PB0328 - New coagulation factor V mutant for measurement of protein S activity

Background: Protein S (PS) deficiency is a risk factor for venous thromboembolism. The PS activity is commonly measured by the clot-based assay, i.e., thrombin and fibrin generation.

Aims: We attempted to develop a new assay for plasma PS activity by quantifying the amount of activated coagulation factor V (FVa) cleaved by activated protein C (APC).

Methods: To obtain a suitable APC substrate, we designed a FV mutant (FVm) that mimics FVa by replacing the B domain with a His tag and adding a Flag tag at the N terminus. FVm was incubated with plasma sample, APC, and phospholipid vesicles. To quantify the cleavage at Arg306 of FVm, we performed a sandwich ELISA using anti-His and anti-FLAG tag antibodies.

Results: Calibration plasma samples prepared by mixing normal and PS-deficient plasma showed ELISA signal intensities dependent on plasma PS concentration. We measured 160 plasma samples purposively selected from the Biobank of the National Cerebral and Cardiovascular Center. The correlation between the FVm-cleavage PS activities and free PS antigen levels was the relatively weak (R2=0.378). We sequenced all exons of PROS1 from 47 subjects with < 60% activity of control plasma 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 and ≥60% activity in the clot-based assay. In contrast, no variant was identified in 16 subjects with < 60% activity in the clot-based assay and ≥60% activity in the FVm-cleavage assay. The FVm-cleavage assay was not affected by rivaroxaban, unlike the clot-based assay.

Conclusion(s): The assay using the FVm substrate may be less susceptible to interference and may provide a more accurate evaluation of plasma PS activity than clot-based assays.