Abstract
Background
Levels of tissue factor (TF)-positive extracellular vesicles (EVs) are increased in plasma from patients with different diseases. However, the low TF levels in plasma are difficult to measure due to the presence of TF pathway inhibitor and the low sensitivity of assays.
Objectives
We evaluated the ability of 3 commercial kits to measure TF activity levels in human plasma.
Methods
We used the Abcam, AssaySense, and Actichrome colorimetric assay kits to measure recombinant TF (Innovin) and procoagulant activity (PCA) in plasma from lipopolysaccharide (LPS)-stimulated human whole blood from 4 healthy donors. We used different anticoagulants and plasma preparations according to the recommendations of the different kits. We examined the effects of depleting EVs and adding an anti-TF antibody on PCA. The Chapel Hill EV TF activity assay was used for comparison.
Results
All 3 kits detected Innovin. The commercial kits detected higher PCA levels in some LPS treated samples but not others compared to controls Higher PCA levels were detected with the Abcam and AssaySense kits compared with the Actichrome kit. Depletion of EVs abolished PCA in both control and LPS plasmas. The Abcam and AssaySense kits detected TF-dependent PCA in LPS samples, but with lower sensitivity compared with the Chapel Hill assay.
Conclusion
In this study, we found that the Abcam and AssaySense kits detected TF-dependent PCA in human plasma. However, these assays had lower sensitivity and specificity compared with the Chapel Hill EV TF activity assay.
Keywords: coagulation, extracellular vesicles, functional assay, plasma, tissue factor
Graphical abstract
Essentials
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Low TF levels are present in the blood in different diseases, but they are difficult to measure.
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We evaluated the ability of 3 commercial kits to measure procoagulant activity in human plasma.
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Two kits were able to measure TF activity in the plasma of a positive control.
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These kits are less sensitive and specific than the extracellular vesicle TF activity assay.
1. Introduction
Tissue factor (TF) is the receptor and cofactor for factor (F)VIIa [1]. The TF-FVIIa complex activates the coagulation protease cascade. Lipopolysaccharide (LPS) stimulation of human monocytes induces TF expression and the release of TF-positive extracellular vesicles (EVs) [2]. In addition, EV TF activity levels are increased in plasma from patients with several diseases, including sepsis, cancer, and viral infection [3]. However, the measurement of TF in plasma is challenging due to its low amount, the presence of coagulation factors and TF pathway inhibitor, and the low sensitivity of assays [4].
Studies have shown that assays measuring TF activity in EVs are superior to those measuring TF antigen, as they have greater sensitivity and specificity [[5], [6], [7]]. Another observation was that assays that use an anti-TF inhibitory antibody to distinguish TF-dependent from TF-independent activities have a higher specificity for measuring TF compared with assays that do not use an anti-TF antibody [6]. We and others have previously analyzed the ability of commercial assays to measure EV TF activity in plasma; we found that 2 assays (ZYMUPHEN MP-TF, Aniara Diagnostics, and CY-QUANT MV-TF Activity, Stago) showed lower sensitivity and specificity compared with our in-house Chapel Hill EV TF activity assay [6,8]. Although the Chapel Hill EV TF activity assay has high sensitivity and specificity for detecting TF, the isolation of EVs by centrifugation is time-consuming.
Commercially available colorimetric assay kits for the measurement of TF use plasma and, therefore, are simpler and faster than assays that isolate EVs. However, they do not use an anti-TF inhibitory antibody. Bogdanov et al. [9] analyzed one of these kits (Actichrome) and found several limitations to its use, including the lack of specificity of the assay and that the results can be influenced by the color of the plasma.
A good assay for the measurement of plasma TF activity should meet the following criteria: have a signal that is higher than the background of the plasma, distinguish positive from negative controls (CTs), demonstrate a loss of signal with the removal of EVs, and demonstrate reduced signal in the presence of an inhibitory anti-TF antibody.
To date, commercial plasma TF activity kits have not been systematically evaluated for TF measurement. We used the Chapel Hill EV TF activity assay, which uses isolated EVs, for comparison. We evaluated the ability of 3 commercial kits to measure recombinant, purified human TF (Innovin), total procoagulant activity, and TF activity in plasma samples from LPS-stimulated and unstimulated human whole blood from healthy donors.
2. Methods
2.1. Innovin
We used 3 kits to measure Innovin (Fisher Scientific, catalog [cat.] #10873566). Innovin was diluted to 1.69, 3.38, 6.76, and 13.5 pM in the sample diluent from the kits. We determined the concentration of TF in Innovin by titrating varying concentrations of FVIIa into a fixed concentration of Innovin. We plotted the rate of substrate cleavage against FVIIa concentration. The substrate curve shows an inflection point at which the rate of substrate cleavage stops due to every FVIIa molecule being bound to a TF molecule. The TF concentration can then be determined from the inflection point.
2.2. Preparation of TF-negative and -positive plasma samples
Whole blood was collected from 4 healthy volunteers who gave written consent, in accordance with a protocol approved by the Institutional Review Board of the University of North Carolina at Chapel Hill. Blood was collected from the antecubital vein into either sodium citrate tubes (BD Bioscience, cat. #366560) or EDTA tubes (BD Bioscience, cat. #367856) using a 21 G Safety-Lok Blood Collection Set (BD Bioscience, cat. #367281). The first 3 mL was discarded to prevent contamination of the blood with TF from the vessel wall. For TF-negative (CT) samples, blood was processed immediately. For TF-positive (LPS) samples, blood was stimulated with bacterial LPS (Escherichia coli serotype O128:B12, Sigma-Aldrich, cat. #L2887, 10 μg/mL) for 6 hours at 37 °C. Platelet-poor plasma (PPP) was prepared from blood by centrifugation at 1500 × g for 15 minutes at room temperature [10]. Platelet-free plasma (PFP) was prepared from blood by centrifugation twice at 2500 × g for 15 minutes at room temperature [11]. Samples were stored immediately at −80 °C until further analysis.
2.3. Chapel Hill EV TF activity assay
EVs were isolated from 100 μL of plasma diluted in 1 mL of HEPES-buffered saline containing bovine serum albumin (HBSA; 137 mmol/L NaCl, 5.38 mmol/L KCl, 5.55 mmol/L glucose, 10 mmol/L HEPES, and 0.1% bovine serum albumin, pH 7.4) by centrifugation at 20,000 × g for 60 minutes at 4 °C. The EV pellet was washed once with 1 mL of HBSA and centrifuged again at 20,000 × g for 60 minutes at 4 °C. The EV pellet was resuspended in 100 μL of HBSA and used for the assay. The assay was performed as described previously [12]. Briefly, EV samples were added to a 96-well microplate and incubated with an anti-TF inhibitory antibody (HTF-1, BD Biosciences, cat. #550252) or a CT immunoglobulin (Ig) G antibody (Sigma-Aldrich, cat. #15381). Next, a mixture of FVIIa, FX, and calcium was added, and the samples were incubated for 2 hours at 37 °C. The reaction was stopped by the addition of HBSA-EDTA. After incubation with a FXa chromogenic substrate (Pefachrome FXa 8595, Pentapharm, cat. #085-27) for 15 minutes at 37 °C, the absorbance was measured at 405 nm. Innovin was used to produce a standard curve (0.008-0.54 pM). TF-dependent procoagulant activity (PCA) was determined by subtracting the PCA in the presence of an anti-TF antibody (TF-independent PCA) from that in the presence of IgG CT (total PCA). Samples were measured in duplicate. PCA was expressed as picomolar (pM).
2.4. Commercial TF activity kits
We tested 3 commercial TF activity kits: Tissue Factor Activity Assay Kit (Abcam, cat. #ab108906, lot #1113742-12); AssaySense, Human Tissue Factor Chromogenic Activity Assay Kit (AssayPro, cat. #CT1002b, lot #07622212R1); Actichrome TF Assay (BioMedica Diagnostics, cat. #846, lot #250121). Table 1 summarizes the details of each kit, including recommendations for anticoagulant, plasma preparation, and incubation times before and after the addition of the chromogenic substrate. It is notable that the Abcam and AssaySense kits are almost identical. The TF activity kits were used according to the manufacturer’s instructions. For the Abcam kit, the optimal time frame was determined as the time at which a steady increase in optical density (OD) was observed in the standards; we used 5 minutes. We then determined the ΔOD/min and calculated the PCA of the samples using the standard curve. For the AssaySense kit, we used the same method as the Abcam assay. For the Actichrome kit, the standards and samples were incubated for 30 minutes at 37 °C, and absorbance was measured at 405 nm and 490 nm. The ΔOD405 to 490 was used to calculate the PCA of the sample. The standards provided in each kit were used as calibrators. Samples were measured in duplicate. The data are presented as PCA (total FXa generation) rather than TF activity because the assays do not distinguish TF-dependent from TF-independent FXa generation.
Table 1.
Detailed information about the commercial tissue factor activity kits.
| Abcam | AssaySense | Actichrome | |
|---|---|---|---|
| Anticoagulant | EDTA or heparin | EDTA or heparin | Sodium citrate |
| Standard curve (pM), range | 7.8-250 | 7.8-250 | 1.88-30 |
| Detection limit, pM | 3.5 | 3.5 | 2 |
| Volume of plasma, μL | 10 | 10 | 25 (+50 of assay buffer) |
| Reaction mix, μL | 70 of FVII + FX | 70 of FVII + FX | 50 of FVIIa + FX |
| Incubation (37 °C), min | 30 | 30 | 15 |
| FXa chromogenic substrate, μL | 20 | 20 | 25 |
| Incubation prior to first read (37 °C), min | 5 | - | 30 |
| Stop reaction, μL | 50 of glacial acetic acid | ||
| First read, nm | 405 | 405 | |
| Incubation (37 °C), min | Up to 30 | Up to 35 | |
| Read | Every 5 min (405 nm) | Every 5 min (405 nm) | Endpoint (405 nm and 490 nm) |
| Analysis | ΔOD/min | ΔOD/min or 405 nm OD | ΔOD405-490 |
FVII, factor VII; FVIIa, activated factor VII; FX, factor X; FXa, activated factor X; OD, optical density.
2.5. Measurement of the absorbance spectrum of plasma samples
Plasma samples were diluted at the same ratios as used for the assays, using the respective assay buffer from each kit. Chromogenic substrate was not added to the samples. The absorbance spectrum of the samples was measured from 300 to 690 nm. Representative plots were constructed using citrated PFP.
2.6. Depletion of EVs
We previously showed that the majority of TF-positive EVs can be removed from plasma by centrifugation at 20,000 × g for 60 minutes [13]. Therefore, plasma samples (100 μL) were centrifuged at 20,000 × g for 60 minutes at 4 °C to remove the majority of TF-positive EVs. The supernatant was collected and considered EV-depleted plasma.
2.7. Determination of TF-dependent and TF-independent PCA
We determined the amounts of TF-dependent and TF-independent PCA by incubating plasma samples with the anti-TF inhibitory monoclonal antibody HTF-1 [14] or a CT IgG antibody (7.84 μg/mL final concentration) for 15 minutes at room temperature, followed by the addition of the reaction mix containing FVII/FVIIa and FX. TF-dependent PCA was determined by subtracting the TF-independent PCA from the total PCA.
2.8. Statistical analysis
Results are presented as median (IQR) or as individual values. Normal distribution of the results was assessed using the Shapiro–Wilk test. Data transformation was applied when necessary. We used a paired t-test to compare values between groups. Data were considered statistically significant when P < .05. Data were analyzed with Prism version 9.4 (GraphPad Software).
3. Results
3.1. Measurement of Innovin using 3 commercial kits
We determined the ability of 3 commercial TF activity kits (Abcam, AssaySense, and Actichrome) to detect different concentrations (1.69-13.5 pM) of Innovin. All 3 assays detected Innovin (Figure 1). However, compared with the standards diluted in each kit, the assays detected higher amounts of Innovin than expected: the means across the different Innovin concentrations were 8.6-, 8.5-, and 2.8-fold for Abcam, AssaySense, and Actichrome, respectively. These differences indicate that all the commercial kits yielded higher PCA values compared with the Chapel Hill assay due to the use of a different TF standard.
Figure 1.
Measurement of recombinant tissue factor (Innovin) using 3 commercial tissue factor activity assays. Different amounts of Innovin (1.69-13.5 pM) were measured using the Abcam, AssaySense, and Actichrome kits.
3.2. Measurement of the background absorption of the different citrated PFPs
A previous study compared the absorbance of buffer, bovine serum albumin, and plasma over 308 to 700 nm [15]. None of the samples was visibly lipemic or showed evidence of obvious hemolysis. Interestingly, the absorbance of both LPS and CT samples decreased with increasing wavelength, but plasma showed an increase in absorbance at ∼410 nm. Therefore, we measured the absorbance of the various plasmas diluted in the buffers from the different kits between 300 nm and 690 nm. We observed substantial variation across the different plasmas (Figure 2). In addition, we observed an increase in absorbance at 405 nm, particularly in LPS samples.
Figure 2.
Measurement of the wavelength spectrum of plasma. Whole blood was collected from 4 healthy donors using sodium citrate. Platelet-free plasma (PFP) was prepared from whole blood with lipopolysaccharide (LPS) or without LPS (control [CT]) stimulation. Plasma was diluted using assay buffers for (A) the Abcam kit, (B) the AssaySense kit, and (C) the Actichrome kit. The wavelength of the plasmas was measured between 300 and 690 nm. OD, optical density.
3.3. Measurement of plasma PCA using 3 commercial kits
LPS induces TF expression in monocytes, which subsequently release TF-positive EVs [10].
We use sodium citrate as an anticoagulant and PFP for plasma preparation in the Chapel Hill assay [10]. However, all the commercial TF activity kits recommend PPP rather than PFP, and the Abcam and AssaySense kits recommend EDTA or heparin as anticoagulants. Therefore, we prepared PPP and PFP from blood collected into either sodium citrate or EDTA from 4 healthy donors, and isolated EVs. First, we isolated EVs from the different plasmas and measured PCA using the Chapel Hill assay. As expected, the median PCA of EVs isolated from plasma of LPS-stimulated whole blood was significantly higher than that of EVs isolated from the respective negative CTs in all conditions analyzed (Figure 3A). EVs isolated from PPP samples had higher PCA levels than those isolated from PFP.
Figure 3.
Measurement of extracellular vesicle procoagulant activity using the Chapel Hill assay and plasma procoagulant activity using commercial tissue factor activity assays. Whole blood was collected from 4 healthy donors using either sodium citrate or EDTA as anticoagulants. Platelet-poor plasma (PPP) and platelet-free plasma (PFP) were prepared from whole blood with lipopolysaccharide (LPS) or without LPS (CT) stimulation. For the Chapel Hill assay, extracellular vesicles were isolated from plasma by centrifugation at 20,000 × g for 60 minutes at 4 °C. Procoagulant activity of the samples was measured using the (A) Chapel Hill assay, (B) Abcam kit, (C) AssaySense kit, and (D) Actichrome kit. Data are shown as median with IQR and individual values. A paired t-test was used to compare values between groups. ∗P < .05; ∗∗P < .01.
Next, we determined the ability of the 3 commercial kits to measure PCA in the different plasmas. The commercial kits detected higher PCA levels in some LPS samples but not in others compared with CTs (Figure 3B–D). The Abcam and AssaySense kits detected higher PCA levels in the LPS samples compared with the Actichrome kit. However, this is partly due to the use of different standards in the various kits. For the Actichrome kit, all LPS samples had higher PCA levels than CTs (Figure 3D). However, all LPS samples, except 1, had values below the detection limit of the assay (2 pM). Therefore, we did not continue the analysis with the Actichrome kit. We observed that the samples clotted with the Abcam and AssaySense kits, but not with the Actichrome kit.
3.4. Effect of depleting EVs on PCA measured using 2 commercial kits
The majority of procoagulant TF in plasma is present in EVs [16,17]. Therefore, we compared the total PCA of plasma without EV removal with that of EV-depleted plasma. We used citrated PFP samples because they provided better discrimination in PCA between LPS and CT samples than EDTA PFP. Depletion of EVs reduced the PCA of CT and LPS samples, as measured using the Abcam and AssaySense kits (Figure 4A, B). This indicates that EVs are required for detecting PCA in plasma using these kits. We also observed higher PCA levels in CT samples using the AssaySense kit than with the Abcam assay (Figure 4A, B).
Figure 4.
Measurement of procoagulant activity in plasma with or without extracellular vesicle (EV) depletion using commercial tissue factor activity assays. Whole blood was collected from 4 healthy donors using sodium citrate. Platelet-free plasma was prepared from whole blood with lipopolysaccharide (LPS) or without LPS (CT) stimulation. EVs were depleted from the plasma by centrifugation at 20,000 × g for 60 minutes at 4 °C. Procoagulant activity of the samples was measured using the (A) Abcam kit and (B) AssaySense kit. Data are shown as median with IQR and individual values.
3.5. Measurement of TF-dependent activity using 2 commercial kits
The addition of an anti-TF inhibitory antibody (HTF-1) allows one to differentiate between TF-dependent and TF-independent PCA. The Chapel Hill EV TF activity assay was used to determine EV TF activity levels in CT and LPS plasma samples from 4 donors. As expected from previous studies showing that LPS induction of TF expression in monocytes varies from donor to donor [10,18], the EV TF activity of citrated plasma samples prepared from LPS-treated whole blood was variable among the 4 donors, ranging from 0.015 to 0.053 pM (Figure 5A). This variation gives the impression of a large difference in the assay when data from different donors are combined. However, we have shown that the coefficient of variation across repeated runs of the same sample for the Chapel Hill assay is around 12% to 30% [6,10]. TF-dependent, but not TF-independent, PCA was significantly higher in LPS samples than in CT samples. We calculated the fold increase in LPS samples for each donor relative to the CT median. The mean fold increase in LPS samples was 33-fold. TF-dependent activity accounted for 97.0% of the total PCA, indicating the high specificity of the assay.
Figure 5.
Measurement of tissue factor (TF)-dependent extracellular vesicle procoagulant activity using the Chapel Hill assay and plasma procoagulant activity using commercial TF activity assays. Whole blood was collected from 4 healthy donors using sodium citrate. Platelet-free plasma was prepared from whole blood with lipopolysaccharide (LPS) or without LPS (CT) stimulation. For the Chapel Hill assay, extracellular vesicles were isolated from plasma by centrifugation at 20,000 × g for 60 minutes at 4 °C. TF-dependent and TF-independent procoagulant activity were measured using the (A) Chapel Hill assay, (B) Abcam kit, and (C) AssaySense kit. Data are shown as individual values.
For the Abcam kit, we observed higher levels of TF-dependent PCA, but not TF-independent PCA, in LPS samples from all 4 healthy donors compared with CT samples (Figure 5B). The mean fold increase in LPS samples was 11-fold. The TF-dependent PCA accounted for an average of 91% of the total PCA in the LPS samples. For the AssaySense kit, higher levels of TF-dependent PCA, but not TF-independent PCA, were observed in LPS samples from all 4 healthy donors compared with CT samples (Figure 5C). The mean fold increase in LPS samples was 5-fold. The AssaySense kit had lower specificity than the Abcam kit, with TF-dependent PCA accounting for 36% of the total PCA. We observed much higher levels of TF-independent PCA in the CT samples using the AssaySense kit compared with the Abcam kit (Figure 5B, C).
4. Discussion
In this study, we evaluated the ability of 3 commercial TF activity kits to measure Innovin and TF present in plasma from LPS-stimulated human whole blood.
The 3 kits detected Innovin in a concentration-dependent manner. However, the values detected by each kit varied widely. This indicates that each kit uses a different form of TF with a distinct specific activity to generate the standard curves. Therefore, the values of each kit cannot be compared. It is also notable that the standard curves for the Abcam and AssaySense kits use a higher range of the standard (7.8-250 pM) than those for the Actichrome kit (1.88-30 pM). We also observed some day-to-day variations in the standard curves for the Abcam and AssaySense kits, which affected the PCA values for a given sample. This may be due to the lower ODs of the standards for the Abcam and AssaySense kits (0.3-0.4 for the highest standard) compared with the Chapel Hill assay (1.2-1-4 for the highest standard).
The PCA of PPP and PFP, measured with the Abcam and AssaySense kits, was higher in samples using citrate than in those using EDTA. This may be due to the higher calcium-chelating activity of EDTA compared with citrate. We also observed higher PCA levels in PPP compared with PFP. Similar results were observed with isolated EVs. We and others have shown higher EV TF activity levels in PPP than in PFP [10,19]. This is likely due to higher phospholipid levels and larger EVs in PPP than in PFP.
Part of the difficulty in measuring substrate cleavage in plasma is that plasma has a significant absorbance at 405 nm, leading to a high background [15]. This background is further worsened by hemolysis of the sample, leading to free hemoglobin that shows a peak at 405 nm [20]. One study noted that the color of plasma could affect the Actichrome kit [9]. We observed that all plasmas had background levels due to the color of the plasma, which varied between individual donors. Moreover, the LPS samples showed higher background levels and a higher peak at 405 nm compared with the CT samples. This may be due to some hemolysis occurring during the 6-hour incubation of the LPS samples.
If the assay analysis does not account for plasma absorbance alone, background absorbance will be included as TF activity. The standards for each kit are in buffer and do not have this high background absorbance. Analysis of the assay results could incorporate a zero-time reading to remove the background. Absorbance for the Actichrome kit substrates was measured at 490 nm in an attempt to remove the background. However, we recommend running a sample without a chromogenic substrate to determine the background. Alternatively, analysis could monitor the change in absorbance due to substrate cleavage kinetically and use the rate of change as the readout. This is the method we used for the Abcam and AssaySense kits.
The Actichrome kit has been used to measure PCA in human plasma since 2007 (Table 2) [[21], [22], [23], [24], [25], [26]]. Studies have reported that healthy individuals have PCA levels of 1.8 to <2 pM using the Actichrome kit (Table 2) [22,23]. These values are similar to our results for healthy donors and are below or close to the detection limit of the assay (2 pM). Other studies have reported higher PCA levels (11.9-55 pM) in different diseases using this kit (Table 2). Although we observed higher PCA levels in LPS samples than in CT samples across all plasma conditions, except citrated PPP, all values, except 1, were below the detection limit of the assay (2 pM). Bogdanov et al. [9] showed that the signal detected by the Actichrome kit in plasma samples with an activity <3 pM could not be inhibited by the use of active site-inactivated FVIIa. We do not recommend using this assay to measure PCA in plasma.
Table 2.
Studies measuring procoagulant activity in plasma using commercial assays.
| Assay | Samples | Disease | Results | Ref. |
|---|---|---|---|---|
| Actichrome | Carotid atherosclerosis | Nondiabetic: 13.3 pM Diabetic: 13.9 pM |
2007 [21] | |
| Actichrome | PPP Sodium citrate |
CAD | PCA is higher in one cohort of CAD and lower in another compared with healthy controls | 2009 [9] |
| Actichrome | PPP Sodium citrate |
HIV | Healthy: <2 pM HIV patients: 55 pM |
2010 [22] |
| Actichrome | PFP Sodium citrate |
Cancer | Controls: 1.8 pM Cancer patients: 3.2 pM |
2013 [23] |
| Actichrome | Obesity and diabetes | Obese nondiabetic subjects: 0.5 pM Obese diabetic subjects: 1.4 pM |
2015 [24] | |
| Actichrome | PPP Sodium citrate |
AMI | STEMI had 20% higher PCA than non-STEMI | 2017 [25] |
| Actichrome | AVR | AVR only: 14.2 pM AVR with drainage: 11.9 pM |
2019 [26] | |
| Abcam | PPP Sodium citrate |
Chronic thromboembolic pulmonary hypertension | Controls: 20 pM Pulmonary thromboembolism: ∼27 pM |
2016 [27] |
| Abcam | PPP Heparin |
Type 1 diabetes | After a low-fat meal: 132 pM After a high-fat meal: 124 pM |
2017 [28] |
| Abcam | COVID-19 | Healthy controls: 135 pM COVID-19 (nonsevere): 92 pM COVID-19 (severe): 82 pM |
2022 [29] | |
| Abcam | PPP Sodium citrate |
Post-AMI | Healthy: ∼30 pM Post-AMI: ∼20 pM |
2022 [30] |
| Abcam | COVID-19 | rNAPc2 treatment: 335.2 pM Heparin treatment: 355.6 pM |
2023 [31] | |
| AssaySense | PFP Sodium citrate |
DVT | Healthy: ∼45 pM Initial DVT: ∼95 pM Recurrent DVT: ∼100 pM |
2012 [32] |
AMI, acute myocardial infarction; AVR, aortic valve replacement; CAD, coronary artery disease; DVT, deep vein thrombosis; PCA, procoagulant activity; PFP, platelet-free plasma; PPP, platelet-poor plasma; rNAPc2, recombinant nematode anticoagulant protein c2; STEMI, ST-elevation myocardial infarction.
There is a wide range of PCA values (20-135 pM) measured in healthy individuals using the Abcam kit (Table 2) [27,29,30]. We detected PCA in citrated PPP (4-37 pM) and citrated PFP (0-16 pM). Other studies have reported higher PCA levels (27-355 pM) in different diseases using this kit (Table 2) [[27], [28], [29], [30], [31]]. One study [29] used an anti-TF inhibitory antibody (HTF-1) in combination with the Abcam kit to measure TF-specific PCA and reported that patients with severe COVID-19 had significantly higher TF activity than healthy CTs (∼0.6 pM vs ∼0.1 pM). However, total PCA levels were considerably higher in healthy donors (∼75-200 pM) than in severe COVID-19 patients (∼50-150 pM). This suggests that the apparent TF-specific activity accounts for less than 1% of the total PCA. Our study indicates that the Abcam kit can measure TF activity in plasma, but it is possible that some of the increase in PCA across different diseases may be independent of TF.
Only 1 study has used the AssaySense kit to measure PCA in plasma (Table 2) [32]. The PCA in healthy individuals (∼45 pM) is similar to our results (0-27 pM). Patients with deep vein thrombosis had higher plasma PCA levels compared with CTs, as measured with this kit. We found that the kit had a high background. However, the kit could detect TF-dependent PCA in LPS samples.
It is notable that plasma clotted with the Abcam and AssaySense kits, but not with the Actichrome kit. This raises concerns about how plasma clotting affects the results.
5. Conclusion
In summary, commercial TF activity assays are attractive to researchers for their practicality. However, it is important to consider their limitations. To ensure rigor and reproducibility in science, it is necessary to test and validate the reagents and assays used, particularly for commercial kits. To measure TF activity in plasma, an anti-TF inhibitory antibody can be used to distinguish TF-dependent from TF-independent PCA. Kinetic readings, rather than endpoint readings, may reduce the contribution of background. The Abcam and AssaySense kits were able to measure TF activity in LPS samples. Although we found that these kits detected TF-dependent PCA in citrated PFP from LPS-stimulated blood, the increase compared with CT samples was considerably lower than that observed with the Chapel Hill EV TF activity assay. The fold increases in LPS samples compared with CT samples for the Chapel Hill assay, Abcam kit, and AssaySense kit were 33-, 11-, and 5-fold, respectively. In addition, the kits had lower specificity compared with the Chapel Hill assay, which uses EVs isolated from plasma.
Note added in proof: A recent study [33] analyzed PCA in samples from patients with active cancer and acute ischemic stroke using the AssaySense kit. The median of PCA of the patients was 32.0 pM. The study showed that patients with low levels of PCA (<32 pM) had a higher survival probability compared with patients with high levels of PCA (>32 pM).
Acknowledgments
We would like to thank Dr Yohei Hisada for helpful comments.
Funding
This work was supported by the National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) R35HL155657 (N.M.) and the John C. Parker professorship (N.M.). A.T.A.S. is an American Heart Association postdoctoral fellow (#24POST1200989).
Author contributions
A.T.A.S. and N.M. designed experiments, interpreted data, and edited the manuscript. A.T.A.S. conducted experiments, analyzed data, and wrote the manuscript. D.M.M. interpreted data and edited the manuscript. All the authors read and approved the final manuscript.
Relationship Disclosure
There are no competing interests to disclose.
Footnotes
Handling Editor: Dr Robert A. Campbell
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