title: Impact of fibrinogen carbamylation on fibrin clot formation and stability.
abstract: 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.
authors.affiliation: Veronika Binder, Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, The Laboratory Building, 5th floor, Bergen, Norway, Tel.: +47 55 97 46 48, Fax: +47 55 97 58 17, E-mail: veronika.binder@k2.uib.no.
authors.forename: Veronika
authors.initials: V
authors.lastname: Binder
authors.forename: Brith
authors.initials: B
authors.lastname: Bergum
authors.forename: Stéphane
authors.initials: S
authors.lastname: Jaisson
authors.forename: Philippe
authors.initials: P
authors.lastname: Gillery
authors.forename: Carsten
authors.initials: C
authors.lastname: Scavenius
authors.forename: Endy
authors.initials: E
authors.lastname: Spriet
authors.forename: Anne Karin
authors.initials: AK
authors.lastname: Nyhaug
authors.forename: Helen M
authors.initials: HM
authors.lastname: Roberts
authors.forename: Iain L C
authors.initials: ILC
authors.lastname: Chapple
authors.forename: Annelie
authors.initials: A
authors.lastname: Hellvard
authors.forename: Nicolas
authors.initials: N
authors.lastname: Delaleu
authors.forename: Piotr
authors.initials: P
authors.lastname: Mydel
mesh_terms: D001777:Blood Coagulation; D002634:Chemotaxis, Leukocyte; D002956:Citrulline / Q000031:analogs & derivatives / Q000097:blood; D003485:Cyanates / Q000737:chemistry / Q000378:metabolism*; D026122:Factor XIIIa / Q000378:metabolism; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D005342:Fibrinolysis; D005344:Fibrinopeptide A / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D058105:Polymerization; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D006435:Renal Dialysis; D051437:Renal Insufficiency / Q000097:blood / Q000628:therapy; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D014508:Urea / Q000097:blood / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003485:Cyanates; C001352:homocitrulline; D005344:Fibrinopeptide A; D002956:Citrulline; D014508:Urea; D005337:Fibrin; D005340:Fibrinogen; D026122:Factor XIIIa; D013917:Thrombin
keywords: Fibrinogen; carbamylation; fibrin structure