Development of an assay using a modified coagulation factor V to measure protein S activity

Abstract

Background

Protein S (PS) is an anticoagulant that functions as a cofactor for activated protein C and the tissue factor pathway inhibitor. PS deficiency is a risk factor for venous thromboembolism. PS activity is commonly measured using clot-based assays involving fibrin and thrombin production, but improvements are needed.

Objectives

To develop a new assay for measuring plasma PS activity by quantifying the amount of activated coagulation factor (F)V cleaved by activated protein C.

Methods

We designed a recombinant, modified FV (FVm) that mimicked FVa. We analyzed 160 purposively selected plasma samples from the Biobank of the National Cerebral and Cardiovascular Center.

Results

The assay using mixed normal and PS-deficient plasma detected FVm cleavage in a PS concentration–dependent manner. The correlation between PS activity, measured using the FVm cleavage assay, and free PS antigen levels was relatively weak. We then sequenced all exons of PROS1 from 47 subjects with <60% activity in either the FVm cleavage assay or the clot-based assay. Nonsynonymous variants were identified in 12 of 24 subjects with <60% activity in both assays and in 2 of 7 subjects with <60% activity in the FVm cleavage assay alone. No variants were identified in 16 subjects with <60% activity in the clot-based assay alone. Unlike the clot-based assay, the FVm cleavage assay was not affected by the presence of rivaroxaban in the plasma.

Conclusion

An assay using the FVm substrate may be less susceptible to interference and provide a more accurate evaluation of plasma PS activity than clot-based assays.