New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma

N. A. Alshaikh, J. Rosing, M. C. L. G. D. Thomassen, E. Castoldi, P. Simioni, T. M. Hackeng*

*Corresponding author for this work

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Background: Protein S plays an important role in the down-regulation of coagulation as cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Aim: To develop functional assays to quantify the APC-and TFPI-cofactor activities of protein S in plasma. Methods: APC-and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELI-SAs. Results: Average APC-cofactor activities of protein S were 113%, 108% and 89% in plasma from normal individuals (n = 15), FV Leiden heterozygotes (n = 14) and FV Leiden homozygotes (n = 7), respectively, whereas the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n = 21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 109%, 115% and 124% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, whereas TFPI-cofactor activities correlated less with protein S antigen levels. Conclusion: We have developed functional protein S assays that measure both the APC-and TFPI-cofactor activities of protein S in plasma, which are hardly if at all affected by the FV Leiden mutation.

Original languageEnglish
Pages (from-to)950-960
Number of pages11
JournalJournal of Thrombosis and Haemostasis
Issue number5
Publication statusPublished - May 2017


  • activated protein C resistance
  • blood coagulation
  • factor V Leiden
  • protein C
  • protein S
  • tissue factor pathway inhibitor
  • TFPI
  • RISK

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