Protein S enhancement of the TFPI-mediated inhibition of thrombin generation in protein S–depleted plasma. Thrombin generation was measured in protein S–depleted plasma supplemented with 50 µM phospholipids and 1 pM TF in the presence or absence of 0 to 2 nM (A) or 1 nM TFPI (B-C) and in the presence or absence of 50 nM WT PS (B-C). (A) A dose-dependent decrease in thrombin generation could be seen after the addition of increasing concentrations of TFPI. (B) Inhibitory antibodies against TFPI (a mix of 120 nM anti-TFPI Kunitz domain 1, 120 nM anti-TFPI Kunitz domain 2, and 120 nM anti-TFPI C-terminus) did not affect thrombin generation in protein S–depleted plasma, whereas they successfully repressed all inhibition of thrombin generation by TFPI in the presence or absence of protein S. In addition, inhibitory antibodies against protein S (2.8 µM; Dako) inhibited the enhancement of TFPI seen in the presence of protein S but had no effect on the absence of protein S. (C) The TFPI cofactor function of WT protein S and protein S variants was evaluated at 1 nM TFPI and 50 nM protein S. Pure WT protein S and WT protein S in concentrated conditioned media gave identical results. Protein S variants with amino acid substitutions in the Gla-TSR-EGF1-EGF2-EGF3-EGF4 domains (see supplemental Table 1 for full details of amino acid substitutions in the protein S variants used in this study) were screened in concentrated conditioned media. Protein S/Gas6 chimeras were purified before all experiments. All protein S variants were compared with WT protein S in concentrated conditioned media or were purified, as suitable. The relative protein S enhancement was determined as the decrease in peak height compared with WT protein S. The decrease in peak height in the presence of WT protein S compared with TFPI alone was set as 100% (n = 2). Results are expressed as mean ± standard deviation (SD). (A-B) Representative experiments are shown (n = 3). PS, protein S.