Báo cáo khoa học: High negative charge-to-size ratio in polyphosphates and heparin regulates factor VII-activating protease pdf

High negative charge-to-size ratio in polyphosphates and
heparin regulates factor VII-activating protease
ă ă

Lars Muhl1, Sebastian P. Galuska1, Katariina Oorni2, Laura Hernandez-Ruiz3, Luminita-Cornelia
4
1
Andrei-Selmer , Rudolf Geyer , Klaus T. Preissner1, Felix A. Ruiz3, Petri T. Kovanen2
and Sandip M. Kanse1
1
2
3
4

Institute for Biochemistry, Justus-Liebig-University, Giessen, Germany
Wihuri Research Institute, Helsinki, Finland
Unidad de Investigacion, Hospital Universidad Puerta del Mar and Universidad de Cadiz, Spain
Philipps University, Marburg, Germany

Keywords
FSAP; heparin; mast cells; platelets;
polyphosphate
Correspondence
S. M. Kanse, Institute for Biochemistry,
Justus-Liebig-University Giessen,
Friedrichstrasse 24, 35392 Giessen,
Germany
Fax: +49 641 9947509
Tel: +49 641 9947521
E-mail:
uni-giessen.de

(Received 12 March 2009, revised 28 May
2009, accepted 29 June 2009)
doi:10.1111/j.1742-4658.2009.07183.x

Factor VII-activating protease (FSAP) circulates as an inactive zymogen in
the plasma. FSAP also regulates fibrinolysis by activating pro-urokinase or
cellular activation via cleavage of platelet-derived growth factor BB
(PDGF-BB). As the Marburg I polymorphism of FSAP, with reduced
enzymatic activity, is a risk factor for atherosclerosis and liver fibrosis, the
regulation of FSAP activity is of major importance. FSAP is activated by
an auto-catalytic mechanism, which is amplified by heparin. To further
investigate the structural requirements of polyanions for controlling FSAP
activity, we performed binding, activation and inhibition studies using heparin and derivatives with altered size and charge, as well as other glycosaminoglycans. Heparin was effective in binding to and activating FSAP in a
size- and charge density-dependent manner. Polyphosphate was more
potent than heparin with regard to its interactions with FSAP. Heparin
was also an effective co-factor for inhibition of FSAP by plasminogen activator inhibitor 1 (PAI-1) and antithrombin, whereas polyphosphate served
as co-factor for the inhibition of FSAP by PAI-1 only. For FSAP-mediated
inhibition of PDGF-BB-induced vascular smooth muscle cell proliferation,
heparin as well as a polyphosphate served as efficient co-factors. Native
mast cell-derived heparin exhibited identical properties to those of unfractionated heparin. Despite the strong effects of synthetic polyphosphate, the
platelet-derived material was a weak activator of FSAP. Hence, negatively
charged polymers with a high charge-to-size ratio are responsible for the
activation of FSAP, and also act as co-factors for its inhibition by serine
protease inhibitors.

Introduction
Factor VII-activating protease (FSAP) is a serine protease that is predominantly expressed in the liver. It
circulates as an inactive zymogen with a concentration

of 12 lgỈmL)1 in the plasma, and is known to activate

factor VII and pro-urokinase [1,2]. It was first purified
by its ability to bind to hyaluronic acid, and was there-

Abbreviations
AT, antithrombin; EGF3, epidermal growth factor like-3; FSAP, factor VII-activating protease; PAI-1, plasminogen activator inhibitor 1;
PDGF-BB, platelet-derived growth factor BB; PolyP, polyphosphate; SERPIN, serine protease inhibitor; SPR, surface plasmon resonance;
TMB, 3,3¢,5,5¢-tetramethylbenzidine; VSMC, vascular smooth muscle cells.

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L. Muhl et al.

fore designated as hyaluronic acid binding protein 2
(HABP-2) [3]. Activation of FSAP requires cleavage
between residues R313 and I314, separating the light
chain and the heavy chain [4].
Negatively charged polyanions such as heparin [4,5],
nucleic acids [6,7] and dextran sulfate [4] bind to
FSAP. This interaction leads to auto-catalytic activation [4,5], followed by auto-proteolysis. This propensity to partial proteolysis has been used to determine
the domain responsible for binding to heparin and
RNA. Multiple regions of FSAP contribute to polyanion binding, but the epidermal growth factor like-3
(EGF3) domain with a cluster of positively charged
amino acids is particularly important [6].
Although FSAP was initially isolated on a hyaluronic acid column [3], no information is available as
to how hyaluronic acid can bind to FSAP, nor
whether it can activate FSAP [4]. The concentration of
hyaluronic acid, as well as its transition from the highto low-molecular-weight form, is related to the regulation of angiogenesis, atherosclerosis, restenosis and

inflammation [8]. FSAP activation is also mediated by
nucleic acids, with RNA having a stronger effect than
DNA [6,7]. Heparin is the most extensively studied
polyanion with respect to FSAP function. It has been
shown that unfractionated heparin is a strong activator
of FSAP, but low-molecular-weight heparin has not
been systematically tested. The role of the more ubiquitous heparan sulfate and other glycosaminoglycans is
also not known.
Polyphosphate (PolyP) is a linear polymer of
orthophosphate (Pi) residues linked by high-energy
phosphoanhydride bonds, found in many cell types
[9]. PolyP, with an approximate chain length from
70–75 phosphate units, is stored in platelet-dense
granules [10] and released upon platelet activation.
PolyP can amplify coagulation by activation of the
contact factor pathway, as well as activation of
factor V, inhibition of the anticoagulant function of
tissue factor pathway inhibitor (TFPI), and enhancing the activity of thrombin-activated fibrinolysis
inhibitor (TAFI) [11].
Once activated, FSAP can be rapidly inhibited by
serine protease inhibitors (SERPINs), such as a1-antitrypsin, a2-antiplasmin, antithrombin (AT) and C1
inhibitor [4,12–14], as well as plasminogen activator
inhibitor 1 (PAI-1) [15] and protease nexin 1 [16]. AT
and a2-antiplasmin were shown to be efficient inhibitors in the presence of heparin [4], whereas PAI-1 was
shown to be an inhibitor only in the presence of RNA
but not heparin [15].
The presence of a naturally occurring polymorphism
in the FSAP gene leading to an amino acid exchange

Polyanions and FSAP

(G534E, or Marburg I polymorphism) results in diminished proteolytic activity towards factor VII, prourokinase [17] and PDGF-BB (platelet-derived growth
factor BB) [18]. The Marburg I polymorphism is associated with a higher risk for carotid stenosis [19], and,
in comparison to wild-type FSAP, is not able to inhibit neointima formation in a mouse model [18]. Similarly, Marburg I FSAP is associated with advanced
liver fibrosis, which may be due to its inability to inhibit PDGF-BB-mediated proliferation of hepatic stellate
cells [21]. These findings indicate the importance of
FSAP enzymatic activity with respect to its function
in vivo. However, it is not clear which polyanions are
relevant for the regulation of FSAP activity. This
prompted us to investigate the requirements for FSAP
interaction with polyanions known to be present in
atherosclerotic arterial wall and ⁄ or fibrotic liver, and
also to define the molecular basis of the binding, activation and regulation mechanisms.

Results
FSAP binding to polyanions
Electrophoretic mobility shift assays were performed
to characterize the interaction between FSAP and various polyanions. Preincubation of FSAP with unfractionated heparin, low-molecular-weight heparin,
PolyP65 or PolyP35 induced a shift in the mobility of
FSAP in polyacrylamide gels with or without urea.
Other polyanions had no influence at all. When BSA
was used as a control, none of the polyanions induced
a shift in the BSA band (Fig. 1A). Concentrationdependent analysis indicated that the EC50 was 95 ±
7 nm for the shift with unfractionated heparin and
28 ± 3 nm for PolyP65 (Fig. 1B and Figs S1 and S2).
To examine whether the various polyanions use the
same region in the FSAP molecule for binding, we
performed competition binding assays in which binding of biotinylated unfractionated heparin to FSAP
was measured (Fig. 1C). Unfractionated heparin competed with biotinylated heparin for binding to FSAP,
whereas low-molecular-weight heparin showed low

competition (Fig. 1C, upper panel). PolyP competed
for this binding in a chain length-dependent manner.
All other heparin derivatives, as well as chondroitin
sulfate, dermatan sulfate, polysialic acid, heparan
sulfate and hyaluronic acid, showed no competition,
indicating no binding to FSAP (Fig. 1C, lower panel,
and Fig. S3A). Thus, using gel-shift and competition
binding assays, it was demonstrated that binding to
FSAP depends on the size and charge density of the
macromolecule.

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L. Muhl et al.

Fig. 1. Binding of FSAP to polyanions. (A) FSAP or BSA (5 lg per
lane) were preincubated with the respective polyanion (10 lg ⁄ lane)
for 30 min. Samples were directly loaded onto gels containing urea
(upper panel) or native polyacrylamide gels (middle and lower panels). Shifted bands (complexed FSAP and polyanions) indicate binding of the particular polyanion to FSAP. (B) In a similar experiment
to that shown in (A), the concentration of unfractionated heparin
and PolyP65 was varied (0.002–2 lM). Complex formation was
quantified by densiometric analysis, and the results from three separate experiments were pooled to determine the EC50 values. (C)
FSAP (10 lgỈmL)1) was immobilized, and heparin derivatives (upper
panel) or other polyanions (lower panel) (0.01–100 lgỈmL)1) were
mixed with biotinylated heparin albumin (0.5 ngỈmL)1) and added to

the plate. Detection of bound biotinylated heparin albumin was
measured using peroxidase-conjugated streptavidin and 3,3¢,5,5¢tetramethylbenzidine (TMB) substrate (mean ± SEM, n = 4).

A

B

C

Fig. 2. Increased auto-activation of FSAP by polyanions. Polyanions
at concentrations in the range 0.01–100 lgỈmL)1 were added to
FSAP (1 lgỈmL)1), and FSAP activity (mmODỈmin)1) was determined (mean ± SEM, n = 4).

Activation of FSAP by various polyanions
We next investigated all the polyanions described
above with respect to their ability to activate FSAP.
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Unfractionated heparin was a strong activator, lowmolecular-weight heparin activated FSAP to a smaller
extent, and all other heparin-derivatives exhibited no
activation (Fig. 2, upper panel). PolyP showed potent
activation of FSAP in a chain length-dependent
manner. There was a 4–6-fold increase in Vmax with
unfractionated heparin and PolyP65, with no change in

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L. Muhl et al.

KM (Fig. S2). Heparan sulfate and dermatan sulfate
showed weak activation of FSAP at high concentrations (Fig. 2, middle panel). Polysialic acid and hyaluronic acid did not activate FSAP (Fig. 2, lower
panel). N-acetyl heparin, de-N-sulfated heparin, N-acetyl-de-O-sulfated heparin, polysialic acid and hyaluronic acid totally failed to increase FSAP activity.
To assess the specificity of the PolyP effect, it was
degraded using calf intestinal phosphatase, which is
also a highly active exopolyphosphatase [11]. The
accelerating effect of PolyP on FSAP activity was
decreased by phosphatase pretreatment in a time- and
dose-dependent manner (Fig. S4). As a control, we
observed that phosphatase treatment did not influence
unfractionated heparin-mediated activation of FSAP
(Fig. S4). Hence, the effect of PolyP was not due to a
contaminant. These studies show that the pattern of
binding of polyanions to FSAP is identical to the
pattern of their ability to activate FSAP.

Polyanions and FSAP

A

B

Polyanions as co-factors for the inhibition of
FSAP by PAI-1 and AT
SERPINs exhibit enhanced or altered substrate specificity in the presence of heparin or other co-factors [22].
To examine the co-factor function of polyanions with
respect to FSAP inhibition, active two-chain FSAP was
preincubated with PAI-1 or AT with or without various
concentrations of polyanions. Inhibition of FSAP by
PAI-1 was increased by unfractionated heparin, lowmolecular-weight heparin and to a lower extent by

N-acetyl heparin (Fig. 3A, upper panel). PolyP exhibits
strong co-factor function for the inhibition of FSAP by
PAI-1 in a chain length-dependent manner. The IC50 of
PAI-1 for the inhibition of FSAP was halved by
unfractionated heparin and PolyP65 (Fig. S5). Heparan
sulfate was a co-factor at high concentrations (Fig. 3A,
lower panel), and dermatan sulfate and polysialic acid

C

Fig. 3. Inhibition of FSAP by PAI-1 and AT; co-factor function of
polyanions. FSAP (1 lgỈmL)1) was preincubated either with PAI-1
(1 lgỈmL)1) (A) or with AT (5 lgỈmL)1) (B) for 30 min with or without heparin derivatives (upper panels) or other polyanions (lower
panels) in the concentration range 0.01–100 lgỈmL)1. FSAP activity
(mmODỈmin)1) was determined, and inhibition was calculated as a
percentage of FSAP activity without inhibitor (mean ± SEM, n = 4).
(C) SPR sensograms showing the association and dissociation of
FSAP–inhibitor complexes in the presence of polyanions. FSAP
(10 lgỈmL)1) was bound to a specific high-affinity antibody to
FSAP, immobilized on a CM5 sensor chip, prior to injection of
either AT (5 lgỈmL)1) or PAI-1 (5 lgỈmL)1), alone (control) or in the
presence of polyphosphate 65 (10 lgỈmL)1) or unfractionated heparin (10 lgỈmL)1). Alignment of SPR sensograms was performed
using the program BIAevaluation 3.2 RC1.

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L. Muhl et al.

at even higher concentrations (Fig. S3B), but hyaluronic acid had no effect at all (Fig. 3A, lower panel).
In the case of FSAP inhibition by AT, only unfractionated heparin and heparan sulfate were able to
serve as co-factors (Fig. 3B). PolyP and other tested
polyanions showed no co-factor properties for the ATdependent FSAP inhibition (Fig. 3B, lower panel and
Fig. S3C). The activity of FSAP was increased by lowmolecular-weight heparin, N-acetyl heparin (Fig. 3B,
upper panel) and PolyP (Fig. 3B, lower panel) even in
the presence of AT.
To consolidate these findings, real-time interaction
studies were performed using surface plasmon resonance (SPR). These results confirm that FSAP interacts
with AT only in the presence of unfractionated heparin
(KA of  2.9 · 107 [1 ⁄ M]) but not in the presence of
PolyP. In contrast, FSAP interacts with PAI-1 without
a co-factor (KA of  1.6 · 107 [1 ⁄ M]) in the presence
of unfractionated heparin (KA of  3.2 · 107 [1 ⁄ M]) as
well as in the presence of PolyP (KA of  97 · 107
[1 ⁄ M]) (Fig. 3C). Hence, polyanions can selectively
promote inhibition of the enzymatic activity of FSAP.
Polyanions as co-factors for the FSAP-dependent
inhibition of VSMC proliferation
A major function of FSAP is the specific proteolytic
cleavage and inactivation of PDGF-BB [23], and this
process is enhanced by heparin and RNA [24]. We
observed that low-molecular-weight heparin and heparan sulfate also increase the inhibitory effect of FSAP
on proliferation of vascular smooth muscle cells
(VSMC), but to a lower extent compared to unfractionated heparin. PolyP also promoted the inhibitory
effect of FSAP on VSMC proliferation, whereas de-Nsulfated heparin and hyaluronic acid were ineffective
(Fig. 4). The ability of each polyanion to inhibit cell

proliferation matched the respective pattern of FSAP
binding and activation.
Assessment of mast cell heparin and platelet
PolyP as co-factors for FSAP function
Mast cell-derived macromolecular heparin and platelet-derived PolyP were isolated as native substances
and tested for their interaction with FSAP. The mast
cell-derived heparin bound to FSAP, as indicated by a
mobility shift in native polyacrylamide gels (Fig. 5A,
upper panel). When compared to unfractionated heparin, mast cell heparin was even more efficient with
respect to competition of biotinylated heparin binding
to immobilized FSAP (Fig. 5A, middle panel) and
FSAP activation (Fig. 5B, upper panel).
4832

no PDGF-BB or FSAP or polyanion
with PDGF-BB and no FSAP or polyanion
Buffer
FSAP

Fig. 4. Polyanion-dependent amplification of the inhibitory effect of
FSAP on VSMC activation. PDGF-BB (20 ngỈmL)1) was preincubated without (light gray columns) or with (dark gray columns) FSAP
(15 lgỈmL)1) and ⁄ or 10 lgỈmL)1 of the various polyanions for 1 h
at 37°C in serum-free medium. Subsequently, VSMC were stimulated for 36 h in medium containing 0.2% fetal calf serum. DNA
synthesis was measured (mean ± SD, n = 3) using a kit that
detects BrdU incorporation into newly synthesized DNA.

In mobility shift assays, platelet-derived PolyP
bound to FSAP weakly (Fig. 5A, upper panel). However, it competed with biotinylated heparin for binding
to immobilized FSAP more strongly than its synthetic
analogue PolyP65 did (Fig. 5A, lower panel). Unexpectedly, activation of FSAP by native platelet-derived

PolyP was much lower when compared to the synthetic
material (Fig. 5B, lower panel). Thus, mast cell-derived
heparin was identical to unfractionated heparin for all
aspects investigated, but there were differences between
platelet-derived and synthetic PolyP.

Discussion
Genetic studies show that the presence of the Marburg I single-nucleotide polymorphism is a risk factor
for carotid stenosis [19] and liver fibrosis [20]. This isoform of FSAP exhibits reduced enzymatic activity [17],
indicating that the local proteolytic activity of FSAP
may play a crucial role in development of the disease
state. Therefore, it is important to understand the regulation of FSAP activity in order to define its pathophysiological role. Polyanions have been shown to
play a key role in regulating FSAP activity by promoting auto-catalytic activation. In the present study, we
systematically characterized the effects of various
polyanions on FSAP activity.

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L. Muhl et al.

Polyanions and FSAP

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Pl

Complexed FSAP

Competition with heparin-albumin-biotin (%)

Free FSAP

120
80
40
0
–40

Mast cell heparin
Unfractionated heparin

//
//
0

0.1

1

Heparin (µg·mL–1)

10

120
80
40
//

//

0
–40

Platelet polyphosphate
Polyphosphate 65

//
0

0.1

1

10

Polyphosphate (µg·mL–1)

B

Mast cell heparin
Unfractionated heparin

20

FSAP-activity (mmOD·min–1 at 405 nm)

15
10

//
5
0 //
0

20

1
0.1
0.01
Heparin (µg·mL–1)

10

Polyphosphate 65
Platelet polyphosphate

15
10
5

//

0 //
0

Fig. 5. Properties of mast cell-derived heparin and platelet-derived
PolyP with respect to FSAP. (A) Upper panel: FSAP (5 lg per lane)
was preincubated with unfractionated heparin (UH), mast cellderived heparin, PolyP65 or platelet-derived PolyP (each 2 lg per
lane), and loaded directly onto native polyacrylamide gel. Shifted

bands (complexed FSAP) indicate binding of the respective polyanion to FSAP. Middle and lower panels: FSAP (10 lgỈmL)1) was
immobilized, and synthetic or mast cell-derived heparin (0.05–
10 lgỈmL)1) and synthetic or platelet-derived PolyP (0.033–
5 lgỈmL)1) were mixed with biotinylated heparin albumin
(0.5 ngỈmL)1) and added to the plate. The amount of bound biotinylated heparin albumin was measured using peroxidase-conjugated
streptavidin and TMB substrate (mean ± SD, n = 3). (B) Unfractionated heparin (0.01–10 lgỈmL)1), mast cell-derived heparin (0.02–
5 lgỈmL)1) (upper panel) or synthetic or platelet-derived PolyP
(0.01–2.5 lgỈmL)1) (lower panel) were added to FSAP (1 lgỈmL)1),
and FSAP activity (mmODỈmin)1) was determined (mean ± SD,
n = 3).

0.01
0.1
1
Polyphosphate (µg·mL–1)

10

lower potential for binding to and activating FSAP.
The heparin homologues N-acetyl heparin, de-N-sulfated heparin and N-acetyl-de-O-sulfated heparin,
which have the same size but reduced negative charge,
neither bind to nor activate FSAP (Fig. 6). The proteoglycan heparan sulfate has an even lower negative
charge, compared to unfractionated heparin, and
exhibits weak FSAP binding and activation. Mast
cell-derived heparin has a higher charge than unfractionated heparin, and exhibits a stronger ability to
bind to and activate FSAP [25].
Chondroitin sulfate, dermatan sulfate and polysialic
acid also have a less negative charge density than unfractionated heparin and show no FSAP binding or
activation potential (Fig. 6). FSAP was first purified
based on its binding to hyaluronic acid [3]. In the present study, we demonstrate that there is no tight interaction between hyaluronic acid and FSAP, most likely

due to the relatively low negative charge density in the
polyanion. Its isolation on hyaluronic acid columns
could be due to altered physical properties of immobilized hyaluronic acid. The significance of these results
is that the very ubiquitous heparan sulfate proteoglycans and other matrix-associated glycosaminoglycans
play no role in the regulation of FSAP activity. This is
rather related to the proximity and activation state of
mast cells that secrete heparin, such as in atherosclerotic plaques [26].
Polyphosphate

Heparin and other glycosaminoglycans
The binding to FSAP and the subsequent activation of
FSAP by heparin depends on its size and overall negative charge. Low-molecular-weight heparin exhibits

PolyP was a more potent activator of FSAP than heparin. PolyP65 was the most active form of PolyP, with
smaller forms showing diminished activity. Degradation by phosphatases decreased its properties with

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L. Muhl et al.

Fig. 6. Structure of the various polyanions used in the study. Potential modifications of the sugar residues by sulfate groups are shown.
The mean numbers of sulfate groups per disaccharide unit (DS) are given for all glycosaminoglycans. The mean acid dissociation constants
(pKa values) for the phosphate, sulfate and carboxyl groups are 1.5, 2.0 and 4.7, respectively [37,38].

respect to FSAP binding and activation, and any influence on FSAP activity was completely neutralized. In

order to put these findings in a pathophysiological context, we compared the activity of synthetic PolyP with
that of native platelet-derived material. Platelet-derived
PolyP exhibited quite anomalous properties compared
to synthetic PolyP. In gel-shift assays, it demonstrated
weak binding, but was as efficient as synthetic PolyP in
4834

competing for heparin binding to FSAP. Native PolyP
was a very weak activator of FSAP compared to the
synthetic version. One reason for this discrepancy
between synthetic and native PolyP could be that
synthetic PolyP65 is a heterogeneous mixture, with
polymers up to 200 units, whereas native PolyP is
extremely pure and has a more homogeneous size with
70–75 units [10,27]. In addition to their difference in

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L. Muhl et al.

size, we cannot exclude the possibility of a contaminant
that has a confounding effect on the interaction of
native PolyP with FSAP. No comparable data exist in
the literature, as this is one of the first studies to compare the activities of synthetic with platelet-derived
PolyP. Given the robust activity of synthetic PolyP, the
role of endogenous platelet-derived material needs to
be investigated further.
Inhibition
SERPINs such as protease nexin 1 and PAI-1 can

efficiently inhibit FSAP. Whereas protease nexin 1
inhibits proteases independently of any co-factor [16],
PAI-1 is known to require heparin as a co-factor for
inhibition of some of its targets such as thrombin
[28]. The co-factor effect of heparin is due to a
change in the conformation of the SERPIN as well
as the ability of heparin to co-join the protease with
the inhibitor. Previously published data showed that
heparin was not a co-factor for PAI-1-dependent
inhibition of FSAP [15]. In this study, we demonstrate that both heparin and polyphosphate are
potent co-factors for the inhibition of FSAP by
PAI-1. A reduction in size and charge density in
heparin led to lower inhibition of FSAP by PAI-1.
AT inhibits FSAP only in the presence of heparin
but not PolyP. The size and negative charge of
heparin has an even greater importance for the interaction with AT, as indicated by the fact that lowmolecular-weight heparin and N-acetyl heparin promote
an increase in FSAP activity rather than inhibiting
it. Thus, polyanion binding to SERPINs, over and
above their binding to FSAP, plays a decisive role
in mediating its inhibition.
PolyP increased the inhibition of FSAP by PAI-1 but
not by AT. Whereas heparin changes the tertiary structure of AT [29], PolyP was shown to be unable to induce
any conformational changes in AT, as determined by
measurement of the intrinsic protein fluorescence of AT
incubated with PolyP (F. A. Ruiz, unpublished results).
Both polyanions decreased the IC50 for the inhibition of
FSAP by PAI-1 twofold. SERPINs inhibit their target
protease by a suicide substrate mechanism that involves
a 1 : 1 formation of an irreversible covalent complex
[30]. Only protease-inactive mutants show reversible

binding to SERPINs [30], and the FSAP–PAI-1 complex demonstrated some dissociation in our experiments
(Fig. 3C), indicating some deviation from the classical
model of protease–inhibitor interactions. Hence, the
overall inhibition of FSAP depends not only on the
inhibitor but also on the presence of an appropriate
co-factor in the vicinity of FSAP.

Polyanions and FSAP

Conclusions
The two major polyanions, heparin and PolyP, use
the same binding region in the FSAP molecule, as
revealed by the competition binding assay. Charge
density, size and also conformational flexibility determine the affinity of this interaction. Other matrixderived polyanions were not effective. Binding to
polyanions was also observed in the presence of a
strong denaturant, urea, indicating a strong charge
interaction. The region of FSAP that is probably
responsible for this binding is the EGF3 domain,
which contains a positively charged cluster of amino
acids, although other regions of FSAP promote this
interaction [6]. Using a recombinant EGF3 domain
deletion mutant of FSAP, no activation of FSAP
was obtained with either heparin or with PolyP [31],
further confirming the involvement of this region in
polyanion binding and activation. Polyanions
strongly reduced the proliferative activity of PDGFBB in the presence of FSAP. This could explain the
influence of polyanions such as heparin on smooth
muscle proliferation in vivo [32], and a similar function is expected for PolyP. As a lowering in FSAP
activity is correlated with diseases [19,20], these new
insights into the regulation of FSAP activity will

lead to increased understanding of FSAP function
under physiological und pathophysiological conditions. Identification of specific size, sequence and
charge requirements may allow rational design of
polyanions with higher specificity for the regulation
of FSAP activity.

Experimental procedures
Materials
FSAP was isolated as described previously [5]. PolyP 65-mer
(molecular mass  6.6 · 103 Da) and PolyP 15-mer (molecular mass  1.5 · 103 Da) were obtained from Sigma
(Munich, Germany), and PolyP 35-mer (molecular mass
 3.5 · 103 Da) was obtained from Roth (Karlsruhe,
Germany). Unfractionated heparin (molecular mass
 15 · 103 Da), heparan sulfate, dermatan sulfate, chondroitin sulfate C, low-molecular-weight heparin (molecular mass
 3 · 103 Da), N-acetyl heparin, de-N-sulfated heparin and
N-acetyl-de-O-sulfated heparin (all molecular masses
hyaluronic
acid
(molecular
mass
 15 · 103 Da),
 1 · 105 Da) from human placenta or rooster comb and
biotinylated heparin albumin were obtained from Sigma. Polysialic acid (molecular mass £ 38 · 103 Da) was separated
from oligosialic acid as described previously [33]. Calf intestinal alkaline phosphatase was obtained from Fermentas

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Polyanions and FSAP

L. Muhl et al.

(St Leon-Rot, Germany). PAI-1 was generously provided by
Dr Paul Declerck (Katholieke Universiteit, Leuven, Belgium).
AT was obtained from CSL Behring (Marburg, Germany).

Isolation of platelet-derived PolyP and mast
cell-derived macromolecular heparin

FSAP enzyme activity assay

Platelet homogenates were prepared as described previously
[34]. After centrifugation at 19 000 g, the pellet was used to
extract native PolyP using perchloric acid [10]. PolyP was
further purified on an OMIX C18 100 lL tip (Varian, Lake
Forest, CA) before use. Native macromolecular heparin
(molecular mass 75 · 104 Da; range 5 · 105–1 · 106) was
purified from granule remnants of rat serosal mast cells, as
described previously [35]. Briefly, granule remnants were
treated with 2 m KCl to release heparin-bound molecules
(notably chymase and other proteases) from heparin proteoglycans and to disintegrate the granule remnants into
heparin proteoglycan monomers [36]. The incubation mixture was then applied to a Sephacryl S-200 column (GE
Healthcare Life Sciences, Uppsala, Sweden) column for isolation and separation of heparin proteoglycans. The residual chymase activity in the heparin proteoglycan fraction
was inhibited using phenylmethanesulfonyl fluoride.

Electrophoretic mobility shift assays to detect
polyanion binding to FSAP
Polyacrylamide–bisacrylamide (37.5:1) native gels (6–10%)

were poured with Tris ⁄ borate ⁄ EDTA (TBE) (90 mm Tris,
90 mm boric acid, 2 mm EDTA, pH 8.3), with or without
6.7 m urea, in a horizontal gel chamber. FSAP (5 lg) was
preincubated for 30 min with or without respective polyanions (10 lg), native sample buffer (TBE with sucrose and
bromphenol blue) was added, and samples were loaded
onto the gel. After separation, the gel was stained either
with toluidine blue to visualize polyanions (not shown) or
with Coomassie brilliant blue to visualize proteins. Densiometric analysis was performed to determine the affinity of
these interactions.

Competition of heparin binding to immobilized
FSAP with various polyanions
Microtiter plates were coated with 50 lL of a 10 lgỈmL)1
FSAP solution in 100 mm sodium carbonate (pH 9.5) overnight at 4°C. Wells were washed, and non-specific binding
sites were blocked with NaCl/Tris (25 mm Tris ⁄ HCl, pH
7.5, 150 mm NaCl) containing 3% w ⁄ v BSA for 1 h. Biotinylated heparin albumin (0.5 ngỈmL)1) mixed with dilutions
of polyanions was allowed to bind for 1 h at room temperature in NaCl/Tris containing 0.1% w ⁄ v BSA, after which
the plates were washed three times with NaCl/Tris containing 0.1% w ⁄ v Tween-20 (NaCl/Tris-T). Bound biotinylated

4836

heparin albumin was detected using peroxidase-conjugated
streptavidin (DAKO, Glostrup, Denmark) and an immunopure TMB substrate kit (Thermo Fischer Scientific, Rockford, IL, USA).

FSAP activity assays were performed as described previously
[16]. In brief, microtiter plates were blocked with NaCl/Tris
containing 3% w ⁄ v BSA for 1 h, and washed with NaCl/
Tris-T. The standard assay system consisted of NaCl/Tris,
1 lgỈmL)1 FSAP and 0.2 mm of the chromogenic substrate
S-2288 (H-d-isoleucyl-l-prolyl-l-arginine-p-nitroanilinedihydrochloride) (Haemochrome, Essen, Germany) and was

followed over a period of 60 min at 37°C at 405 nm in an
EL 808 microplate reader (BioTek Instruments, Winooski,
VT, USA). If an inhibitor was used, this was added together
with FSAP to the plates with and without polyanionic
co-factor 30 min before adding the chromogenic substrate.

Characterization of FSAP–inhibitor interaction
using surface plasmon resonance (SPR)
technology
Immobilization on sensor chips, and association and dissociation of interacting biomolecules, were followed in real
time by monitoring the change in SPR signal expressed in
resonance units (RU). All experiments were performed at
25°C. To prepare the sensor chip surface, antibodies to
FSAP or isotype controls were immobilized on a CM5
research-grade chip (Biacore/GE Healthcare, Freiburg,
Germany) at 10 000 RU, via amino coupling (Biacore)
and using HBS-N (20 mm Hepes, pH 7.4, 100 mm NaCl),
as running buffer. Interaction analysis experiments were
performed at a flow rate of 20 lLỈmin)1 using HBS-P
[20 mm Hepes, pH 7.4, 100 mm NaCl, 0.05% Surfactant
P20 (Biacore cat.nr.:BR-1000-54)] supplemented with 2 mm
CaCl2 as running buffer. FSAP (25 lL, 10 lgỈmL)1) was
captured on the immobilized antibodies, and then AT or
PAI-1 (25 lL, 0–5 lgỈmL)1) were injected alone and in the
presence of unfractionated heparin or PolyP (10 lgỈmL)1).
Sensorgrams were analyzed using BIAevaluation software
version 3.2 RC1. Kinetic constants were obtained using the
Langmuir binding model 1:1.

Cell culture

Mouse vascular smooth muscle cells (VSMC) were cultured
in Iscove’s modified medium (Invitrogen, Karlsruhe, Germany) with 10% v ⁄ v fetal calf serum (HyClone, Logan,
UT, USA), 10 mL)1 penicillin, 10 lgỈmL)1 streptomycin,
2 mm l-glutamine and 1 mm sodium pyruvate (Invitrogen).
Cells were growth-arrested in serum-free medium for 18 h
prior to experiments.

FEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBS


L. Muhl et al.

Polyanions and FSAP

DNA synthesis assays
VSMC were stimulated for 36 h with the test substances in
medium containing 0.2% fetal calf serum. For the last
24 h, 5-bromo-2-deoxyuridine (BrdU) was added, and the
cells were processed using a BrdU detection kit (Roche
Diagnostics, Mannheim, Germany) as described by the
manufacturer.

8

9

10

Acknowledgements
The assistance of Susanne Tannert-Otto is greatly

appreciated. We are grateful to Dr Paul Declerck
(Department of Pharmaceutical Sciences, Katholieke
Universiteit, Leuven, Belgium) for providing PAI-1.
This study was financed by a grant from the Deutsche
Forschungsgemeinschaft to S.M.K. (SFB 547: C14).
Wihuri Research Institute is maintained by the Jenny
and Antti Wihuri Foundation (Helsinki, Finland).

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Supporting information
The following supplementary material is available:
Fig. S1. Analysis of the binding between FSAP
and polyanions using electrophoretic mobility shift
assay.
Fig. S2. Determination of the kinetic constants for
FSAP.
Fig. S3. Interaction of polysialic acid or dermatan
sulfate with FSAP.
Fig. S4. Inhibition of the effect of PolyP by phosphatase.
Fig. S5. Determination of the IC50 value for PAI-1mediated inhibition of FSAP.

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L. Muhl et al.

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online article.
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Polyanions and FSAP

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4839