Differential binding of factor XII and activated factor XII
to soluble and immobilized fibronectin – localization of
the Hep-1/Fib-1 binding site for activated factor XII
Inger Schousboe
1
, Birthe T. Nystrøm
1
and Gert H. Hansen
2
1 Department of Biomedical Sciences, The Panum Institute, University of Copenhagen, Denmark
2 Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Denmark
Several studies have suggested that in the cardio-
vascular system, the interaction between the vessel wall
and the contact activation system of blood coagula-
tion, including factor XII (FXII), high molecular mass
kininogen (HK) and prekallikrein, involves Zn
2+
-
dependent and receptor-mediated binding of FXII and
HK. Thus, investigations of FXII and HK binding to
endothelial cells in the vascular wall mimicked by
Keywords
association; factor XII; factor XIIa;
fibronectin
Correspondence
I. Schousboe, Department of Biomedical
Sciences, Heart and Circulatory Research
Section, The Panum Institute, University of
Copenhagen, Blegdamsvej 3C, DK-2200
Copenhagen, Denmark
Fax: +45 35367980

of FXII in an anti-FN immunoprecipitate of plasma indicated that some
FXII in plasma circulates bound to FN. The binding of FXIIa to FN
was inhibited by gelatine and fibrin but not by heparin, indicating that
FXIIa binds to immobilized FN through the type I repeat modules.
Accordingly, FXIIa was found to bind to immobilized fragments of FN
containing the type I repeat modules in the N-terminal domain to which
fibrin and gelatine bind.
Abbreviations
CTI, corn trypsin inhibitor; DS, dextran sulfate; ECM, extracellular matrix; Fib-1, the N-terminal fibrinogen binding domain on fibronectin; FN,
fibronectin; FXII, factor XII; FXIIa, activated factor XII; Hep-1, the N-terminal heparin binding domain on fibronectin; Hep-2, the C-terminal
heparin binding domain on fibronectin; HK, high molecular mass kininogen; HRP, horseradish peroxidase; HUVEC, human umbilical vein
endothelial cell; OPD, o-phenylenediamine.
FEBS Journal 275 (2008) 5161–5172 ª 2008 The Authors Journal compilation ª 2008 FEBS 5161
cultures of human umbilical vein endothelial cells
(HUVECs) have shown that FXII and HK interact by
multiprotein assembly [1–3].
FXII is a precursor of the proteolytically active acti-
vated FXII (FXIIa). FXII and FXIIa bind equally well
to a confluent layer of HUVECs [4]. However, a recent
investigation has shown that the binding might have
been artefactual, and that FXII in the presence but
not in the absence of a negatively charged surface
bound rather to the extracellular matrix (ECM) gener-
ated during growth of HUVECs. The presence of
negatively charged surfaces appeared to serve two
purposes: (a) it induced and enhanced the autoactiva-
tion of FXII, generating FXIIa; and (b) it abrogated
nonspecific binding of FXIIa [5].
The binding of FXIIa to the ECM showed several
differences from the binding to HUVECs. Thus, the

FXII/FXIIa binding to FN
The association of FXIIa with the ECM was assumed
to take place through binding to FN. Therefore, inves-
tigations were first performed to determine whether it
could be shown that FXIIa associated with FN depos-
ited on the surface of the culture dish after depletion
of HUVECs by EDTA extraction. Immunofluores-
cence clearly showed that FXIIa bound to the depos-
ited FN (Fig. 1). No FN was deposited on and no
FXIIa bound to surfaces incubated with growth
medium under the same conditions and for the same
periods of time as the cells but in the absence of cells.
To obtain more information about this association,
the interaction between FXIIa and FN was subse-
quently analyzed by measuring the binding of FXIIa
to FN immobilized on a plastic surface.
Using a solid-phase binding assay, the binding
of FXIIa to FN was visualized by reactions with an
A
B
Fig. 1. Colocalization of the ECM-bound FXII and FN. HUVECs
were grown to near confluence, and the generated ECM was
exposed by detaching the cells with EDTA. After washing, the
ECM was incubated for 1 h with 20 n
M FXIIa. The ECM was then
washed again and incubated first with a mixture of goat anti-FXII
IgG (1 : 100) and rabbit anti-FN IgG (1 : 100) for 1 h, and second
with a mixture of Alexa 594-conjugated donkey anti-(goat IgG)
(1 : 800) and Alexa 488-conjugated goat anti-(mouse IgG) (1 : 800).
(A) Red indicates the presence of FXIIa. (B) Green indicates the

wells. After subtraction of nonspecific binding from
the total binding, saturated binding to immobilized
FN was observed at FXIIa concentrations ‡ 20 nm
(Fig. 2). Linear transformation of the the binding iso-
therm (Fig. 2 insert) obtained in one of three indepen-
dent experiments, each performed in triplicate, showed
high-affinity binding, the K
D
of which was estimated
to be 8.5 ± 0.9 nm, using all available data.
To determine whether the binding of FXIIa to
immobilized FN was mediated through the N-terminal
surface binding sequence in FXIIa, investigations were
performed to determine whether the presence of nega-
tively charged compounds such as sulfatides would
affect the binding of FXIIa to FN. This showed that
sulfatides neither inhibited nor enhanced the binding
to immobilized FN. The apparently higher-affinity
binding of FXIIa in the present experiment in the
absence than in the presence of sulfatides was due to
parallel higher nonspecific binding. However, if FXIIa
was exchanged with FXII, the presence of sulfatides
induced binding of FXII, which in the absence of
0
0.5
1
1.5
2
2.5
3

(con-
trol), respectively, and subsequently blocked with blocking buffer. Then, it was incubated for 1 h with increasing concentrations of FXIIa in
blocking buffer. The amount of bound FXIIa was determined by sequential incubation with goat anti-FXII IgG and HRP-conjugated rabbit anti-
(goat IgG) and visualized by reactions with OPD as described in Experimental procedures. d, total amount of FXIIa bound to wells coated
with FN; s, total amount of FXIIa bound to control wells (devoid of FN but ‘coated’ overnight with NaCl ⁄ P
i
; , binding of FXIIa to FN, calcu-
lated as the difference between binding of FXIIa to the former and the latter. Linear transformation of the results shown in the figure, which
is representative of three experiments performed in triplicate, gave a K
D
of 8.7 nM. Results are means ± SD (n = 3), shown by vertical bars
when extending beyond the symbols.
I. Schousboe et al. Factor XII binding to fibronectin
FEBS Journal 275 (2008) 5161–5172 ª 2008 The Authors Journal compilation ª 2008 FEBS 5163
sulfatides was negligible (Fig. 3). The sulfatide-depen-
dent binding of FXII was most likely due to a sulfat-
ide-induced and sulfatide-enhanced autoactivation of
FXII [16,17]. Accordingly, the presence of corn trypsin
inhibitor (CTI), which inhibits the activity of FXIIa,
and thus the autoactivation of FXII, almost com-
pletely blocked the sulfatide-induced binding of FXII
to FN. As compared to FXIIa, a small amount of
FXII bound to FN. Binding of the activated form of
FXII was shown by western blots of extracts of immo-
bilized FN incubated with FXII in the presence of sulf-
atides (Fig. 4).
As FXII and FXIIa bind equally well to sulfatides
[5], the lack of binding to immobilized FN of FXII
and the lack of inhibition of FXIIa by sulfatides indi-
cate that the binding is not brought about by the

0
0.5
1
1.5
2
2.5
FXII –
sulfatide
FXII +
sulfatide
FXII +
sulfatide +
CTI
FXIIa –
sulfatide
FXIIa +
sulfatide
FXIIa + anti-
FXII
antibody
Block buffer
FXIIa bound, absorbance units
Fig. 3. The effect of sulfatide on the binding of FXIIa to immobilized FN. The microtiter plate, coated overnight with FN (10 lgÆmL
)1
) and
NaCl ⁄ P
i
(control), respectively, was blocked with blocking buffer and incubated for 1 h with FXII (20 nM) and FXIIa (20 nM) in the presence
(+sulfatide) and absence ()sulfatide) of sulfatides (20 lgÆmL
)1

activity of FXIIa and thus the conversion of FXII to
FXIIa. Furthermore, in order to prevent FXII from
activation during the incubation with FN, CTI was
added to the incubation mixture. This did not affect
the binding of FN (results not shown). Thus, these
results clearly show that soluble FN interacts directly
with FXII in the absence of sulfatides. To determine
whether this interaction also occurs in plasma, the
presence of FXII was analyzed in immunoprecipitates
of FN. Plasma was immunoprecipitated with antibod-
ies against FN and adsorbed to protein G–Sepharose,
from which FXII was extracted. The plasma was not
preabsorbed to protein G–Sepharose, as binding of
FXII to the Sepharose could disturb the equilibrium
for the binding of FXII to FN. Instead, the amount of
FXII bound to protein G–Sepharose in the absence of
antibodies against FN was simultaneously analyzed
(Fig. 7). A much greater amount of FXII could be
21FXIIaFXII 43
80
50
Fig. 4. Western blot of extracts of bound protein after incubation
of FXII on immobilized FN in the absence and presence of sul-
fatides. The microtiter plate was coated overnight with FN
(10 lgÆmL
)1
) and subsequently blocked with blocking buffer. Then,
it was incubated for 1 h with 20 n
M FXII in blocking buffer in the
presence and absence of 20 lgÆmL

FXII – sulf + globular FN
FXII + sulf
FXII + sulf + globular FN
FXIIa – sulf
FXIIa – sulf + globular FN
FXIIa bound, absorbance units
FN
Control
**
*
Fig. 5. The effect of soluble FN on the binding of FXII and FXIIa to
immobilized FN. The microtiter plate was coated overnight with FN
(10 lgÆmL
)1
) and NaCl ⁄ P
i
, respectively. Then, it was blocked with
blocking buffer and incubated for 1 h with FXII (20 n
M) or FXIIa
(20 n
M) in blocking buffer in the absence ()sulf) and presence (+sulf)
of sulfatides (20 lgÆmL
)1
) and in the absence and presence of solu-
ble FN (10 lgÆmL
)1
), as indicated. The amount of FXIIa bound to FN
was measured by sequential incubation with goat anti-FXII IgG and
HRP-conjugated rabbit anti-(goat IgG) as described in Experimental
procedures. The amounts of FXIIa bound to FN and control wells are

that its interaction with immobilized FN would be
inhibited by heparin. Thus, the lack of inhibition by
heparin indicated that FXIIa did not bind to the
C-terminal high-affinity heparin-binding domain in
FN (Hep-2). However, the inhibition by high concen-
trations of DS and gelatine may indicate that FXIIa
binds to the N-terminal region of FN, including the
low-affinity Hep-1-binding domain. DS is a heparin-
like molecule and may, as such, be assumed to bind
to the heparin-binding sites on FN. To investigate
this further, the binding of FXIIa to commercially
available proteolytic fragments of FN was analyzed.
Each of these fragments contains binding domains
for heparin, gelatine and cells, respectively. Surpris-
ingly, the binding of FXIIa to these fragments
showed that although heparin was unable to inhibit
the binding of FXIIa to intact FN, FXIIa bound pri-
marily to the 30 kDa low-affinity heparin-binding
fragment (Hep-1), less to the 45 kDa gelatine-binding
fragment, and not at all to the 120 kDa fragment
containing the cell-binding domain (Fig. 10). The
amount of FXIIa that bound to the 30 kDa Hep-1-
binding fragment was similar to the amount of
FXIIa bound to FN. The N-terminal 30 kDa Hep-1-
binding domain has also been identified as a binding
site for fibrinogen and fibrin [25,26]. Further evidence
for FXIIa binding to this domain was therefore
provided, showing that the binding of FXIIa to
immobilized FN was inhibited in a concentration-
dependent manner by both fibrin generated by incu-

)1
). The amounts of FN bound to
FXII and FXIIa, respectively, were determined by sequential incuba-
tion with rabbit anti-FN IgG, HRP-conjugated swine anti-(rabbit IgG)
and OPD, as described in Experimental procedures. Results are
means ± SD (n = 3), shown by vertical bars.
Fig. 7. Western blots of FXII present in FN immunoprecipitates of
plasma. FN was isolated from plasma by immunoprecipitation with
a rabbit antibody against FN and protein G–Sepharose. The pres-
ence of FXII in the immunoprecipitate (lane 2) was analyzed by
western blotting using goat anti-FXII IgG as primary antibodies and
HRP-conjugated rabbit anti-(goat IgG) as secondary antibody. To
assure that the presence of FXII in the immunoprecipitate was not
due to adsorption of FXII to the protein G–Sepharose, the amount
of adsorbed FXII in the absence of the antibody against FN was
analyzed simultaneously (lane 1).
Factor XII binding to fibronectin I. Schousboe et al.
5166 FEBS Journal 275 (2008) 5161–5172 ª 2008 The Authors Journal compilation ª 2008 FEBS
tion is still not known. For the past 15 years it has
been assumed that its function is connected with
Zn
2+
-dependent binding to a surface or a receptor.
The present study has demonstrated that in purified
systems, activated FXII (FXIIa), but not its zymogen
(FXII), binds with high affinity to immobilized FN.
The binding is independent of the presence of Zn
2+
,is
not affected by the presence of a negatively charged

Gelatine Cell Hep-2 Fib-2
S
Type I
Type IIIType II
30 kDa 120 kDa45 kDa
Fig. 8. Schematic diagram of the modular
structure of the FN monomer. The FN dimer
is formed through interchain disulfide bonds
at the C-terminus. Each subunit consists of
type I, type II and type III repeating
modules. Sets of repeats form domains of
regions implicated in adhesion of different
ligands. The squares show the positions and
the sizes of the different fragments.
0.0
0.5
1.0
1.5
2.0
Block buffer
Heparin, 20 µg·mL
–1
Heparin, 40 µg·mL
–1
Gelatine, 33 µg·
mL
–1
Gelatine, 330 µg·mL
–1
DS, 2

serum present in the cell culture medium. However,
the lack of appearance of deposited FN on culture
dishes incubated with the medium using the same con-
ditions and periods of time as in the presence of cells
but in their absence showed that the deposited FN in
the present investigation was generated by a cell-medi-
ated process. This process induces conformational
changes in FN, exposing cryptic sites of importance
for fibril generation and elongation [28–30].
The high-affinity binding of FXIIa to the ECM with
a K
D
of 12.8 nm [5] and the binding of FXIIa to the
immobilized FN with a K
D
of 8.5 nm make it probable
that FN, whether deposited during growth of
HUVECs or coated on a plastic surface, constitutes a
binding site for FXIIa. Indeed, this binding site was
found not to be present in soluble FN, as soluble FN
was unable to inhibit the binding of FXIIa to immobi-
lized FN. Together with the observed lack of inhibi-
tion by an antibody against soluble FN, this suggests
that the association between FXIIa and FN involves a
cryptic site in FN. Such a binding site has been shown
to be also responsible for the interaction of FN with
fibrinogen and fibrin [27]. Hence, fibrinogen and fibrin
inhibited the binding of FXIIa. The binding of fibrino-
gen and fibrin has been mapped to type I modules of
FN present both N-terminally and C-terminally

1.6
1.8
PBS buffer 30 kDa 45 kDa
coatin
g
120 kDa FN
Ab
sor
b
ance un
it
s
Fig. 10. The binding of FXIIa to immobilized fragments of FN. The
microtiter plate was incubated overnight with the 30 kDa heparin-
and fibrin-binding fragment, the 45 kDa gelatine-binding fragment,
the 120 kDa cell-binding fragment, and FN, respectively. The frag-
ments, as well as FN, were coated at a concentration of
10 lgÆmL
)1
in NaCl ⁄ P
i
. The plate was then washed, blocked with
blocking buffer, and incubated for 1 h with FXIIa (20 n
M) in blocking
buffer. The amount of bound FXIIa was determined by sequential
incubation with goat anti-FXII IgG and HRP-conjugated rabbit anti-
(goat IgG) and visualized by reactions with OPD, as described in
Experimental procedures. Results are means ± SD (n = 3), shown
by vertical bars.
0.0

amounts of FXIIa bound to FN in the presence of fibrinogen (
)
and fibrin (d), and the amount of FXIIa bound to control wells (s),
were determined by sequential incubation with goat anti-FXII IgG
and HRP-conjugated rabbit anti-(goat IgG) and visualized by reac-
tions with OPD, as described in Experimental procedures. Results
are mean ± SD (n = 3), shown by vertical bars when extending
beyond the symbols.
Factor XII binding to fibronectin I. Schousboe et al.
5168 FEBS Journal 275 (2008) 5161–5172 ª 2008 The Authors Journal compilation ª 2008 FEBS
activation of FXII. The binding of FXIIa to the
same domain as fibrin and fibrinogen indicates, how-
ever, that FXIIa may interfere with fibril formation
and elongation during fibrillogenesis and not with the
binding of FN to its cellular receptors. Further stud-
ies are needed to determine whether and how the
binding of FXIIa to immobilized FN regulates these
processes.
FXII was observed not to bind to immobilized FN,
but soluble FN bound to immobilized FXII, and
immunoprecipitates of plasma FN revealed the pres-
ence of FXII. This indicates a role of FN in the activa-
tion and function of FXII. The general concept of the
function of FXII is connected to its binding to a sur-
face. This generates FXIIa, which circumstantially can
cleave FXI and prekallikrein. However, the mechanism
of this activation in vivo has still not been elucidated.
Furthermore, the significance of FXIIa for the activa-
tion of FXI and prekallikrein in vivo has been ques-
tioned, as FXII deficiency is not associated with

from Chemicon (AH Diagnostics, Aarhus, Denmark).
YHKCTHKGR(39–47), the surface-binding region of
FXII ⁄ FXIIa, was a gift from A. H. Schmaier (Case Wes-
tern Reserve University, Cleveland, OH, USA). Fibrinogen
from bovine serum was obtained lyophilized from citrate
buffer (pH 7.4). It was purchased from Calbiochem (La
Jolla, CA, USA). The concentration of fibrinogen in
solution was determined at 280 nm absorbance using an
extinction coefficient (E
1%
280 nm
) of 15.1. Heparin [sodium
salt; H3125; Grade 1 from porcine intestinal mucosa
(181 USP unitsÆmg
)1
)] was from Sigma Chemicals, and DS
(sodium salt; M
r
 500 000) was from Pharmacia Fine
Chemicals (Uppsala, Sweden). All other chemicals were of
the purest grade commercially available.
Affinity-purified goat anti-(human FXII) IgG (GAFXII-
AP) was from Affinity Biologicals Inc. (Hamilton, ON,
Canada). Rabbit anti-FN IgG (ab 299) and monoclonal
antibody to FN, (Fn-3, ab 18265), which reacts with human
cellular fibronectin but not with plasma fibronectin, were
from Abcam (Cambridge, UK). HRP-conjugated rabbit
anti-(goat IgG) (P-0449), HRP-conjugated swine anti-
(rabbit IgG) (P-0399) and o-phenylenediamine (OPD) were
from DAKOCytomation (Ejby, Denmark). Secondary

presence or absence of 20 l gÆmL
)1
sulfatides. Bound FXII
antigens were measured following washing of the wells with
washing buffer [Tween-20 0.05% v ⁄ v in NaCl ⁄ Tris (50 mm
Tris, 0.15 mm NaCl, pH 8.0)]. The wells were then incu-
bated for 1 h with goat anti-(human FXII) IgG, diluted
1 : 2000 in 1% (w ⁄ v) skimmed milk in washing buffer, and
for 1 h with HRP-conjugated secondary antibodies diluted
1 : 2500 in the skimmed milk solution. Extensive washing
with washing buffer was performed between each change of
I. Schousboe et al. Factor XII binding to fibronectin
FEBS Journal 275 (2008) 5161–5172 ª 2008 The Authors Journal compilation ª 2008 FEBS 5169
incubation conditions. Finally, the plates were incubated
for 10–30 min with OPD, dissolved in water according to
the manufacturer’s recommendations. The peroxidase reac-
tion was stopped by twofold dilution with 0.5 m H
2
SO
4
,
and the relative amount of bound FXII antigen was
determined as absorbance units at 490 nm. All experiments
were performed in triplicate and repeated at least twice. To
obtain estimates of affinity constants, the data were
analyzed according to the isotherm
A ¼ A
max
[FXIIa]/(K
D

continued for another night. Following centrifugation
(1 min, 2000 g) and 10-fold washing of the precipitate with
0.5 mL of NaCl ⁄ Tris (10 mm Tris, 1 mm EDTA, 1 mm
EGTA, 0.2 m NaCl, pH 7.4), the protein adsorbed to the
protein G–Sepharose was extracted by boiling for 10 min
with 100 lL of SDS ⁄ glycerol ⁄ dithiothreitol according to the
standard procedure for SDS ⁄ PAGE electrophoresis.
SDS/PAGE and immunoblotting
For western blot analysis, bound proteins were extensively
washed with Locke’s buffer and then extracted with electro-
phoresis buffer containing 2% (w ⁄ v) SDS and 0.1 m dith-
iothreitol. Aliquots of the extracts and FXII, FXIIa and
molecular weight markers were run simultaneously. Pro-
teins were separated on 4–12% SDS ⁄ polyacrylamide gels,
and transferred to poly(vinylidene difluoride) membranes
according to standard procedures. The membrane was then
incubated for 1 h with NaCl ⁄ Tris blocking buffer (50 mm
Tris, 0.15 mm NaCl, pH 8.0, containing 0.1% v ⁄ v Tween-
20 and 0.1% w ⁄ v BSA) and probed with goat anti-FXII
IgG (diluted 1 : 5000) ⁄ HRP-conjugated rabbit anti-(goat
IgG) (diluted 1 : 5000). Dilutions of antibodies were per-
formed in 1% nonfat skimmed milk in NaCl ⁄ Tris blocking
buffer. Detection was carried out using the chemilumines-
cence enhancer SuperSignal West Femto Maximum Sensi-
tivity Substrate (Pierce Biotechnology, Rockford, IL, USA)
as recommended by the manufacturer, and the results were
monitored on a Las Chemiluminator.
Immunofluorescence microscopy
For immunofluorescence microscopy of FXIIa bound to
the ECM, HUVECs were plated on eight chamber slides

Acknowledgements
The work was supported by grants 2005-1-192 and
2006-1-0247 from the Carlsberg Foundation.
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