New Functional Assays to Selectively Quantify APC- and TFPI-Cofactor Activities of Protein S in Plasma

Background: Protein S is an important anticoagulant in the down-regulation of blood coagulation by acting as cofactor for two main regulators of coagulation. Protein S acts as a cofactor for activated protein C (APC) in the inactivation of the procoagulants FVa and FVIIIa and for TFPI in the formation of the TFPI-FXa encounter complex that subsequently inactivates TF-FVIIa. Several studies have indicated that protein S deficiency is associated with increased risks of venous thrombosis.

Aims: Current protein S functional assays are influenced by plasma determinants such as FVLeiden. The aim of the study was to develop thrombin generation based assays that enable quantification of both the APC- and TFPI-cofactor activities of protein S in plasma whereas the outcome of the assays should not be affected by the presence of FVLeiden or other plasma variables.

Methods: APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography (CAT) 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 ELISA's.

Results: Average APC-cofactor activities of protein S were 113%, 122%, and 87% in plasma from normal individuals (n=10), FV Leiden heterozygotes (n=4), and FV Leiden homozygotes (n=2), respectively, while 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 108%, 112%, and 97% 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, while TFPI-cofactor activities correlated less with protein S antigen levels.

Summary/Conclusion: We have developed functional protein S assays that measure both the TFPI- and APC-cofactor activities of protein S in plasma and that are not affected by the FV Leiden mutation or other plasma influences.