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 oth....

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.......

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