Recombinant antithrombin Lys227 variant expressed in HEK-EBNA cells. (A) Glycoforms generated in the β context (Ala169) revealed an impaired N-glycosylation in Lys227 antithrombin compared with Glu227 antithrombin (residue present in the wild-type molecule). The study was done with (+) or without (−) treatment with PNGase F. (B) Forms secreted to the conditioned medium of cells transfected with the Glu227 (residue present in the wild-type molecule) plasmid and the mutated plasmids Lys227 and Ala227 in the β context (Ala169). The proposed number of N-glycans of each form of antithrombin is indicated. (C) Antithrombin glycoforms secreted to the conditioned medium of HEK-EBNA cells transfected with plasmids in the wild-type context (Ser169). A more complex heterogeneity of forms was observed for both Glu227 (residue present in the wild-type molecule) and mutated Lys227 plasmids, the last one generating increased levels of 3N-hypoglycosylated form and a new 2N-glycan variant form. The proposed number of N-glycans of each form of antithrombin is indicated. (D) Anticoagulant activity of the β AT (Glu227, residue present in the wild-type molecule) and mutant (Lys227) recombinant antithrombins. Anti-FXa activity was determined by a chromogenic assay. The activity of a pool of plasma of 100 healthy blood donors was taken as a reference value of 100%. (E) T-AT complexes were detected by Western blotting after adding thrombin (T) with or without UFH. The abnormal antithrombin is pointed by a red arrow.