Red blood cell–derived transglutaminase 2 influences thrombus formation

Abstract

Background: Red blood cells (RBCs) are important constituents of venous clots and contribute to thrombus size and stability. However, it remains unclear whether and how RBCs affect the thrombus. Transglutaminase (TG)2), a protein with similar activity to factor XIIIa, may influence thrombus characteristics. Objectives: We explored whether RBC-derived TG2 influences thrombus characteristics using a novel approach involving TG2 knockout (KO) RBCs. Methods: Inhibitors and TG2 KO RBCs were used in clotting assays. In vitro–generated clots were analyzed using advanced microscopy techniques to quantify the fibrin network. Whole blood clotting kinetics were assessed by thrombin generation assay and thromboelastography. Vesiculation was assessed using microparticle flow cytometry. Results: Our study showed clots formed with TG2 KO RBCs or upon TG2 inhibition displayed fewer and thinner fibrin fibers at the clot surface. Inhibition of TG2 further revealed a redistribution of fibrin away from the clot surface toward deeper regions. Functionally, TG2 inhibition accelerated thrombin generation and clot formation, as shown by shortened lag time in thrombin generation assay and faster kinetics in thromboelastography. A normal fibrin density was observed in clots treated with the TG2 inhibitor after addition of RBC-derived extracellular vesicles (EVs). Moreover, TG2 inhibition increased EV formation, associated with more phosphatidylserine exposure on RBC membranes. Conclusion: TG2 activity within RBCs plays a specific role in modulating EV formation, which in turn influences the fibrin structure and spatial distribution within blood clots. Absence of TG2 activity leads to more EV formation, promoting faster thrombin generation and clot formation, suggesting a regulatory role for TG2 in coagulation kinetics.