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

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
<ol>
	<li>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.</li>
	<li>Draw 2.7 or 1.8 mL (depending on the size of the tubes) of blood into a vacutainer containing 3...

Improved Sensitivity for Tissue Factor Activity Assay in Extracellular Vesicles

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

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL tube for 20,000 x g centrifuge</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Fisher Scientific (Catalog number: 05-408-129).</td>
		</tr>
		<tr>
			<td>1.5 mL tube for ultracentrifuge</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Beckman Coulter (Catalog number: 357448)</td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from VWR (Catalog number: 89039-666)</td>
		</tr>
		<tr>
			<td>21 G x .75 in. BD Vacutainer Safety-Lok Blood Collection Set with 12 in. tubing and luer adapter</td>
			<td>BD</td>
			<td>367281</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Globe Scientific (Catalog number: 120338).</td>
		</tr>
		<tr>
			<td>BD Vacutainer Citrate Tubes</td>
			<td>BD</td>
			<td>363083</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 1.5 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the Centrifuge 5417R (Eppendorf).</td>
		</tr>
		<tr>
			<td>Centrifuge for 15 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the Centrifuge 5810R (Eppendorf).</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>E6511</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FVIIa</td>
			<td>Enzyme Research Laboratory</td>
			<td>HFVIIa</td>
			<td>The solution should be diluted with HBSA-Ca(+).</td>
		</tr>
		<tr>
			<td>Human FX</td>
			<td>Enzyme Research Laboratory</td>
			<td>HFX1010</td>
			<td>The solution should be diluted with HBSA-Ca(+).</td>
		</tr>
		<tr>
			<td>Inhibitory mouse anti-human tissue factor IgG, clone HTF-1</td>
			<td>Fisher Scientific</td>
			<td>550252</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Escherichia coli O111:B4</td>
			<td>Sigma Aldrich</td>
			<td>L2630</td>
			<td>There are several lipopolysaccharide from different E. coli. Different lipopolysaccharide have different potential to activate monocytes.</td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Sigma Aldrich</td>
			<td>I5381</td>
			<td></td>
		</tr>
		<tr>
			<td>Pefachrome FXa 8595</td>
			<td>Enzyme Research Laboratory</td>
			<td>085-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the SpectraMax i3x from Molecular Devices</td>
		</tr>
		<tr>
			<td>Re-lipidated recombinant tissue factor, Dade Innovin</td>
			<td>Siemens</td>
			<td>10873566</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima TLX</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>TLA-55</td>
			<td></td>
		</tr>
	</tbody>
</table>

extracellular vesicle tissue factor activity assay

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.

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.

A successful result gives a positive control value of &#8805;0.5 pg/mL and a negative control value of &#60;0.5 pg/mL. It is best to find a high LPS responder with &#62;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...

Watch this Scientific Journal Video about High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis at JoVE.com

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 ...

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Research

JoVE Journal

Medicine

501 ビュー.  University of North Carolina at Chapel Hill. 低レベルの組織因子陽性細胞外小胞は、さまざまな疾患の患者の血漿中に存在します。しかし、ELISAやフローサイトメトリーなど、ほとんどの抗原ベースのアッセイには、これらの低レベルを検出する感度がありません。多くの論文で、血漿中の組織因子を検出するためにELISAまたはフローサイトメトリーが採用されています。私たちのプロトコルは、血漿中の組織...

ビデオ: 著者スポットライト:血漿診断のための高感度組織因子活性アッセイ 

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...

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measurement of factor v activity in human plasma using a microplate coagulation assay

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human...

extracellular vesicle tissue factor assay with blood plasma derived extracellular vesicles

Extracellular Vesicle Tissue Factor Assay with Blood Plasma Derived Extracellular Vesicles  

high sensitivity measurement of transcription factor-dna binding affinities by competitive titration using fluorescence microscopy

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery...

flow cytometry analysis of tissue factor expression in human platelets

Author Spotlight: Standardized Protocol for Measuring Platelet-Associated Tissue Factor via Whole Blood Flow Cytometry in Clinical Settings

This article outlines the protocol for quantifying the percentage of Tissue Factor (TF)-positive platelets using whole blood flow cytometry, assessing the protein: (1) intracellularly in resting conditions, and (2) on the cell surface, in both resting and activated conditions. Guidance is also provided for evaluating TF-positive platelets in platelet-rich...

Assessing Intracellular and Surface TF-Positive Platelets via Flow Cytometry

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corneal sensitivity testing procedure for ophthalmologic and optometric patients

 Author Spotlight: Establishing a Practical and Cost-Effective Protocol for Corneal Sensitivity Testing in Clinical Settings

Corneal sensitivity testing provides insight into the health of the corneal nerves and helps diagnose ocular surface diseases. We present a concise protocol to qualitatively assess corneal sensitivity that can be readily used by eye care providers across clinical settings.

Assessing the Health of the Corneal Nerves in Ocular Surface Diseases

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high sensitivity 5-hydroxymethylcytosine detection in balb/c brain tissue

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable...

the 4 mountains test: a short test of spatial memory with high sensitivity for the diagnosis of pre-dementia alzheimer's disease

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer&#039;s Disease

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease

quantifying the modulation of elastase enzyme activity through colorimetric analysis

Author Spotlight: Quantifying Activity of Natural Plant Extracts for Developing Efficient Bioactive Molecules

This protocol describes the development of a colorimetric assay method for determining the ability of compounds to inhibit or activate elastase...

Measuring Elastase Modulation: A Colorimetric Assay for Therapeutic Insights

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non-equilibrium microwave plasma for efficient high temperature chemistry

Non-equilibrium Microwave Plasma for Efficient High Temperature Chemistry

This article describes a flowing microwave reactor that is used to drive efficient non-equilibrium chemistry for the application of conversion/activation of stable molecules such as CO2, N2 and CH4. The goal of the procedure described here is to measure the in situ gas temperature and gas conversion.

high-speed video microscopy analysis for first-line diagnosis of primary ciliary dyskinesia

High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia

High-speed video microscopy analysis is a relatively easy-to-perform, fast, cost-effective, and, in experienced hands, a considerably reliable tool for first-line diagnostics of primary ciliary dyskinesia, which should be available in every center involved in diagnostics and the treatment of severe lung...

citrate synthase activity assay: a colorimetric assay to measure mitochondrial citrate synthase activity in drosophila tissue homogenate

This video describes a colorimetric assay to measure the mitochondrial citrate synthase activity assay present in the tissue homogenate of...

Citrate Synthase Activity Assay: A Colorimetric Assay to Measure Mitochondrial Citrate Synthase Activity in Drosophila Tissue Homogenate

Aliquot 10 microliters of each homogenized sample into a new tube for protein content measurement, keeping these tubes on ice as well. Add 400 microliters of ice-cold extraction buffer to each remaining sample for a total volume of 500 microliters, and pipette up and down gently to...

examination of oral candida infection in primary sj&#246;gren&#39;s syndrome patients

Author Spotlight: Oral &lt;i&gt;Candida&lt;/i&gt; Diagnosis to Advance Clinical Treatment Regimen for pSS Patients

Here, we present a protocol for the examination of oral Candida infection in patients with primary Sj&#246;gren's syndrome, which can be used for timely treatment and, thereafter, avoiding related complications.

Diagnosis and Strain Identification of pSS-Related Oral &lt;i&gt;Candida&lt;/i&gt;

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basophil activation test for allergy diagnosis

Basophil Activation Test for Allergy Diagnosis

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

fabrication of silica ultra high quality factor microresonators

Fabrication of Silica Ultra High Quality Factor Microresonators

We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids.  The reflow method removes surface imperfections, allowing long photon lifetimes within both devices.  The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to...

real-time, two-color stimulated raman scattering imaging of mouse brain for tissue diagnosis

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue...

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic...

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.