from increased intrinsic enzymatic activity and
inhibitor susceptibility as compared with wild-type FVIIa,
this mutant has a diminished requirement for calcium ions
and a more deeply buried protease domain N-terminus
Correspondence to E. Persson, Haemostasis Biology, Novo Nordisk
A/S, Novo Nordisk Park, DK-2760 Ma
˚løv, Denmark.
Fax: + 45 44434417, Tel.: + 45 44434351,
E-mail: egpe@novonordisk.com
Abbreviations: FVIIa, coagulation factor VIIa; FX, coagulation factor
X; sTF, soluble tissue factor (residues 1–219); TF, tissue factor;
AT, antithrombin (III).
(Received 20 June 2002, revised 2 October 2002,
accepted 22 October 2002)
Eur. J. Biochem. 269, 5950–5955 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03323.x