Extracellular Vesicle Tissue Factor Activity Assay

Summary

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.

Abstract

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.

Introduction

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 state^2,3,4. 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 TF^6,7,8. asTF is also present in the circulation but lacks the transmembrane domain and is therefore soluble^9,10. Importantly, asTF has very low levels of procoagulant activity compared to full-length active TF^10,11.

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 circulation^13,14,15. 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 assays^{17,18,19,20,21}. 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 mortality^{23,24,25,26,27,28}. Here, we will describe this in-house two-stage EVTF activity assay.

Protocol

The research was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill (protocol number: 14-2108).

1. Blood collection from donors

1. Collect whole blood using clean venipuncture into the antecubital vein with a 21 G needle. Discard the first 3 mL of blood because this portion of blood may contain TF from perivascular cells.
2. Draw 2.7 or 1.8 mL (depending on the size of the tubes) of blood into a vacutainer containing 3.2% sodium citrate (0.109 mol/L). Do not over- or underfill the tubes. Gently invert tubes immediately after blood collection to disperse the sodium citrate.
3. Avoid agitation if the tubes are transported before processing. Prepare plasma within 2 h of blood collection.

2. Preparation of negative control plasma

NOTE: Samples should not be placed on ice at any time before the final freeze.

1. Prepare negative control plasma using whole blood from healthy volunteers. Immediately after blood collection, prepare platelet-free plasma as follows.
   1. Transfer blood samples from the vacutainer tubes to 15 mL tubes.
   2. Centrifuge blood samples at 2,500 × g for 15 min at room temperature (20-24 °C) without brake.
   3. Transfer the supernatant (platelet-poor plasma) to a new tube and repeat the spin under the same conditions.
   4. Transfer the supernatant (platelet-depleted plasma) to a new tube.
   5. Aliquot the sample into ≥100 µL aliquots. Leave approximately 100 µL at the bottom of the tube.
   6. Immediately freeze the platelet-depleted plasma at −80 °C (Figure 1).

3. Preparation of positive control plasma

NOTE: Response to lipopolysaccharide is variable between individuals.

1. Transfer blood samples from the vacutainers to 15 mL tubes.
2. Prepare positive control plasma using whole blood from healthy volunteers stimulated with lipopolysaccharide (LPS) from Escherichia coli O111:B4 (10 µg/mL) for 5 h at 37 °C with agitation.
3. After 5 h incubation, follow the steps 2.1.2 to 2.1.6.

4. Isolation of extracellular vesicles from plasma

NOTE: EV pellets may not be visible. The defrosting time is dependent on sample volume but we usually defrost 100 µL plasma for 30 min at 37 °C.

1. Defrost plasma samples at 37 °C.
2. Add 1 mL of HBSA without calcium [HBSA-Ca(-)] buffer [137 mM NaCl, 5.38 mM KCl, 5.55 mM glucose, 10 mM HEPES, 0.1% (w/v) bovine serum albumin] to each 100 µL of plasma sample in a 1.5 mL tube.
3. Centrifuge plasma samples at 20,000 × g for 15 min at 4 °C.
4. Aspirate the supernatant down to 20 µL without disturbing the EV pellet.
5. Reconstitute the EV pellet by adding 1 mL of HBSA-Ca(-) buffer to each tube, pipette up and down at the location of the EV pellet, and vortex each tube before centrifuging for a second time at 20,000 × g for 15 min at 4 °C.
6. Aspirate the supernatant down to 20 µL without disturbing the EV pellet.
7. Reconstitute the EV pellet in 80 µL of HBSA-Ca(-) and pipette up and down at the location of the EV pellet (Figure 2).

5. Option: Isolation of small extracellular vesicles using ultracentrifuge

1. After step 4.3, collect the supernatant and ultracentrifuge at 100,000 × g for 70 min at 4 °C.
2. Aspirate the supernatant down to 20 µL without disturbing the EV pellet.
3. Reconstitute the EV pellet by adding 1 mL of HBSA-Ca(-) buffer to each tube and vortex each tube before ultracentrifuging for a second time at 100,000 × g for 70 min at 4 °C.
4. Aspirate the supernatant down to 20 µL without disturbing the EV pellet.
5. Reconstitute the EV pellet in 80 µL of HBSA-Ca(-).

6. Measurement of extracellular vesicle tissue factor activity

NOTE: EV samples should be pipetted and vortexed well to mix before adding to wells. EDTA-disodium will inhibit the ability of FXa to cleave the chromogenic substrate. Therefore, EDTA-disodium cannot be used as a replacement for EDTA-tetrasodium in step 6.7.

1. Add 40 µL of an EV sample to two wells of a 96-well plate.
2. Add 11 µL of an inhibitory mouse anti-human TF IgG [(36.4 µg/mL, final concentration 7.8 µg/mL)] to one well of an EV sample and 11 µL of control mouse IgG [(36.4 µg/mL, final concentration 7.8 µg/mL)] to the other well.
3. Incubate the 96-well plate for 15 min at room temperature.
4. During the incubation time, prepare 50 µL/well of TF standards (0, 0.32, 0.63, 1.25, 2.5, 5, 10, and 20 pg/mL) in duplicate using relipidated recombinant TF.
5. Prepare factor mix by mixing 4 mL of HBSA with calcium [HBSA-Ca(+)] [HBSA-Ca(-) + 10 mM CaCl₂, final concentration 10 mM], 800 µL of 900 nM FX in HBSA-Ca(+) (final concentration: 146.4 nM), and 120 µL of 200 nM FVIIa in HBSA-CA(+) (final concentration: 4.8 nM).
6. Add 50 µL of the factor mix solution to each well, cover the plate with film, and incubate for 2 h in an incubator at 37 °C.
7. After 2 h, stop FXa generation by adding 25 µL of HBSA ethylenediaminetetraacetic acid (EDTA) buffer [HBSA-Ca(-) + 25 mM EDTA-tetrasodium, final concentration 5 mM] and incubate for 5 min at room temperature.
8. Reconstitute the chromogenic substrate that is cleaved by FXa to 4mM (add 8.7 mL of distilled water to a 25 mg vial).
9. Add 25 µL of the chromogenic substrate solution to each well, cover the plate with film and then with aluminum foil to protect it from light, and incubate for 15 min at 37 °C.
10. After 15 min of incubation, remove bubbles with a needle or by centrifugation at 1,500 × g for 1 min.
11. Read the plate at 405 nm using a plate reader with one mix before reading.
12. Convert the FXa generation of each well to TF activity using the standard curve generated using relipidated recombinant TF.
13. Calculate TF-dependent FXa generation (EVTF activity) using equation (1):
    TF-dependent FXa generation (EVTF activity [pg/mL]) = Total FXa generation (control IgG well) - TF-independent FXa generation (anti-TF IgG well) (Figure 3) (1)

Results

A successful result gives a positive control value of ≥0.5 pg/mL and a negative control value of <0.5 pg/mL. It is best to find a high LPS responder with >1.0 pg/mL EVTF activity for a positive control. The representative result shows the EVTF activity of EVs isolated from plasma from the whole blood of 11 healthy donors, with and without LPS activation (Figure 4). Six out of eleven donors (Donors 2, 4, 5, 8, 10, 11) were medium to high LPS responders, whereas five out of eleve...

Discussion

Here, the protocol of our in-house EVTF activity assay is presented. There are three critical steps in the protocol. When reconstituting the EV pellet, it is important to pipette up and down at the location of the EV pellet even if it is not visible. Incomplete reconstitution of the EV pellet will result in a false negative or underestimation of EVTF activity values of the samples. Second, using HBSA-Ca(+) is critical in protocol step 6.5 because FXa generation cannot be generated without calcium. Third, using EDTA-tetra...

Materials

Name	Company	Catalog Number	Comments
1.5 mL tube for 20,000 x g centrifuge	any company	N/A	We use the one from Fisher Scientific (Catalog number: 05-408-129).
1.5 mL tube for ultracentrifuge	any company	N/A	We use the one from Beckman Coulter (Catalog number: 357448)
15 mL tube	any company	N/A	We use the one from VWR (Catalog number: 89039-666)
21 G x .75 in. BD Vacutainer Safety-Lok Blood Collection Set with 12 in. tubing and luer adapter	BD	367281
96-well plate	any company	N/A	We use the one from Globe Scientific (Catalog number: 120338).
BD Vacutainer Citrate Tubes	BD	363083
Bovine serum albumin	Sigma Aldrich	A9418
Calcium chloride	Fisher Scientific	C69-500
Centrifuge for 1.5 mL tube	any company	N/A	We use the Centrifuge 5417R (Eppendorf).
Centrifuge for 15 mL tube	any company	N/A	We use the Centrifuge 5810R (Eppendorf).
D-(+)-Glucose	Sigma Aldrich	G7021
Ethylenediaminetetraacetic acid tetrasodium salt dihydrate	Sigma Aldrich	E6511
Hepes	Sigma Aldrich	H4034
Human FVIIa	Enzyme Research Laboratory	HFVIIa	The solution should be diluted with HBSA-Ca(+).
Human FX	Enzyme Research Laboratory	HFX1010	The solution should be diluted with HBSA-Ca(+).
Inhibitory mouse anti-human tissue factor IgG, clone HTF-1	Fisher Scientific	550252
Lipopolysaccharide from Escherichia coli O111:B4	Sigma Aldrich	L2630	There are several lipopolysaccharide from different E. coli. Different lipopolysaccharide have different potential to activate monocytes.
Mouse IgG	Sigma Aldrich	I5381
Pefachrome FXa 8595	Enzyme Research Laboratory	085-27
Plate reader	any company	N/A	We use the SpectraMax i3x from Molecular Devices
Re-lipidated recombinant tissue factor, Dade Innovin	Siemens	10873566
Sodium chloride	Fisher Scientific	S271-500
Ultracentrifuge	Beckman Coulter	Optima TLX
Ultracentrifuge rotor	Beckman Coulter	TLA-55