Interaction of bovine coagulation factor X and its
glutamic-acid-containing fragments with phospholipid membranes
A surface plasmon resonance study
Eva-Maria Erb
1
, Johan Stenflo
1
and Torbjo¨ rn Drakenberg
2
1
Department of Clinical Chemistry, University Hospital Malmo
¨
, Lund University, Malmo
¨
, Sweden;
2
Department of Biophysical
Chemistry, Lund University, Lund, Sweden
The interaction of blood coagulation factor X and its
Gla-containing fragments with negatively charged phos-
pholipid membranes composed of 25 mol% phosphatidyl-
serine (PtdSer) and 75 mol% phosphatidylcholine (PtdCho)
was studied by surface plasmon resonance. The binding to
100 mol% PtdCho membranes was negligible. The calcium
dependence in the membrane binding was evaluated for
intact bovine factor X (factor X) and the fragment con-
taining the Gla-domain and the N-terminal EGF (epidermal
growth factor)-like domain, Gla–EGF
N
,fromfactorX.
Both proteins show the same calcium dependence in the

Blood coagulation factor X belongs to the family of vitamin
K-dependent proteins. It consists of an NH
2
-terminal
c-carboxyglutamic acid (Gla)-containing domain, followed
by two epidermal growth factor (EGF)-like domains and a
serine protease (SP) domain [1]. The Gla-domain mediates
Ca
2+
-dependent binding to biological membranes, for
example the platelet membrane [2]. Binding of factor X
and other Gla domain-containing coagulation factors is
greatly enhanced after platelet activation, due to the
exposure of negatively charged phosphatidylserine (PtdSer)
on the cell surface. The crystal structure of the Ca
2+
-loaded
form of prothrombin fragment 1 showed that six or seven of
the Gla residues ligate four to five Ca
2+
in the interior of the
protein and that three conserved residues with hydrophobic
side-chains, Phe4, Leu5 and Val8 in bovine factor X, form a
hydrophobic patch on the surfase of the domain [3–5].
These residues are thought to mediate membrane-binding
by inserting their side-chains into the membrane. This
hypothesis gained support from site directed mutagenesis
studies. In protein C the Leu5 fi Gln mutation reduces
membrane affinity and biological activity [5,6]. NMR
studies have illustrated how Ca

-bound Gla
domain and phosphate head groups in the phospholipid
membrane. This notion also gains support from numerous
studies where site-directed mutagenesis was employed to
establish the functional role of individual amino acids in Gla
domains [9–11].
Membrane binding of vitamin K-dependent coagulation
factors has previously been studied by ellipsometry [12,13],
light scattering [9,14–16] and fluorescence polarization [17].
The K
d
values determined for the same coagulation factor
Correspondence to T. Drakenberg, Department of Biophysical
Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Fax: + 46 46 222 45 43, Tel.: + 46 46 222 44 70,
E-mail:
Abbreviations: PtdSer, phosphatidylserine; PtdCho,
phosphatidtylcholine; Gla, c-carboxy glutamic acid; EGF-like,
epidermal growth factor-like; Gla–EGF
N
, a fragment comprising the
Gla domain and the first EGF domain of factor X; Gla-EGF
NC
,a
fragment comprising the Gla domain, the first and the second EGF
domain of factor X; RU, response units.
Note: this work was funded in part by the EU Biotechnology program
(contract no BIO4-CT96-0662).
(Received 20 December 2001, revised 23 April 2002,
accepted 7 May 2002)

(Darmstadt, Germany) or Sigma (St Louis, MO, USA). The
peptide corresponding to the Gla domain (residues 1–46) of
factor X, was chemically synthesized using standard Fmoc
chemistry. The fragments Gla–EGF
N
(residues 1–86) Gla–
EGF
NC
(residues 1–140, 154–183) were generated by
digestion of bovine factor X with trypsin [22]. Bovine
factor X, factor Xa and DEGR-factor Xa were purchased
from Haematologic Technologies Inc. (Burlington, VT,
USA). All surface plasmon resonance experiments were
performed on either a BIAcore X or a BIA2000 together
with L1 pioneer sensor chips (Biacore AB, Uppsala,
Sweden).
Membrane generation
Liposomes were prepared by the extruder technique and
bound to the L1 sensor chip as described previously [18].
In brief, liposomes containing either 100 mol% PtdCho,
10 mol% PtdSer/90 mol% PtdCho, 25% mol% PtdSer/
75 mol% PtdCho or 40 mol% PtdSer/60 mol% PtdCho
were injected into a Biacore instrument equipped with a L1
sensor chip. The flow rate was 10 lLÆmin
)1
. Liposomes
were captured on the sensor chip and spontaneously fused
to generate a flat lipid membrane surface. Excess liposomes
were removed by two 60 s pulses with 5 m
M

M
CaCl
2
to a final
concentration of 39 n
M
and 2 l
M
CaCl
2
, respectively. The
running buffer always had the same Ca
2+
concentration as
the protein containing buffer. Association was followed for
180 s at a flow rate of 10 lLÆmin
)1
, followed by a 600-s
dissociation phase using the same flow rate. The membrane
was regenerated by two 60 s pulses with 5 m
M
EDTA
pH 8.0 at a flow rate of 5 lLÆmin
)1
. The binding data were
fittedtoEqn(1).
Y ¼ R ½Ca
2þ

n

concentration used here would be expected to
almost completely saturate the Ca
2+
binding sites in the
Gla domain. The response signal, when using membranes
containing 25 mol% PtdSer, was corrected for the back-
ground binding to membranes composed of 100%
PtdCho. Data were evaluated with the program
BIAEVAL-
UATION
3.0 using either the simple bimolecular interaction
model or a two-step binding model as described by the
following equations. The rate equation for the bivalent
analyte model:
A þ B )
*
k
on;1
k
off;1
AB ð2Þ
AB þ B )
*
k
on;2
k
off;2
AB
2
ð3Þ

off;2
½AB
2
ð6Þ
The rate equations for the conformational change model:
A þ B )
*
k
on;1
k
off;1
AB ð7Þ
AB )
*
k
on;2
k
off;2
AB
Ã
ð8Þ
where
d½B=dt ¼Àk
on;1
½A½Bþk
off;1
½ABð9Þ
3042 E M. Erb et al.(Eur. J. Biochem. 269) Ó FEBS 2002
d½AB=dt ¼ k
on;1

]
0
¼ 0.
The total response signal is the sum of the initial response
signal R
i
plus the signals from the complexes AB and AB
2
or
AB* for the bivalent model or for the conformational
change model, respectively.
Equilibrium response signals
Equilibrium response signals were plotted vs. the protein
concentration. The K
d
values were determined by fitting the
data to Eqn (2) assuming a single class of binding sites:
saturation ¼½protein=ð½proteinþK
d
Þ: ð12Þ
The equilibrium response signal is the sum of the signals
from the intermediate complex AB and the final complex
AB
2
. However, the contribution of the second binding step
to the total response is about 15%, and therefore the
evaluation of the equilibrium response signals by Eqn (2)
gives a good approximation for the K
d
values of the first

maximal binding occurred at a calcium concentration of 1.5
and 1.4 m
M
for factor X and Gla–EGF
N
, respectively,
which is close to the concentration of free calcium in blood of
1.2 m
M
. The best fit to the data in Fig. 1 was obtained
assuming three cooperatively bound Ca
2+
ions. As shown in
Fig. 1 the membrane binding of intact factor X and the Gla–
EGF
N
fragment, showed very similar Ca
2+
-dependencies,
indicating that neither the second EGF domain nor the
serine protease domain alter those Ca
2+
-binding properties
of factor X that are relevant to membrane binding. Experi-
ments using membranes containing either 10 mol% PtdSer/
90 mol% PtdCho or 40 mol% PtdSer/60 mol% PtdCho
showedthesameCa
2+
-dependence as 25 mol% PtdSer/
75 mol% PtdCho for binding intact factor X and Gla–

) K
d
(
M
) (I) K
d
(
M
) (II)
Gla (8.0 ± 2.2) · 10
3
(3.7 ± 0.2) · 10
)2
(4.6 ± 1.3) · 10
)6
(9.4 ± 1.4) · 10
)6
Gla–EGF
N
(4.5 ± 1.1) · 10
4
(3.8 ± 0.2) · 10
)2
(8.4 ± 2.1) · 10
)7
(1.7 ± 0.3) · 10
)6
Gla–EGF
N,C
(6.7 ± 2.1) · 10

)8
Fig. 1. Ca
2+
-dependence in the membrane binding of factor X (A) and
the fragment Gla–EGF
N
(B) as determined by surface plasmon reson-
ance. Binding experiments were performed on 25 mol% PtdSer-con-
taining membranes (solid symbols) and 100 mol% PtdCho-containing
membranes (open symbols). The solid curve is the best fit to the
experimental data points obtained by Eqn (1), assuming n ¼ 3
(c
2
¼ 359.2); the dotted line assuming n ¼ 4(c
2
¼ 595.7); the dashed
line assuming n ¼ 2(c
2
¼ 715.3).
Ó FEBS 2002 Membrane binding of coagulation factor X (Eur. J. Biochem. 269) 3043
phospholipid membrane at various protein concentrations.
Similar sensorgrams were obtained for the other forms of
factor X and fragments, although with different concentra-
tions for half maximum binding (data not shown). In a first
attempt the association and dissociation processes were
treated as simple one step processes. However, with this
approach it was not possible to obtain a reasonable agree-
ment between observed and calculated sensorgrams. Mod-
els with two on-rates and two off-rates improved the fit
significantly. Moreover, a model including a conformation-

The concentration dependence of factor X binding is shown
in Fig. 2. It is apparent that the adsorption is rapid and that
a plateau is reached within 100–200 s. Figure 3 shows the
binding isotherms of factor X and its peptides. Their mem-
brane binding affinities increase in the order Gla < Gla–
EGF
N
¼ Gla–EGF
NC
<factor X ¼ factor Xa ¼ DEGR-
factor Xa (Table 1). Although both the first and second
binding step contribute to the equilibrium response signal,
the first binding step is the dominating process and the
influence from the second one, whether a conformational
change or a bifunctional ligand, has been neglected. The
consistency of the K
d
values resulting from the evaluation of
the equilibrium response signals and those obtained by
evaluating the first step in the association phase of the
sensorgrams justifies this assumption.
DISCUSSION
Calcium binding to the Gla domain is known to be crucial
for the induction of a conformation in the domain that
mediates membrane binding. Early studies employing
equilibrium dialysis established the existence of about 10
Ca
2+
-binding sites, at least three of which mediate cooper-
ative binding [23–26]. By studies of the binding of divalent

evident that unlike Ca
2+
-binding, Mg
2+
-binding to the
Fig. 3. Equilibrium isotherms of factor X and its Gla-containing frag-
ments binding to membranes containing 25 mol% PtdSer in the presence
of 10 m
M
Ca
2+
. The measured equilibrium binding signal is plotted
against the solution phase concentration of factor X (d), factor Xa
(m), DEGR-factor Xa (n), Gla–EGF
NC
(e), Gla–EGF
N
(r)andGla
(.). Solid lines indicate the least-square fit of the Langmuir model to
this data as described in Materials and methods. The estimated binding
parameters are listed in Table 1.
Fig. 2. Adsorption and desorption kinetics of factor X to 25 mol%
PtdSer containing membranes. Experiments were performed using
10 m
M
Tris/HCl,pH7.5,150m
M
NaCl, 10 m
M
CaCl

that the metal ion binding translocated the residues that
constitute the hydrophobic patch from the interior of the
domain to the surface, allowing them to interact with the
phospholipid membrane [7]. Furthermore, these results
support the notion that the nature of this drastic conform-
ational transition must be highly cooperative with respect
to Ca
2+
due to noncompensated electrostatic repulsion
between carboxylate groups with, for instance, only one
Ca
2+
bound in this region.
We have now found that the Ca
2+
concentration that
induces half-maximal membrane binding of factor X and
the fragment Gla–EGF
N
to PtdSer-containing membranes
is about 1.5 m
M
. This is consistent with results from light
scattering experiments with other Gla domain-containing
proteins. Thus the Ca
2+
-concentration necessary to
induce half-maximal binding has been determined to be
0.55 m
M

in blood (1.2 m
M
). It is thus
possible that binding of at least some Gla domain-
containing proteins to biological membranes will be
sensitive to local variations in the Ca
2+
concentration in
the immediate vicinity of the membrane.
We found that the isolated factor X Gla domain exhibits
low affinity binding to PtdSer-containing membranes with a
K
d
of 4.6 l
M
. This agrees well with the value of 2.4 l
M
for
factor IX (1–47) [8] and 3.7 l
M
for human protein C (1–48)
[34] measured under similar conditions (1 l
M
Ca
2+
,40%
PtdSer) by resonance energy transfer and circular dichro-
ism, respectively. The C-terminal helix of the factor X Gla
domain of Gla–EGF
N

evaluations of the experiments are in the same range as
observed previously [13,35]. The K
d
determined for factor X
is consistent with the value determined by McDonald
et al.[9].
The effect of the serine protease domain upon the
membrane affinity of the intact protein is enigmatic. It could
be due to a long distance conformational change in the
protein mediated through the two EGF-domains. In this
context it should be noted that mutation of Ca
2+
ligating
amino acids in the N-terminal part of the first EGF-like
domain of factor X influences the amidolytic activity of the
intact protein [36]. However, direct interactions between the
Gla and serine protease domains, intra or intermolecular,
might also explain the difference in binding affinities.
Another factor contributing to the higher on-rate for the
intact protein is the net charge. The Gla–EGF
NC
fragment is
highly negatively charge, especially when not saturated
with Ca
2+
()29 without Ca
2+
and )15 with 7Ca
2+
). The

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