Evaluation of a new commercial assay for the measurement of tissue factor activity of extracellular vesicles isolated from human plasma

Abstract

Background

Tissue factor (TF)-positive extracellular vesicles (EVs) are released into the circulation and activate coagulation in several diseases, such as sepsis, viral infections, and cancer.

Objectives

We compared a new commercial assay, called “CY-QUANT MV-TF” (CY), with an in-house assay (Chapel Hill [CH]) for the measurement of EV TF activity.

Methods

TF-positive EVs were generated by stimulating citrated human whole blood from 4 healthy donors with lipopolysaccharide for 5 hours. EVs were isolated from platelet-low plasma by centrifugation at 20,000 × g for 60 minutes at 4 °C.

Results

Lower levels of EV TF activity were detected in the samples using the CY assay compared with the CH assay (mean ± SD, 0.54 ± 0.30 pg/mL, n = 4 vs 1.59 ± 0.43 pg/mL, n = 4; P = .01). Interestingly, the CY assay used a lower ratio of plasma to wash buffer compared with the CH assay for EV isolation. This led to higher levels of TF pathway inhibitor (TFPI) in EVs isolated using the CY protocol compared with the CH protocol. Importantly, the addition of an inhibitory anti-TFPI antibody increased EV TF activity in both assays and eliminated the difference in EV TF activity between the 2 assays.

Conclusion

In this study, we found that the CY assay detected TF activity in EVs isolated from plasma. However, significant amounts of TFPI were present in the EV preparations using the CY EV isolation protocol, which inhibited TF activity, leading to lower apparent EV TF levels.