APC and TFPI cofactor function of N-linked glycan protein S variants determined using CAT. (A) Spatial distribution of amino acids which were substituted to obtain additional N-linked glycosylations in the protein S SHBG-like region are highlighted in red. Naturally occurring N-linked glycolysations are highlighted in blue. The model of the SHBG-like region is adapted from Villoutreix et al.⁴²  (B-E) The generated variants were investigated for their abilities to function as cofactors for APC (B) or TFPIα (C-E) using CAT. For this, thrombin generation was initiated by 1 pM TF in protein S-depleted plasma supplemented with 50 μM phospholipids. (B) Screening assay for thrombin generation quantified in plasma supplemented with 9 nM APC in the presence or absence of 50 nM WT or variant protein S. The relative APC enhancement by the protein S variants were determined as the reduction in thrombin peak height compared with that of WT protein S. The decrease in peak height in the presence of WT protein S compared with APC alone was set as 100%. (C) Screening assay for TFPI cofactor function of the protein S variants, using thrombin generation measured in plasma supplemented with 1 nM TFPIα in the presence or absence of 50 nM WT or variant protein S. The relative enhancement of TFPIα was calculated as that of APC above. Here, the decrease in peak height in the presence of both WT protein S and TFPIα compared with that caused by TFPIα alone was set as 100%. (D-E) Titrations of TFPI cofactor function were performed for WT protein S, protein S D253T, and Q427N/K429T (0-50 nM), and the normalized lag times (D) and peak thrombin (E) were plotted against protein S concentration. The peak height and lag time observed in the presence of TFPIα alone represents 100%. The results are presented as mean ± SEM (n = 3-6). PS, protein S.