Thrombin generation‐based assays to measure the activity of the TFPI–protein S pathway in plasma from normal and protein S‐deficient individuals

Summary

Background: Protein S acts as a cofactor for full‐length tissue factor pathway inhibitor (TFPI) in the downregulation of thrombin formation. Objective:To develop a functional test to measure the activity of the TFPI–protein S system in plasma. Methods/Patients: Using calibrated automated thrombography, we quantified the activity of the TFPI–protein S system in plasma by measuring thrombin generation in the absence and presence of neutralizing antibodies against protein S or TFPI. Moreover, we designed an enzyme‐linked immunosorbent assay (ELISA) to determine the level of full‐length TFPI in plasma. The performance of these assays was examined in plasma from 85 normal individuals and from 35 members of protein S‐deficient families. Results: The ratio of thrombin peaks determined in the absence and presence of anti‐protein S antibodies (protein S ratio = 0.5 in normal plasma) is a measure of the TFPI cofactor activity of protein S, whereas the ratio of thrombin peaks determined in the absence and presence of anti‐TFPI antibodies (TFPI ratio = 0.25 in normal plasma) is a measure of the overall activity of the TFPI–protein S system. Protein S and TFPI ratios were elevated in protein S‐deficient individuals, indicating an impairment of the TFPI–protein S system. Both ratios correlated well with full‐length TFPI levels, which were significantly lower in protein S‐deficient patients than in normal family members. Conclusions: Functional assays for the TFPI–protein S system and an ELISA for full‐length TFPI were developed. These assays show that the activity of the TFPI–protein S anticoagulant pathway is impaired in individuals with congenital protein S deficiency.