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High negative charge-to-size ratio in polyphosphates andheparin regulates factor VII-activating proteaseLars Muhl1, Sebastian P. Galuska1, Katariina O¨o¨rni2, Laura Herna´ndez-Ruiz3, Luminita-CorneliaAndrei-Selmer4, Rudolf Geyer1, Klaus T. Preissner1, Felix A. Ruiz3, Petri T. Kovanen2and Sandip M. Kanse11 Institute for Biochemistry, Justus-Liebig-University, Giessen, Germany2 Wihuri Research Institute, Helsinki, Finland3 Unidad de Investigacion, Hospital Universidad Puerta del Mar and Universidad de Cadiz, Spain4 Philipps University, Marburg, GermanyIntroductionFactor VII-activating protease (FSAP) is a serine pro-tease that is predominantly expressed in the liver. Itcirculates as an inactive zymogen with a concentrationof 12 lgÆmL)1in the plasma, and is known to activatefactor VII and pro-urokinase [1,2]. It was first purifiedby its ability to bind to hyaluronic acid, and was there-KeywordsFSAP; heparin; mast cells; platelets;polyphosphateCorrespondenceS. M. Kanse, Institute for Biochemistry,Justus-Liebig-University Giessen,Friedrichstrasse 24, 35392 Giessen,GermanyFax: +49 641 9947509Tel: +49 641 9947521E-mail: sandip.kanse@biochemie.med.uni-giessen.de(Received 12 March 2009, revised 28 May2009, accepted 29 June 2009)doi:10.1111/j.1742-4658.2009.07183.xFactor VII-activating protease (FSAP) circulates as an inactive zymogen inthe plasma. FSAP also regulates fibrinolysis by activating pro-urokinase orcellular activation via cleavage of platelet-derived growth factor BB(PDGF-BB). As the Marburg I polymorphism of FSAP, with reducedenzymatic activity, is a risk factor for atherosclerosis and liver fibrosis, theregulation of FSAP activity is of major importance. FSAP is activated byan auto-catalytic mechanism, which is amplified by heparin. To furtherinvestigate the structural requirements of polyanions for controlling FSAPactivity, 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 asize- and charge density-dependent manner. Polyphosphate was morepotent than heparin with regard to its interactions with FSAP. Heparinwas also an effective co-factor for inhibition of FSAP by plasminogen acti-vator inhibitor 1 (PAI-1) and antithrombin, whereas polyphosphate servedas co-factor for the inhibition of FSAP by PAI-1 only. For FSAP-mediatedinhibition of PDGF-BB-induced vascular smooth muscle cell proliferation,heparin as well as a polyphosphate served as efficient co-factors. Nativemast cell-derived heparin exhibited identical properties to those of unfrac-tionated heparin. Despite the strong effects of synthetic polyphosphate, theplatelet-derived material was a weak activator of FSAP. Hence, negativelycharged polymers with a high charge-to-size ratio are responsible for theactivation of FSAP, and also act as co-factors for its inhibition by serineprotease inhibitors.AbbreviationsAT, 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.4828 FEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBSfore designated as hyaluronic acid binding protein 2(HABP-2) [3]. Activation of FSAP requires cleavagebetween residues R313 and I314, separating the lightchain and the heavy chain [4].Negatively charged polyanions such as heparin [4,5],nucleic acids [6,7] and dextran sulfate [4] bind toFSAP. This interaction leads to auto-catalytic activa-tion [4,5], followed by auto-proteolysis. This propen-sity to partial proteolysis has been used to determinethe domain responsible for binding to heparin andRNA. Multiple regions of FSAP contribute to polyan-ion binding, but the epidermal growth factor like-3(EGF3) domain with a cluster of positively chargedamino acids is particularly important [6].Although FSAP was initially isolated on a hyal-uronic acid column [3], no information is available asto how hyaluronic acid can bind to FSAP, norwhether it can activate FSAP [4]. The concentration ofhyaluronic acid, as well as its transition from the high-to low-molecular-weight form, is related to the regula-tion of angiogenesis, atherosclerosis, restenosis andinflammation [8]. FSAP activation is also mediated bynucleic acids, with RNA having a stronger effect thanDNA [6,7]. Heparin is the most extensively studiedpolyanion with respect to FSAP function. It has beenshown that unfractionated heparin is a strong activatorof FSAP, but low-molecular-weight heparin has notbeen systematically tested. The role of the more ubiq-uitous heparan sulfate and other glycosaminoglycans isalso not known.Polyphosphate (PolyP) is a linear polymer oforthophosphate (Pi) residues linked by high-energyphosphoanhydride bonds, found in many cell types[9]. PolyP, with an approximate chain length from70–75 phosphate units, is stored in platelet-densegranules [10] and released upon platelet activation.PolyP can amplify coagulation by activation of thecontact factor pathway, as well as activation offactor V, inhibition of the anticoagulant function oftissue factor pathway inhibitor (TFPI), and enhanc-ing the activity of thrombin-activated fibrinolysisinhibitor (TAFI) [11].Once activated, FSAP can be rapidly inhibited byserine protease inhibitors (SERPINs), such as a1-anti-trypsin, a2-antiplasmin, antithrombin (AT) and C1inhibitor [4,12–14], as well as plasminogen activatorinhibitor 1 (PAI-1) [15] and protease nexin 1 [16]. ATand a2-antiplasmin were shown to be efficient inhibi-tors in the presence of heparin [4], whereas PAI-1 wasshown to be an inhibitor only in the presence of RNAbut not heparin [15].The presence of a naturally occurring polymorphismin 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 growthfactor 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 advancedliver fibrosis, which may be due to its inability to inhi-bit PDGF-BB-mediated proliferation of hepatic stellatecells [21]. These findings indicate the importance ofFSAP enzymatic activity with respect to its functionin vivo. However, it is not clear which polyanions arerelevant for the regulation of FSAP activity. Thisprompted us to investigate the requirements for FSAPinteraction with polyanions known to be present inatherosclerotic arterial wall and ⁄ or fibrotic liver, andalso to define the molecular basis of the binding, acti-vation and regulation mechanisms.ResultsFSAP binding to polyanionsElectrophoretic mobility shift assays were performedto characterize the interaction between FSAP and vari-ous polyanions. Preincubation of FSAP with unfrac-tionated heparin, low-molecular-weight heparin,PolyP65or PolyP35induced a shift in the mobility ofFSAP in polyacrylamide gels with or without urea.Other polyanions had no influence at all. When BSAwas used as a control, none of the polyanions induceda shift in the BSA band (Fig. 1A). Concentration-dependent analysis indicated that the EC50was 95 ±7nm for the shift with unfractionated heparin and28 ± 3 nm for PolyP65(Fig. 1B and Figs S1 and S2).To examine whether the various polyanions use thesame region in the FSAP molecule for binding, weperformed competition binding assays in which bind-ing of biotinylated unfractionated heparin to FSAPwas measured (Fig. 1C). Unfractionated heparin com-peted with biotinylated heparin for binding to FSAP,whereas low-molecular-weight heparin showed lowcompetition (Fig. 1C, upper panel). PolyP competedfor this binding in a chain length-dependent manner.All other heparin derivatives, as well as chondroitinsulfate, dermatan sulfate, polysialic acid, heparansulfate and hyaluronic acid, showed no competition,indicating no binding to FSAP (Fig. 1C, lower panel,and Fig. S3A). Thus, using gel-shift and competitionbinding assays, it was demonstrated that binding toFSAP depends on the size and charge density of themacromolecule.L. Muhl et al. Polyanions and FSAPFEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBS 4829Activation of FSAP by various polyanionsWe next investigated all the polyanions describedabove with respect to their ability to activate FSAP.Unfractionated heparin was a strong activator, low-molecular-weight heparin activated FSAP to a smallerextent, and all other heparin-derivatives exhibited noactivation (Fig. 2, upper panel). PolyP showed potentactivation of FSAP in a chain length-dependentmanner. There was a 4–6-fold increase in Vmaxwithunfractionated heparin and PolyP65, with no change inA B C Fig. 1. Binding of FSAP to polyanions. (A) FSAP or BSA (5 lg perlane) 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 experimentto that shown in (A), the concentration of unfractionated heparinand PolyP65was varied (0.002–2 lM). Complex formation wasquantified by densiometric analysis, and the results from three sep-arate experiments were pooled to determine the EC50values. (C)FSAP (10 lgÆmL)1) was immobilized, and heparin derivatives (upperpanel) or other polyanions (lower panel) (0.01–100 lgÆmL)1) weremixed with biotinylated heparin albumin (0.5 ngÆmL)1) and added tothe plate. Detection of bound biotinylated heparin albumin wasmeasured 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. Polyanionsat concentrations in the range 0.01–100 lgÆmL)1were added toFSAP (1 lgÆmL)1), and FSAP activity (mmODÆmin)1) was deter-mined (mean ± SEM, n = 4).Polyanions and FSAP L. Muhl et al.4830 FEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBSKM(Fig. S2). Heparan sulfate and dermatan sulfateshowed weak activation of FSAP at high concentra-tions (Fig. 2, middle panel). Polysialic acid and hyal-uronic acid did not activate FSAP (Fig. 2, lowerpanel). 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 wasdegraded using calf intestinal phosphatase, which isalso a highly active exopolyphosphatase [11]. Theaccelerating effect of PolyP on FSAP activity wasdecreased by phosphatase pretreatment in a time- anddose-dependent manner (Fig. S4). As a control, weobserved that phosphatase treatment did not influenceunfractionated heparin-mediated activation of FSAP(Fig. S4). Hence, the effect of PolyP was not due to acontaminant. These studies show that the pattern ofbinding of polyanions to FSAP is identical to thepattern of their ability to activate FSAP.Polyanions as co-factors for the inhibition ofFSAP by PAI-1 and ATSERPINs 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 withrespect to FSAP inhibition, active two-chain FSAP waspreincubated with PAI-1 or AT with or without variousconcentrations of polyanions. Inhibition of FSAP byPAI-1 was increased by unfractionated heparin, low-molecular-weight heparin and to a lower extent byN-acetyl heparin (Fig. 3A, upper panel). PolyP exhibitsstrong co-factor function for the inhibition of FSAP byPAI-1 in a chain length-dependent manner. The IC50ofPAI-1 for the inhibition of FSAP was halved byunfractionated heparin and PolyP65(Fig. S5). Heparansulfate was a co-factor at high concentrations (Fig. 3A,lower panel), and dermatan sulfate and polysialic acidABCFig. 3. Inhibition of FSAP by PAI-1 and AT; co-factor function ofpolyanions. 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 (lowerpanels) in the concentration range 0.01–100 lgÆmL)1. FSAP activity(mmODÆmin)1) was determined, and inhibition was calculated as apercentage of FSAP activity without inhibitor (mean ± SEM, n = 4).(C) SPR sensograms showing the association and dissociation ofFSAP–inhibitor complexes in the presence of polyanions. FSAP(10 lgÆmL)1) was bound to a specific high-affinity antibody toFSAP, immobilized on a CM5 sensor chip, prior to injection ofeither AT (5 lgÆmL)1) or PAI-1 (5 lgÆmL)1), alone (control) or in thepresence of polyphosphate 65 (10 lgÆmL)1) or unfractionated hepa-rin (10 lgÆmL)1). Alignment of SPR sensograms was performedusing the program BIAevaluation 3.2 RC1.L. Muhl et al. Polyanions and FSAPFEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBS 4831at 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 toserve as co-factors (Fig. 3B). PolyP and other testedpolyanions showed no co-factor properties for the AT-dependent FSAP inhibition (Fig. 3B, lower panel andFig. 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 inthe presence of AT.To consolidate these findings, real-time interactionstudies were performed using surface plasmon reso-nance (SPR). These results confirm that FSAP interactswith AT only in the presence of unfractionated heparin(KAof  2.9 · 107[1 ⁄ M]) but not in the presence ofPolyP. In contrast, FSAP interacts with PAI-1 withouta co-factor (KAof  1.6 · 107[1 ⁄ M]) in the presenceof unfractionated heparin (KAof  3.2 · 107[1 ⁄ M]) aswell as in the presence of PolyP (KAof  97 · 107[1 ⁄ M]) (Fig. 3C). Hence, polyanions can selectivelypromote inhibition of the enzymatic activity of FSAP.Polyanions as co-factors for the FSAP-dependentinhibition of VSMC proliferationA major function of FSAP is the specific proteolyticcleavage and inactivation of PDGF-BB [23], and thisprocess is enhanced by heparin and RNA [24]. Weobserved that low-molecular-weight heparin and hepa-ran sulfate also increase the inhibitory effect of FSAPon proliferation of vascular smooth muscle cells(VSMC), but to a lower extent compared to unfrac-tionated heparin. PolyP also promoted the inhibitoryeffect of FSAP on VSMC proliferation, whereas de-N-sulfated heparin and hyaluronic acid were ineffective(Fig. 4). The ability of each polyanion to inhibit cellproliferation matched the respective pattern of FSAPbinding and activation.Assessment of mast cell heparin and plateletPolyP as co-factors for FSAP functionMast cell-derived macromolecular heparin and plate-let-derived PolyP were isolated as native substancesand tested for their interaction with FSAP. The mastcell-derived heparin bound to FSAP, as indicated by amobility shift in native polyacrylamide gels (Fig. 5A,upper panel). When compared to unfractionated hepa-rin, mast cell heparin was even more efficient withrespect to competition of biotinylated heparin bindingto immobilized FSAP (Fig. 5A, middle panel) andFSAP activation (Fig. 5B, upper panel).In mobility shift assays, platelet-derived PolyPbound to FSAP weakly (Fig. 5A, upper panel). How-ever, it competed with biotinylated heparin for bindingto immobilized FSAP more strongly than its syntheticanalogue PolyP65did (Fig. 5A, lower panel). Unex-pectedly, activation of FSAP by native platelet-derivedPolyP was much lower when compared to the syntheticmaterial (Fig. 5B, lower panel). Thus, mast cell-derivedheparin was identical to unfractionated heparin for allaspects investigated, but there were differences betweenplatelet-derived and synthetic PolyP.DiscussionGenetic studies show that the presence of the Mar-burg I single-nucleotide polymorphism is a risk factorfor carotid stenosis [19] and liver fibrosis [20]. This iso-form of FSAP exhibits reduced enzymatic activity [17],indicating that the local proteolytic activity of FSAPmay play a crucial role in development of the diseasestate. Therefore, it is important to understand the reg-ulation of FSAP activity in order to define its patho-physiological role. Polyanions have been shown toplay a key role in regulating FSAP activity by promot-ing auto-catalytic activation. In the present study, wesystematically characterized the effects of variouspolyanions on FSAP activity.no PDGF-BB or FSAP or polyanionBufferFSAPwith PDGF-BB and no FSAP or polyanionFig. 4. Polyanion-dependent amplification of the inhibitory effect ofFSAP 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)1of the various polyanions for 1 hat 37°C in serum-free medium. Subsequently, VSMC were stimu-lated for 36 h in medium containing 0.2% fetal calf serum. DNAsynthesis was measured (mean ± SD, n = 3) using a kit thatdetects BrdU incorporation into newly synthesized DNA.Polyanions and FSAP L. Muhl et al.4832 FEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBSHeparin and other glycosaminoglycansThe binding to FSAP and the subsequent activation ofFSAP by heparin depends on its size and overall nega-tive charge. Low-molecular-weight heparin exhibitslower 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 negativecharge, compared to unfractionated heparin, andexhibits weak FSAP binding and activation. Mastcell-derived heparin has a higher charge than unfrac-tionated heparin, and exhibits a stronger ability tobind to and activate FSAP [25].Chondroitin sulfate, dermatan sulfate and polysialicacid also have a less negative charge density than un-fractionated heparin and show no FSAP binding oractivation potential (Fig. 6). FSAP was first purifiedbased 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 likelydue to the relatively low negative charge density in thepolyanion. Its isolation on hyaluronic acid columnscould be due to altered physical properties of immobi-lized hyaluronic acid. The significance of these resultsis that the very ubiquitous heparan sulfate proteogly-cans and other matrix-associated glycosaminoglycansplay no role in the regulation of FSAP activity. This israther related to the proximity and activation state ofmast cells that secrete heparin, such as in atheroscle-rotic plaques [26].PolyphosphatePolyP was a more potent activator of FSAP than hepa-rin. PolyP65was the most active form of PolyP, withsmaller forms showing diminished activity. Degrada-tion by phosphatases decreased its properties withFree FSAP Complexed FSAP Unfractionated heparin Mast cell heparin Polyphosphate 65 Platelet polyphosphate 80120Unfractionated heparinMast cell heparin–40040// // Heparin (µg·mL–1)// 120Competition with heparin-albumin-biotin (%)080Platelet polyphosphate40// // 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 0.01 0.1 1 10 Heparin (µg·mL–1)FSAP-activity (mmOD·min–1 at 405 nm)5 10 15 Polyphosphate (µg·mL–1)0.1 0 0.01 1 10 0 // // A B Fig. 5. Properties of mast cell-derived heparin and platelet-derivedPolyP with respect to FSAP. (A) Upper panel: FSAP (5 lg per lane)was preincubated with unfractionated heparin (UH), mast cell-derived heparin, PolyP65or platelet-derived PolyP (each 2 lg perlane), and loaded directly onto native polyacrylamide gel. Shiftedbands (complexed FSAP) indicate binding of the respective polyan-ion to FSAP. Middle and lower panels: FSAP (10 lgÆmL)1) wasimmobilized, 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-conjugatedstreptavidin 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).L. Muhl et al. Polyanions and FSAPFEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBS 4833respect to FSAP binding and activation, and any influ-ence on FSAP activity was completely neutralized. Inorder to put these findings in a pathophysiological con-text, we compared the activity of synthetic PolyP withthat of native platelet-derived material. Platelet-derivedPolyP exhibited quite anomalous properties comparedto synthetic PolyP. In gel-shift assays, it demonstratedweak binding, but was as efficient as synthetic PolyP incompeting for heparin binding to FSAP. Native PolyPwas a very weak activator of FSAP compared to thesynthetic version. One reason for this discrepancybetween synthetic and native PolyP could be thatsynthetic PolyP65is a heterogeneous mixture, withpolymers up to 200 units, whereas native PolyP isextremely pure and has a more homogeneous size with70–75 units [10,27]. In addition to their difference inFig. 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].Polyanions and FSAP L. Muhl et al.4834 FEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBSsize, we cannot exclude the possibility of a contaminantthat has a confounding effect on the interaction ofnative PolyP with FSAP. No comparable data exist inthe literature, as this is one of the first studies to com-pare the activities of synthetic with platelet-derivedPolyP. Given the robust activity of synthetic PolyP, therole of endogenous platelet-derived material needs tobe investigated further.InhibitionSERPINs such as protease nexin 1 and PAI-1 canefficiently inhibit FSAP. Whereas protease nexin 1inhibits proteases independently of any co-factor [16],PAI-1 is known to require heparin as a co-factor forinhibition of some of its targets such as thrombin[28]. The co-factor effect of heparin is due to achange in the conformation of the SERPIN as wellas the ability of heparin to co-join the protease withthe inhibitor. Previously published data showed thatheparin was not a co-factor for PAI-1-dependentinhibition of FSAP [15]. In this study, we demon-strate that both heparin and polyphosphate arepotent co-factors for the inhibition of FSAP byPAI-1. A reduction in size and charge density inheparin led to lower inhibition of FSAP by PAI-1.AT inhibits FSAP only in the presence of heparinbut not PolyP. The size and negative charge ofheparin 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 promotean increase in FSAP activity rather than inhibitingit. Thus, polyanion binding to SERPINs, over andabove their binding to FSAP, plays a decisive rolein mediating its inhibition.PolyP increased the inhibition of FSAP by PAI-1 butnot by AT. Whereas heparin changes the tertiary struc-ture of AT [29], PolyP was shown to be unable to induceany conformational changes in AT, as determined bymeasurement of the intrinsic protein fluorescence of ATincubated with PolyP (F. A. Ruiz, unpublished results).Both polyanions decreased the IC50for the inhibition ofFSAP by PAI-1 twofold. SERPINs inhibit their targetprotease by a suicide substrate mechanism that involvesa 1 : 1 formation of an irreversible covalent complex[30]. Only protease-inactive mutants show reversiblebinding to SERPINs [30], and the FSAP–PAI-1 com-plex demonstrated some dissociation in our experiments(Fig. 3C), indicating some deviation from the classicalmodel of protease–inhibitor interactions. Hence, theoverall inhibition of FSAP depends not only on theinhibitor but also on the presence of an appropriateco-factor in the vicinity of FSAP.ConclusionsThe two major polyanions, heparin and PolyP, usethe same binding region in the FSAP molecule, asrevealed by the competition binding assay. Chargedensity, size and also conformational flexibility deter-mine the affinity of this interaction. Other matrix-derived polyanions were not effective. Binding topolyanions was also observed in the presence of astrong denaturant, urea, indicating a strong chargeinteraction. The region of FSAP that is probablyresponsible for this binding is the EGF3 domain,which contains a positively charged cluster of aminoacids, although other regions of FSAP promote thisinteraction [6]. Using a recombinant EGF3 domaindeletion mutant of FSAP, no activation of FSAPwas obtained with either heparin or with PolyP [31],further confirming the involvement of this region inpolyanion binding and activation. Polyanionsstrongly reduced the proliferative activity of PDGF-BB in the presence of FSAP. This could explain theinfluence of polyanions such as heparin on smoothmuscle proliferation in vivo [32], and a similar func-tion is expected for PolyP. As a lowering in FSAPactivity is correlated with diseases [19,20], these newinsights into the regulation of FSAP activity willlead to increased understanding of FSAP functionunder physiological und pathophysiological condi-tions. Identification of specific size, sequence andcharge requirements may allow rational design ofpolyanions with higher specificity for the regulationof FSAP activity.Experimental proceduresMaterialsFSAP was isolated as described previously [5]. PolyP 65-mer(molecular mass  6.6 · 103Da) and PolyP 15-mer (mole-cular mass  1.5 · 103Da) 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 · 103Da), heparan sulfate, dermatan sulfate, chondroi-tin sulfate C, low-molecular-weight heparin (molecular mass 3 · 103Da), N-acetyl heparin, de-N-sulfated heparin andN-acetyl-de-O-sulfated heparin (all molecular masses 15 · 103Da), hyaluronic acid (molecular mass 1 · 105Da) from human placenta or rooster comb andbiotinylated heparin albumin were obtained from Sigma. Poly-sialic acid (molecular mass £ 38 · 103Da) was separatedfrom oligosialic acid as described previously [33]. Calf intesti-nal alkaline phosphatase was obtained from FermentasL. Muhl et al. Polyanions and FSAPFEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBS 4835(St Leon-Rot, Germany). PAI-1 was generously provided byDr Paul Declerck (Katholieke Universiteit, Leuven, Belgium).AT was obtained from CSL Behring (Marburg, Germany).Isolation of platelet-derived PolyP and mastcell-derived macromolecular heparinPlatelet homogenates were prepared as described previously[34]. After centrifugation at 19 000 g, the pellet was used toextract native PolyP using perchloric acid [10]. PolyP wasfurther purified on an OMIX C18 100 lL tip (Varian, LakeForest, CA) before use. Native macromolecular heparin(molecular mass 75 · 104Da; range 5 · 105–1 · 106) waspurified from granule remnants of rat serosal mast cells, asdescribed previously [35]. Briefly, granule remnants weretreated with 2 m KCl to release heparin-bound molecules(notably chymase and other proteases) from heparin prote-oglycans and to disintegrate the granule remnants intoheparin proteoglycan monomers [36]. The incubation mix-ture was then applied to a Sephacryl S-200 column (GEHealthcare Life Sciences, Uppsala, Sweden) column for iso-lation and separation of heparin proteoglycans. The resid-ual chymase activity in the heparin proteoglycan fractionwas inhibited using phenylmethanesulfonyl fluoride.Electrophoretic mobility shift assays to detectpolyanion binding to FSAPPolyacrylamide–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 without6.7 m urea, in a horizontal gel chamber. FSAP (5 lg) waspreincubated for 30 min with or without respective polya-nions (10 lg), native sample buffer (TBE with sucrose andbromphenol blue) was added, and samples were loadedonto the gel. After separation, the gel was stained eitherwith toluidine blue to visualize polyanions (not shown) orwith Coomassie brilliant blue to visualize proteins. Densio-metric analysis was performed to determine the affinity ofthese interactions.Competition of heparin binding to immobilizedFSAP with various polyanionsMicrotiter plates were coated with 50 lLofa10lgÆmL)1FSAP solution in 100 mm sodium carbonate (pH 9.5) over-night at 4°C. Wells were washed, and non-specific bindingsites were blocked with NaCl/Tris (25 mm Tris ⁄ HCl, pH7.5, 150 mm NaCl) containing 3% w ⁄ v BSA for 1 h. Bioti-nylated heparin albumin (0.5 ngÆmL)1) mixed with dilutionsof polyanions was allowed to bind for 1 h at room temper-ature in NaCl/Tris containing 0.1% w ⁄ v BSA, after whichthe plates were washed three times with NaCl/Tris contain-ing 0.1% w ⁄ v Tween-20 (NaCl/Tris-T). Bound biotinylatedheparin albumin was detected using peroxidase-conjugatedstreptavidin (DAKO, Glostrup, Denmark) and an immuno-pure TMB substrate kit (Thermo Fischer Scientific, Rock-ford, IL, USA).FSAP enzyme activity assayFSAP activity assays were performed as described previously[16]. In brief, microtiter plates were blocked with NaCl/Triscontaining 3% w ⁄ v BSA for 1 h, and washed with NaCl/Tris-T. The standard assay system consisted of NaCl/Tris,1 lgÆmL)1FSAP and 0.2 mm of the chromogenic substrateS-2288 (H-d-isoleucyl-l-prolyl-l-arginine-p-nitroanilinedi-hydrochloride) (Haemochrome, Essen, Germany) and wasfollowed over a period of 60 min at 37°C at 405 nm in anEL 808 microplate reader (BioTek Instruments, Winooski,VT, USA). If an inhibitor was used, this was added togetherwith FSAP to the plates with and without polyanionicco-factor 30 min before adding the chromogenic substrate.Characterization of FSAP–inhibitor interactionusing surface plasmon resonance (SPR)technologyImmobilization on sensor chips, and association and disso-ciation of interacting biomolecules, were followed in realtime by monitoring the change in SPR signal expressed inresonance units (RU). All experiments were performed at25°C. To prepare the sensor chip surface, antibodies toFSAP or isotype controls were immobilized on a CM5research-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 wereperformed at a flow rate of 20 lLÆmin)1using HBS-P[20 m m Hepes, pH 7.4, 100 mm NaCl, 0.05% SurfactantP20 (Biacore cat.nr.:BR-1000-54)] supplemented with 2 mmCaCl2as running buffer. FSAP (25 lL, 10 lgÆmL)1) wascaptured on the immobilized antibodies, and then AT orPAI-1 (25 lL, 0–5 lgÆmL)1) were injected alone and in thepresence of unfractionated heparin or PolyP (10 lgÆmL)1).Sensorgrams were analyzed using BIAevaluation softwareversion 3.2 RC1. Kinetic constants were obtained using theLangmuir binding model 1:1.Cell cultureMouse vascular smooth muscle cells (VSMC) were culturedin 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,2mml-glutamine and 1 mm sodium pyruvate (Invitrogen).Cells were growth-arrested in serum-free medium for 18 hprior to experiments.Polyanions and FSAP L. Muhl et al.4836 FEBS Journal 276 (2009) 4828–4839 ª 2009 The Authors Journal compilation ª 2009 FEBSDNA synthesis assaysVSMC were stimulated for 36 h with the test substances inmedium containing 0.2% fetal calf serum. For the last24 h, 5-bromo-2-deoxyuridine (BrdU) was added, and thecells were processed using a BrdU detection kit (RocheDiagnostics, Mannheim, Germany) as described by themanufacturer.AcknowledgementsThe assistance of Susanne Tannert-Otto is greatlyappreciated. We are grateful to Dr Paul Declerck(Department of Pharmaceutical Sciences, KatholiekeUniversiteit, Leuven, Belgium) for providing PAI-1.This study was financed by a grant from the DeutscheForschungsgemeinschaft to S.M.K. (SFB 547: C14).Wihuri Research Institute is maintained by the Jennyand Antti Wihuri Foundation (Helsinki, Finland).References1 Romisch J (2002) Factor VII activating protease(FSAP): a novel protease in hemostasis. Biol Chem 383,1119–1124.2 Kanse SM, Parahuleva M, Muhl L, Kemkes-MatthesB, Sedding D & Preissner KT (2008) Factor VII-activat-ing protease (FSAP): vascular functions and role inatherosclerosis. Thromb Haemost 99, 286–289.3 Choi-Miura NH, Tobe T, Sumiya J, Nakano Y, SanoY, Mazda T & Tomita M (1996) Purification and char-acterization of a novel hyaluronan-binding protein(PHBP) from human plasma: it has three EGF, a krin-gle and a serine protease domain, similar to hepatocytegrowth factor activator. 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(2004) Factor VII-activating protease (FSAP) inhibits growth factor- mediated cell proliferation and migration of vascular smooth muscle cells FASEB J 18, 728–730 24 Shibamiya A, Muhl L, Tannert-Otto S, Preissner KT & Kanse SM (2007) Nucleic acids potentiate Factor VII-activating protease (FSAP)-mediated cleavage of platelet-derived growth factor- BB and inhibition of vascular smooth muscle cell proliferation... the inactivation of factor Xa by antithrombin by promoting the assembly of a Michaelis-type intermediate complex Demonstration by rapid kinetic, surface plasmon resonance, and competitive binding studies Biochemistry 45, 5324–5329 4838 31 Muhl L, Hersemeyer K, Preissner KT, Weimer T & Kanse SM (2009) Structure–function analysis of factor VII activating protease (FSAP): sequence determinants for heparin. .. for heparin binding and cellular functions FEBS Lett 583, 1994–1998 32 Garg HG, Cindhuchao N, Quinn DA, Hales CA, Thanawiroon C, Capila I & Linhardt RJ (2002) Heparin oligosaccharide sequence and size essential for inhibition of pulmonary artery smooth muscle cell proliferation Carbohydr Res 337, 2359–2364 33 Galuska SP, Geyer R, Muhlenhoff M & Geyer H (2007) Characterization of oligo- and polysialic... concentration, ionic strength and the structure and negative charge density of polysaccharides Biochem J 248, 821–827 26 Kovanen PT (2007) Mast cells: multipotent local effector cells in atherothrombosis Immunol Rev 217, 105–122 27 Clark JE & Wood HG (1987) Preparation of standards and determination of sizes of long-chain polyphosphates by gel electrophoresis Anal Biochem 161, 280–290 28 Ehrlich HJ, Gebbink... distinctions between heparan sulphate and heparin Analysis of sulphation patterns indicates that heparan sulphate and heparin are separate families of N-sulphated polysaccharides Biochem J 230, 665–674 38 Miller MJ, Costello CE, Malmstrom A & Zaia J (2006) A tandem mass spectrometric approach to determination of chondroitin ⁄ dermatan sulfate oligosaccharide glycoforms Glycobiology 16, 502–513 Supporting...Polyanions and FSAP L Muhl et al 20 Wasmuth HE, Tag CG, Van deLeurE, Hellerbrand C, Mueller T, Berg T, Puhl G, Neuhaus P, Samuel D, Trautwein C et al (2008) The Marburg I variant (G534E) of the factor VII-activating protease determines liver fibrosis in hepatitis C infection by reduced proteolysis of platelet-derived growth factor BB Hepatology 49, 775–780 21 Roderfeld... 16, 502–513 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 . High negative charge-to-size ratio in polyphosphates and heparin regulates factor VII-activating protease Lars Muhl1, Sebastian. Low-molecular-weight heparin exhibitslower potential for binding to and activating FSAP.The heparin homologues N-acetyl heparin, de-N-sul-fated heparin and N-acetyl-de-O-sulfated
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