Name: Extracellular Vesicle Tissue Factor Assay with Blood Plasma Derived Extracellular Vesicles
Uploaded: 2023-12-29T12:30:00
Description: Watch this Scientific Journal Video about Extracellular Vesicle Tissue Factor Assay with Blood Plasma Derived Extracellular Vesicles at JoVE.com

extracellular vesicle tissue factor assay with blood plasma derived extracellular vesicles

Improved Sensitivity for Tissue Factor Activity Assay in Extracellular Vesicles

Blood coagulation is initiated with the binding of factor (F) VII/VIIa to tissue factor (TF)1. The TF/FVIIa complex activates both FIX and FX to activate blood coagulation1. There are two forms of full-length, membrane-bound TF: encrypted and active. In addition, there is an alternatively spliced form of TF (asTF). Sphingomyelin and phosphatidylcholine in the outer leaflet of the cell membrane maintain TF in an encrypted state2,3,4. When cells are activated or damaged, phospholipid scramblase transfers phosphatidylserine and other negatively charged phospholipids to the outer leaflet1. The activation of cells also results in the translocation of acid sphingomyelinase to the outer leaflet where it degrades sphingomyelin to ceramide5. These two mechanisms convert encrypted TF to the active form. It is also proposed that protein disulfide isomerase mediates disulfide bond formation between Cys186 and Cys209 in encrypted TF, which results in the de-encryption of TF6,7,8. asTF is also present in the circulation but lacks the transmembrane domain and is therefore soluble9,10. Importantly, asTF has very low levels of procoagulant activity compared to full-length active TF10,11.
Extracellular vesicles (EVs) are released from resting, activated, and dying host cells, as well as cancer cells12. EVs express proteins from their parental cells12. Active TF-bearing EVs are released from activated monocytes, endothelial cells, and tumor cells into the circulation13,14,15. Levels of TF in plasma can be measured by activity- and antigen-based assays. Antigen-based assays include ELISA and flow cytometry16. There are two different activity-based assays: one and two-stage TF activity assays. The one-stage assay is based on a plasma-based clotting assay. The TF-containing sample is added to plasma and the time to form a clot is measured after re-calcification. The two-stage assay measures FXa generation of samples by adding FVII or FVIIa, FX, and calcium. FXa levels are determined using a substrate that is cleaved by FXa.
In both the one- and two-stage TF activity assays, the TF concentration is determined using a standard curve generated with recombinant TF. Two-stage assays have higher sensitivity and specificity than the one-stage assay. Many studies have confirmed that activity-based assays have higher sensitivity and specificity than antigen-based assays17,18,19,20,21. In addition, our in-house activity assay has higher sensitivity and specificity than a commercial activity assay22. Healthy individuals have very low or undetectable levels of EVTF activity in plasma. In contrast, individuals with pathologic conditions, such as cancer, cirrhosis, sepsis, and viral infection, have detectable levels of EVTF activity and this is associated with thrombosis, disseminated intravascular coagulation, disease severity, and mortality23,24,25,26,27,28. Here, we will describe this in-house two-stage EVTF activity assay.

Author Spotlight: High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis 

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.

Watch this Scientific Journal Video about Extracellular Vesicle Tissue Factor Assay with Blood Plasma Derived Extracellular Vesicles at JoVE.com

Extracellular Vesicle Tissue Factor Assay with Blood Plasma Derived Extracellular Vesicles  

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 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 Xa 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 Xa generation of each well. Then calculate the tissue factor-dependent factor Xa generation. Six out of 11 donors were medium to high lipopolysaccharide responders, whereas five out of 11 donors were low lipopolysaccharide responders.

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Research

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Medicine

Tissue factor (TF) is a transmembrane receptor for factor (F) VII and FVIIa. The TF/FVIIa complex initiates the coagulation cascade by activating both FIX and FX. TF is released from cells into the circulation in the form of extracellular vesicles (EVs). The level of TF-positive (+) EVs is increased in various diseases, including cancer, bacterial and viral infections, and cirrhosis, and is associated with thrombosis, disseminated intravascular coagulation, disease severity, and mortality. There are two ways to measure TF+ EVs in plasma: antigen- and activity-based assays. Data indicates that activity-based assays have higher sensitivity and specificity than antigen-based assays. This paper describes our in-house EVTF activity assay based on a two-stage FXa generation assay. FVIIa, FX, and calcium are added to the TF+ EV-containing samples to generate FXa in the presence and absence of anti-TF antibody to distinguish TF-dependent FXa generation from TF-independent FXa generation. A chromogenic substrate cleaved by FXa is used to determine the FXa level, while a standard curve generated with a relipidated recombinant TF is used for the determination of the TF concentration. This in-house EVTF activity assay has higher sensitivity and specificity than a commercial TF activity assay.

Here we describe an in-house extracellular vesicle tissue factor activity assay. Activity-based assays and antigen-based assays have been used to measure tissue factor in extracellular vesicles from human plasma samples. Activity-based assays have higher sensitivity and specificity than antigen-based assays.

Tissue factor (TF) is a transmembrane receptor for factor (F) VII and FVIIa. The TF/FVIIa complex initiates the coagulation cascade by activating both FIX ...