Extracellular Vesicle Tissue Factor Activity Assay

Low levels of tissue factor positive extracellular vesicles are present in plasma from patients with different diseases. However, most antigen based assays, including ELISA and flow cytometry, do not have the sensitivity to detect these low levels. Numerous papers have employed ELISA or flow cytometry to detect tissue factor in plasma.

Our protocol measures tissue factor activity in plasma with high sensitivity and specificity, although there is some variability due to the use of a functional assay. There are commercial assay kits that measure levels of tissue factor activity in plasma. However, these kits do not contain anti-tissue factor antibodies, and therefore cannot distinguish between tissue factor-dependent and tissue factor-independent Factor X generation.

To begin, defrost the isolated blood plasma samples at 37 degrees Celsius. Transfer 100 microliters of each plasma sample into 1.5 milliliter tubes. Add one milliliter of calcium free HBSA buffer to each tube.

Centrifuge the samples at 20, 000 G for 15 minutes at four degrees Celsius. Aspirate the supernatant to 20 microliters without disturbing the pellet. Next, add one milliliter of the calcium-free HBSA buffer to each tube.

Then pipette up and down to reconstitute the pellet. Vortex each tube for a few seconds to ensure equal distribution of the extracellular vesicles. Then centrifuge it at 20, 000 G for 15 minutes at four degrees Celsius.

Again, aspirate the supernatant to 20 microliters without disturbing the pellet. Then reconstitute the pellet in 80 microliters of calcium-free HBSA buffer. Pipette the suspension up and down to obtain an even suspension.

To measure the extracellular vesicle tissue factor activity, first pipette 40 microliters of the extracellular vesicle sample into two wells of a 96 well plate. Into the first well add 11 microliters of an inhibitory mouse anti-human TF IgG. To the other well, add 11 microliters of control mouse IgG.

After incubation, add 50 microliters of the factor mix solution into each well. Cover the plate with film, then incubate the plate for two hours at 37 degrees Celsius. Add 25 microliters of HBSA EDTA buffer to stop the Factor 10a generation.

Reconstitute the chromogenic substrate in distilled water to a concentration of four millimoles. Now add 25 microliters of the chromogenic substrate into each well. Cover the plate with film and then wrap it in aluminum foil.

Incubate the plate at 37 degrees Celsius for 15 minutes. Next, centrifuge the plate at 1, 500 G for one minute to remove any bubbles. Then place the plate in a plate reader at 405 nanometers.

Use the standard curve generated with relipidated recombinant tissue factor to convert the Factor 10a generation of each well. Then calculate the tissue factor dependent Factor 10a generation. Six out of 11 donors were medium to high lipopolysaccharide responders, whereas five out of 11 donors were low lipopolysaccharide responders.