Neutrophil Factor XIII-A transglutaminase contributes to Neutrophil Extracellular Trap (NET)-mediated fibrin network formation and crosslinking
Fatemeh Soltani1, Alain Pacis2,3, Marie Lordkipanidzé4,5, Mari T. Kaartinen1,6
1Division of Experimental Medicine, Department of Medicine, Faculty of Medicine and Health Sciences, McGill University, Montreal, Canada 2Victor Phillip Dahdaleh Institute of Genomic Medicine at McGill University, Montréal, Québec, Canada 3Canadian Centre for Computational Genomics, McGill University, Montréal, Québec, Canada 4Faculté de pharmacie, Université de Montréal, Montréal, Québec, Canada. 5Research Center, Montreal Heart Institute, Montréal, Québec, Canada. 6Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, Canada
Neutrophil extracellular traps (NET), fibrillar networks composed of decondensed DNA, histones, and antimicrobial protein, are known to contribute to thrombotic events via interacting with endothelium and promoting fibrin clot formation. Factor XIII-A (FXIII-A) is a plasma transglutaminase produced by bone marrow monocytes and macrophages. It stabilizes the fibrin clot in a thrombin-inducible process that increases clot resistance to fibrinolysis. Our recent Tabula Muris and Tabula Sapiens RNAseq Atlas mining shows that F13A1/F13a1 mRNA is also expressed by circulating neutrophils, and bone marrow and adipose tissue neutrophils. Here, we have confirmed the production of FXIII-A by bone marrow neutrophils and investigated its function in NETosis mediated fibrinogenesis. FXIII-A was produced in normal CD11b+, CD45+ and Ly6G+ neutrophils, and at a comparable level to monocytes and macrophages. FXIII-A was externalized during phorbol myristate acetate (PMA)-induced
NETosis where it colocalized with NET DNA, and citrullinated histone H3. The in vitro transglutaminase activity assays using neutrophil extracts and transglutaminase activity-probe biotin-primary amine showed its covalent incorporation to FXIII-A substrates fibrinogen and plasma fibronectin. Incubation of neutrophil extracts with in vitro generated fibrin scaffold and ATTO488-primary amine also showed incorporation of the probe to fibrin network, indicative of the presence of FXIII-A in the preparations. Neutrophil extracts also showed cleavage of fibrinogen a-chain suggesting capacity to induce fibrin clot formation. Indeed, induction of NETosis in the presence of soluble fibrinogen resulted in dramatic fibrin network formation. Our data reveals neutrophils as a novel cellular source of FXIII-A, shows it participation to NETosis-mediated fibrinogenesis and fibrin stabilization. This can have consequences to initiation of the thromboinflammatory responses occurring in adipose tissue in obesity and resolution of inflammation-induced thrombosis where FXIII-A-crosslinked NET-fibrin may increase clot resistance to fibrinolysis.