Antithrombin glycoforms from type II antithrombin deficient patients selectively adsorb onto the surface of plasma extracellular vesicles

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 the EV membrane. Furthermore, 2D-electrophoresis conducted on plasma and EVs of healthy subjects highlighted that specific AT glycoforms are selectively enriched onto the EVs with respect to whole plasma, suggesting that glycosylation plays a role in the partitioning of AT between the EV surface and liquid plasma phase, and ultimately on the EV exofacial topology. Finally, we separated EVs from the plasma of 8 patients affected by type II AT defect. The comparison of the AT 2D-electrophoretic pattern of patients and healthy subjects highlighted a difference in AT adsorption onto EV surface, supporting the role of EVs in coagulation and suggesting a promising approach to improve diagnosis and management of type II AT deficiencies. Key pointsO_LISpecific AT glycoforms are enriched in EV preparations with respect to the whole plasma. C_LIO_LIEV-AT glycoforms promise to discriminate healthy or type II deficient patients C_LI Visual abstract O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/452649v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1a00fadorg.highwire.dtl.DTLVardef@1655f15org.highwire.dtl.DTLVardef@456fe7org.highwire.dtl.DTLVardef@b5e046_HPS_FORMAT_FIGEXP M_FIG C_FIG