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Platelet Factor 4 Binds to Vascular Proteoglycans and Controls Both Growth Factor Activities and Platelet Activation*

  • Megan S. Lord
    Correspondence
    To whom correspondence should be addressed: Graduate School of Biomedical Engineering, University of New South Wales, Sydney NSW 2052, Australia. Tel.: 61-2-9385-3910; Fax: 61-2-9663-2108; .
    Affiliations
    Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales 2052, Australia
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  • Bill Cheng
    Footnotes
    Affiliations
    Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales 2052, Australia
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  • Brooke L. Farrugia
    Affiliations
    Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales 2052, Australia
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  • Simon McCarthy
    Affiliations
    Tricol Biomedical, Portland, Oregon 97205
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  • John M. Whitelock
    Affiliations
    Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales 2052, Australia
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  • Author Footnotes
    * This work was supported by Australian Research Council Linkage Project LP0776293. The authors declare that they have no conflicts of interest with the contents of this article.
    2 Present address: Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, R.O.C.
    3 The abbreviations used are: PF4platelet factor 4GAGglycosaminoglycanC'asechondroitinaseCSchondroitin sulfateDSdermatan sulfateHSheparan sulfateFGFRFGF receptorHepIIIheparinase IIIBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolBicineN,N-bis(2-hydroxyethyl)glycineDPBSDulbecco's PBSANOVAanalysis of variance.
Open AccessPublished:January 23, 2017DOI:https://doi.org/10.1074/jbc.M116.760660
      Platelet factor 4 (PF4) is produced by platelets with roles in both inflammation and wound healing. PF4 is stored in platelet α-granules bound to the glycosaminoglycan (GAG) chains of serglycin. This study revealed that platelet serglycin is decorated with chondroitin/dermatan sulfate and that PF4 binds to these GAG chains. Additionally, PF4 had a higher affinity for endothelial-derived perlecan heparan sulfate chains than serglycin GAG chains. The binding of PF4 to perlecan was found to inhibit both FGF2 signaling and platelet activation. This study revealed additional insight into the ways in which PF4 interacts with components of the vasculature to modulate cellular events.

      Introduction

      Platelet factor 4 (PF4),
      The abbreviations used are: PF4
      platelet factor 4
      GAG
      glycosaminoglycan
      C'ase
      chondroitinase
      CS
      chondroitin sulfate
      DS
      dermatan sulfate
      HS
      heparan sulfate
      FGFR
      FGF receptor
      HepIII
      heparinase III
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      Bicine
      N,N-bis(2-hydroxyethyl)glycine
      DPBS
      Dulbecco's PBS
      ANOVA
      analysis of variance.
      or chemokine (CXC motif) ligand 4 (CXCL4), is a 7.8-kDa protein synthesized by megakaryocytes and packaged in the α-granules of platelets as a tetrameric complex bound to serglycin GAG chains (
      • Handin R.I.
      • Cohen H.J.
      Purification and binding properties of human platelet factor four.
      ). Upon platelet activation PF4 is released into the circulation where it has roles in both inflammation and wound healing, and these activities are thought to be modulated through interactions with the GAG chains of proteoglycans.
      PF4 has been found in the blood vessel wall within minutes following removal of the endothelium and platelet attachment to the denuded basement membrane (
      • Goldberg I.D.
      • Stemerman M.B.
      • Handin R.I.
      Vascular permeation of platelet factor 4 after endothelial injury.
      ). It binds to endothelial cells through integrins αvβ3, αvβ5, and α5β1 (
      • Aidoudi S.
      • Bujakowska K.
      • Kieffer N.
      • Bikfalvi A.
      The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.
      ) and also to thrombomodulin on the endothelial cell surface through its chain (
      • Dudek A.Z.
      • Pennell C.A.
      • Decker T.D.
      • Young T.A.
      • Key N.S.
      • Slungaard A.
      Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C: a potential mechanism for enhancing generation of activated protein C.
      ). PF4 inhibits heparin binding growth factors, including FGF2 and VEGF165, binding to their receptors through both heparin-dependent and -independent mechanisms (
      • Sato Y.
      • Abe M.
      • Takaki R.
      Platelet factor 4 blocks the binding of basic fibroblast growth factor to the receptor and inhibits the spontaneous migration of vascular endothelial cells.
      ,
      • Gengrinovitch S.
      • Greenberg S.M.
      • Cohen T.
      • Gitay-Goren H.
      • Rockwell P.
      • Maione T.E.
      • Levi B.-Z.
      • Neufeld G.
      Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms.
      ,
      • Jouan V.
      • Canron X.
      • Alemany M.
      • Caen J.P.
      • Quentin G.
      • Plouet J.
      • Bikfalvi A.
      Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action.
      ) with downstream effects on endothelial cell migration and proliferation (
      • Sato Y.
      • Abe M.
      • Takaki R.
      Platelet factor 4 blocks the binding of basic fibroblast growth factor to the receptor and inhibits the spontaneous migration of vascular endothelial cells.
      ,
      • Maione T.E.
      • Gray G.S.
      • Petro J.
      • Hunt A.J.
      • Donner A.L.
      • Bauer S.I.
      • Carson H.F.
      • Sharpe R.J.
      Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides.
      ). PF4 promotes pro-coagulant activities by preventing the formation of a stable heparin·anti-thrombin III·thrombin ternary complex (
      • Lane D.A.
      • Denton J.
      • Flynn A.M.
      • Thunberg L.
      • Lindahl U.
      Anticoagulant activities of heparin oligosaccharides and their netrualization by platelet factor 4.
      ).
      The presence of heparin·PF4 immune complexes suggests that PF4 has a high affinity for heparin and that it is not a normally occurring complex in the blood stream. Heparin-induced thrombocytopenia can occur via both non-immune and immune mediated complications of heparin administration in some patients (
      • Baroletti S.A.
      • Goldhaber S.Z.
      Heparin-induced thrombocytopenia.
      ). Immune-mediated heparin-induced thrombocytopenia is caused by the formation of heparin·PF4 complexes that are recognized as foreign, resulting in the formation of antibodies against the complex. These antibodies bind to the heparin·PF4 complex and platelets themselves, increasing coagulation and reducing levels of circulating platelets.
      Serglycin is an intracellular proteoglycan produced by hematopoietic cells including neutrophils, mast cells, macrophages, and platelets (
      • Kolset S.O.
      • Gallagher J.T.
      Proteoglycans in haemopoietic cells.
      ), as well as cells of non-hematopoietic origin including smooth muscle and endothelial cells (
      • Lemire J.M.
      • Chan C.K.
      • Bressler S.
      • Miller J.
      • LeBaron R.G.
      • Wight T.N.
      Interleukin-1β selectively decreases the synthesis of versican by arterial smooth muscle cells.
      ,
      • Schick B.P.
      • Gradowski J.F.
      • San Antonio J.D.
      Synthesis, secretion, and subcellular localization of serglycin proteoglycan in human endothelial cells.
      ). The name serglycin was derived from the serine-glycine repeat structure where up to eight GAG chains can decorate the protein core of ∼18 kDa. The type and sulfation of GAG chains that decorate serglycin are cell type-dependent mechanisms in which connective tissue mast cells decorate serglycin with heparin (
      • Kolset S.O.
      • Pejler G.
      Serglycin: a structural and functional chameleon with wide impact on immune cells.
      ), whereas mucosal tissue mast cells decorate serglycin with CS (
      • Kolset S.O.
      • Tveit H.
      Serglycin-structure and biology.
      ), neutrophils and platelets decorate serglycin with CS, and macrophages decorate serglycin with CS and HS (
      • Kolset S.O.
      • Mann D.M.
      • Uhlin-Hansen L.
      • Winberg J.O.
      • Ruoslahti E.
      Serglycin-binding proteins in activated macrophages and platelets.
      ).
      Serglycin is located in the α-granules of platelets where it is bound to many cytokines and proteases, including PF4, until they become activated (
      • Kolset S.O.
      • Tveit H.
      Serglycin-structure and biology.
      ). Serglycin knock-out mice exhibit a reduced platelet aggregation response and reduced levels of platelet α-granule proteins including PF4, β-thromboglobulin, and platelet-derived growth factor that are thought to be related to defects in packaging and secretion of α-granule proteins, including proteases, that are required for platelet activation (
      • Woulfe D.S.
      • Lilliendahl J.K.
      • August S.
      • Rauova L.
      • Kowalska M.A.
      • Abrink M.
      • Pejler G.
      • White J.G.
      • Schick B.P.
      Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice.
      ).
      Perlecan is an HS proteoglycan present in the basement membrane that binds and supports the signaling of heparin binding growth factors including FGFs and VEGFs (
      • Whitelock J.M.
      • Murdoch A.D.
      • Iozzo R.V.
      • Underwood P.A.
      The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases.
      ,
      • Zoeller J.J.
      • Whitelock J.M.
      • Iozzo R.V.
      Perlecan regulates developmental angiogenesis by modulating the VEGF-VEGFR2 axis.
      ). In addition, perlecan supports platelet adhesion via the integrin α2β1 (
      • Lord M.S.
      • Yu W.
      • Cheng B.
      • Simmons A.
      • Poole-Warren L.
      • Whitelock J.M.
      The modulation of platelet and endothelial cell adhesion to vascular graft materials by perlecan.
      ,
      • Bix G.
      • Iozzo R.A.
      • Woodall B.
      • Burrows M.
      • McQuillan A.
      • Campbell S.
      • Fields G.B.
      • Iozzo R.V.
      Endorepellin, the C-terminal angiostatic module of perlecan, enhances collagen-platelet responses via the α2β1-integrin receptor.
      ) but does not activate platelets (
      • Lord M.S.
      • Cheng B.
      • McCarthy S.J.
      • Jung M.
      • Whitelock J.M.
      The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.
      ).
      This study demonstrated that platelet serglycin is decorated with CS and DS and that PF4 binds to the CS/DS chains. Additionally, PF4 has a higher affinity for perlecan HS chains than serglycin GAG chains and can be competed from complexes with serglycin in the presence of perlecan. The binding of PF4 to perlecan inhibits both FGF signaling and platelet activation.

      Results

      Characterization of Platelet-derived Serglycin

      Peptide identification of proteins present in the proteoglycan-enriched platelet extract by mass spectrometry identified two cell surface proteoglycans, betaglycan and syndecan-1, as well as the intracellular proteoglycan serglycin (Table 1). This fraction also contained PF4, indicating that it may be closely associated with the proteoglycans in this fraction.
      TABLE 1Proteoglycans and PF4 identified in proteoglycan enriched platelet extract detected by peptide LC-MS2 from an in-solution tryptic digest and ranked by molecular mass search score (MOWSE score)
      Peptide identifiedProtein molecular massMOWSE score
      MOWSE score as determined by Mascot query. This is the value (P) that is a measure of the probability that the match is a random event expressed as −10log(P). The higher the score, the more confidence that the match is not due to a random event.
      Swiss-Prot accession numberb
      Betaglycan100,000171P26342
      Syndecan-132,473133P18827
      PF47,69590P02776
      Serglycin17,80051P10124
      a MOWSE score as determined by Mascot query. This is the value (P) that is a measure of the probability that the match is a random event expressed as −10log(P). The higher the score, the more confidence that the match is not due to a random event.
      Platelet-derived serglycin was analyzed by Western blotting and detected as a smear between apparent relative molecular mass (Mr) of 75,000 and more than 250,000, which corresponded to the expected size of serglycin decorated with GAGs (Fig. 1A, first lane). It should be noted that the protein standards migrate faster than glycosylated proteins; therefore the Mr reported are estimates. Treatment of the serglycin with chondroitinase (C'ase) ABC to digest GAG chains containing both CS and DS disaccharides resulted in a reduction of the Mr of the smear to ∼25,000, corresponding to the protein core indicating that platelet-derived serglycin contained ∼45,000–225,000 CS and/or DS (Fig. 1A, second lane). Treatment of the serglycin with C'ase ACII that digests only terminal CS disaccharides resulted in a shift in Mr of the platelet-derived serglycin in the range of 70,000 to more than 250,000, but not the generation of the protein core (Fig. 1A, third lane). Similarly C'ase B only removes terminal DS disaccharides and resulted in a shift in Mr of the platelet derived serglycin to 65,000 to more than 250,000, but not the generation of the protein core (Fig. 1B, fourth lane). Because C'ases ACII and B are exoglycosidases, it indicated that the GAG chains that decorated this form of serglycin contained both CS and DS disaccharides (Fig. 1B). This would account for the small shift in Mr when treated with either C'ase ACII or B, because once a terminal disaccharide was encountered that the enzyme could not digest, then digestion would cease. Treatment of the sample with heparinase III (HepIII) indicated that the platelet-derived serglycin was not decorated with HS because there was no change in Mr compared with the undigested sample (Fig. 1A, first and fifth lanes).
      Figure thumbnail gr1
      FIGURE 1Characterization of platelet-derived serglycin. A, Western blot of proteoglycan-enriched platelet extract probed for the presence of serglycin using a rabbit polyclonal antibody. Samples were analyzed either without (−) or with (+) C'ase ABC, ACII, or B digestion or HepIII digestion. B, schematic of serglycin GAG chains without treatment or treatment with either C'ase ACII or B. C, ELISA for the presence of serglycin in proteoglycan-enriched platelet extract or in the extract after either C'ase ABC, C'ase B, or HepIII digestion. D, ELISA for the presence of 4-sulfated CS stub in the proteoglycan-enriched platelet extract or in the proteoglycan-enriched extract following digestion with either C'ase ABC or B. E, ELISA for the presence of CS chain structures using clone CS-56. Individual data points are shown (n = 3); bars indicate the means. * indicates significant differences (p < 0.05) compared with undigested samples analyzed by one-way ANOVA.
      Proteoglycan-enriched platelet extract was also analyzed by ELISA for the presence of serglycin (Fig. 1C). Low levels of serglycin were detected in the undigested sample; however, upon digesytion with either C'ase ABC or C'ase B, significantly higher (p < 0.05) levels of serglycin were detected, indicating that CS and DS disaccharides decorated the protein core of serglycin and that these GAG chains hindered the access of the antibody to the epitopes present on the protein core. Treatment of the proteoglycan-enriched platelet extract with HepIII did not alter the level of serglycin detected compared with the undigested sample, confirming that platelet-derived serglycin was not decorated with HS (Fig. 1C).
      Serglycin was found to contain the 4-sulfated stub structure after C'ase ABC digestion, but not C'ase B digestion, indicating that only CS disaccharides were present close to the protein core and were decorated with 4-sulfated CS stubs (Fig. 1D). Un- and 6-sulfated CS stubs were not detected after digestion with either C'ase ABC or B (data not shown). The CS chain structures present in the proteoglycan-enriched platelet extract were reactive with the CS antibody clone CS-56 (Fig. 1E), indicating the presence of CS disaccharides with the sequence GlcUA-GalNAc(4S)-GlcUA(2S)-GalNAc(6S) (
      • Ito Y.
      • Hikino M.
      • Yajima Y.
      • Mikami T.
      • Sirko S.
      • von Holst A.
      • Faissner A.
      • Fukui S.
      • Sugahara K.
      Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library.
      ). There was a background level of reactivity with the CS chain antibody clones LY-111 and MO-225 (data not shown).

      PF4 Binds to CS/DS and HS

      The types of GAG chains attached to serglycin that were involved in the binding of PF4 were investigated using the proteoglycan-enriched platelet extract. Proteoglycan-enriched platelet extract was probed for the presence of PF4 and found to contain immunoreactive bands as a smear between 70,000 and more than 250,000, which corresponded to the expected Mr of serglycin (Fig. 2A, first lane 1). Additionally, PF4 was detected at Mr 8,000, corresponding to its monomer form. PF4 was present at Mr 8,000 in the extract following either C'ase ABC, ACII, or B digestion, indicating that PF4 was released from the proteoglycans in the extract following digestion of the CS/DS chains (Fig. 2A, second, third, and fourth lanes). Together with the data presented in Fig. 1A, which demonstrated a minimal level of GAG digestion when either C'ase ACII or B was used alone, this indicated that the majority of PF4 bound to the non-reducing end of the GAG chains.
      Figure thumbnail gr2
      FIGURE 2PF4 is bound to platelet serglycin via CS and DS. A, Western blot of PF4 in proteoglycan-enriched platelet extract. Samples were analyzed either without (−) or with (+) C'ase ABC, ACII, or B digestion. B, sandwich ELISA using a rabbit polyclonal anti-serglycin antibody capture and detected with a mouse monoclonal anti-PF4 antibody. The samples were analyzed without and with C'ase ABC, ACII, or B digestion. Individual data points are shown (n = 3); bars indicate the means. * indicates significant differences (p < 0.05) compared with undigested samples analyzed by one-way ANOVA.
      To confirm that PF4 was bound to serglycin via its CS/DS chains, a sandwich ELISA was performed where a serglycin polyclonal antibody was used to immobilize serglycin from the proteoglycan-enriched platelet extract and a monoclonal PF4 antibody was used to detect the presence of PF4 in the fraction or in the fraction following C'ase ABC, ACII, or B digestion (Fig. 2B). PF4 bound to serglycin in the undigested extract, whereas the levels of PF4 bound to serglycin were significantly (p < 0.05) reduced following digestion with either C'ase ABC, ACII or B, indicating that PF4 was bound to platelet serglycin via CS/DS chains.
      To further explore the interaction between PF4 and serglycin, recombinantly expressed serglycin decorated with CS/DS, HS/heparin (
      • Lord M.S.
      • Cheng B.
      • Tang F.
      • Lyons J.G.
      • Rnjak-Kovacina J.
      • Whitelock J.M.
      Bioengineered human heparin with anticoagulant activity.
      ) was used. Recombinant serglycin did not contain PF4 (Fig. 3A, lane 2) when compared with a positive control of PF4 (Fig. 3A, lane 1). Incubation of recombinant serglycin with PF4 resulted in complexes with Mr of 100,000 to greater than 250,000, as well as PF4 at Mr of 8,000 when analyzed by Western blotting with a PF4 antibody (Fig. 3B, lane 1), indicating similar-sized complexes were formed as for platelet-derived serglycin with PF4. Incubation of recombinant serglycin with PF4 followed by digestion with either C'ase ABC or B resulted in a reduction in level of complexes with Mr of 100,000 to greater than 250,000 and the presence of PF4 monomers at Mr 8,000 (Fig. 3B, lanes 2 and 4). Incubation of recombinant serglycin with PF4 followed by digestion with either C'ase ACII or HepIII resulted in a broader range of complexes with Mr of 50,000 to greater than 250,000 and the presence of PF4 monomers at Mr 8,000 (Fig. 3B, lanes 3 and 5). Incubation of recombinant serglycin with PF4 followed by digestion with both C'ase ABC and HepIII resulted in the presence of PF4 monomers at Mr 8,000 (Fig. 3B, lane 6). These data indicated that PF4 bound to the recombinant serglycin predominantly via by CS, DS, and HS/heparin. These data also indicated differences in the binding of PF4 to recombinant serglycin compared with platelet serglycin because of the presence of the HS/heparin.
      Figure thumbnail gr3
      FIGURE 3PF4 binds to recombinant serglycin via CS/DS and HS/heparin. A, Western blot for the presence of PF4 using a mouse monoclonal anti-PF4 antibody in PF4 (lane 1) and recombinant serglycin (lane 2). B, Western blot for the presence of PF4 in fractions of recombinant serglycin incubated with PF4 followed by digestion with either C'ase ABC, ACII, or B. C, sandwich ELISA for PF4 bound to serglycin using a rabbit polyclonal serglycin antibody capture and detected with a mouse monoclonal PF4 antibody. Samples were analyzed without and with C'ase ABC, ACII, or B or HepIII digestion. Individual data points are shown (n = 3); bars indicate the means. * indicates significant differences (p < 0.05) compared with undigested samples analyzed by one-way ANOVA. ** indicates significant differences (p < 0.05) for C'ase B-treated samples compared with C'ase ACII-treated samples analyzed by one-way ANOVA.
      A sandwich ELISA was also performed following incubation of recombinant serglycin with PF4 and GAG digestion. This assay revealed that PF4 bound to the recombinant serglycin via both CS and DS, with DS involved to a significantly greater (p < 0.05) extent than CS (Fig. 3C). Additionally, PF4 was found to bind to the HS/heparin chains attached to the recombinant serglycin.

      PF4 Binds to HS Proteoglycans with a Higher Affinity than CS Proteoglycans

      The ability of PF4 to bind to HS was further explored using endothelial-derived perlecan that was exclusively decorated with HS and as perlecan is abundant in the vascular basement membrane. PF4 bound to endothelial-derived perlecan to a significantly greater (p < 0.05) extent than recombinant serglycin when coated at the same concentration (Fig. 4A). PF4 did not bind to endothelial perlecan that had been treated with HepIII to remove its HS chains (Fig. 4A), indicating that PF4 only bound to the HS that decorated the protein core. PF4 bound to recombinantly expressed perlecan domain V via both its CS and HS chains (Fig. 4B).
      Figure thumbnail gr4
      FIGURE 4PF4 binds to perlecan via HS. A, ELISA for PF4 bound to recombinant serglycin, endothelial perlecan, or endothelial perlecan predigested with HepIII. B, ELISA for PF4 bound to recombinant perlecan domain V either without treatment or predigested with C'ase ABC, HepIII, or both C'ase ABC and HepIII. Individual data points are shown (n = 3); bars indicate the means. * indicates significant differences (p < 0.05) compared with undigested samples analyzed by one-way ANOVA.
      Surface plasmon resonance studies indicated that PF4 bound to immobilized heparin, perlecan, and serglycin in a dose-dependent fashion (Fig. 5). The association rate was similar for each condition with equilibrium reached rapidly followed by a slow dissociation (Fig. 5, A–C). The equilibrium constants were 25, 100, and 50 nm for heparin, serglycin, and perlecan, respectively, indicating that PF4 had the highest affinity for heparin followed by perlecan and serglycin (Fig. 5D). Control experiments performed with perlecan predigested with Hep III to remove HS and serglycin predigested with both C'ase ABC and HepIII to remove CS/DS and HS gave baseline sensorgrams indicating that growth factor binding was only to the GAG chains (data not shown). Analysis of the level of PF4 binding to heparin (Fig. 5A) indicated that one PF4 tetramer bound per heparin chain. This analysis is not possible for the proteoglycans because the relative contribution of the protein and GAG chains to the response units measured by the instrument cannot be determined.
      Figure thumbnail gr5
      FIGURE 5Sensorgrams of surface plasmon resonance-based analysis of PF4 binding to perlecan and recombinant serglycin. A–C, 1000 RU of biotinylated heparin (A), recombinant serglycin (B), or endothelial perlecan (C) was immobilized on a streptavidin chip and perfused at 20 μl/min. At 10 s (start of association phase), equilibrium buffer containing either 0.1, 0.5, or 1 μm PF4 was perfused across the chip, and at 160 s buffer was perfused across the chip. One representative curve (of triplicates) at each concentration is shown. For analysis, all curves were utilized for each concentration. D, equilibrium dissociation constant, KD, for PF4 interacting with heparin, perlecan, and serglycin.
      The relative affinity of PF4 for perlecan and serglycin was analyzed in an ELISA assay where serglycin was immobilized on the ELISA plate and incubated with PF4 followed by incubation with perlecan. Perlecan was able to release PF4 from serglycin as detected by a significant (p < 0.05) reduction in the level of PF4 bound to serglycin after incubation with perlecan compared with without soluble perlecan treatment (Fig. 6). However, when the PF4·serglycin complex was incubated with perlecan that had been digested with HepIII to remove its HS chains, there was no change in the level of PF4 bound to serglycin, indicating that the protein core of perlecan did not release PF4 from serglycin. Incubation of the PF4·serglycin complex with perlecan domain V was also able to release PF4 from serglycin, and this interaction was found to be dependent on the presence of the HS chains. Treatment of the PF4·serglycin complex with GAG digestion enzymes alone under the same conditions had no effect on the level of PF4 bound to serglycin because of the short incubation times used in this assay. This indicated that PF4 had a higher affinity for perlecan HS than serglycin GAG chains.
      Figure thumbnail gr6
      FIGURE 6PF4 has a higher affinity for perlecan and serglycin. ELISA for PF4 that was removed from serglycin after exposure to perlecan or perlecan domain V either untreated or treated with C'ase ABC, HepIII, or both. As controls, the amount of PF4 removed from serglycin without additional treatment or treatment with either C'ase ABC, HepIII, or both was determined. The data are presented as percentages of PF4 removed from serglycin by treatment (individual data points are shown (n = 3); bars indicate the means). * indicates significant differences (p < 0.05) compared with no treatment analyzed by one-way ANOVA.

      PF4 Inhibits the Activity of FGF2 Bound to Heparin and Perlecan

      The ability of ternary complexes to form between heparin, FGF2, and FGF receptor 1c (FGFR1c) and signal in the presence of PF4 was analyzed in BaF-32 cells transfected with FGFR1c (Fig. 7). Heparin and FGF2 were used as a positive control for the assay, whereas cells in the presence of medium or growth factor alone were used as negative controls. BaF-32 cells expressing FGFR1c responded to FGF2 in the presence of heparin to a level significantly above (p < 0.05) the medium only control; however, in the presence of PF4 at concentrations of 0.64–2.56 μm, the level of signaling was significantly reduced (p < 0.05) compared with the positive control (Fig. 7A). Perlecan in the presence of FGF2 was able to signal to a similar extent as the positive control; however, in the presence of PF4, the level of signaling was significantly reduced (p < 0.05) and at a level comparable to the negative control as well as perlecan in the absence of growth factor (Fig. 7B). These data indicated that PF4 could inhibit the binding and signaling of FGF2 through both heparin and perlecan HS.
      Figure thumbnail gr7
      FIGURE 7Proliferation of BaF-32 cells expressing FGFR1c in the presence of heparin (30 nm, A) or perlecan (2 μg/ml, B) with FGF2 (0.03 nm) and PF4 (0.13–2.56 μm, A; 2.56 μm, B). Cells exposed to medium or the growth factor only were used as negative controls. Individual data points are shown (n = 3); bars indicate the means. * denotes significant differences compared with the negative control analyzed by one-way ANOVA. ** indicates significant differences compared with the heparin + FGF2 condition.

      PF4 Activates Platelets, Whereas GAG Bound PF4 Does Not Activate Platelets

      Platelet activation was determined by measuring P-selectin expression. Type I collagen was used as a positive control for the assay, whereas freshly isolated platelets were used as the negative control (Fig. 8A). Exposure of platelets to PF4 was found to increase P-selectin expression, but not to the same extent as exposure to collagen type I. Exposure of platelets to heparin, endothelial perlecan, or recombinant serglycin did not activate platelets (Fig. 8B). Preincubation of PF4 with perlecan, serglycin, or heparin before exposure to the platelets inhibited the PF4 mediated activation of platelets (Fig. 8C), suggesting that the GAG chains modulated the activity of PF4.
      Figure thumbnail gr8
      FIGURE 8Flow cytometric analysis of P-selectin (CD62P) expression on platelet membranes in the presence of PF4 (0. 5 μm, A), collagen type I (30 μg/ml), endothelial perlecan (10 μg/ml, B), recombinant serglycin (10 μg/ml), heparin (10 μg/ml), or PF4 (0.5 μm, C) alone or in combination with endothelial perlecan (10 μg/ml), recombinant serglycin (10 μg/ml), or heparin (10 μg/ml). The profiles are representative of three experiments for each condition.

      Discussion

      This study demonstrated that platelets contain serglycin decorated with GAG chains that consist of both CS and DS disaccharides with 4-sulfated CS closer to the protein core and part of the linkage tetrasaccharide structure. This finding extends previous reports that platelet serglycin is decorated with CS containing 4-sulfated disaccharides (
      • Woulfe D.S.
      • Lilliendahl J.K.
      • August S.
      • Rauova L.
      • Kowalska M.A.
      • Abrink M.
      • Pejler G.
      • White J.G.
      • Schick B.P.
      Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice.
      ).
      PF4 was found to bind to platelet serglycin through both CS and DS as well as perlecan through HS chains. Additionally, PF4 bound to recombinant serglycin via CS, DS, and HS/heparin chains further supporting the affinity of PF4 for a variety of GAG chains. Previous analyses of the affinity of PF4 for various GAG chains revealed that PF4 had the highest affinity for heparin followed by CSD or E, HS, and CSA, B, or C (
      • Vandercappellen J.
      • Van Damme J.
      • Struyf S.
      The role of CXC chemokines platelet factor-4 (CVXCL4/PF-4) and its variant (CCXCL4L1/PF-4var) in inflammation, angiogenesis and cancer.
      ,
      • Petersen F.
      • Brandt E.
      • Lindahl U.
      • Spillmann D.
      Characterization of a neutrophil cell surface glycosaminoglycan that mediates binding of platelet factor 4.
      ) and that this binding involved lysine residues in PF4 binding to the sulfate groups on the GAG chains (
      • Handin R.I.
      • Cohen H.J.
      Purification and binding properties of human platelet factor four.
      ). Heparin had the highest affinity for PF4 in this study with a KD of 30 nm that was similar to previous reports in the range of 16–60 nm (
      • Handin R.I.
      • Cohen H.J.
      Purification and binding properties of human platelet factor four.
      ,
      • Stringer S.E.
      • Gallagher J.T.
      Specific binding of the chemokine platelet factor 4 to heparan sulfate.
      ,
      • Loscalzo J.
      • Melnick B.
      • Handin R.I.
      The interaction of platelet factor four and glycosaminoglycans.
      ). The affinity of PF4 for proteoglycans was explored for the time in this study and indicated that PF4 had a higher affinity for perlecan decorated with HS than serglycin predominantly decorated with CS/DS. PF4 has been established to be a tetrameric complex at physiological pH and ionic strength via NMR and neutron scattering (
      • Ibel K.
      • Poland G.A.
      • Baldwin J.P.
      • Pepper D.S.
      • Luscombe M.
      • Holbrook J.J.
      Low-resolution structure of the complex of human blood platelet factor 4 with heparin determined by small-angle neutron scattering.
      ,
      • Mayo K.H.
      • Ilyina E.
      • Roongta V.
      • Dundas M.
      • Joseph J.
      • Lai C.K.
      • Maione T.
      • Daly T.J.
      Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding.
      ). Models of these studies support the hypothesis that the heparin chain wraps around the PF4 tetramer (
      • Mayo K.H.
      • Ilyina E.
      • Roongta V.
      • Dundas M.
      • Joseph J.
      • Lai C.K.
      • Maione T.
      • Daly T.J.
      Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding.
      ). Although this arrangement is likely with exposure of PF4 tetramers to single GAG chains, physiologically it is likely that the PF4 will be exposed to serglycin with eight GAG chains and perlecan with three GAG chains. Given the close proximity of the GAG chains on both serglycin and perlecan, there is limited flexibility for the chains to wrap around the PF4 tetramers as is possible with an isolated GAG chain. Thus there is the possibility of multiple GAG chains binding the PF4 tetramers as depicted in Fig. 9; however, this model remains to be experimentally verified.
      Figure thumbnail gr9
      FIGURE 9Schematic of proposed mechanism of PF4 binding to proteoglycan GAG chains. The proposed model involves multiple GAG chains on proteoglycans, including serglycin and perlecan, binding PF4.
      Following vascular injury, platelets are known to adhere to the denuded vascular basement membrane, particularly to collagen, inducing platelet aggregation, activation, and the release of PF4. PF4 is released from platelets in complex with serglycin (
      • Capitanio A.M.
      • Niewiarowski S.
      • Rucinski B.
      • Tuszynski G.P.
      • Cierniewski C.S.
      • Hershock D.
      • Kornecki E.
      Interaction of platelet factor 4 with human platelets.
      ). Thus the release of PF4 from serglycin GAG chains by perlecan HS, as demonstrated in this study, is a likely mechanism by which PF4 is transferred to the vascular basement membrane as observed in vivo (
      • Goldberg I.D.
      • Stemerman M.B.
      • Handin R.I.
      Vascular permeation of platelet factor 4 after endothelial injury.
      ). Additionally, under physiological conditions, endothelial cell surface GAG chains bind basal levels of platelet-derived PF4 (
      • Dawes J.
      • Pumphrey C.W.
      • McLaren K.M.
      • Prowse C.V.
      • Pepper D.S.
      The in vivo release of human platelet factor 4 by heparin.
      ).
      PF4 binds to the surface of resting and activated platelets and supports platelet aggregation (
      • Capitanio A.M.
      • Niewiarowski S.
      • Rucinski B.
      • Tuszynski G.P.
      • Cierniewski C.S.
      • Hershock D.
      • Kornecki E.
      Interaction of platelet factor 4 with human platelets.
      ). This study also demonstrated that PF4 promotes platelet activation; however, platelets did not become activated when exposed to PF4 bound to either serglycin or perlecan GAG chains, suggesting that the binding of PF4 to proteoglycans can modulate platelet activation events.
      PF4 is reported to be angiostatic as demonstrated in a chicken chorioallantoic membrane assay (
      • Maione T.E.
      • Gray G.S.
      • Petro J.
      • Hunt A.J.
      • Donner A.L.
      • Bauer S.I.
      • Carson H.F.
      • Sharpe R.J.
      Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides.
      ). The angiostatic effect of PF4 has been ascribed to its binding to the same regions of HS as growth factors involved in angiogenesis including FGF2 and VEGF (
      • Jouan V.
      • Canron X.
      • Alemany M.
      • Caen J.P.
      • Quentin G.
      • Plouet J.
      • Bikfalvi A.
      Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action.
      ). Additionally, PF4 can inhibit the binding of FGF2 to both fibroblast cell ECM and cell surface receptors (
      • Sato Y.
      • Abe M.
      • Takaki R.
      Platelet factor 4 blocks the binding of basic fibroblast growth factor to the receptor and inhibits the spontaneous migration of vascular endothelial cells.
      ) through binding to HS. The present study supported these findings using the BaF32 cell assay where the addition of PF4 could dose-dependently inhibit the binding and signaling of FGF2 that require the formation of ternary complexes with FGF2, the FGFR1c cell surface receptor, and either heparin or perlecan HS. FGF2 is produced by vascular endothelial cells and acts as a both a mitogenic and migratory signal for these cells. Indeed the addition of PF4 to cultures of endothelial cells inhibited their migration (
      • Sato Y.
      • Abe M.
      • Takaki R.
      Platelet factor 4 blocks the binding of basic fibroblast growth factor to the receptor and inhibits the spontaneous migration of vascular endothelial cells.
      ). Together these data suggest that both PF4 and FGF2 bind to similar regions of HS to inhibit the HS-dependent signaling of FGF2 via FGF receptors.
      PF4 also exerts its angiostatic activity via HS-independent mechanisms as it was able to inhibit VEGF121 induced cell proliferation but did not interfere with its binding to the VEGF receptor, flk-1 (
      • Gengrinovitch S.
      • Greenberg S.M.
      • Cohen T.
      • Gitay-Goren H.
      • Rockwell P.
      • Maione T.E.
      • Levi B.-Z.
      • Neufeld G.
      Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms.
      ). HS-independent mechanisms include binding integrins on the endothelial cell surface (
      • Aidoudi S.
      • Bujakowska K.
      • Kieffer N.
      • Bikfalvi A.
      The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.
      ), inhibiting growth factor binding to receptors and binding directly to FGF2 and preventing dimerization (
      • Jouan V.
      • Canron X.
      • Alemany M.
      • Caen J.P.
      • Quentin G.
      • Plouet J.
      • Bikfalvi A.
      Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action.
      ).
      PF4 preferentially binds to newly formed blood vessels providing a mechanism to control their growth (
      • Hansell P.
      • Maione T.E.
      • Borgström P.
      Selective binding of platelet factor 4 to regions of active angiogenesis in vivo.
      ). Thus one of the physiological roles of PF4 may be to control normal and neoplastic vascularization through controlling the activities of growth factors. Thus it is hypothesized that the binding of PF4 to various GAG chains that decorate proteoglycans in the vasculature controls angiogenesis in wound healing through modulating the activity of growth factors and platelet activation.

      Experimental Procedures

      Materials

      Chondroitinase ABC ((EC 4.2.2.4) purified from Proteus vulgaris proteinase free lyase I), B (EC 4.2.2) purified from Flavobacterium heparinum, ACII purified from Arthrobacter aurescens, and heparinase III (EC 4.2.2.8) purified from F. heparinum were purchased from Seikagaku Corporation (Tokyo, Japan). Mouse mAbs reactive to the stubs of CS chains following chondroitinase ABC digestion (clones 1B5, 2B6, and 3B3) were provided by Prof. Bruce Caterson (Cardiff University, Cardiff, UK). The rabbit polyclonal anti-serglycin antibody was a gift from Prof. Achilleas Theocharis (University of Patras, Patras, Greece), ascites 1:1000) (
      • Theocharis A.D.
      • Seidel C.
      • Borset M.
      • Dobra K.
      • Baykov V.
      • Labropoulou V.
      • Kanakis I.
      • Dalas E.
      • Karamanos N.K.
      • Sundan A.
      • Hjerpe A.
      Serglycin constitutively secreted by myeloma plasma cells is a potent inhibitor of bone mineralisation in vitro.
      ). Mouse monoclonal antibodies CS clones LY111 and MO-225 were purchased from Seikagaku Corporation. Anti-mouse and anti-rabbit secondary antibodies were purchased from Merck-Millipore (Sydney, Australia). Secondary HRP-conjugated antibodies were purchased from Dako (Sydney, Australia). PF4 was purchased from R&D Systems (Minneapolis, MN). All other chemicals were purchased from Sigma-Aldrich.

      Isolation of Proteoglycans from Platelets

      Platelet lysate was prepared by incubating platelets with 1% (w/v) Triton X-100 for 1 h at 4 °C followed by filtration with a 0.45-μm syringe filter. Anion exchange chromatography using a diethylaminoethyl column (1 ml of DEAE-Sepharose Fast Flow; GE Healthcare) attached to a FPLC (AKTA purifier; GE Healthcare) was used to enrich samples for proteoglycans. Briefly, the DEAE column was equilibrated at 1 ml/min with running buffer (250 mm NaCl, 20 mm Tris, 10 mm EDTA, pH 7.5) before the addition of platelet lysate and baseline absorbance re-established with running buffer. Proteoglycans were eluted using an eluting buffer (1 m NaCl, 20 mm Tris, 10 mm EDTA, pH 7.5). Proteoglycan-enriched fractions were subsequently concentrated and analyzed for protein concentration using a Coomassie Blue protein assay (Thermo Scientific, Scoresby, Australia).

      Mass Spectrometry

      DEAE-enriched samples (50 μg/ml) were prepared for LC-MS2 analysis by in-solution digestion, which involved reduction with 10 mm DTT for 10 min at 95 °C and alkylation with 25 mm iodoacetamide for 20 min at 25 °C. Samples were then incubated with 20 μg/ml sequencing grade Trypsin in 50 mm NH4HCO3 at 30 °C for 16 h and then subjected to peptide analysis by LC-MS2. Samples were analyzed by LC-MS2 using an LTQ mass spectrometer (Thermo Fisher Scientific). The results were analyzed with XcaliburTM software (Bioworks version 3.1; Thermo Fisher Scientific) and the MASCOT database with a National Center for Biotechnology Information protein (homosapian) database.

      Glycosaminoglycan Digestion

      Samples of proteoglycan-enriched material were digested with 50 milliunits/ml chondroitinase (C'ase) ABC, ACII, or B in 0.1 m Tris acetate, pH 8, at 37 °C for 16 h to confirm the presence and structure of the CS. Samples of proteoglycan-enriched material were digested with 10 milliunits/ml HepIII in PBS, pH 7.2, for 16 h at 37 °C to confirm the presence and structure of the HS.

      Western Blotting Analysis

      Proteoglycan-enriched samples (200 μg/ml based on Coomassie protein assay), with and without glycosaminoglycan digestion, were electrophoresed in 4–12% (w/v) BisTris gels (Life Technologies, Sydney, Australia) under non-reducing conditions using MES buffer (50 mm MES, 50 mm Tris, 0.1% (w/v) SDS, 1 mm EDTA, pH 7.3) at 200 V for 45 min. A series of molecular mass markers (Precision Plus All Blue; Bio-Rad) were electrophoresed on each gel. Samples were then transferred to PVDF membrane using transfer buffer (5 mm Bicine, 5 mm BisTris, 0.2 mm EDTA, 50 μg/ml SDS, 10% (v/v) methanol, pH 7.2) in a semi-dry blotter at 300 mA and 20 V for 60 min. The membrane was blocked with 1% (w/v) BSA in TBS (20 mm Tris base, 136 mm NaCl, pH 7.6) with 0.1% (v/v) Tween 20 (TBST) for 2 h at 25 °C followed by incubation with primary antibody diluted in 1% (w/v) BSA/TBST for 2 h at 25 °C. Membranes were subsequently rinsed with TBST, incubated with secondary HRP conjugated antibodies (1:50,000) for 45 min at 25 °C, and rinsed with TBST and TBS before being imaged using chemiluminescence reagent (Femto reagent kit; Pierce) and X-ray film.

      ELISA

      Proteoglycan-enriched samples (10 μg/ml based on Coomassie Blue protein assay), with and without glycosaminoglycan digestion, were coated onto high binding 96-well ELISA plates (Greiner) for 2 h at 25 °C. Wells were rinsed twice with Dulbecco's PBS, pH 7.4 (DPBS), followed by blocking with 0.1% (w/v) casein in DPBS for 1 h at 25 °C. Wells were rinsed twice with DPBS with 1% (v/v) Tween 20 (PBST) followed by incubation with primary antibodies diluted in 0.1% (w/v) casein in DPBS for 2 h at 25 °C. Wells were rinsed twice with PBST followed by incubation with biotinylated secondary antibodies (1:1000) diluted in 0.1% (w/v) casein in DPBS for 1 h at 25 °C, rinsed again twice with PBST, and then incubated with streptavidin-HRP (1:500) for 30 min at 25 °C. Binding of the antibodies to the samples was detected using the colorimetric substrate, 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid), and absorbance was measured at 405 nm.
      A sandwich ELISA was performed by coating ELISA plates with a rabbit polyclonal anti-serglycin capture antibody in 0.1 m sodium carbonate buffer, pH 9.6, for 16 h at 4 °C. Wells were rinsed twice with DPBS followed by blocking with 0.1% (w/v) casein in DPBS for 2 h at 25 °C. Wells were rinsed with PBST followed by incubation with the proteoglycan-enriched samples (10 μg/ml based on Coomassie Blue protein assay) for 2 h at 25 °C and subsequent detection with primary and secondary antibodies as for the standard ELISA. Data for both the ELISA and sandwich ELISA were corrected for background absorbance.

      PF4 Binding Assays

      The binding of PF4 to recombinant serglycin prepared as described previously (
      • Lord M.S.
      • Cheng B.
      • Tang F.
      • Lyons J.G.
      • Rnjak-Kovacina J.
      • Whitelock J.M.
      Bioengineered human heparin with anticoagulant activity.
      ), immunopurified endothelial derived perlecan prepared as described previously (
      • Whitelock J.M.
      • Graham L.D.
      • Melrose J.
      • Murdoch A.D.
      • Iozzo R.V.
      • Underwood P.A.
      Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.
      ,
      • Knox S.
      • Melrose J.
      • Whitelock J.
      Electrophoretic, biosensor, and bioactivity analyses of perlecans of different cellular origins.
      ), and recombinant perlecan domain V prepared as described previously (
      • Jung M.
      • Lord M.S.
      • Cheng B.
      • Lyons J.G.
      • Alkhouri H.
      • Hughes J.M.
      • McCarthy S.J.
      • Iozzo R.V.
      • Whitelock J.M.
      Mast cells produce novel shorter forms of perlecan that contain functional endorepellin a role in angiogenesis and wound healing.
      ) was assessed by Western blotting, ELISA, and surface plasmon resonance. For Western blotting, PF4 (5 μg/ml) was incubated with serglycin (2 μg/ml) in solution for 16 h at 37 °C and either analyzed without further modification or treated with glycosaminoglycan digestion enzymes prior to analysis.
      For ELISA, plates were coated with either serglycin, endothelial perlecan, or perlecan domain V at a concentration of 10 μg/ml for 16 h at 4 °C prior to blocking with 1% (w/v) casein in PBS for 2 h at 25 °C. This was followed by incubation with 1 μg/ml PF4 for 2 h at 37 °C and then detection with the mouse monoclonal anti-PF4 antibody. The binding of PF4 to the proteoglycans was also analyzed after glycosaminoglycan digestion prior to immobilizing on the ELISA plates. The background level of PF4 binding to casein was also measured, and absorbance values were subtracted from sample absorbance values.
      For the displacement assay, ELISA plates were coated with 10 μg/ml serglycin that was either untreated or treated with C'ase ABC, HepIII, or both C'ase ABC and HepIII for 16 h at 4 °C. The plates were then blocked with 1% (w/v) casein in PBS for 2 h at 25 °C followed by incubation with 8 μg/ml PF4 for 30 min at 37 °C. Plates were then incubated with either 10 μg/ml perlecan or 10 μg/ml perlecan domain V that was untreated or after glycosaminoglycan digestion for 1 h at 37 °C. As a control, selected wells were incubated with 0.05 unit/ml glycosaminoglycan digestion enzymes alone for 1 h at 37 °C. The plates were then analyzed for bound PF4 using the mouse monoclonal anti-PF4 antibody by the ELISA technique.

      Surface Plasmon Resonance

      Heparin (Sigma H3393; 17–19 kDa), recombinant serglycin, and endothelial perlecan were biotinylated as described previously (
      • Melrose J.
      Cartilage and smooth muscle cell proteoglycans detected by affinity blotting using biotinylated hyaluronan.
      ,
      • Melrose J.
      • Roughley P.
      • Knox S.
      • Smith S.
      • Lord M.
      • Whitelock J.
      The structure, location, and functin of perlecan, a prominent pericellular proteoglycan of fetal, postnatal, and mature hyaline cartilages.
      ). The interaction between PF4 and either serglycin or endothelial perlecan or heparin was analyzed with a BiaCore 2000 (GE Healthcare). Streptavidin-coated sensor surfaces (GE Healthcare) were coated by injection of 100 μl of 10 μg/ml biotinylated heparin, endothelial perlecan, or recombinant serglycin at 5 μl/min (diluted in DPBS, pH 7.4). The surfaces were then blocked by injection of 150 μl of 1% (w/v) BSA in DPBS at 5 μl/min. The binding of PF4 (0.1, 0.5, and 1 μm) was conducted in DPBS, pH 7.4, at a flow rate of 20 μl/min at 25 °C using an injection volume of 50 μl. Sensorgrams were analyzed using BIAcore 2000 evaluation software 3.0. Sensorgrams were fitted with differential rate equations for the association and dissociation rates. Control experiments were performed with samples that had been predigested with either C'ase ABC or HepIII and PF4 binding to blocked streptavidin chips.

      BaF32 Cell Proliferation Assays

      Baf32 cells are from an IL-3-dependent and HSPG-deficient myeloid B cell line that has been stably transfected with FGFR1c (
      • Ornitz D.M.
      • Xu J.
      • Colvin J.S.
      • McEwen D.G.
      • MacArthur C.A.
      • Coulier F.
      • Gao G.
      • Goldfarb M.
      Receptor specificity of the fibroblast growth family.
      ,
      • Ornitz D.M.
      • Yayon A.
      • Flanagan J.G.
      • Svahn C.M.
      • Levi E.
      • Leder P.
      Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells.
      ). Baf32 cells are a model system developed to identify heparan sulfate and heparin structures that interact with FGFs and their receptors. The readout of this assay is cell proliferation, which indicates the formation of ternary complexes in situ. Baf32 cells were maintained in RPMI 1640 medium containing 10% (v/v) FBS, 10% (v/v) WEHI-3BD conditioned medium, 100 units/ml penicillin, and 100 μg/ml streptomycin. WEHI-3BD cells were maintained in RPMI 1640 medium supplemented with 2 g/liter sodium bicarbonate, 10% (v/v) FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, and the conditioned medium was collected three times per week and stored at −20 °C until required. For the mitogenic assays, the BaF32 cells were transferred into IL-3 depleted medium for 24 h prior to experimentation and seeded into 96-well plates at a density of 2 × 104 cells/well in the presence of FGF2 (0.03 nm), heparin (30 nm), or immunopurified endothelial perlecan (4 pm) and PF4 (0.125–2.5 μm). The cells were incubated for 96 h in 5% CO2 at 37 °C, and the amount of cells present was assessed using the MTS reagent (Promega, Madison, WI) by adding to the cell cultures for 6 h prior to measuring the absorbance at 490 nm.

      Platelet Activation

      Platelets were harvested from human donors under ethics approval from the University of New South Wales. Blood was collected in acid citrate dextrose anticoagulant-treated vacutainers. Platelet-rich plasma was prepared by centrifugation of the blood at 350 × g for 20 min at 25 °C followed by careful removal of the upper platelet-rich layer. Platelet-rich plasma was then centrifuged at 1200 × g for 10 min to yield a platelet pellet with platelet poor plasma as the supernatant. The platelet pellet was resuspended in Tyrode's buffer (1.8 mm CaCl2, 1 mm MgCl2, 2.7 mm KCl, 136.9 mm NaCl, 0.4 mm NaH2PO4, 11.9 mm NaHCO3, 5.6 mm d-glucose, and 0.1 unit/ml apyrase) and centrifuged again at 1200 × g for 10 min. The supernatant was discarded, and the platelets were resuspended in Tyrode's buffer to a concentration of 2 × 107 platelets/ml. Platelet suspensions (2 × 107 platelets/ml) in Tyrode's buffer were exposed to either PF4 (4 μg/ml), collagen type I (30 μg/ml), 10 μg/ml recombinant serglycin, 10 μg/ml endothelial perlecan, 10 μg/ml heparin, or mixtures of 10 μg/ml serglycin and 4 μg/ml PF4, 10 μg/ml perlecan and 4 μg/ml PF4, or 10 μg/ml heparin and 4 μg/ml PF4 for 5 min at 37 °C in the presence of FITC-labeled anti-CD62P (1 μg/ml, clone 9E1; Bio-Scientific Pty. Ltd., Gymea, Australia) detects P-selectin on the surface of activated platelets. The reaction was stopped by adding paraformaldehyde to a final concentration of 1% (w/v). The fluorescence intensity of 10,000 platelets was analyzed using flow cytometry (FACScan; Becton Dickinson).

      Statistical Analysis

      A one-way analysis of variance (ANOVA) was performed, and the results of p < 0.05 were considered significant. The experiments were performed in triplicate and repeated.

      Author Contributions

      M. S. L. and J. M. W. conceived and coordinated the study. M. S. L. designed and performed the experiments shown in Figs. 5 and 8. B. C. designed and performed the experiments shown in FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4 and 6. B. L. F. designed and performed the experiments shown in Fig. 7. M. S. L. wrote the paper. All authors analyzed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank B. Caterson (Cardiff University, Cardiff, UK) for the gift of the1B5, 2B6, and 3B3 hybridoma culture supernatants and T. Achilleas (University of Patras, Patras, Greece) for the supply of rabbit polyclonal anti-serglycin antibody. We also acknowledge the technical assistance of Marie Labeye.

      References

        • Handin R.I.
        • Cohen H.J.
        Purification and binding properties of human platelet factor four.
        J. Biol. Chem. 1976; 251: 4273-4282
        • Goldberg I.D.
        • Stemerman M.B.
        • Handin R.I.
        Vascular permeation of platelet factor 4 after endothelial injury.
        Science. 1980; 209: 611-612
        • Aidoudi S.
        • Bujakowska K.
        • Kieffer N.
        • Bikfalvi A.
        The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.
        PLoS One. 2008; 3: e2657
        • Dudek A.Z.
        • Pennell C.A.
        • Decker T.D.
        • Young T.A.
        • Key N.S.
        • Slungaard A.
        Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C: a potential mechanism for enhancing generation of activated protein C.
        J. Biol. Chem. 1997; 272: 31785-31792
        • Sato Y.
        • Abe M.
        • Takaki R.
        Platelet factor 4 blocks the binding of basic fibroblast growth factor to the receptor and inhibits the spontaneous migration of vascular endothelial cells.
        Biochem. Biophys. Res. Commun. 1990; 172: 595-600
        • Gengrinovitch S.
        • Greenberg S.M.
        • Cohen T.
        • Gitay-Goren H.
        • Rockwell P.
        • Maione T.E.
        • Levi B.-Z.
        • Neufeld G.
        Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms.
        J. Biol. Chem. 1995; 270: 15059-15065
        • Jouan V.
        • Canron X.
        • Alemany M.
        • Caen J.P.
        • Quentin G.
        • Plouet J.
        • Bikfalvi A.
        Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action.
        Blood. 1999; 94: 984-993
        • Maione T.E.
        • Gray G.S.
        • Petro J.
        • Hunt A.J.
        • Donner A.L.
        • Bauer S.I.
        • Carson H.F.
        • Sharpe R.J.
        Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides.
        Science. 1990; 247: 77-79
        • Lane D.A.
        • Denton J.
        • Flynn A.M.
        • Thunberg L.
        • Lindahl U.
        Anticoagulant activities of heparin oligosaccharides and their netrualization by platelet factor 4.
        Biochem. J. 1984; 218: 725-732
        • Baroletti S.A.
        • Goldhaber S.Z.
        Heparin-induced thrombocytopenia.
        Circulation. 2006; 114: e355-e356
        • Kolset S.O.
        • Gallagher J.T.
        Proteoglycans in haemopoietic cells.
        Biochim. Biophys. Acta. 1990; 1032: 191-211
        • Lemire J.M.
        • Chan C.K.
        • Bressler S.
        • Miller J.
        • LeBaron R.G.
        • Wight T.N.
        Interleukin-1β selectively decreases the synthesis of versican by arterial smooth muscle cells.
        J. Cell Biochem. 2007; 101: 753-766
        • Schick B.P.
        • Gradowski J.F.
        • San Antonio J.D.
        Synthesis, secretion, and subcellular localization of serglycin proteoglycan in human endothelial cells.
        Blood. 2001; 97: 449-458
        • Kolset S.O.
        • Pejler G.
        Serglycin: a structural and functional chameleon with wide impact on immune cells.
        J. Immunol. 2011; 187: 4927-4933
        • Kolset S.O.
        • Tveit H.
        Serglycin-structure and biology.
        Cell. Mol. Life Sci. 2008; 65: 1073-1085
        • Kolset S.O.
        • Mann D.M.
        • Uhlin-Hansen L.
        • Winberg J.O.
        • Ruoslahti E.
        Serglycin-binding proteins in activated macrophages and platelets.
        J. Leukocyte Biol. 1996; 59: 545-554
        • Woulfe D.S.
        • Lilliendahl J.K.
        • August S.
        • Rauova L.
        • Kowalska M.A.
        • Abrink M.
        • Pejler G.
        • White J.G.
        • Schick B.P.
        Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice.
        Blood. 2008; 111: 3458-3467
        • Whitelock J.M.
        • Murdoch A.D.
        • Iozzo R.V.
        • Underwood P.A.
        The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases.
        J. Biol. Chem. 1996; 271: 10079-10086
        • Zoeller J.J.
        • Whitelock J.M.
        • Iozzo R.V.
        Perlecan regulates developmental angiogenesis by modulating the VEGF-VEGFR2 axis.
        Matrix Biol. 2009; 28: 284-291
        • Lord M.S.
        • Yu W.
        • Cheng B.
        • Simmons A.
        • Poole-Warren L.
        • Whitelock J.M.
        The modulation of platelet and endothelial cell adhesion to vascular graft materials by perlecan.
        Biomaterials. 2009; 30: 4898-4906
        • Bix G.
        • Iozzo R.A.
        • Woodall B.
        • Burrows M.
        • McQuillan A.
        • Campbell S.
        • Fields G.B.
        • Iozzo R.V.
        Endorepellin, the C-terminal angiostatic module of perlecan, enhances collagen-platelet responses via the α2β1-integrin receptor.
        Blood. 2007; 109: 3745-3748
        • Lord M.S.
        • Cheng B.
        • McCarthy S.J.
        • Jung M.
        • Whitelock J.M.
        The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.
        Biomaterials. 2011; 32: 6655-6662
        • Ito Y.
        • Hikino M.
        • Yajima Y.
        • Mikami T.
        • Sirko S.
        • von Holst A.
        • Faissner A.
        • Fukui S.
        • Sugahara K.
        Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library.
        Glycobiology. 2005; 15: 593-603
        • Lord M.S.
        • Cheng B.
        • Tang F.
        • Lyons J.G.
        • Rnjak-Kovacina J.
        • Whitelock J.M.
        Bioengineered human heparin with anticoagulant activity.
        Metab. Eng. 2016; 38: 105-114
        • Vandercappellen J.
        • Van Damme J.
        • Struyf S.
        The role of CXC chemokines platelet factor-4 (CVXCL4/PF-4) and its variant (CCXCL4L1/PF-4var) in inflammation, angiogenesis and cancer.
        Cytokine Growth Factor Rev. 2011; 22: 1-18
        • Petersen F.
        • Brandt E.
        • Lindahl U.
        • Spillmann D.
        Characterization of a neutrophil cell surface glycosaminoglycan that mediates binding of platelet factor 4.
        J. Biol. Chem. 1999; 274: 12376-12382
        • Stringer S.E.
        • Gallagher J.T.
        Specific binding of the chemokine platelet factor 4 to heparan sulfate.
        J. Biol. Chem. 1997; 272: 20508-20514
        • Loscalzo J.
        • Melnick B.
        • Handin R.I.
        The interaction of platelet factor four and glycosaminoglycans.
        Arch. Biochem. Biophys. 1985; 240: 446-455
        • Ibel K.
        • Poland G.A.
        • Baldwin J.P.
        • Pepper D.S.
        • Luscombe M.
        • Holbrook J.J.
        Low-resolution structure of the complex of human blood platelet factor 4 with heparin determined by small-angle neutron scattering.
        Biochim. Biophys. Acta. 1986; 870: 58-63
        • Mayo K.H.
        • Ilyina E.
        • Roongta V.
        • Dundas M.
        • Joseph J.
        • Lai C.K.
        • Maione T.
        • Daly T.J.
        Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding.
        Biochem. J. 1995; 312: 357-365
        • Capitanio A.M.
        • Niewiarowski S.
        • Rucinski B.
        • Tuszynski G.P.
        • Cierniewski C.S.
        • Hershock D.
        • Kornecki E.
        Interaction of platelet factor 4 with human platelets.
        Biochim. Biophys. Acta. 1985; 839: 161-173
        • Dawes J.
        • Pumphrey C.W.
        • McLaren K.M.
        • Prowse C.V.
        • Pepper D.S.
        The in vivo release of human platelet factor 4 by heparin.
        Thrombosis Res. 1982; 27: 65-76
        • Hansell P.
        • Maione T.E.
        • Borgström P.
        Selective binding of platelet factor 4 to regions of active angiogenesis in vivo.
        Am. J. Physiol. 1995; 269: H829-H836
        • Theocharis A.D.
        • Seidel C.
        • Borset M.
        • Dobra K.
        • Baykov V.
        • Labropoulou V.
        • Kanakis I.
        • Dalas E.
        • Karamanos N.K.
        • Sundan A.
        • Hjerpe A.
        Serglycin constitutively secreted by myeloma plasma cells is a potent inhibitor of bone mineralisation in vitro.
        J. Biol. Chem. 2006; 281: 35116-35128
        • Whitelock J.M.
        • Graham L.D.
        • Melrose J.
        • Murdoch A.D.
        • Iozzo R.V.
        • Underwood P.A.
        Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.
        Matrix Biol. 1999; 18: 163-178
        • Knox S.
        • Melrose J.
        • Whitelock J.
        Electrophoretic, biosensor, and bioactivity analyses of perlecans of different cellular origins.
        Proteomics. 2001; 1: 1534-1541
        • Jung M.
        • Lord M.S.
        • Cheng B.
        • Lyons J.G.
        • Alkhouri H.
        • Hughes J.M.
        • McCarthy S.J.
        • Iozzo R.V.
        • Whitelock J.M.
        Mast cells produce novel shorter forms of perlecan that contain functional endorepellin a role in angiogenesis and wound healing.
        J. Biol. Chem. 2013; 288: 3289-3304
        • Melrose J.
        Cartilage and smooth muscle cell proteoglycans detected by affinity blotting using biotinylated hyaluronan.
        Methods Mol. Biol. 2001; 171: 53-66
        • Melrose J.
        • Roughley P.
        • Knox S.
        • Smith S.
        • Lord M.
        • Whitelock J.
        The structure, location, and functin of perlecan, a prominent pericellular proteoglycan of fetal, postnatal, and mature hyaline cartilages.
        J. Biol. Chem. 2006; 281: 36905-36914
        • Ornitz D.M.
        • Xu J.
        • Colvin J.S.
        • McEwen D.G.
        • MacArthur C.A.
        • Coulier F.
        • Gao G.
        • Goldfarb M.
        Receptor specificity of the fibroblast growth family.
        J. Biol. Chem. 1996; 271: 15292-15297
        • Ornitz D.M.
        • Yayon A.
        • Flanagan J.G.
        • Svahn C.M.
        • Levi E.
        • Leder P.
        Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells.
        Mol. Cell Biol. 1992; 12: 240-247