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

摘要

Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC‐ and TFPI‐cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation. Summary 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 ELISAs. 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.