Comparison of the Use of TFPI Inhibition and Plasma Dilution to Increase the Sensitivity of Thrombin Generation Assay to Measure the Procoagulant Activity of Microvesicles

Patients with cancer have a 7- to 10- fold increased risk of developing venous thromboembolism. Circulating microvesicles (MVs) could be a predictive biomarker for venous thromboembolism in cancer. Thrombin generation assay is a useful technique to detect procoagulant activity of MVs. However, thrombin generation assay suffers from a lack of sensitivity due to the presence of Tissue Factor Pathway Inhibitor (TFPI) in plasma.

To improve the sensitivity of thrombin generation assay to tissue factor (TF) by limiting the interference of TFPI.

Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate MVs. Samples were then centrifuged and supernatants which contain MVs were used for thrombin generation assay. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of thrombin generation assay to detect procoagulant activity of MVs.

i) Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intra-assay variability. ii) Plasma dilution had no impact on the thrombin generation assay sensitivity when thrombin generation assay was triggered by MVs derived from MDA-MB-231.

Thrombin generation is a very sensitive method to study the procoagulant activity of TF-MVs. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz Domain I. A twice plasma dilution is an interesting alternative to study the procoagulant activity of MVs by thrombin generation assay with a good sensitivity, especially when low plasma quantities are available.

No relevant conflicts of interest to declare.