Impact of fibrinogen carbamylation on fibrin clot formation and stability

Summary

Carbamylation is a non-enzymatic post-translational modification induced upon exposure of free amino groups to urea-derived cyanate leading to irreversible changes of protein charge, structure and function. Levels of carbamylated proteins increase significantly in chronic kidney disease and carbamylated albumin is considered as an important biomarker indicating mortality risk. High plasma concentrations and long half-life make fibrinogen a prime target for carbamylation. As aggregation and cross-linking of fibrin monomers rely on lysine residues, it is likely that carbamylation impacts fibrinogen processing. In this study we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerisation and cross-linking in vitro. In conjunction, all these factors determine clot structure and stability and thus control biochemical and mechanical properties. LC-MS/MS analyses revealed significantly higher homocitrulline levels in patient fibrinogen than in fibrinogen isolated from control plasma. In our in vitro studies we found that although carbamylation does not affect thrombin cleavage per se, it alters fibrin polymerisation kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with decreased mechanical stability. Using mass spectroscopy, we discovered that N-terminally carbamylated fibrinopeptide A was generated in this process and acted as a strong neutrophil chemoattractant potentially mediating recruitment of inflammatory cells to sites of fibrin(ogen) turnover. Taken together, carbamylation of fibrinogen seems to play a role in aberrant fibrin clot formation and might be involved in haemostatic disorders associated with chronic inflammatory diseases.

Keywords

Fibrinogen - carbamylation - fibrin structure

^* Equally contributing authors.

• References

• 1 Walsh CT, Garneau-Tsodikova S, Gatto Jr. GJ. Posttranslational Modificaiton of Proteins. Angew Chem Int Ed Engl 2006; 44: 7342-7372.
• 2 Karve TM, Cheema AK. Small changes huge impact: the role of protein posttranslational modifications in cellular homeostasis and disease. J Amino Acids 2011; 2011: 207691.
• 3 Verbrugge FH, Wilson Tang WH, Hazen SL. Protein Carbamylation and cardiovascular disease. Kidney International 2015; 88: 474-478.
• 4 Gajjala RP, Fliser D, Speer T. et al. Emerging role of post-translational modifications in chronic kidney disease and cardiovascular disease. Nephrol Dial Transplant 2015; 30: 1814-1824.
• 5 Shi J, Knevel R, Suwannalai P. et al. Autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage. PNAS 2011; 108: 17372-17377.
• 6 Gorisse L, Pietrement C, Vuiblet V. et al. Protein carbamylation is a hallmark of aging. PNAS 2016; 113: 1191-1196.
• 7 Trepanier DJ, Thibert RJ, Draisey DF. et al. Carbamylation of erythrocyte membrane proteins: an in vitro and in vivo study. Clinical Biochemistry 1996; 29: 347-355.
• 8 Berg AH, Drechsler C, Wenger J. et al. Carbamylation of Serum Albumin as a Risk Factor for Mortality in Patients with Kidney Failure. Sci Transl Med 2013; 05: 175ra29.
• 9 El-Gamal D, Holzer M, Gauster M. et al. Cyanate Is a Novel Inducer of Endothelial ICAM-1 Expression. Antioxid Redox Signal 2012; 16: 129-137.
• 10 Hawkins CL. Role of cyanate in the induction of vascular dysfunction during uremia: more than protein carbamylation?. Kidney Int 2014; 86: 875-877.
• 11 Nilsson L, Lundquist P, Kågedal R. et al. Plasma Cyanate Concentrations in Chronic Renal Failure. Clin Chem 1996; 42: 482-483.
• 12 Kraus LM, Kraus AP. Carabmoylation of amino acids and proteins in uremia. Kidney Int Suppl 2001; 78: S102-S107.
• 13 Pietrement C, Gorisse L, Jaisson S. et al. Chronic increase of urea leads to carbamylated proteins accumulation in tissues in a mouse model of CKD. PLoS One 2013; 08: e82506.
• 14 Jaisson S, Lorimier S, Ricard-Blum S. et al. Impact of Carbamylation on Type I Collagen Conformational Structure and Its Ability to Activate Human Polymorphonuclear Neutrophils. Chem Biol 2006; 13: 149-159.
• 15 Oimomi M, Hatanaka H, Yoshimura Y. et al. Carbamylation of insulin and its biological activity. Nephron 1987; 46: 63-66.
• 16 Koro C, Hellvard A, Delaleu N. et al. Carbamylated LL-37 as a modulator of the immune response. Innate Immun 2016; 22: 218-229.
• 17 Koro C, Bielecka E, Dahl-Knudsen A. et al. Carbamylation of immunoglobulin abrogates the classical pathway of complement activation. Eur J Immunol 2014; 44: 3403-3412.
• 18 Gillery P, Jaisson S. Post-translational modification derived products (PTMDPs): toxins in chronic diseases?. Clin Chem Lab Med 2014; 52: 33-38.
• 19 Bergum B, Koro C, Delaleu N. et al. Antibodies against carbamylated proteins are present in primary Sjögren’s syndrome and are associated with disease severity. Ann Rheum Dis 2016; 75: 1494-1500.
• 20 Jiang X, Trouw LA, van Wesemael TJ. et al. Anti-CarP antibodies in two large cohorts of patients with rheumatoid arthritis and their relationship to genetic risk factors, cigarette smoking and other autoantibodies. Ann Theum Dis 2014; 73: 1761-1768.
• 21 Shi J, Willemze A, Janssen GMC. et al. Recognition of citrullinated and carbamylated proteins by human antibodies: specificity, cross-reactivity and the ”AMC-Senshu” method. Ann Rheum Dis 2012; 0: 1-3
• 22 Hill JA, Al-Bishri J, Gladman DD. et al. Serum autoantibodies that bind citrullinated fibrinogen are frequently found in patients with theumatoid arthritis. J Rheumatol 2006; 33: 2115-2119.
• 23 Jennewein C, Tran N, Paulus P. et al. Novel Aspects of Fibrino(gen) Fragments during Inflammation. Mol Med 2011; 17: 568-573.
• 24 Stein TP, Leskiw MJ, Wallace HW. Measurement of half-life human plasma fibrinogen. Am J Physiol 1987; 234: D504-510.
• 25 Weisel JW, Litvinov RI. Mechanisms of fibrin polymerisation and clinical implications. Blood 2013; 121: 1712-1719.
• 26 Riedel T, Suttnar J, Brynda E. et al. Fibrinopeptides A and B release in the process of surface fibrin formation. Blood 2011; 117: 1700-1706.
• 27 Davalos D, Akassoglou K. Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol 2012; 34: 43-62.
• 28 Sidelmann JJ, Gram J, Jespersen J. et al. Fibrin clot formation and lysis: basic mechanisms. Semin Thromb Haemost 2000; 26: 605-618.
• 29 Ongeri Millien V, Lu W, Shaw J. et al. Cleavage of Fibrinogen by Proteinases Elicits Allergic Responses Through Toll-Like Receptor 4. Science 2013; 341: 792-796.
• 30 Fattouh M, Alkady O. Inflammatory biomarkers in chronic obstructive pulmonary disease. EJCDT 2014; 63: 799-804.
• 31 Martinez M, Weisel JW, Ischiropoulos H. Functional Impact of oxidative post-translational modifications on fibrinogen and fibrin clots. Free Radic Biol Med 2013; 65: 411-418.
• 32 Wang W. Identification of Respective Lysine Donor and Glutamine Acceptor Sites Involved in Factor XIIIa-catalysed Fibrin a Chain Cross-linking. J Biol Chem 2011; 286: 44952-44964.
• 33 Cesarman-Maus G, Hajjar KA. Molecular mechanisms of fibrinolysis. Brit J Haematol 2005; 129: 307-321.
• 34 MacLean B, Tomazela D, Shulman N. et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 2010; 26: 966-968.
• 35 Hazeldine J, Harris P, Chapple IL. et al. Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals. Aging Cell 2014; 13: 690-698.
• 36 Jaisson S, Gorisse L, Pietrement C. et al. Quantification of plasma homocitrulline using hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry. Anal Bioanal Chem 2012; 402: 1635-1641.
• 37 Sobel J, Gawinowicz MA. Identification of the a Chain Lysine Donor Sites Involved in Factor XIIIa Fibrin Cross-linking. J Biol Chem 1996; 271: 19288-19297.
• 38 Wolberg AS, Campbell RA. Thrombin generation, fibrin clot formation and haemostasis. Transfus Apher Sci 2008; 38: 15-23.
• 39 Pretorius E, Oberholzer HM, van der Spuy WJ. et al. Scanning electron microscopy of fibrin networks in rheumatoid arthritis: a quantitative analysis. Rheumatol Int 2012; 32: 1611-1615.
• 40 Sugo T, Nakamikawa C, Yoshida N. et al. End-linked homodimers in fibrinogen Osaka VI with a Bβ-chain extension lead to fragile clot structure. Blood 2012; 96: 3779-3785.
• 41 Stopelli P. The Plasminogen Activation System in Cell Invasion 2013. Retrieved from Marie Curie Science Database http://www.ncbi.nlm.nih.gov/books/NBK6146
• 42 Lord S. Molecular mechanisms affecting fibrin structure and stability. Arterioscler Thromb Vasc Biol 2011; 31: 494-499.
• 43 Senior MR, Skogen WF, Griffin GL. et al. Effects of Fibrinogen Derivatives Upon the Inflammatory Response. J Clin Invest 1986; 77: 1014-1019.
• 44 Kalim S, Karumanchi SA, Thadhani RI. et al. Protein Carbamylation in Kidney Disease: Pathogenesis and Clinical Implications. Am J Kidney Dis 2014; 64: 793-803.
• 45 Holzer M, Zangger K, El-Gamal D. et al. Myeloperoxidase-derived chlorinating species induce protein carbamylation through decomposition of thiocyanate and urea: novel pathways generating dysfunctional high-density lipoprotein. Antioxid Redox Signal 2012; 17: 1043-1052.
• 46 Fleury V, Angles-Cano E. Characterisation of the binding of plasmin to fibrin surfaces: the role of carboxy-terminal lysines. Biochemistry 1991; 30: 7630-7638.
• 47 Voskuilen M, Vermond A, Veeneman GH. et al. Fibrinogen lysine residue Aa 157 plays a crucial role in the fibrin-induced acceleration of plasminogen activation, catalysed by tissue-type plasminogen activator. J Biol Chem 1987; 262: 5944-5946.
• 48 Scinocca M, Bell DA, Racapé M. et al. Antihomocitrullinated Fibrinogen Antibodies are Specific to Rheumatoid Arthritis and Frequently Bind Citrullinated Proteins/peptides. J Rheumatol 2014; 41: 270-279.
• 49 Jaisson S, Larreta-Garde V, Bellon G. et al. Carbamylation differentially alters type I collagen sensitivity to various collagenases. Matrix Biology 2007; 26: 190-196.
• 50 Jalal D, Chonchol M, Targher G. Disorders of Haemostasis Associated with Chronic Kidney Disease. Semin Thromb Haemost 2010; 36: 34-40.
• 51 Vadseth C, Souza JM, Thomson L. et al. Pro-thrombotic State Induced by Post-translational Modification of Fibrinogen by Reactive Nitrogen Species. JBC 2004; 279: 8820-8826.