Fibrinogen Carbamylation as a Modulator of Blood Coagulation

Introduction The human body responds to injury with a temporary inflammatory reaction, but in certain diseases including chronic kidney disease, rheumatoid arthritis and chronic obstructive pulmonary disease inflammation remains persistent. Interestingly, these pathological conditions are also associated with a pro-thrombotic tendency. The inflammatory milieu promotes modification of endogenous proteins leading to structural and functional alterations that can result in complete loss of function. Modified proteins have been found to indicate increased cardiovascular risk in chronic kidney disease patients and to trigger the autoimmune- reactions in rheumatoid arthritis. Content of the Research Project The present project aims at identifying correlations between disease specific modifications of fibrinogen, the protein that helps in blood clot formation, and malfunction of the coagulation cascade. The ultimate goal is to discover modifications that can be used as a biological marker to estimate the risk for thrombosis in the respective patient population. Beyond that, we want to investigate how fibrinogen modification changes the inflammatory potential of the peptides released by proteolytic cleavage. Hypotheses We hypothesize that modification of fibrinogen, has an impact on the progression and the outcome of coagulation and might contribute to establish the pro-thrombotic state associated with various inflammatory diseases. Moreover, we believe that peptides from modified fibrinogen differ from their unmodified counterparts regarding their ability to augment inflammation. MethodsIn the proposed project, we will employ mass spectrometry to define modification patterns of fibrinogen in clinical samples with special focus on the positions whose originality is essential for the individual steps within the coagulation cascade. Clot formation will be measured based on turbidity changes and characterized by selected parameters, while scanning electron microscopy will be used to visualize structural features of the fibrin clots. The release of inflammatory mediators in response to fibrinopeptides will be tested in cell culture and quantified by ELISA kits. Innovation The above-mentioned diseases involve both, an inflammatory component and a predisposition to thrombosis. Fibrinogen modification, leading to aberrant blood clot formation and increased pro-inflammatory signaling of the cleavage peptides, might be a common element. Consequently, the use of disease specific modification patterns of fibrinogen as a biological marker could potentially help to identify patients at increased risk for thrombosis. Moreover, counteracting fibrinogen modification by administration of free amino acids could be a measure to prevent thrombotic events as well as the exacerbation of inflammation.

Carbamylation is a non-enzymatic posttranslational 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 project we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerization, cross-linking and fibrinolysis in vitro. In conjunction, all these factors determine clot structure and stability and 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 polymerization kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with both, decreased mechanical stability and increased resistance to fibrinolysis. Decreased mechanical strength can lead to detachment of the clot from the vascular wall and induce embolism and ischemia. Clots generated from patient fibrinogen showed a heavily altered structure characterized by a lack of recognizable fibers and pores potentially being related to the prolonged fibrinolysis time observed. Using mass spectroscopy, we discovered that processing of modified fibrinogen yielded N-terminally carbamylated fibrinopeptide A, which acted as a strong chemoattractant for neutrophils and may thus contribute to the 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 may be involved in hemostatic disorders associated with chronic inflammatory diseases.