Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove
JoVE Journal Medicine

Medicine

Extracellular Vesicle Tissue Factor Activity Assay

Published: December 29, 2023 doi: 10.3791/65840
Automatic Translation
1, 1
1Division of Hematology, UNC Blood Research Center, University of North Carolina at Chapel Hill

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 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.

Subscription Required. Please recommend JoVE to your librarian.

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)

Subscription Required. Please recommend JoVE to your librarian.

Representative 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 eleven donors (Donors 1, 3, 6, 7, 9) were low LPS responders.

Figure 1
Figure 1: Preparation of platelet-depleted plasma. The figure was modified from Hisada and Mackman29. Abbreviation: PDP = platelet-depleted plasma. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Isolation of extracellular vesicles from platelet-depleted plasma. The figure was modified from Hisada and Mackman29. Abbreviations: PDP = platelet-depleted plasma; EVs = extracellular vesicles. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Measurement of extracellular vesicle tissue factor activity. The figure was modified from Hisada and Mackman29. Abbreviations: TF = tissue factor; EVs = extracellular vesicles. Please click here to view a larger version of this figure.

Figure 4
Figure 4: EVTF activity of plasma from whole blood of 11 healthy donors, with and without LPS activation. LPS activation was performed under the same condition as the positive control. The positive control was from a known LPS responder whereas the responses to LPS of 11 healthy donors were not known at the time of the experiment. White and black bars indicate with and without LPS activation, respectively. Abbreviations: EVTF = extracellular vesicle tissue factor; LPS = lipopolysaccharide. Please click here to view a larger version of this figure.

Table 1: A summary of the studies comparing this in-house EVTF activity assay, a commercial TF ELISA, and an activity assay. Abbreviations: EVTF = extracellular vesicle tissue factor; ELISA = enzyme-linked immunosorbent assay. Please click here to download this Table.

Subscription Required. Please recommend JoVE to your librarian.

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-tetrasodium but not EDTA-disodium to stop FXa generation is critical because the latter inhibits the ability of FXa to cleave the chromogenic substrate.

We recommend the following storage and use method for each reagent. We store assay buffers and the reconstituted substrate at 4 °C and repeatedly use them until we run out of them. If we do not run out of the substrate within 2-3 weeks, we discard it because it turns yellow. We store recombinant TF, antibodies, FVIIa, and FX at -80 °C and recommend making aliquots for one assay to avoid a freeze-thaw cycle.

In a preliminary study, we left plasma samples for 9 h at room temperature, refroze them at -80 °C overnight, and performed the assay the next day. As a control, we thawed the same plasma and refroze it immediately. We found that the sample left for 9 h at room temperature had a 16% decrease in EVTF activity compared to the one that was thawed and refrozen immediately (Hisada and Mackman, unpublished data 2017).

In addition to the difference in response to LPS, the difference in the concentration of EVs in plasma from individual donors may explain the different EVTF activity of different donors. Indeed, we observed some variations in the concentrations of EVs in plasma from LPS-stimulated whole blood (1.9 ± 0.6 × 1010 particles/mL, n = 6, mean ± standard deviation)30.

This in-house EVTF activity assay has several features that commercial assays do not have. First, the assay uses an anti-TF antibody to distinguish TF-dependent FXa generation from TF-independent FXa generation. Three commercial activity assays are claimed to measure TF activity in plasma but none of them uses anti-TF antibodies16. Therefore, these commercial assays simply measure total FXa generation but not TF-dependent FXa generation. Second, we use 2.4 nM of FVIIa (the final concentration in the reaction) to minimize TF-independent FXa generation by FVIIa. One commercial activity assay uses 12 nM of FVIIa in the reaction, which gives a high background22. Indeed, FVIIa activates FX linearly depending on FVIIa concentration in the absence of TF31. We summarized the studies that compared this in-house EVTF activity assay and commercial TF ELISA and an activity assay in Table 1.

Four response classifications of EVTF activity for citrated platelet-poor plasma are proposed: zero (0 to <0.5 pg/mL); weak (0.5 to <1.0 pg/mL), moderate (1.0 to <2.0 pg/mL), and strong (≥2.0 pg/mL)23. Notably, different anticoagulants affect the results of EVTF activity assays. For instance, higher levels of EVTF activity were observed using heparin after LPS stimulation compared to sodium citrate (Donor A: 4.6 (citrate) vs 28.6 (heparin) pg/mL and Donor B: 3.4 (citrate) vs 26.0 (heparin) pg/mL, respectively, Hisada and Mackman unpublished data 2017). In contrast, lower levels of EVTF activity were observed using EDTA after LPS stimulation compared to sodium citrate (Donor C: 3.9 (citrate) vs 1.4 (EDTA) pg/mL, Donor D: 3.8 (citrate) vs 0.4 (EDTA) pg/mL, respectively, Tatsumi and Mackman unpublished data 2016). These results indicate that EVTF activity data are not comparable when different anticoagulants are used.

There are a few limitations to the method. The coefficient of variation (CV) is relatively high compared to clinical assays. Indeed, the CV for the positive control in seven independent studies was 24%17,29. Isolation of EVs using centrifugation pellets not only EVs but also cellular debris. We previously analyzed the pellet of platelet-rich plasma by transmission electron microscopy and observed platelets, EVs, and cellular debris32. We use a fixed-angle rotor and have not experienced a swing-out rotor for 20,000 × g centrifugation. We recently found that small EVs that can be pelleted with 100,000 × g but not with 20,000 × g have EVTF activity in patients with pancreatic cancer and COVID-1930. This indicates that this protocol does not measure EVTF activity from small EVs, such as exosomes. Notably, exosomes are sphingomyelin-rich EVs and the presence of TF-bearing exosomes has been reported30,33,34,35. We recommend pelleting EVs in plasma using 100,000 × g to more accurately determine the total amount of EVTF activity in the sample. Of note, it requires an ultracentrifuge and a longer assay time because the centrifugation time increases from 15 min to 70 min.

This method can be applied to plasma samples from any kind of disease. EVTF activity is associated with disease severity and survival in patients with different diseases, including cancer, COVID-19, bacterial infection, and cirrhosis23,26,27.

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

There are no competing interests to disclose.

Acknowledgments

This work was supported by the NIH NHLBI R35HL155657 (N.M.) and the John C. Parker professorship (N.M.). We would like to thank Ms. Sierra J. Archibald for her helpful comments

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

DOWNLOAD MATERIALS LIST

References

  1. Grover, S. P., Mackman, N. Tissue factor: an essential mediator of hemostasis and trigger of thrombosis. Arteriosclerosis, Thrombosis, and Vascular Biology. 38 (4), 709-725 (2018).
  2. Shaw, A. W., Pureza, V. S., Sligar, S. G., Morrissey, J. H. The local phospholipid environment modulates the activation of blood clotting. Journal of Biological Chemistry. 282 (9), 6556-6563 (2007).
  3. Tavoosi, N., et al. Molecular determinants of phospholipid synergy in blood clotting. Journal of Biological Chemistry. 286 (26), 23247-23253 (2011).
  4. Wang, J., Pendurthi, U. R., Rao, L. V. M. Sphingomyelin encrypts tissue factor: ATP-induced activation of A-SMase leads to tissue factor decryption and microvesicle shedding. Blood Advances. 1 (13), 849-862 (2017).
  5. Kornhuber, J., Rhein, C., Muller, C. P., Muhle, C. Secretory sphingomyelinase in health and disease. Biological Chemistry. 396 (6-7), 707-736 (2015).
  6. Versteeg, H. H., Ruf, W. Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity. Journal of Biological Chemistry. 282 (35), 25416-25424 (2007).
  7. Reinhardt, C., et al. Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation. Journal of Clinical Investigation. 118 (3), 1110-1122 (2008).
  8. Langer, F., et al. Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase. Blood. 121 (12), 2324-2335 (2013).
  9. Bogdanov, V. Y., et al. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein. Nature Medicine. 9 (4), 458-462 (2003).
  10. Mackman, N. Alternatively spliced tissue factor - one cut too many. Thrombosis and Haemostasis. 97 (1), 5-8 (2007).
  11. Censarek, P., Bobbe, A., Grandoch, M., Schror, K., Weber, A. A. Alternatively spliced human tissue factor (asHTF) is not pro-coagulant. Thrombosis and Haemostasis. 97 (1), 11-14 (2007).
  12. Gyorgy, B., et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cellular and Molecular Life Sciences. 68 (16), 2667-2688 (2011).
  13. Osterud, B., Bjorklid, E. Tissue factor in blood cells and endothelial cells. Frontiers in Bioscience (Elite Edition). 4 (1), 289-299 (2012).
  14. Hisada, Y., Mackman, N. Cancer cell-derived tissue factor-positive extracellular vesicles: biomarkers of thrombosis and survival. Current Opinion in Hematology. 26 (5), 349-356 (2019).
  15. Vatsyayan, R., Kothari, H., Pendurthi, U. R., Rao, L. V. 4-Hydroxy-2-nonenal enhances tissue factor activity in human monocytic cells via p38 mitogen-activated protein kinase activation-dependent phosphatidylserine exposure. Arteriosclerosis, Thrombosis, and Vascular Biology. 33 (7), 1601-1611 (2013).
  16. Mackman, N., Sachetto, A. T. A., Hisada, Y. Measurement of tissue factor-positive extracellular vesicles in plasma: strengths and weaknesses of current methods. Current Opinion in Hematology. 29 (5), 266-274 (2022).
  17. Lee, R. D., et al. Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity. Thrombosis Research. 129 (1), 80-85 (2012).
  18. Claussen, C., et al. Microvesicle-associated tissue factor procoagulant activity for the preoperative diagnosis of ovarian cancer. Thrombosis Research. 141, 39-48 (2016).
  19. Mackman, N., Hisada, Y., Archibald, S. J., et al. Tissue factor and its procoagulant activity on cancer-associated thromboembolism in pancreatic cancer: Comment by Mackman et al. Cancer Science. 113 (5), 1885-1887 (2022).
  20. Sachetto, A. T. A., et al. Evaluation of four commercial ELISAs to measure tissue factor in human plasma. Research and Practice in Thrombosis and Haemostasis. 7 (3), 100133 (2023).
  21. Archibald, S. J., Hisada, Y., Bae-Jump, V. L., Mackman, N. Evaluation of a new bead-based assay to measure levels of human tissue factor antigen in extracellular vesicles in plasma. Research and Practice in Thrombosis and Haemostasis. 6 (2), e12677 (2022).
  22. Tatsumi, K., et al. Evaluation of a new commercial assay to measure microparticle tissue factor activity in plasma: communication from the SSC of the ISTH. Journal of Thrombosis and Haemostasis. 12 (11), 1932-1934 (2014).
  23. Hisada, Y., et al. Measurement of microparticle tissue factor activity in clinical samples: A summary of two tissue factor-dependent FXa generation assays. Thrombosis Research. 139, 90-97 (2016).
  24. Tatsumi, K., Hisada, Y., Connolly, A. F., Buranda, T., Mackman, N. Patients with severe orthohantavirus cardiopulmonary syndrome due to Sin Nombre Virus infection have increased circulating extracellular vesicle tissue factor and an activated coagulation system. Thrombosis Research. 179, 31-33 (2019).
  25. Schmedes, C. M., et al. Circulating Extracellular Vesicle Tissue Factor Activity During Orthohantavirus Infection Is Associated With Intravascular Coagulation. Journal of Infectious Diseases. 222 (8), 1392-1399 (2020).
  26. Rosell, A., et al. Patients with COVID-19 have elevated levels of circulating extracellular vesicle tissue factor activity that is associated with severity and mortality-Brief report. Arteriosclerosis, Thrombosis, and Vascular Biology. 41 (2), 878-882 (2021).
  27. Campbell, R. A., et al. Comparison of the coagulopathies associated with COVID-19 and sepsis. Research and Practice in Thrombosis and Haemostasis. 5 (4), e12525 (2021).
  28. Guervilly, C., et al. Dissemination of extreme levels of extracellular vesicles: tissue factor activity in patients with severe COVID-19. Blood Advances. 5 (3), 628-634 (2021).
  29. Hisada, Y., Mackman, N. Measurement of tissue factor activity in extracellular vesicles from human plasma samples. Research and Practice in Thrombosis and Haemostasis. 3 (1), 44-48 (2019).
  30. Sachetto, A. T. A., et al. Tissue factor activity of small and large extracellular vesicles in different diseases. Research and Practice in Thrombosis and Haemostasis. 7 (3), 100124 (2023).
  31. Bom, V. J., Bertina, R. M. The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII. Biochemical Journal. 265 (2), 327-336 (1990).
  32. Tilley, R. E., Holscher, T., Belani, R., Nieva, J., Mackman, N. Tissue factor activity is increased in a combined platelet and microparticle sample from cancer patients. Thrombosis Research. 122 (5), 604-609 (2008).
  33. Gurung, S., Perocheau, D., Touramanidou, L., Baruteau, J. The exosome journey: from biogenesis to uptake and intracellular signalling. Cell Communication and Signaling. 19 (1), 47 (2021).
  34. Garnier, D., et al. Cancer cells induced to express mesenchymal phenotype release exosome-like extracellular vesicles carrying tissue factor. Journal of Biological Chemistry. 287 (52), 43565-43572 (2012).
  35. Park, J. A., et al. Tissue factor-bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo. Journal of Allergy and Clinical Immunology. 130 (6), 1375-1383 (2012).

Tags

Medicine
Extracellular Vesicle Tissue Factor Activity Assay
Play Video
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Hisada, Y., Mackman, N.… More

Hisada, Y., Mackman, N. Extracellular Vesicle Tissue Factor Activity Assay. J. Vis. Exp. (202), e65840, doi:10.3791/65840 (2023).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter