DSpace at VNU: Fibronectin unfolded by adnt but not suspended platelets: An in vitro explanation for its dual role in haemostasis

DSpace at VNU: Fibronectin unfolded by adnt but not suspended platelets: An in vitro explanation for its dual role in ha...

Ờ Å ỊÙ× Ư Ờ Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis Khon Huynh, Marianna Gyenes, Cornelis P Hollenberg, Thi-Hiep Nguyen, Toi Van Vo, Volker R Stoldt PII: DOI: Reference: S0049-3848(15)30084-0 doi: 10.1016/j.thromres.2015.08.003 TR 6060 To appear in: Thrombosis Research Received date: Revised date: Accepted date: May 2015 30 June 2015 August 2015 Please cite this article as: Huynh Khon, Gyenes Marianna, Hollenberg Cornelis P., Nguyen Thi-Hiep, Van Vo Toi, Stoldt Volker R., Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis, Thrombosis Research (2015), doi: 10.1016/j.thromres.2015.08.003 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Fibronectin unfolded by adherent but not suspended platelets: an in vitro explanation for its dual role in haemostasis SC RI Vo 5, and Volker R Stoldt 1,2,3 PT Khon Huynh 1,3,5,*, Marianna Gyenes 1,2, Cornelis P Hollenberg 4, Thi-Hiep Nguyen 5, Toi Van Department of Experimental and Clinical Haemostasis, Haemotherapy, and Transfusion NU Medicine, Heinrich Heine University Medical Center, Dusseldorf, Germany Biological Medical Research Center, Heinrich Heine University, Dusseldorf, Germany NRW Research School Biostruct, Heinrich Heine University, Dusseldorf, Germany Institute of Microbiology, Heinrich Heine University, Dusseldorf, Germany Biomedical Engineering Department, International University, Vietnam National University, Ho AC CE P Chi Minh City, Vietnam TE D MA *Corresponding author: Khon Huynh, Ph.D Biomedical Engineering Department International University – Vietnam National University HCMC Quarter 6, Linh Trung ward, Thu Duc district, Ho Chi Minh city, Vietnam Email: hckhon@hcmiu.edu.vn Tel: (+84-8) 3724 4270 Ext 3236 Fax: (+84-8) 3724 4271 ACCEPTED MANUSCRIPT Summary Fibronectin (FN), a dimeric adhesive glycoprotein, which is present both in plasma and the PT extracellular matrix can interact with platelets and thus contribute to platelet adhesion and RI aggregation It has been shown that FN can decrease platelet aggregation but enhance platelet SC adhesion, suggesting a dual role of FN in haemostasis The prevalent function(s) of FN may be determined by its fibril form To explore the suggested dual role of this adhesive protein for NU haemostasis in further detail, we now tested for any differences of adherent and suspended platelets with regard to their effect to unfold and assemble FN upon interaction Platelet MA aggregation and adhesion assays were performed using washed platelets in the presence of exogenous FN Addition of plasma FN reduced platelet aggregation in response to collagen or TE D PMA by 50% or 25% but enhanced platelet adhesion onto immobilized collagen, as compared to control experiments Analyses by fluorescence resonance energy transfer (FRET) demonstrated AC CE P that adherent platelets but not suspended platelets were capable of unfolding FN during h incubation Fluorescence microscopy and deoxycholate (DOC) solubility assays demonstrated that FN fibrils formed only on the surfaces of adherent platelets In addition, platelets adherent onto FN revealed a significantly higher activity of specific Src phosphorylation (pY418) than platelets in suspension These data suggest (1) that the function of FN in haemostasis is prevalent to its assembly, unfolding and subsequent fibril formation on the surface of adherent platelets and (2) that outside-in signaling contributes to the interaction of platelets and FN Keywords: Fibronectin, platelet adhesion and aggregation, fibronectin assembly, fibronectin unfolding and fibril formation Abbreviations: FN, fibronectin; FG, fibrinogen; ADP, adenosine diphosphate; PMA: phorbol 12- myristate-13-acetate; FRET, fluorescence resonance energy transfer; DOC: deoxycholate ACCEPTED MANUSCRIPT Highlights Fibronectin decreases platelet aggregation but enhances platelet adhesion  Adherent but not suspended platelets induce fibronectin fibrillogenesis  Adherent platelets show higher Src phosphorylation than suspended platelets  Function of FN in haemostasis is prevalent to its fibrillogenesis AC CE P TE D MA NU SC RI PT  ACCEPTED MANUSCRIPT Introduction Platelet adhesion and subsequent aggregation are the crucial steps in preventing blood loss after PT vascular injury These processes are highly regulated by interactions between platelets and RI extracellular matrix and/or plasma proteins Fibronectin (FN) is a dimeric glycoprotein of 230 to SC 250 kDa subunits which is present in soluble form in plasma and in insoluble form in the extracellular matrix (1, 2) Plasma FN has been suggested to contribute to platelet adhesion and NU aggregation (3-5) However, various studies have reported controversial results about the nature of its role during these processes (6-12) Defining the role of plasma FN in platelet adhesion and MA aggregation will lead to a better understanding of platelet biology and pathology TE D Plasma FN has a compact conformation that contains several buried protein binding sites for interacting with cell surface receptors, collagen, proteoglycans, or other FN molecules Many of AC CE P them are involved in the assembly of FN into fibrillar matrix that supports cell adhesion, growth, migration and differentiation (13) Previous studies have reported that fibril assembly is dependent on the interaction of FN with integrins (14-17) Upon interacting with integrins, the FN molecule becomes unfolded with subsequent exposure of the buried binding sites Interaction of FN with integrins can also induce receptor clustering, which in turn, brings together bound and unfolded FN to promote fibril assembly FN is then organized into detergent insoluble fibrils which are formed by the overlapping of unfolded FN dimers These fibrils were reported to contribute to platelet adhesion and aggregation (18) In general, unfolding is a cell-dependent process that turns FN into its active fibrilar form (19) Binding of FN to cell surface integrins is necessary for the FN assembly process but is not sufficient Instead, cells must generate intracellular signals to induce reorganization of the ACCEPTED MANUSCRIPT cytoskeleton which exerts the biomechanical forces via integrins to promote FN unfolding and fibril assembly (20, 21) Integrins activate several protein kinases including protein tyrosine PT kinases (focal adhesion kinase, FAK, Src family kinases, Abelson tyrosine kinase, Abl) (22, 23) Src family tyrosine kinases are reported to control signaling pathways involved in cytoskeletal RI reorganization indirectly through binding to FAK or directly through binding to the β NU SC cytoplasmic tails of integrins (24) In the present study, we examined the effect of soluble plasma FN on platelet adhesion and MA aggregation in vitro We observed that plasma FN can play a dual effect in platelet adhesion and aggregation To explore the nature of the two opposite effects, we examined conformational D changes of FN when interacting with platelets in suspension or adherent platelets Moreover, we AC CE P suspension TE tested for any differences in specific Src phosphorylation of adherent platelets and platelets in ACCEPTED MANUSCRIPT Materials and methods Materials PT Gelatin-sepharose and collagen type I were purchased from Sigma Aldrich (Hamburg, Germany) RI Sephadex G-25 gel filtration columns were obtained from Amersham Pharmacia (GE Healthcare, SC Munich, Germany) Alexa Fluor® 488 succinimidyl ester (AF488), Alexa Fluor® 546 maleimide (AF546) and CellTrackerTM Green CMFDA were from Molecular Probes (Darmstadt, NU Germany) Rotilabo® PMMA disposable cuvettes were purchased from Carl Roth GmbH MA (Karlsruhe, Germany) All other chemicals were from major suppliers Isolation and fluorescent labeling of FN D Isolation of plasma FN TE Human plasma FN was isolated by a modified procedure using gelatin-sepharose AC CE P chromatography (25) Briefly, frozen human plasma obtained from Heinrich Heine University Blood Center in Dusseldorf was thawed at 37ºC and supplemented with 10 mM ethylenediaminetetraacetic acid (EDTA) and 0.02% sodium azide (NaN3) Plasma was then applied to a gelatin-sepharose packed column The column was washed with 50 mM Tris pH 7.4 until there was no detectable protein in the eluant (absorbance at 280 nm) Washing was continued with M NaCl followed by M urea Finally, FN was eluted by M urea and immediately subjected to dialysis against PBS pH 7.3 containing 10% glycerol overnight at ºC Fractions were analyzed by SDS-PAGE (6% gel) Purity was further confirmed by dot blot experiments using specific monoclonal antibodies directed against FN, PLG, or FG Protein concentrations were determined by absorbance at 280 nm using E1 mg/mL = 1.28 ACCEPTED MANUSCRIPT FN labeling for FRET (Fluorescence resonance energy transfer) Isolated plasma FN was doubly labeled with AF488 (donor) and AF546 (acceptor) for FRET PT experiments, as previously described (26) Briefly, isolated FN was denatured by M GdnHCl to RI expose the two cryptic free cysteine residues AF546 was then added to the protein solution at a molar ratio of 30:1 (dye/FN molecule) to label the four free cysteine residues in the FN dimer SC specifically The incubation was performed in dark for h at room temperature with gentle NU rotation After that, unbound dyes were removed by dialysis against PBS pH 7.3 overnight AF546-conjugated FN (FN546) protein was collected, and the concentration was measured by MA reading the absorption at 280 nm Next, 0.1 M sodium bicarbonate pH 8.7 was added to the FN546 solution for amine labeling according to the user manual Labeling was performed by D adding AF488 in an 80-fold molar excess to the FN546 solution The mixture was incubated for TE h in dark at room temperature with gentle rotation Free dyes were again removed by dialysis AC CE P against PBS pH 7.3 overnight Concentrations and corresponding conjugation ratios (dye/FN molecule) were determined by reading the absorption at 280 nm, 496 nm, and 556 nm, respectively The calculation was performed according to the user manual Batches of doubly labeled FN conjugated with 3-4 acceptors and 6-9 donors were chosen for further experiments Sensitivity of FRET to changes in FN conformation To examine the sensitivity of FRET signals indicative of changes in FN conformation, labeled FN was exposed to solutions of GdnHCl at stepwise increasing concentrations (0 through M) Fluorescence signals were recorded at 517 nm (donor emission wavelength) and 570 nm (acceptor emission wavelength) with an excitation wavelength at 488 nm using a LS55 fluorescence spectrometer (Perkin Elmer, Rodgau, Germany) FRET signals were determined as ACCEPTED MANUSCRIPT ratio of acceptor fluorescence intensity to donor fluorescence intensity (IA/ID) Highest FRET PT signals of soluble FN in solution without GdnHCl were shown as 100% RI Platelet preparation Citrated blood was collected from healthy volunteers, who had given informed consent according SC to the Helsinki declaration Washed platelets were prepared from PRP as previously described NU (27) The platelets were finally resuspended in HEPES Tyrode buffer (NaCl 136.5 mM, KCl 2.7 mM, MgCl2.6H2O mM, NaH2PO4.H2O 3.3 mM, HEPES 10 mM, dextrose 5.5 mM and fatty MA acid-free albumin g/L, pH 7.4) Platelet suspension was adjusted to a final concentration of 2.5×108 platelets/mL The platelet suspension was supplemented with mM CaCl2 which was AC CE P Platelet aggregation assay TE D added immediately before the experiments Washed platelets (2.5ì108/mL) were mixed with 300 àg/mL plasma FN Aggregation was induced by 10 µg/mL collagen or 40 nM PMA For control experiments, washed platelets were tested for aggregation in the absence of exogenous plasma FN and/or agonists Platelet aggregation was monitored by recording changes in light transmission over using aggregometer (DiaSys Greiner, Flacht, Germany) Platelet adhesion assay Wells of a 96-well plate were coated with collagen type I (50 µg/mL) or FN (50 µg/mL) and subsequently blocked with 1% bovine serum albumin (BSA) Washed platelets (5ì108/mL) were labeled with 10 àM CMFDA for h at room temperature HEPES Tyrode buffer (200 µL) containing 107 CMFDA-labeled platelets and mM CaCl2 was added and incubated for 30 at ACCEPTED MANUSCRIPT 37°C in the absence or presence of added plasma FN (300 µg/mL) In parallel experiments, platelet adhesion was performed in the presence of 10 µM ADP Nonadherent platelets were PT washed away Adherent platelets were quantified by fluorescence intensity of CMFDA recorded SC Unfolding of FN by platelets assessed by FRET RI by a microplate fluorometer (Fluoroskan Acent, Thermo Scientific (Langenselbold, Germany) NU Labeled FN was mixed with 10-fold excess of unlabeled FN to prevent energy transfer between adjacent protein molecules For experiments with platelets in suspension, PMMA cuvettes were MA coated with 1% BSA at 37ºC for h to prevent any possible interaction of platelets with PMMA A 10 µg/mL of FN mixture (labeled FN: unlabeled FN, 1:10 ratio) was added to cuvettes D containing washed platelets (106/mL) in mL HEPES Tyrode buffer supplemented with mM TE CaCl2 and 40 nM PMA Gentle stirring was applied to ensure that platelets were kept in AC CE P suspension For experiments with adherent platelets, washed platelets (108/mL) in mL of HEPES Tyrode buffer supplemented with mM CaCl2 were allowed to adhere for h at 37°C onto PMMA cuvette surfaces precoated with FN (50 µg/mL) Unbound platelets were washed away and 10 µg/mL of FN mixture with 40 nM PMA was added to the cuvettes In both settings, FRET signals were recorded after h, h, h and h of incubation For control, FRET signals of a FN mixture without platelets were recorded Fluorescence measurement of deposited FN fibrils on platelets For suspended platelets, PMMA cuvettes were coated with 1% BSA at 37ºC for 1h FN488 (60 µg/mL) was mixed with suspension of washed platelets (106/mL) in HEPES Tyrode buffer supplemented with mM CaCl2 and 10 µM ADP The samples (2 mL) were then applied into BSA pre-coated cuvettes and incubated for 0-3 h For adherent platelets, PMMA cuvettes were ACCEPTED MANUSCRIPT 21 References AC CE P TE D MA NU SC RI PT Mosher DF Fibronectin San Diego: Academic Press; 1989 xviii, 474 p p Hynes RO Fibronectins New York: Springer-Verlag; 1990 xv, 546 p p Mosher DF Fibronectin Prog Hemost Thromb 1980;5:111-51 PubMed PMID: 6999530 Epub 1980/01/01 eng Clark RA Fibronectin matrix 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7174679 Epub 1982/12/25 eng 44 Tanabe J, Fujita H, Iwamatsu A, Mohri H, Ohkubo T Fibronectin inhibits platelet aggregation independently of RGD sequence J Biol Chem 1993 Dec 25;268(36):27143-7 PubMed PMID: 8262952 Epub 1993/12/25 eng 45 Leng L, Kashiwagi H, Ren XD, Shattil SJ RhoA and the function of platelet integrin alphaIIbbeta3 Blood 1998 Jun 1;91(11):4206-15 PubMed PMID: 9596668 Epub 1998/05/30 eng 46 Baugh L, Vogel V Structural changes of fibronectin adsorbed to model surfaces probed by fluorescence resonance energy transfer J Biomed Mater Res A 2004 Jun 1;69(3):525-34 PubMed PMID: 15127399 Epub 2004/05/06 eng 47 Somers CE, Mosher DF Protein kinase C modulation of fibronectin matrix assembly J Biol Chem 1993 Oct 25;268(30):22277-80 PubMed PMID: 8226736 Epub 1993/10/25 eng ACCEPTED MANUSCRIPT 24 Figure legends Figure Dual effect of FN on platelet adhesion and aggregation PT Washed platelets were stimulated with 10 µg/mL collagen (A) or 40 nM PMA (B) in the absence RI or presence of 300 µg/mL plasma FN Platelets aggregation was measured by using SC aggregometer Resting platelets did not aggregate without treatment with agonist (data not shown) NU 200 µL of CMFDA-labeled platelets (5ì107/mL) without or with 300 àg/mL plasma FN in MA HEPES Tyrode’s buffer containing mM CaCl2 were placed on collagen- (C) or FN-coated wells (D) of 96-well plate and incubated for 30 at 37°C In parallel experiments, platelets were D allowed to adhere onto immobilized ligand in the presence of 10 µM ADP Adhesion was TE quantified by fluorescence intensity of CMFDA as described in “Materials and methods” Values AC CE P represent the mean ± SD of three individual experiments (*) p< 0.05 Figure Fluorescence conjugation and characterization of labeled FN (A) Schematic sketch of the putative positions of fluorophores conjugated to FN molecule for FRET FN was doubly labeled with AF 488 (donor) and AF546 (acceptor) as described in “Materials and methods” (B) Schematic sketch of FN conformations in GdnHCl solutions correlated with FRET FN in solution of M GdnHCl is in a compact structure so that it exhibits highest FRET signal As GdnHCl concentration in solution increases to > M or > 4M causing FN to partially unfolded or unfolded, respectively, FRET signal decreases (C) Fluorescence emission spectra of doubly labeled FN exposed to solutions with increasing concentrations of GdnHCl Spectra have been normalized to the donor peak so that changes in FRET are indicated solely by changes in the acceptor peak ACCEPTED MANUSCRIPT 25 (D) Reference curve probing FN unfolding in GdnHCl solutions Labeled FN was exposed to 0-4 M GdnHCl and fluorescence intensities of donors and acceptors were recorded FRET PT was calculated as ratio of IA/ID FRET of FN in M GdnHCl solution was shown as RI 100% SC Figure FN unfolding and assembly by platelets monitored by FRET analysis and DOC- NU solubility assay MA (A) FN unfolding by suspended and adherent platelets monitored by FRET Labeled FN was mixed with at least 10 fold excess of unlabeled FN to prevent energy transfer between D adjacent protein molecules FN mixtures (10 µg/mL) were incubated for h at room TE temperature with washed platelets in suspension or with platelets adherent onto immobilized FN (50 µg/mL) In both settings, platelets were stimulated by 40 nM PMA AC CE P For control, FRET signals of FN mixtures without platelets were recorded (B) Representative fluorescence emission spectra of labeled FN incubated with adherent platelets over h Spectra have been normalized to the donor peak so that changes in FRET are indicated solely by changes in the acceptor peak (C) & (D) Fluorescence measurement of deposited FN fibrils on platelets Platelets in suspension or adherent onto immobilized FN were incubated with FN488 (60 µg/mL) for 1-3 h in the presence of ADP (C) or PMA (D) DOC-solubility assays were performed to compared the amount of FN fibrils deposition on platelet surfaces as described in “Materials and Methods” Values represent the mean ± SD of three individual experiments (*) p