Citrullinated Fibrinogen Renders Clots Mechanically Less Stable, but Lysis-Resistant
2021; Lippincott Williams & Wilkins; Volume: 129; Issue: 2 Linguagem: Inglês
10.1161/circresaha.121.319061
ISSN1524-4571
AutoresImre Varjú, Nicoletta Sorvillo, Deya Cherpokova, A Farkas, Veronika J. Farkas, Erzsébet Komorowicz, Tímea Feller, Balázs Kiss, Miklós Kellermayer, László Szabó, András Wacha, Attila Bóta, Colin Longstaff, Denisa D. Wagner, Krasimir Kolev,
Tópico(s)Wound Healing and Treatments
ResumoHomeCirculation ResearchVol. 129, No. 2Citrullinated Fibrinogen Renders Clots Mechanically Less Stable, but Lysis-Resistant Open AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialOpen AccessLetterPDF/EPUBCitrullinated Fibrinogen Renders Clots Mechanically Less Stable, but Lysis-Resistant Imre Varjú, Nicoletta Sorvillo, Deya Cherpokova, Ádám Z. Farkas, Veronika J. Farkas, Erzsébet Komorowicz, Tímea Feller, Balázs Kiss, Miklós Z. Kellermayer, László Szabó, András Wacha, Attila Bóta, Colin Longstaff, Denisa D. Wagner and Krasimir Kolev Imre VarjúImre Varjú https://orcid.org/0000-0001-6592-4800 Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA (I.V., N.S., D.C., D.D.W.). Department of Pediatrics, Harvard Medical School, Boston, MA (I.V., N.S., D.C., D.D.W.). Department of Biochemistry (I.V., Á.Z.F., V.J.F., E.K., L.S., K.K.), Semmelweis University, Budapest, Hungary. Department of Sociomedical Sciences, Mailman School of Public Health, Columbia University, NY (I.V.). , Nicoletta SorvilloNicoletta Sorvillo Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA (I.V., N.S., D.C., D.D.W.). Department of Pediatrics, Harvard Medical School, Boston, MA (I.V., N.S., D.C., D.D.W.). , Deya CherpokovaDeya Cherpokova Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA (I.V., N.S., D.C., D.D.W.). Department of Pediatrics, Harvard Medical School, Boston, MA (I.V., N.S., D.C., D.D.W.). , Ádám Z. FarkasÁdám Z. Farkas Department of Biochemistry (I.V., Á.Z.F., V.J.F., E.K., L.S., K.K.), Semmelweis University, Budapest, Hungary. , Veronika J. FarkasVeronika J. Farkas Department of Biochemistry (I.V., Á.Z.F., V.J.F., E.K., L.S., K.K.), Semmelweis University, Budapest, Hungary. , Erzsébet KomorowiczErzsébet Komorowicz Department of Biochemistry (I.V., Á.Z.F., V.J.F., E.K., L.S., K.K.), Semmelweis University, Budapest, Hungary. , Tímea FellerTímea Feller https://orcid.org/0000-0002-9137-5881 Department of Biophysics and Radiation Biology (T.F., B.K., M.Z.K.), Semmelweis University, Budapest, Hungary. , Balázs KissBalázs Kiss Department of Biophysics and Radiation Biology (T.F., B.K., M.Z.K.), Semmelweis University, Budapest, Hungary. , Miklós Z. KellermayerMiklós Z. Kellermayer https://orcid.org/0000-0002-5553-6553 Department of Biophysics and Radiation Biology (T.F., B.K., M.Z.K.), Semmelweis University, Budapest, Hungary. , László SzabóLászló Szabó Department of Biochemistry (I.V., Á.Z.F., V.J.F., E.K., L.S., K.K.), Semmelweis University, Budapest, Hungary. Department of Functional and Structural Materials (L.S.), Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. , András WachaAndrás Wacha Biological Nanochemistry Research Group (A.W., A.B.), Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. , Attila BótaAttila Bóta Biological Nanochemistry Research Group (A.W., A.B.), Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. , Colin LongstaffColin Longstaff National Institute for Biological Standards and Control, South Mimms, United Kingdom (C.L.). , Denisa D. WagnerDenisa D. Wagner Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA (I.V., N.S., D.C., D.D.W.). Department of Pediatrics, Harvard Medical School, Boston, MA (I.V., N.S., D.C., D.D.W.). Division of Hematology/Oncology, Boston Children's Hospital, MA (D.D.W.). and Krasimir KolevKrasimir Kolev Correspondence to: Krasimir Kolev, PhD, Semmelweis University, Department of Biochemistry, 1094 Budapest, Tűzoltó utca 37-47, Hungary. Email E-mail Address: [email protected] https://orcid.org/0000-0002-5612-004X Department of Biochemistry (I.V., Á.Z.F., V.J.F., E.K., L.S., K.K.), Semmelweis University, Budapest, Hungary. Originally published26 May 2021https://doi.org/10.1161/CIRCRESAHA.121.319061Circulation Research. 2021;129:342–344is related toMeet the First AuthorsOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: May 26, 2021: Ahead of Print Meet the First Author, see p 220Clot stability, the resistance to mechanical stress and fibrinolytic dissolution, might affect the clinical behavior of deep vein thrombi and the success of thrombolysis or pharmacomechanical thrombectomy. Clot stability is sensitive to chemical modifications of the underlying fibrin matrix and its precursor fibrinogen. Citrullination is a posttranslational modification of peptidyl-arginine by PADs (peptidyl-arginine-deiminases).1 Both PAD2 and 4—proven to catalyze fibrinogen citrullination—have been detected in human serum and may be released from neutrophils during—or independently of—the formation of the highly procoagulant neutrophil extracellular traps.1 However, (1) the effect of citrullinated fibrinogen (CitFg) on clot stability is poorly understood and (2) no study has investigated CitFg in intravascular thrombi. Here, we provide evidence that citrullination greatly impacts the stability of human model thrombi and that CitFg is present in a murine analogue of human deep vein thrombi.To incorporate CitFg in model thrombi, we incubated human fibrinogen with PAD4 in 100 mmol/L TRIS-HCl 10 mmol/L CaCl2 1 mmol/L DTT pH 7.5 at 37 °C (concentrations and incubation times were adjusted for different experiments). PAD4 activity was halted with 60 mg/L Cl-amidine (in controls immediately at 0 hours).We studied the mechanical properties of clots using atomic-force microscopy-based nano-thromboelastography2 as well as oscillation rheometry.3 In nano-thromboelastography, the forming fibrin deflects the vertically traveling cantilever immersed into the clot (6 µmol/L fibrinogen incubated with 0.25 mg/L PAD4 for 0/3 hours, clotted with 10 nmol/L thrombin). Fibrin strength is calculated from the deflection as the force difference (ΔF) between the onset and end of cantilever pulls. ΔF was markedly decreased throughout the clotting of CitFg, and the ratio of times elapsed from start until the maximal slope of force-curves was 3.10±0.68 (3/0 hours citrullinated fibrin, n=3), indicating delayed clotting (Figure [A]). This was in line with electromechanical coagulometry results: 17 nmol/L thrombin yielded clotting times 37.6±2.6/77.8±6.9*/87.4±6.0** s with 4 µmol/L fibrinogen citrullinated with 0.4 mg/L PAD4 for 0/3/4 hours, respectively (n=7, Kruskal-Wallis test P=0.0002, *P=0.0152, **P=0.0017 versus 0 hours, Dunn-test; mean±SE of mean shown throughout, statistical analyses performed with GraphPad Prism 9.00).Download figureDownload PowerPointFigure. Citrullinated fibrin(ogen): effects on mechanical (A and B) and fibrinolytic (C and D) stability, presence in murine samples (E and F). Mean±SE of mean shown. Red/green: control/citrullinated. Animal work was approved by the Institutional Animal Care and Use Committee (No. 17-01-3308R). A, Nano-thromboelastography (nanoTEG). Fibrin strength during clotting.2B, Microrheology. 0.015 oscillatory strain at 1 Hz imposed 2 min after clotting initiation.3 G′, G″: storage and loss moduli after 15 min clotting. Curves: fibrin viscosity (η) vs increasing shear stress (τ) postclotting.3 Factor XIIIa-inhibitor ZED1301 added at 20 µmol/L. C, Photoscanning. tPA (tissue-type plasminogen activator)-induced lysis of plasma clots spiked with fibrinogen (citrullinated for indicated time). Lysis-front run distances after 45 min were normalized for channel length (relative units, RU). D, Turbidimetry. Clotted fibrinogen (PAD4 [peptidyl-arginyl-deiminase-4]-treated for 0/8 h, or their 1:1 mixture) lysed with plasmin (1 µmol/L).3 Time until half-maximal turbidity at 340 nm (t50) was normalized using control t50 as reference. E/F, Polyacrylamide-gelelectrophoresis, western blot. 0.05 g/L PAD4 applied for 3h where indicated. E, upper row: ≈1 kDa shift in the apparent size of Aα chain upon citrullination of 0.3 μmol/L murine fibrinogen, resembling the shift previously shown in citrullinated human fibrinogen. E, lower row: citrullination patterns are similar in hirudin-anticoagulated recalcified murine vs human plasma in vitro, absent from native plasma (±clotted with thrombin). Note: molecular weights of murine fibrinogen chains are lower than human. F, Homogenized inferior vena cava (IVC) thrombi and corresponding plasma 48 h postligation. Citrullinated Aα chains detected by anticitrullinated fibrinogen (CitFg)/anti-alpha antibodies (upper/lower). To calculate normalized citrullination intensity, citrullinated Aα signals (upper) were normalized by citrullination nonspecific fibrinogen antibody signal intensity from stripped and reblotted membranes (not shown).Assuming that the nano-thromboelastography cantilever behaves as a Hookean spring, the maximal ΔF is analogous with the maximal clot elasticity measured by conventional thromboelastometry.2 The ratio of maximal ΔF of 3/0 hours citrullinated fibrin was 0.52±0.08 (n=3), indicating reduced clot elasticity,2 in line with rheometry3 results of clots formed with 10 nmol/L thrombin and fibrinogen (8 µmol/L citrullinated with 0.6 mg/L PAD4). At the end of clotting, storage and loss moduli (G′, G″) stabilized at lower values in citrullinated clots, indicating decreased elastic and viscous properties, respectively, while the loss tangent (G″/G′) increased, revealing overall decreased plastic stability. Compared with control, flow-curves of citrullinated clots presented with markedly lower dynamic viscosity and a lack of strain hardening as increasing shear stress was applied (Figure [B]). Gel/fluid transition (marking clot disassembly3) occurred at 41.2±2.5/62.8±5.8 Pa in 3/0 hours citrullinated clots (n=10/11, P=0.0033, Kolmogorov-Smirnov test), indicating decreased resistance to shear stress upon citrullination.Besides fibrin biomechanics, the persistence of thrombi also depends on their susceptibility to plasmin, a serine protease formed from plasminogen by plasminogen activators (eg, tPA [tissue-type plasminogen activator]). Therefore, we investigated the lysis of clotted recalcified human plasma spiked with 1 µmol/L fibrinogen (pretreated with 1.15 mg/L PAD4 for 0/3/4 hours) in transparent microfluid channels. Fibrin/fluid interface migration was followed by photoscanning after applying tPA (0.3 µmol/L).3 CitFg-supplemented clots lysed slower (Figure [C]). Similarly, turbidimetry showed decelerated plasmin-mediated degradation of clots formed with 15 nmol/L thrombin and 6 µmol/L fibrinogen (pretreated with 1.15 mg/L PAD4, Figure [D]).To test the presence of CitFg in thrombi, we performed the inferior vena cava stenosis model in 8- to 10-week old wild-type C57BL/6J male mice. In this model, thrombus development (under restricted flow without direct endothelial damage) closely resembles human deep vein thrombi.4 First, we established that citrullinated murine fibrinogen shows a pattern similar to that of human fibrinogen on Western blot (Figure [E]). Next, we blotted processed inferior vena cava thrombi (samples were encoded upon blinded harvest), which showed a strong citrullination signal corresponding the Aα-chain (Figure [E] and [F]). Normalized citrullination intensity was 0.53±0.19 (n=6) within 1-3 h inferior vena cava ligation, with no statistically significant change at later timepoints (0.31±0.11 at 6-12 h, n=9; 0.49±0.14 at 24-48 h, n=7). In plasma from thrombus-bearing mice and in vitro clots generated thereof, CitFg was hardly detectable: normalized citrullination intensity was 0.04±0.02 for plasma; 0.04±0.01 for plasma clots (n=4, Kolmogorov-Smirnov test P<0.05 when either is compared with any thrombus age group).Here, we demonstrate that CitFg—previously identified in rheumatoid arthritis and atherosclerotic plaques, but not in intravascular clots—(1) markedly reduces clot stability and increases resistance to lysis in vitro and (2) is abundant in a murine analogue of human deep vein thrombi. Since microrheological parameters of clots are associated with thromboembolism recurrence,5 our findings warrant future studies to test how our proposed novel mechanism regulating clot stability translates into clinical characteristics of thrombi.Nonstandard Abbreviations and AcronymsCitFgcitrullinated fibrinogenPADpeptidyl-arginyl-deiminasetPAtissue-type plasminogen activatorData availabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.AcknowledgmentsWe are grateful to Györgyi Oravecz, Krisztián Bálint, Long Chu, and Steve Cifuni for excellent technical assistance.Sources of FundingThis work was supported by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary for the Molecular Biology thematic programme of Semmelweis University and STIA-2020-OTKA/137209 to K. Kolev, National Heart, Lung, and Blood Institute of the National Institutes of Health (grant R35 HL135765 to D.D. Wagner), the Hungarian National Research, Development and Innovation Office (NKFIH) No. PD124451 to A. Wacha and the Excellence Program BIONANO_GINOP-2.3.2-15-2016-00017 to A. Bóta. I. Varjú was supported by postdoctoral fellowships from the Hungarian Academy of Sciences, the Fulbright Commission as well as the Tempus Public Foundation, and a scholarship by the Rosztoczy Foundation. D. Cherpokova was supported by a postdoctoral fellowship of the German Research Foundation (CH 1734/1-1). A.Z. Farkas and V.J. Farkas received EFOP (Human Resource Development Operational Program) scholarship (EFOP-3.6.3-VEKOP-16-2017-00009).Disclosures None.FootnotesFor Sources of Funding and Disclosures, see page 343.Correspondence to: Krasimir Kolev, PhD, Semmelweis University, Department of Biochemistry, 1094 Budapest, Tűzoltó utca 37-47, Hungary. Email krasimir.kolev@eok.sote.huReferences1. Döring Y, Libby P, Soehnlein O. Neutrophil extracellular traps participate in cardiovascular diseases: recent experimental and clinical insights.Circ Res. 2020; 126:1228–1241. doi: 10.1161/CIRCRESAHA.120.315931LinkGoogle Scholar2. Feller T, Kellermayer MS, Kiss B. Nano-thrombelastography of fibrin during blood plasma clotting.J Struct Biol. 2014; 186:462–471. doi: 10.1016/j.jsb.2014.04.002CrossrefMedlineGoogle Scholar3. Longstaff C, Varjú I, Sótonyi P, Szabó L, Krumrey M, Hoell A, Bóta A, Varga Z, Komorowicz E, Kolev K. Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones.J Biol Chem. 2013; 288:6946–6956. doi: 10.1074/jbc.M112.404301CrossrefMedlineGoogle Scholar4. von Brühl ML, Stark K, Steinhart A, Chandraratne S, Konrad I, Lorenz M, Khandoga A, Tirniceriu A, Coletti R, Köllnberger M, et al. 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Authors of the article cited in the comment will be invited to reply, as appropriate. Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page. Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetailsCited By Green R and Thompson P (2023) Current insights into the role of citrullination in thrombosis, Current Opinion in Chemical Biology, 10.1016/j.cbpa.2023.102313, 75, (102313), Online publication date: 1-Aug-2023. Herre M, Cedervall J, Mackman N and Olsson A (2023) Neutrophil extracellular traps in the pathology of cancer and other inflammatory diseases, Physiological Reviews, 10.1152/physrev.00062.2021, 103:1, (277-312), Online publication date: 1-Jan-2023. 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Westerlind H, Kastbom A, Rönnelid J, Hansson M, Alfredsson L, Mathsson-Alm L, Serre G, Cornillet M, Holmdahl R, Skriner K, Bang H, Klareskog L, Saevarsdottir S, Lundberg K, Grönwall C and Askling J (2022) The association between autoantibodies and risk for venous thromboembolic events among patients with rheumatoid arthritis, Rheumatology, 10.1093/rheumatology/keac601 Wagner D and Heger L (2022) Thromboinflammation: From Atherosclerosis to COVID-19, Arteriosclerosis, Thrombosis, and Vascular Biology, 42:9, (1103-1112), Online publication date: 1-Sep-2022. Mao L, Mostafa R, Ibili E and Fert-Bober J (2022) Role of protein deimination in cardiovascular diseases: potential new avenues for diagnostic and prognostic biomarkers, Expert Review of Proteomics, 10.1080/14789450.2021.2018303, 18:12, (1059-1071), Online publication date: 2-Dec-2021. Related articlesMeet the First AuthorsCirculation Research. 2021;129:218-220 July 9, 2021Vol 129, Issue 2 Advertisement Article Information Metrics © 2021 The Authors. Circulation Research is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited.https://doi.org/10.1161/CIRCRESAHA.121.319061PMID: 34037437 Originally publishedMay 26, 2021 KeywordscitrullinationfibrinfibrinolysisplasminogenthrombosisthromboelastographyPDF download Advertisement Subjects Animal Models of Human Disease Basic Science Research Mechanisms Pathophysiology Thrombosis
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