To further investigate the structural requirements of polyanions for controlling FSAP activity, we performed binding, activation and inhibition studies using hep-arin and derivatives wit
Trang 1heparin regulates factor VII-activating protease
Lars Muhl1, Sebastian P Galuska1, Katariina O¨ o¨rni2
, Laura Herna´ndez-Ruiz3, Luminita-Cornelia Andrei-Selmer4, Rudolf Geyer1, Klaus T Preissner1, Felix A Ruiz3, Petri T Kovanen2
and Sandip M Kanse1
1 Institute for Biochemistry, Justus-Liebig-University, Giessen, Germany
2 Wihuri Research Institute, Helsinki, Finland
3 Unidad de Investigacion, Hospital Universidad Puerta del Mar and Universidad de Cadiz, Spain
4 Philipps University, Marburg, Germany
Introduction
Factor VII-activating protease (FSAP) is a serine
pro-tease that is predominantly expressed in the liver It
circulates as an inactive zymogen with a concentration
of 12 lgÆmL)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-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: sandip.kanse@biochemie.med.
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 hep-arin and derivatives with altered size and charge, as well as other glycosa-minoglycans 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 acti-vator 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 unfrac-tionated 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
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.
Trang 2fore 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
activa-tion [4,5], followed by auto-proteolysis This
propen-sity to partial proteolysis has been used to determine
the domain responsible for binding to heparin and
RNA Multiple regions of FSAP contribute to
polyan-ion 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
hyal-uronic 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
high-to low-molecular-weight form, is related high-to the
regula-tion 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
ubiq-uitous 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
enhanc-ing the activity of thrombin-activated fibrinolysis
inhibitor (TAFI) [11]
Once activated, FSAP can be rapidly inhibited by
serine protease inhibitors (SERPINs), such as
a1-anti-trypsin, 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
inhibi-tors 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
(G534E, or Marburg I polymorphism) results in dimin-ished proteolytic activity towards factor VII, pro-urokinase [17] and PDGF-BB (platelet-derived growth factor BB) [18] The Marburg I polymorphism is asso-ciated with a higher risk for carotid stenosis [19], and,
in comparison to wild-type FSAP, is not able to inhi-bit neointima formation in a mouse model [18] Simi-larly, Marburg I FSAP is associated with advanced liver fibrosis, which may be due to its inability to inhi-bit 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, acti-vation and regulation mechanisms
Results
FSAP binding to polyanions Electrophoretic mobility shift assays were performed
to characterize the interaction between FSAP and vari-ous polyanions Preincubation of FSAP with unfrac-tionated 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) Concentration-dependent 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 bind-ing of biotinylated unfractionated heparin to FSAP was measured (Fig 1C) Unfractionated heparin com-peted 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
Trang 3Activation of FSAP by various polyanions
We next investigated all the polyanions described
above with respect to their ability to activate FSAP
Unfractionated heparin was a strong activator, low-molecular-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
A
B
C
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 pan-els) Shifted bands (complexed FSAP and polyanions) indicate bind-ing of the particular polyanion to FSAP (B) In a similar experiment
to that shown in (A), the concentration of unfractionated heparin and PolyP 65 was varied (0.002–2 l M ) Complex formation was quantified by densiometric analysis, and the results from three sep-arate experiments were pooled to determine the EC50 values (C) FSAP (10 lgÆmL)1) was immobilized, and heparin derivatives (upper panel) or other polyanions (lower panel) (0.01–100 lgÆmL)1) were mixed with biotinylated heparin albumin (0.5 ngÆmL)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).
Fig 2 Increased auto-activation of FSAP by polyanions Polyanions
at concentrations in the range 0.01–100 lgÆmL)1 were added to FSAP (1 lgÆmL)1), and FSAP activity (mmODÆmin)1) was deter-mined (mean ± SEM, n = 4).
Trang 4KM (Fig S2) Heparan sulfate and dermatan sulfate
showed weak activation of FSAP at high
concentra-tions (Fig 2, middle panel) Polysialic acid and
hyal-uronic acid did not activate FSAP (Fig 2, lower
panel) N-acetyl heparin, de-N-sulfated heparin,
N-ace-tyl-de-O-sulfated heparin, polysialic acid and
hyal-uronic 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 as co-factors for the inhibition of
FSAP by PAI-1 and AT
SERPINs exhibit enhanced or altered substrate
specific-ity 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,
low-molecular-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 IC50of
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
A
B
C
Fig 3 Inhibition of FSAP by PAI-1 and AT; co-factor function of
polyanions FSAP (1 lgÆmL)1) was preincubated either with PAI-1
(1 lgÆmL)1) (A) or with AT (5 lgÆmL)1) (B) for 30 min with or
with-out heparin derivatives (upper panels) or other polyanions (lower
panels) in the concentration range 0.01–100 lgÆmL)1 FSAP activity
(mmODÆmin)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 lgÆmL)1) was bound to a specific high-affinity antibody to
FSAP, immobilized on a CM5 sensor chip, prior to injection of
either AT (5 lgÆmL)1) or PAI-1 (5 lgÆmL)1), alone (control) or in the
presence of polyphosphate 65 (10 lgÆmL)1) or unfractionated
hepa-rin (10 lgÆmL)1) Alignment of SPR sensograms was performed
using the program BIAevaluation 3.2 RC1.
Trang 5at even higher concentrations (Fig S3B), but
hyal-uronic acid had no effect at all (Fig 3A, lower panel)
In the case of FSAP inhibition by AT, only
unfrac-tionated 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
AT-dependent FSAP inhibition (Fig 3B, lower panel and
Fig S3C) The activity of FSAP was increased by
low-molecular-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
reso-nance (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 (KAof 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
hepa-ran sulfate also increase the inhibitory effect of FSAP
on proliferation of vascular smooth muscle cells
(VSMC), but to a lower extent compared to
unfrac-tionated heparin PolyP also promoted the inhibitory
effect of FSAP on VSMC proliferation, whereas
de-N-sulfated 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
plate-let-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
hepa-rin, 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)
In mobility shift assays, platelet-derived PolyP bound to FSAP weakly (Fig 5A, upper panel) How-ever, it competed with biotinylated heparin for binding
to immobilized FSAP more strongly than its synthetic analogue PolyP65 did (Fig 5A, lower panel) Unex-pectedly, 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 Mar-burg I single-nucleotide polymorphism is a risk factor for carotid stenosis [19] and liver fibrosis [20] This iso-form 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 reg-ulation of FSAP activity in order to define its patho-physiological role Polyanions have been shown to play a key role in regulating FSAP activity by promot-ing auto-catalytic activation In the present study, we systematically characterized the effects of various polyanions on FSAP activity
no PDGF-BB or FSAP or polyanion Buffer
FSAP with PDGF-BB and no FSAP or polyanion
Fig 4 Polyanion-dependent amplification of the inhibitory effect of FSAP on VSMC activation PDGF-BB (20 ngÆmL)1) was
preincubat-ed without (light gray columns) or with (dark gray columns) FSAP (15 lgÆmL)1) and ⁄ or 10 lgÆmL)1 of the various polyanions for 1 h
at 37C in serum-free medium Subsequently, VSMC were stimu-lated 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.
Trang 6Heparin and other glycosaminoglycans
The binding to FSAP and the subsequent activation of
FSAP by heparin depends on its size and overall
nega-tive charge Low-molecular-weight heparin exhibits
lower potential for binding to and activating FSAP The heparin homologues N-acetyl heparin, de-N-sul-fated 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 pro-teoglycan 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 unfrac-tionated 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 un-fractionated 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 pres-ent study, we demonstrate that there is no tight inter-action 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 immobi-lized hyaluronic acid The significance of these results
is that the very ubiquitous heparan sulfate proteogly-cans and other matrix-associated glycosaminoglyproteogly-cans 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 atheroscle-rotic plaques [26]
Polyphosphate PolyP was a more potent activator of FSAP than hepa-rin PolyP65 was the most active form of PolyP, with smaller forms showing diminished activity Degrada-tion by phosphatases decreased its properties with
Free FSAP
Complexed FSAP
Unfractionated heparin Mast cell heparin Polyphosphate 65 Platelet polyphosphate
80
120
Unfractionated heparin Mast cell heparin –40
0
40
//
//
Heparin (µg·mL–1)
//
120
0
80
Platelet polyphosphate
40
//
//
0 0.1 1 10
0 0.1 1 10
Polyphosphate (µg·mL–1)
Polyphosphate 65 –40 //
//
Unfractionated heparin
Mast cell heparin
10
15
20
0
0
5
//
//
Polyphosphate 65
Platelet polyphosphate
20
Heparin (µg·mL–1)
5
10
15
Polyphosphate (µg·mL–1)
0.1
0 //
//
A
B
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 cell-derived 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 polyan-ion to FSAP Middle and lower panels: FSAP (10 lgÆmL)1) was immobilized, and synthetic or mast cell-derived heparin (0.05–
10 lgÆmL)1) and synthetic or platelet-derived PolyP (0.033–
5 lgÆmL)1) were mixed with biotinylated heparin albumin (0.5 ngÆmL)1) and added to the plate The amount of bound biotiny-lated heparin albumin was measured using peroxidase-conjugated streptavidin and TMB substrate (mean ± SD, n = 3) (B) Unfraction-ated heparin (0.01–10 lgÆmL)1), mast cell-derived heparin (0.02–
5 lgÆmL)1) (upper panel) or synthetic or platelet-derived PolyP (0.01–2.5 lgÆmL)1) (lower panel) were added to FSAP (1 lgÆmL)1), and FSAP activity (mmODÆmin)1) was determined (mean ± SD,
n = 3).
Trang 7respect to FSAP binding and activation, and any
influ-ence on FSAP activity was completely neutralized In
order to put these findings in a pathophysiological
con-text, 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
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
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].
Trang 8size, 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
com-pare 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
demon-strate 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
inter-action with AT, as indicated by the fact that
low-molecular-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
struc-ture 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 IC50for 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
com-plex 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
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 deter-mine the affinity of this interaction Other matrix-derived 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
PDGF-BB 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 func-tion 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 condi-tions 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 (mole-cular mass 1.5 · 103
Da) were obtained from Sigma (Munich, Germany), and PolyP 35-mer (molecular mass
3.5 · 103Da) was obtained from Roth (Karlsruhe, Germany) Unfractionated heparin (molecular mass
15 · 103
Da), heparan sulfate, dermatan sulfate, chondroi-tin sulfate C, low-molecular-weight heparin (molecular mass
3 · 103Da), N-acetyl heparin, de-N-sulfated heparin and N-acetyl-de-O-sulfated heparin (all molecular masses
15 · 103Da), hyaluronic acid (molecular mass
1 · 105
Da) from human placenta or rooster comb and biotinylated heparin albumin were obtained from Sigma Poly-sialic acid (molecular mass£ 38 · 103
Da) was separated from oligosialic acid as described previously [33] Calf intesti-nal alkaline phosphatase was obtained from Fermentas
Trang 9(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
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· 104Da; 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
prote-oglycans and to disintegrate the granule remnants into
heparin proteoglycan monomers [36] The incubation
mix-ture was then applied to a Sephacryl S-200 column (GE
Healthcare Life Sciences, Uppsala, Sweden) column for
iso-lation and separation of heparin proteoglycans The
resid-ual 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
polya-nions (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
Densio-metric 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 lgÆmL)1
FSAP solution in 100 mm sodium carbonate (pH 9.5)
over-night at 4C 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
Bioti-nylated heparin albumin (0.5 ngÆmL)1) mixed with dilutions
of polyanions was allowed to bind for 1 h at room
temper-ature in NaCl/Tris containing 0.1% w⁄ v BSA, after which
the plates were washed three times with NaCl/Tris
contain-ing 0.1% w⁄ v Tween-20 (NaCl/Tris-T) Bound biotinylated
heparin albumin was detected using peroxidase-conjugated streptavidin (DAKO, Glostrup, Denmark) and an immuno-pure TMB substrate kit (Thermo Fischer Scientific, Rock-ford, IL, USA)
FSAP enzyme activity assay
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 lgÆmL)1 FSAP and 0.2 mm of the chromogenic substrate S-2288 (H-d-isoleucyl-l-prolyl-l-arginine-p-nitroanilinedi-hydrochloride) (Haemochrome, Essen, Germany) and was followed over a period of 60 min at 37C 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 disso-ciation 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 25C 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 lLÆmin)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 lgÆmL)1) was captured on the immobilized antibodies, and then AT or PAI-1 (25 lL, 0–5 lgÆmL)1) were injected alone and in the presence of unfractionated heparin or PolyP (10 lgÆmL)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, Ger-many) with 10% v⁄ v fetal calf serum (HyClone, Logan,
UT, USA), 10 UÆmL)1penicillin, 10 lgÆmL)1streptomycin,
2 mm l-glutamine and 1 mm sodium pyruvate (Invitrogen) Cells were growth-arrested in serum-free medium for 18 h prior to experiments
Trang 10DNA 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
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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