However, soluble FN could bind to FXII, and this binding inhibited the surface-induced autoactivation of FXII and subse-quent binding of the generated FXIIa to immobilized FN.. As our pr
Trang 1to soluble and immobilized fibronectin – localization of the Hep-1/Fib-1 binding site for activated factor XII
Inger Schousboe1, Birthe T Nystrøm1and Gert H Hansen2
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 Zn2+ -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
Tel: +45 35327800
E-mail: schousboe@imbg.ku.dk
(Received 7 May 2008, revised 8 July 2008,
accepted 18 August 2008)
doi:10.1111/j.1742-4658.2008.06647.x
Fibronectins (FNs) are dimeric glycoproteins that adopt a globular con-formation when present in plasma and solution and an extended confor-mation in the extracellular matrix Factor XII (FXII) is a zymogen of the proteolytically active FXIIa that plays a role in thrombus stabiliza-tion by enhancing clot formastabiliza-tion and in inflammastabiliza-tion by enhancing bradykinin formation To investigate whether the extracellular matrix could play a role in these events, we have recently shown that FXIIa, but not FXII, binds to the extracellular matrix (ECM), and suggested that FN may be the target for the binding Immunofluorescence micros-copy has in the present investigation confirmed that FXIIa added to the ECM colocalizes with FN deposited during growth of human umbilical vein endothelial cells The aim of the present study, therefore, was to fur-ther elucidate the interaction between FXIIa and FN by the use of a solid face binding assay This showed, like the binding to the ECM, that FXIIa, but not FXII, binds in a Zn2+-independent manner to immo-bilized FN The KD for the binding was 8.5 ± 0.9 nm (n = 3) The binding was specific for the immobilized FN, as the binding could not be inhibited by soluble FN Furthermore, soluble FN did not bind to immobilized FXIIa However, soluble FN could bind to FXII, and this binding inhibited the surface-induced autoactivation of FXII and subse-quent binding of the generated FXIIa to immobilized FN The presence
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.
Trang 2cultures 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
binding to the ECM was: (a) specific for FXIIa;
(b) Zn2+-independent; (c) not inhibited by HK; and
(d) nonelectrostatic As proteolytic degradation of the
ECM abrogated the binding of FXIIa, it was assumed
that a matrix protein was the target for the binding
Therefore, it was tentatively analyzed and found that
FXIIa binds to fibronectin (FN) [5]
FN is a dimeric high molecular mass glycoprotein
that is found both as a circulating soluble molecule in
the blood and as insoluble molecules forming
elon-gated multimeric structures in the ECM [6,7] The
monomer of the dimeric soluble molecule is a mosaic
protein composed of modular subunits generating
different domains [8], which harbor binding sites for
glycosaminoglycans, collagen or gelatine, fibrin, and
integrin receptors Some of these binding sites become
available only in the multimeric, elongated forms in
which internal sequences of amino acid residues
become exposed [9,10] Several factors mediate the
transition from the soluble to an elongated form,
including adsorption of FN to plastic surfaces [11–14]
As our previous studies have shown that the binding
of FXIIa to the ECM could be due to binding of
FXIIa to FN generated during growth of HUVECs
[5], we here report on studies of the interactions of
FXIIa with FN using a solid-phase binding assay in
which either FN or FXIIa is immobilized
Results
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 deposdepos-ited 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 presence of FN Bar: 20 lm.
Trang 3antibody against FXII and a horseradish peroxidase
(HRP)-labeled secondary antibody Neither the
anti-body against FXII nor the secondary antianti-body was
observed to bind to FN in the absence of FXIIa This
excludes the possibility that the response was
nonspe-cific and due to a direct interaction between the
immo-bilized FN and the immunoglobulins, as previously
noted [15] Furthermore, preincubation of FXIIa for
1 h with a two-fold molar excess of the antibody
against FXII prior to incubation with FN abolished
the binding Surprisingly, the binding could not be
inhibited if the immobilized FN had been preincubated
with a polyclonal antibody against soluble FN (data
not shown) This could be due to lack of recognition
of the binding site on the immobilized FN for FXIIa,
but it could also be due to a nonspecific interaction
between FXIIa and the plastic surface However, very
little FXIIa bound to wells devoid of FN (controls)
Moreover, nonspecific binding is nonsaturable The
binding of FXIIa to immobilized FN was saturable
even at low concentrations of FXIIa This was
demon-strated by analyzing the binding of varying
concentra-tions of FXIIa At low concentraconcentra-tions of FXIIa,
considerably more FXIIa bound to FN than to control
wells At high concentrations of FXIIa, the binding to
FN increased linearly with the concentration of FXIIa, and in parallel with the binding of FXIIa to control 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 KD 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 3.5
20
Concentration of FXIIa, n M
FN Control
FN - Control
y = 1.1214x + 9.7607
R2 = 0.993
0 10 20 30 40 50 60 70
Concentration of FXIIa, n M
Fig 2 Concentration-dependent binding of FXIIa to FN The microtiter plate was coated overnight with FN (10 lgÆmL)1) and NaCl ⁄ P i (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 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 KDof 8.7 n M Results are means ± SD (n = 3), shown by vertical bars when extending beyond the symbols.
Trang 4sulfatides 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
N-terminal surface-binding region in FXIIa To confirm
this, it was investigated whether the binding of FXIIa
to FN could be inhibited by the nine amino acid
pep-tide YHKCTHKGR(39–47), containing the
surface-binding sequence [18] The presence of this peptide did
not inhibit the binding of FXIIa to immobilized FN
(data not shown)
In plasma and in solution, FN adopts a compact soluble conformation in which the two subunits of the dimer are thought to be folded upon each other [7] Several studies have reported a change in the FN con-formation upon binding to plastic [11–14], exposing a cryptic binding site by transition from the soluble to the immobilized form [19,20] To determine whether these conformational changes were of significance for the binding of FXIIa, subsequent investigations were performed to determine whether the presence of solu-ble FN could inhibit the binding to immobilized FN This was shown not to be the case The amount of FXIIa that bound to immobilized FN was the same regardless of the presence of soluble FN In contrast, the presence of soluble FN reduced the sulfatide-induced binding of FXII (P < 0.001) (Fig 5) How-ever, as sulfatides had hardly any effect on the binding
of FXIIa to immobilized FN, and FXII did not bind
to immobilized FN in the absence of sulfatides (Fig 3), the inhibition could be due to an inhibition of the interaction between FXII and sulfatides To inves-tigate this further, the solid-phase binding assay was turned around and the microtiter plate was coated
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
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 n M ) and FXIIa (20 n M ) in the presence (+sulfatide) and absence ( )sulfatide) of sulfatides (20 lgÆmL )1) To ensure that possible sulfatide-dependent binding of FXII could not be explained by autoactivation of FXII, incubation of FXII in the presence of sulfatides was additionally performed in the presence of CTI (10 lgÆmL)1) FN was also incubated for 1 h with FXIIa, which had been preincubated for 1 h with a twofold molar excess of goat anti-FXII IgG At the end of the incubation, the incubation mixtures were removed, and the microtiter plate was washed extensively Then, the microtiter plate was incubated sequentially with goat anti-FXII IgG, and HRP-conjugated rabbit anti-(goat IgG) in 1% skimmed milk, and the amount of bound FXIIa was visualized by reaction with OPD The combination of primary and secondary antibodies did not bind to either FN-coated or control wells in the absence of FXIIa ⁄ FXII + sulfatides, as indicated by the column showing the binding of blocking buffer The total amount of FXIIa bound to FN and control wells is indicated by gray and white, respectively Results are means ± SD (n = 3), shown by vertical bars.
Trang 5with FXII and FXIIa instead of FN Then, the
bind-ing of soluble FN to immobilized FXII and FXIIa was
visualized by incubation with rabbit anti-(soluble FN)
IgG as the primary antibody and HRP-conjugated
swine anti-(rabbit IgG) as secondary antibody
Figure 6 shows that whereas almost no FN could bind
to immobilized FXIIa, it could bind to FXII The
presence of sulfatides increased only slightly the
bind-ing to both FXII and FXIIa Although it seemed most
unlikely, these differences in the amount of bound FN
could be due to differences in the amount of FXII and
FXIIa coated on the plate This was found not to be
the case, as the immunochemical response was
analyzed and observed to be identical using goat
anti-FXII IgG Moreover, to ensure that FXII had not been activated during the coating period, the wells were coated in the presence of CTI, which inhibits the 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
2 1 FXIIa
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)1sulfatide After washing, the
proteins bound to immobilized FN were extracted with SDS under
reducing conditions (SDS containing dithiothreitol) and subjected to
reduced SDS⁄ PAGE and western blotting FXII, FXIIa and standard
samples of molecular mass markers were run simultaneously
Anti-body-reacting bands were visualized by sequential incubation with
goat (human FXII) IgG (1 : 2500), HRP-conjugated rabbit
anti-(goat IgG) (1 : 2500) and SuperSignal West Femto Maximum
Sensi-tivity Substrate FXII; FXIIa; Lane 1: proteins extract from control
wells devoid of FN in which FXII had been incubated in the
absence of sulfatides Lane 2: proteins extracted from immobilized
FN after incubation with FXII in the absence of sulfatides Lane 3:
proteins extracted from control wells in which FXII had been
incu-bated in the presence of sulfatides Lane 4: proteins extracted from
immobilized FN after incubation with FXII in the presence of
sulfati-des The positions of 50 kDa and 80 kDa proteins are indicated to
the left The blot shows that only FXIIa binds to FN.
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
FXII – sulf
FXII – sulf
+ globular FN
FXII + sulf
FXII + sulf
FXIIa – sulf + globular
FN
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 indicated by gray and white, respectively Statistically significant dif-ferences in binding of FXIIa to FN coated on the microtiter plate when incubated in the absence and presence of soluble FN are indi-cated by asterisks (*not significant and **P < 0.001) Results are means ± SD (n = 3), shown by vertical bars.
Trang 6extracted from FN immunoprecipitates of plasma than
from the plasma alone This indicates that FXII also
forms a complex with FN in plasma
Further characterization of FXIIa binding to
immobilized FN
The high-affinity interaction between FXIIa and
immobilized FN and the lack of interference by
solu-ble FN indicated that the binding site on FN for
FXIIa may be buried in the compact soluble form of
FN [7] FN has binding sites for a series of ligands
such as glycosaminoglycans, collagens or gelatine, fibrin and integrins [21–27] Figure 8 shows a sketch
of FN and the localization of the different binding sites used in our attempt to identify the binding site for FXIIa Thus, concentration-dependent inhibition
of FXIIa binding to immobilized FN was observed with gelatine and high concentrations of dextran sul-fate (DS) but not with heparin (Fig 9) As shown in Fig 8, FN has two binding sites for heparin The Hep-1-binding site is a low-affinity binding site, and the Hep-2-binding site is a high-affinity binding site [21–23] If FXIIa bound to the C-terminal high-affin-ity Hep-2-binding site, it would have been expected 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 Hep-1-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-bation of fibrinogen with thrombin and fibrinogen
As compared to the inhibition by fibrin, however, an approximately 100-fold higher fibrinogen concentra-tion was needed to yield an identical amount of inhibition (Fig 11)
Discussion
Although the presence in the blood of FXII has been known for more than 50 years, its physiological
func-0
0.5
1
1.5
2
2.5
3
FXII FXIIa
Fig 6 Binding of soluble FN to immobilized FXII and FXIIa The
microtiter plate was coated overnight as indicated with FXII (20 n M )
and FXIIa (20 n M ), respectively, diluted in NaCl ⁄ P i Then, the
micro-titer plate was blocked with blocking buffer and incubated for 1 h
with FN (10 lgÆmL)1) in blocking buffer or in blocking buffer
con-taining sulfatides (+sulf; 20 lgÆmL)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).
Trang 7tion is still not known For the past 15 years it has
been assumed that its function is connected with
Zn2+-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 Zn2+, is
not affected by the presence of a negatively charged
surface represented by sulfatides, and is not inhibited
by soluble FN Accordingly, soluble FN did not
bind to immobilized FXIIa The binding of FXIIa to
immobilized FN occurs through type I modules in the
30 kDa N-terminal heparin (Hep-1)-binding and fibrin
(Fib-1)-binding domain of FN
Immunohistochemical visualization of the
interac-tion between FXIIa and FN deposited on the surface
of the culture dish during 3 days of growth of HUVECs clearly showed that FXIIa associated with
FN left behind on the plastic surface after removal of the cells The visualization showed that FN had been deposited in a sparse and patchy manner, which may reflect the conditions under which the cells had been cultivated and subsequently removed by EDTA extrac-tion Indeed, the majority of the deposited FN was attached to the cells and was thus removed during extraction of the cells Furthermore, experiments with cultures of arterial endothelial cells have shown that the amount of FN deposited on the surface of the cells varied dramatically when preconfluent, newly confluent and postconfluent cultures were analyzed Thus, whereas sparse patches of FN were generated in pre-confluent and newly pre-confluent cultures, a massive net
Fib-1/Hep-1
S
FXIIa binding
COOH
NH2
S
Type I Type II Type III
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
0 µg·mL –1
DS, 40 µg·mL
–1
*
Fig 9 The effect of gelatine and heparin on binding of FXIIa to immobilized FN The microtiter plate was incubated overnight with FN (10 lgÆmL)1) and NaCl ⁄ P i , respectively, and blocked with blocking buffer Then, it was incubated for 1 h with FXIIa (20 n M ) in blocking buffer containing heparin (20 and 40 lgÆmL)1), gelatine (33 and 330 lgÆmL)1) or DS (20 and 40 lgÆmL)1) The amount of bound FXIIa was deter-mined 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 mean ± SD (n = 3), shown by vertical bars Statistically significant differences between FXIIa bound to FN incubated in the presence and in the absence of effectors are indicated by asterisks (*P < 0.001).The binding to control wells was less than 0.05 absorbance units.
Trang 8of FN covering the entire surface of the cells was
formed only in postconfluent cultures [28] It may be
claimed that the deposited FN originates from the
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 KD of 12.8 nm [5] and the binding of FXIIa to the
immobilized FN with a KDof 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 (Fig 8) Binding of FXIIa to the 30 kDa N-terminal fragment of FN indicates that FXIIa binds to FN through the type I modules in the cryptic N-terminal end of FN but does not exclude the possibility that FXIIa may also interact with the C-terminal Fib-2-binding site
The binding site in FXIIa is unknown, but lack of inhibition of the binding of FXIIa to FN by sulfatides and the surface-binding peptide of FXII strongly indi-cates that the binding does not involve the surface-binding region in FXIIa [18] The lack of inhibition of FXIIa binding to immobilized FN by the surface-bind-ing peptide strengthens the statement that FN is the target for the binding of FXIIa to the ECM, as this binding also could not be inhibited by the peptide [5] Thus, the affinities for FXIIa binding to ECM and to immobilized FN were the same, and neither one of the binding events could be inhibited by the surface-binding peptide of FXIIa
The binding to immobilized FN was specific for FXIIa, as FXII did not bind This indicates that the binding is of no physiological relevance for the
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
coating
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)1in 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 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 0.5 1.0 1.5 2.0
Concentration of fibrinogen/fibrin, n M
Fig 11 Fibrin inhibition of FXIIa binding to immobilized FN The microtiter plate was incubated overnight with FN (10 lgÆmL)1) and NaCl ⁄ P i , respectively, and blocked with blocking buffer Mean-while, 1.74 l M fibrinogen dissolved in blocking buffer was incu-bated overnight with 90 mUÆmL)1 thrombin or blocking buffer at room temperature, and subsequently diluted with blocking buffer containing hirudin (100 UÆmL)1) to give the indicated final concen-trations of fibrinogen and fibrin after mixing with FXIIa (final con-centration: 20 n M ) The presence of hirudin did not affect the binding of FXIIa to FN, and the concentration of hirudin was suffi-ciently high to completely block the activity of thrombin The 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.
Trang 9activation 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 funcactiva-tion 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
hemophilia In addition, FXI can be activated by
thrombin [31], and prekallikrein by a
prolylcarboxy-peptidase [32] and the HSP90 protein [33] However,
recent investigations have shown that FXII in vivo
plays an important role in thrombus formation, being
activated on the surface of activated platelets [34] to
which FN binds [35] The mechanism for this
activa-tion is unknown, but although speculative, the present
investigation may be of importance in understanding
the impact of FXII in thrombus formation Thus, the
binding of FXII to soluble FN may be of relevance
for the activation of FXII on the surface of activated
platelets, but this remains to be established
Experimental procedures
Materials
FXII and thrombin were obtained as 50% glycerol
solu-tions from Haematologic Technologies Inc (Essex Junction,
band with a molecular mass of 80 kDa in reduced
Enzyme Research Laboratories (Swansea, UK) FXIIa was
dissolved in water as recommended by the company,
Siliconized test tubes were likewise used for subsequent
dilutions of FXII and FXIIa, and excess dilutions were
dis-carded Human plasma FN was from Gibco (Invitrogen,
Carlsbad, CA, USA) CTI, hirudin, the N-terminal 29 kDa
heparin-binding fragment and the 45 kDa gelatine-binding
fragment were from Sigma Chemicals (St Louis, MO,
USA) The 120 kDa cell-binding fragment was obtained
from Chemicon (AH Diagnostics, Aarhus, Denmark)
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
280 nm) of 15.1 Heparin [sodium salt; H3125; Grade 1 from porcine intestinal mucosa
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 (goat IgG) (P-0449), HRP-conjugated swine anti-(rabbit IgG) (P-0399) and o-phenylenediamine (OPD) were
immunofluores-cence microscopy were from Invitrogen (Copenhagen, Denmark)
Solid-phase binding assay
The solid-phase binding assay was performed in 96-well maximum-binding polystyrene microtiter plates (NUNC, Roskilde, Denmark) The plates were coated with 150 lL
with Locke’s buffer (154 mm NaCl, 5.6 mm KCl, 3.6 mm
pH 7.4), and unoccupied binding sites were blocked by incubation for a minimum of 30 min at room temperature
Sigma Chemicals) dissolved in Locke’s buffer] The wells were then incubated for 60 min with FXII or FXIIa added
in a final volume of 100 lL in blocking buffer in the
antigens were measured following washing of the wells with
Tris, 0.15 mm NaCl, pH 8.0)] The wells were then incu-bated for 1 h with goat anti-(human FXII) IgG, diluted
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
Trang 10incubation conditions Finally, the plates were incubated
for 10–30 min with OPD, dissolved in water according to
the manufacturer’s recommendations The peroxidase
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
where [FXIIa] is the molar concentration of FXIIa, A is
the absorbance of the oxidized HRP substrate, which is
assumed to be proportional to the amount of FXIIa bound,
concen-trations of FXIIa
Alternatively, the microtiter plate was coated with 20 nm
immobi-lized FXII or FXIIa was visuaimmobi-lized by sequential incubation
with rabbit FN IgG and HRP-conjugated swine
milk in washing buffer, and OPD, as described above
Immunoprecipitation
Ten microliters of rabbit anti-FN IgG was added to one of
two aliquots containing 200 lL of plasma, 2 lL of hirudin
slurry of protein G–Sepharose (Sigma-Aldrich, St Louis,
MO, USA) was added to each aliquot, and the rotation was
continued for another night Following centrifugation
(1 min, 2000 g) and 10-fold washing of the precipitate with
EGTA, 0.2 m NaCl, pH 7.4), the protein adsorbed to the
protein G–Sepharose was extracted by boiling for 10 min
SDS/PAGE and immunoblotting
For western blot analysis, bound proteins were extensively
washed with Locke’s buffer and then extracted with
dith-iothreitol Aliquots of the extracts and FXII, FXIIa and
molecular weight markers were run simultaneously
and transferred to poly(vinylidene difluoride) membranes
according to standard procedures The membrane was then
IgG) (diluted 1 : 5000) Dilutions of antibodies were
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 (Nalgene Nunc International Corp., Roskilde, Denmark) at
medium on the second day On day 4, the cells were detached by EDTA The ECM was incubated with 20 nm FXIIa in blocking buffer for 1 h After the washing proce-dure described above for the solid-phase binding assay, the slides were incubated with antibodies The primary anti-bodies were a mixture of goat anti-FXII IgG (diluted
1 : 100) and mouse anti-FN IgG (Fn-3) (diluted 1 : 100) The secondary antibodies were a mixture of Alexa 594-con-jugated donkey anti-(goat IgG) (diluted 1 : 800) and
Finally, the slides were mounted in antifade medium (DAKOCytomation, Ejby, Denmark) and examined in a Leica DM 4000 B microscope equipped with a Leica
DC 300 FX digital camera
Specificity analyses of the antibodies showed no reaction
of the secondary antibodies with the ECM incubated in the absence of the primary antibodies
Sulfatide preparation
Sulfatides extracted from bovine brain were from Sigma Chemicals Vesicles of sulfatides were prepared as previ-ously described [5]
Statistics
The results are shown as means ± SD, and statistically significant differences were calculated using Student’s t-test
Acknowledgements
The work was supported by grants 2005-1-192 and 2006-1-0247 from the Carlsberg Foundation
References
1 Hasan AA, Cines DB, Ngaiza JR, Jaffe EA & Schmaier
AH (1995) High-molecular-weight kininogen is exclu-sively membrane bound on endothelial cells to influence activation of vascular endothelium Blood 85, 3134– 3143