PB0291 - Citrullination of plasma proteins in immune-mediated thrombotic thrombocytopenic purpura and cancer-related thrombosis

Background: Peptidyl Arginine Deiminase-4 (PAD4) mediated citrullination affects coagulation and fibrinolysis and is a known driver of autoimmune disease. Importantly, PAD4 is released in large quantities from neutrophil extracellular traps (NETs), which have been linked to autoimmune diseases as well as thrombo-inflammatory disorders. Citrullinated ADAMTS13 has been found in sepsis patients as well as in patients with cardiovascular comorbidities.

Aims: In this project, we investigate plasma protein citrullination in immune-mediated thrombotic thrombocytopenic purpura (iTTP) and sepsis as a link between inflammation and iTTP.

Methods: Citrulline-specific labeling of proteins is possible by targeting the modified guanidinium group with phenylglyoxal (PG) and PG derivatives. We are developing mass-spectrometry based methods for the assessment of plasma protein citrullination. Using Biotin coupled PG we enrich for citrullinated proteins to determine target proteins in patient plasma. Furthermore we employ mass-spectrometry to determine the specific citrullination sites of plasma proteins after PG labeling to gain insight on protein structure-function effects.

Results: Neutrophil proteins are expected citrullinated targets in sepsis and iTTP patient samples. We have successfully established a protocol for producing NETs in vitro using Ionomycin as a stimulus. NET-formation coincided with citrullination of histone H3 and vimentin. We are currently optimizing our Biotin-PG assay for the analysis of the plasma protein citrullination. Initial results show citrullination of coagulation pathway proteins and complement components. We successfully validated the labeling specificity of PG by labeling synthetic peptides containing citrullines and arginines.

Conclusion(s): We have successfully established assays to monitor PAD4 mediated citrullination of histone H3 and vimentin during in vitro formation of NETs. Determining targets for citrullination in patient plasma is facilitated by biotin-PG. Using PG, we selectively label citrulline residues and detect the modification using mass spectrometry. We will further establish these methods and utilize them to assess citrullination of plasma proteins in patients with sepsis and iTTP.