Antithrombin glycoforms from Type II deficient subjects selectively adsorb onto plasma extracellular vesicles

Abstract Antithrombin (AT) is a glycoprotein produced by the liver and acts as the most important antagonist of clotting factors. A deficit in AT production or function leads to coagulation disorders. Two kinds of AT deficiencies are reported, named quantitative (or type I) and qualitative (or type II) defects. The first is characterized by low levels of AT in the bloodstream, the latter by impaired AT activity related to dysfunctional domains of AT and it is challenging to diagnose. Although being a soluble protein, evidence of AT transported by plasma EVs has been found but the physicochemical features of the association of AT to EVs are missing. We separated and characterized EVs from the plasma of healthy subjects, focusing on AT association. We found AT is localized on the external leaflet of EV membrane, contributing to form a “EV protein corona”. Moreover, 2D-electrophoresis highlighted specific AT glycoforms are selectively enriched onto EVs with respect to whole plasma, suggesting glycosylation plays a role in the partitioning of AT between EV surface and liquid plasma phase, and ultimately on EV exofacial topology. In the light of these findings, we investigated EVs from the plasma of 8 patients affected by type II AT defect. The comparison of the AT 2D-electrophoretic patterns highlighted a difference in AT glycoforms enrichment onto the surface of EVs from patients and healthy subjects, supporting the evidences of the key role of EVs in coagulation and suggesting a promising approach to improve diagnosis and management of type II AT deficiencies. Key points Specific AT glycoforms are enriched on on the surface of plasma derived extracellular vesicles Analysis of extracellular vesicle-AT glycoforms promise to discriminate healthy or type II deficient patients TOC Antithrombin (AT) is a soluble glycoprotein involved in coagulation and produced by the liver. Evidence of AT transported by plasma extracellular vesicles (EVs) has been found, but the features and the nature of this association are missing. We present our investigation, focusing on EV AT localization. We noticed the presence of selectively enriched AT glycoforms onto EV, compared to whole plasma. Moreover, using 2D SDS-PAGE, we also observed a different migration pattern between the EV-associated AT of healthy subjects and the EV-associated AT of patients affected by AT T2 defect, a rare coagulation disorder.