A sensitive tissue factor activity assay determined by an optimized thrombin generation method

Søren Risom Kristensen; Jette Nybo

doi:10.1371/journal.pone.0288918

. 2023 Jul 19;18(7):e0288918. doi: 10.1371/journal.pone.0288918

A sensitive tissue factor activity assay determined by an optimized thrombin generation method

Søren Risom Kristensen ^1,^2,^*, Jette Nybo ¹

Editor: Heather Faith Pidcoke³

PMCID: PMC10355404 PMID: 37467256

Abstract

Background

Tissue factor (TF) is the principal activator of the coagulation system, but an increased concentration in the blood in cancer and inflammatory diseases has been suggested to play a role increasing the risk of venous thromboembolism. However, measurement of the TF concentration is difficult, and quantitation of activity is the most valid estimation. The objective of this study was to establish a sensitive method to measure TF activity based on thrombin generation.

Methods

The assay is based on thrombin generation (TG) measured on the Calibrated Automated Thrombogram (CAT). Various low concentrations of TF were prepared from reagents containing 1 pM TF and 4 μM phospholipid (PPL), and no TF and 4 μM PPL, and a calibration curve was produced from Lagtime vs TF concentration. TF in blood samples was measured after isolation and resuspension of extracellular vesicles (EVs) in a standard plasma from which EVs had been removed. The same standard plasma was used for the calibrators.

Results

Contact activation of the coagulation system was avoided using CTI plasma samples in Monovette tubes. EVs contain procoagulant phospholipids but addition of PPL only reduced lagtime slightly at very low concentrations of TF resulting in overestimation to a lesser extent at 10 fM but no interference at 30 fM or higher. Addition of EVs to the TG analysis induced a small unspecific TF-independent activity (i.e., an activity not inhibited by antibodies against TF) which also may result in a smaller error in estimation of TF activity at very low levels but the effect was negligible at higher concentrations. It was possible to measure TF activity in healthy controls which was found to be 1–6 fM (EVs were concentrated, i.e. solubilized in a lower volume than the original volume plasma). Coefficient of variation (CV) was below 20% at the low level, and below 10% at a level around 100 fM TF. However, the step with isolation of EVs have a higher inherent CV.

Conclusion

A sensitive and rather precise one-stage TG-based method to measure TF activity has been established.

Introduction

Tissue factor (TF) is the primary coagulation factor that initiates activation of the coagulation system which leads to fibrin formation [1]. This is crucial to avoid bleeding after traumas, but an increased formation has also been ascribed a role in the increased risk of venous thromboses (VTE) in e.g. cancer and inflammatory diseases [2, 3]. TF is present in all vascular walls and is normally absent or present in very low concentrations in plasma [4, 5]. However, cancer cells and monocytes may synthesize TF and release extracellular vesicles (EVs) containing TF as a transmembranal protein (EV-TF), and this may contribute to thrombosis [2, 6]. Therefore, measurement of TF is interesting in order to describe a potentially increased risk of VTE in patients.

However, determination of tissue factor is difficult. It can be measured as the antigenic level, but these methods measure very different levels including non-active forms [4, 7]. Therefore, activity assays are preferred as more reliable tests. The probably most used method is a two-stage method where EVs are isolated with a centrifugation at 20,000 g for 15 min [8, 9] (later improved with a centrifugation step for 60 min [10]) and incubated with Factor X (FX) and FVIIa [8, 9] or FVII [10], and the TF activity in combination with FVIIa will activate FX to FXa; in the second stage a chromogenic substrate is added to quantitate FXa [8–10]. The assay is performed in the presence and absence of antibodies against TF to ensure specificity of TF-driven formation of FXa. Several other methods as one-stage quantitations or use of antibody-capture of EVs have been described, but according to Mackman et al. [7] none of these methods are neither as sensitive nor specific to quantitate TF as the two-stage method mentioned above. Østerud et al. [5] indicated very recently, however, that this method is not completely specific, mainly because of a long incubation time where FXa itself may activate FX, thus not specific for TF activity. In the paper of Østerud et al. a new sensitive and specific method was described [5]. It has a similar principle but includes also addition of FV and procoagulant phospholipids (PPL) and prothrombin, and the generation of thrombin is followed using a chromogenic substrate. This reduces the incubation time to a few minutes.

The thrombin generation assay (TG), Calibrated Automated Thrombogram (CAT) developed by Hemker and colleagues [11, 12] has also been demonstrated to be able to detect TF in several papers [13–16]. Ollivier et al. [13] showed that especially lagtime depends on the TF concentration, i.e. a higher concentration of TF reduces the lagtime. However, an optimized method measuring low levels of TF has not been described. The aim of the present study was to establish a sensitive one-stage method to determine TF activity based on the CAT method. The principle is to isolate EVs from plasma which are added to a standard plasma where contact activation has been eliminated making the assay fully dependent on activation from TF activity. To optimize the method, we examined various important factors and additions to make it sensitive and specific for TF activity. Linearity was demonstrated and variation of the optimized method was estimated. Furthermore, we tested the effect of addition of procoagulant phospholipids, which also is present in EVs.

Materials and methods

Materials

Reagents for TG measured by the CAT method (Thrombinoscope BV, Maastricht, The Netherlands) were MP-reagent containing 4μM phospholipids (PL), PPP-reagent Low containing 1 pM TF and 4 μM PL, and Fluca-buffer containing a fluorescent substrate (Z-Gly-Gly-Arg-AMC) and CaCl₂ (Stago, Thrombinoscope BV, Netherlands). Corn Trypsin Inhibitor (CTI) and antibodies against TFPI (aTFPI) (Sheep Anti-Human Tissue Factor Pathway Inhibitor) were from Haematologic Technologies. Antibodies against TF (aTF) (HTF-1, Purified Mouse Anti-Human CD142) were from BD Biosciences. Antibodies against FXII (aFXII) were from Sanquin, Amsterdam, the Netherlands (clone CLB OT-2). Lipopolysaccharides (LPS) and soybean trypsin inhibitor were from Sigma-Aldrich, St. Louis.

Experiments

The method was developed successively by first testing surface activation, i.e. effects of sample tube (Vacutainer and Monovette), and possible inhibition of surface activation by adding antibodies against FXII, and of addition of CTI. TFPI binds to and inactivates TF or rather TF:FVIIa:FXa complex reducing the signal from TF [17], and we therefore tested addition of antibodies against TFPI (aTFPI) in these experiments in order to maintain the TF activity in the sample, i.e. increase the signal. A concentration of 100 μg/mL has been used previously [16], and the effect of a higher concentration was tested to ensure complete inactivation. Addition of antibodies against TF was used to ensure that the measured activity is TF-activity; a concentration published previously [18] was used, and higher concentrations were tested to ensure full TF inhibition. Since TF is cofactor to FVIIa which activates FX to FXa, we tested whether addition of FVIIa could increase the signal. EVs contain procoagulant phospholipids (PPL). The effect of increasing the concentration of phospholipids was tested to make sure that this will not affect the measured TF activity. With the established method, linearity was demonstrated and variation calculated, measuring several samples with various concentrations including EVs from 7 normal individuals and also plasma from blood activated with LPS which increases the TF activity.

Blood samples

To prepare standard plasmas (SP) for the assay: Blood was sampled from healthy volunteers which included 5–10 persons; it was performed several times during the period and the number was adjusted after the planned series of experiments. After oral and written information and signed consent (the study was approved by the local Ethical Committee, N-20220011) blood was sampled using a 21-gauge needle in 3.2% (w/v) trisodium citrate. The first tube was discarded. The samples were centrifuged twice at 2500*g and 20°C in 15 min as recommended [6]; subsequently plasma was centrifuged at 20.000*g in 1 hour at 4°C to remove EVs, and the supernatants were pooled. In the initial experiments blood from Vacutainer tubes (BD, Plymouth, UK) were used but since these tubes have a rather high level of surface activation, we changed to Monovette tubes (Sarstedt, Nümbrecht, Germany). After testing for the effect of CTI, CTI was added to the Monovette tubes (18,3 μg CTI per mL citrated whole blood as suggested by Luddington and Baglin [19]).

Samples for measurement of TF: Blood samples were centrifuged twice at 2500*g and 20°C in 15 min [20], and EVs were isolated after centrifugation of plasma at 20.000*g for 1 hour at 4°C; the sediment was washed with phosphate buffered saline and centrifuged once more at 20.000*g in 1 hour at 4°C. The final sediment was resuspended in SP with aTFPI added (100 μg/mL).

LPS stimulation of blood samples: 10 μg/mL LPS were added to the blood sample which was incubated at 37°C for five hours. Following that, the samples were centrifuged and EVs isolated as described above.

In seven healthy volunteers TF activity was measured in EVs from plasma and from plasma from LPS-stimulated blood. The volunteers included 6 women and 1 man, age 25–68 years.

Thrombin generation

For all experiments were used SP to which aTFPI was added (100 μg/mL plasma) and incubated 15 min at 37°C before use. TG measurements were conducted according to the manufacturer’s instruction using 80 μl sample mixed with 20 μl of trigger solution (i.e., calibration curve: 80 μL SP + 20 μL dilution of TF (dilution of PPP-reagent Low with MP-reagent to get the various concentrations of TF); samples: 60 μL SP + 20 μL EVs suspension (in SP) + 20 μL MP-reagent), and subsequently coagulation was initiated by the addition of 20 μl FluCa-buffer. All measurements were performed in duplicates. Each plasma measurement was calibrated against the same plasma mixed with 20 μl Thrombin Calibrator (Thrombinoscope BV, Maastricht, The Netherlands). Fluorescence of AMC (7-amino-4methylcoumarin) was measured on a Fluoroskan Ascent (Thermo Scientific, Waltham, MA, USA), 390 nm excitation and 460 nm emission. The dedicated Thrombinoscope software (Thrombinoscope BV) was used to calculate: Lagtime (LT), Endogenous thrombin potential (ETP), Peak, and time to Peak of which mainly LT was used in this study. All samples were run without and with addition of aTF (7,84 μg/mL) incubated 15 min at 37°C before analysis to ensure inhibition of TF.

Trigger solutions: The calibration-curve was constructed from mixtures of the trigger solutions PPP-reagent Low and MP-reagent in various ratios to make solutions with TF concentrations from 0–0.1 or 0.5 pM TF (dependent on the range of expected TF concentrations). For measurement of TF concentrations, the solutions of isolated EVs in SP were used. When examining the effect of an increased concentration of PPL, the MP-kit reconstituted with half volume water was used (0 pM TF, 8 μM PPL) to achieve PPL concentrations of 5–8 μM.

The coefficient of variations (CVs) of measurements are described at the end of the Results section.

Statistical analysis

All experiments were performed several times using duplicates, and representative experiments are shown in the figures. Graph Pad was used to construct the figures. To test for differences in the various tests of different concentrations etc., a Students t-test was used.

Imprecision was determined as coefficient of variation (CV) i.e., the standard deviation of the measurements of each sample divided by the mean, and the pooled CV was calculated as the square root of the mean of the squared CVs.

Results

When measuring low levels of TF it is important to avoid contact activation, and therefore the choice of sample tube is important. Fig 1A shows thrombin generation in plasma sampled in Monovette tubes and Vacutainer tubes, respectively. Contact activation is obviously higher in Vacutainer tubes, but it is also present in Monovette tubes at a lower level. We, therefore tested addition of antibodies against FXIIa [18, 21] and addition of corn trypsin inhibitor (CTI) to inhibit the contact activation (S1 Fig). LT increased in the presence of aFXII, but after addition of 18.3 μg/L CTI [19] LT increased much more indicating a high degree of inhibition of the contact activation. Using Monovette tubes with addition of CTI (Fig 1B) virtually no thrombin was generated if no TF was added, and in samples with a low concentration of TF the thrombin generation was totally inhibited by antibodies against TF (aTF). A complete inhibition of aTF at this concentration was ensured by testing a double and triple concentration (S2 Fig). A higher concentration of CTI may be needed for complete inhibition of FXII [22, 23], and consequently we tested and compared addition of 18.3 and 36.6 μg/mL CTI (S3 Fig). A slightly longer LT was found in samples with 36.6 μg/mL, but the differences were minimal resulting in almost the same levels of TF concentration, and addition of 18.3 μg/L was chosen. TFPI will inactivate some TF and therefore antibodies against TFPI (aTFPI) was added in a concentration of 100 μg/mL [16] which increased the signals of the low TF concentrations (shorter LT) with a large difference between LT in the presence and absence of aTFPI (S4 Fig)). A higher concentration of 150 μg/L did not increase the inhibition, and consequently, we used a concentration of 100 μg/mL in the following experiments. The signals also had a lower variation of the duplicates after addition of aTFPI.

It was tested whether addition of FVIIa could improve the signals since the activity of TF is performed by the complex between TF and FVIIa. High concentrations of FVIIa to as much as 1000 pM had an effect on LT which decreased showing an apparent higher TF activity. However, an effect was also seen in samples with aTF added, indicating that this effect was not through complexing with TF (S5 Fig). Consequently, this addition was not beneficial and was not used in the following experiments.

Using the final design with Monovette blood with CTI added and addition of aTFPI to plasma, a calibration curve with TF concentration vs LT was linear in a log-log system, Fig 2. A calibration curve to determine the TF concentrations was created in all the following experiments, always with the same appearance.

Fig 2 — A shows a calibration curve for the TF concentration vs the Lagtime (and the resulting equation), and B shows that the relationship is linear in a log/log system.

Linearity of the method was ensured measuring dilutions of plasma from LPS stimulated blood (an example is shown in Fig 3).

To measure TF in a plasma sample, EVs were isolated by centrifugation at 20,000*g for one hour and resuspended in a lower volume SP to increase the concentration of TF (dependent on the expected concentration). TF concentrations of EVs in healthy persons were measurable if they were concentrated, i.e. EVs from 1500 μl plasma (or higher) were resuspended in 300 μl SP (Fig 4A) before mixing with SP in the TG analysis. The levels in 7 healthy controls were between 1 and 6 fM (TG curves for all 7 individuals are shown in S6 Fig). Higher concentrations were achieved in blood samples incubated with LPS (Fig 4B). The undiluted levels of TF in EVs from plasma from LPS stimulated blood were between 100 and 800 fM, where EVs from 500 μl plasma were resuspended in 300 μl SP. The samples with EVs showed a small TG activity in samples with aTF added (Fig 4A and 4B), i.e. an unspecific TF-independent activity, possibly surface activation by the EVs in spite of addition of CTI. This was most marked in samples with a low TF activity with a concentrated amount of EVs in the TG sample. It has been suggested that a similar effect of EVs from red blood cells can be inhibited by soybean trypsin inhibitor (SBTI) [24]. We, therefore, tested the effect of SBTI (S7 Fig). At a concentration of 1 μg/mL there was no effect. Higher concentrations of SBTI inhibited the TF activity as well as activity in the presence of aTF, and, therefore, we could not use this addition. In the calculation of TF activity from the calibration curves (Fig 2) we calculated the concentration of TF activity in the samples without aTF as well as with aTF and subtracted the apparent concentration in samples with aTF added. The premise for this calculation is that the activations by TF activity and the unspecific TF-independent activity are additive which may not be true. But this unspecific TF-independent activity was always substantially smaller than the concentration without aTF. For the low values it was 10–25%, mainly 15–20%, of the measured activity, but for the higher activities above 100 fM, it was less than 1%. Thus, this TF-independet activity did not interfere much with the result although not negligible at the low level.

Fig 4 — A depicts the thrombin generation measured in EV samples from 4 normal controls having 1 (red, n-EV3), 2 (blue, nEV2), 3 (black, n-EV1) and 6 fM TF (purple, n-EV6). The EVs are concentrated from 7500 μl plasma sampled resuspended in 300 μl SP before mixing with SP in the TG analysis. The dashed lines show the thrombin generation in samples with aTF added, i.e. there is a small TF independent activation in these samples. B shows the thrombin generation in four LPS stimulated blood samples. EV-TF concentrations (EV5-EV8) were between 300 and 800 fM. Below the figures are shown LT and the corresponding TF activity.

EVs contain procoagulant phospholipids (PPL) as well as TF, and, therefore, the content of PPL may affect TG. The effect of higher concentrations of PPL was tested by addition of 1, 2 and 4 μmol/L PPL to the trigger solution (i.e, final concentrations of 5, 6 and 8 μmol/L). Fig 5 shows that although TG increased at a higher PPL concentration with an increased ETP and Peak, LT was almost unchanged at these higher concentrations of PPL, although slightly shorter at a concentration of 10 fM TF, where the shortened LT corresponds to measurements of 11 fM at 5 μM PPL, 13 fM at 6 μM, and 15 fM at 8 μM PPL. Therefore, when measuring TF using EVs with procoagulant phospholipids, a potential increase of PPL concentrations has a very small effect on the measurement, but very low concentrations of TF may be overestimated.

Fig 5 — Thrombin generation in samples where the phospholipid (PPL) concentration is increased from the normal level (4 μM) to 5, 6 or 8 μM. The TF concentrations were from 0.01 to 0.1 pM. Below the figures are shown LT. At the higher concentrations of TF the effect of PL is negligible whereas there is a small effect at the lower concentrations.

Variation

In the final optimized method, the coefficient of variation (CV) of LT for the duplicates of the calibrators (i.e. CV_within) were 3,7% for 0.01 pM TF, and 1.9–3.4% for the calibrators between 0.02 pM and 0.5 pM. The total variation for all the calibrators during 3 months analyses were 6–8%. The total variation (CV_total) of measurements of TF activity at a very low TF concentration was below 20% when measured on three different plates (duplicates on each). At the higher level in LPS stimulated samples CV was below 10% determined in the same way. It has been shown earlier that the major variation originates from the isolation of EVs [10]. We did not test this extensively but from three centrifugations of 4 samples having a concentration of 200–300 fM, the variation was 10–33%. From three centrifugations of 4 samples at a concentration of 1–4 fM, the variation was 50–100%, a considerable variation, but demonstrating that very low concentrations can repeatedly be measured as very low concentrations.

Discussion

We have optimized a method to measure TF activity based on thrombin generation. It is important to avoid contact activation and to add antibodies against TFPI to get reproducible results of low TF concentrations.

Loeffen [25] has described the difference of TG measured in various different sample tubes, and in accordance with previous findings [26] we also found that Monovette tubes have a much lower contact activation than Vacutainer tubes. However, Monovette tubes still induce some contact activation because the thrombin generation cannot be inhibited totally by aTF (Fig 1A). Addition of antibodies against FXIIa which formerly has been used to avoid contact activation [18, 21] was not sufficient. Therefore, we tested addition of Corn Trypsin inhibitor (CTI) at a concentration of 18.3 μg/mL to the blood sample, Fig 1B. With this addition we saw no activation when no TF was added, and the differences of LT between 0.01 pM and 0.1 pM was much more marked. According to Luddington and Baglin [19] this concentration is sufficient to inhibit contact activation, and a higher concentration only had a small effect (S3 Fig). Therefore, addition of 18.3 μg/L was chosen, also because of the expense of CTI (CTI additions are costly). The basis for the TG activity is a SP. Normal plasma is usually produced from samples from at least 20 normal persons. We used a lower number of persons, but we always used the same SP when we made comparisons. Although the differences between different stocks of the SP that we used actually were not marked (the TF activity is calculated from the calibration curves), it is probably preferable to use the same SP to a particular project where TF activity should be measured, but not mandatory.

TG has been used before to estimate the presence of TF. Bidot et al. [14] showed in 2008 that TG, where EVs from various patients were added to a SP, could differentiate between patients with recurrent VTE, patients with a single event of VTE, and controls. LT was shortest and peak highest in patients with recurrent VTE, intermediary in patients with a single VTE and LT was longest and the peak smallest in controls. They concluded that EVs had a procoagulant effect, probably because EVs contained TF, but they did not quantitate TF. Debaugnies [15] compared cancer patients and controls using TG and found a difference but did not quantitate the TF concentration. Ollivier [13] described a method to measure TF based on TG where plasma samples with CTI added could detect a concentration of TF on 0.025 pM and higher although it was not calibrated to estimate the concentrations. They saw an increasing TG when PPL was included in the reaction up to 4 μM. Boknäs et al. demonstrated the importance of CTI especially when the reagent contain PPL [23]. Hellum et al. [16] used TG to detect TF in patients with pancreatic cancer and found that addition of aTFPI improved the signals considerably—we could confirm that this concentration was sufficient to inhibit TFPI (S4 Fig). The present method has included several of these findings in an optimized method. Important points are addition of CTI to avoid contact activation, and addition of aTFPI [16] which increased the signals of the low TF concentrations (shorter LT). The calibration curves are based on the reagents from Stago containing 1 pM TF. aTF were added in a concentration used previously [18] which ensures that the activation is TF dependent, and higher concentrations had no further effect (S2 Fig). This resulted in a linear calibration curve in a log-log system (Fig 2), and dilutions were linear (Fig 3). The procoagulant effect of EVs on TG is because of the presence of TF but may also be promoted by PPL of the EVs. Hemker et al. described that the effect of an increasing concentration of PPL on TG was small at concentrations > 3 μmol/L which is the reason for using 4 μmol/L in the reagents for CAT [11]. The effect of higher concentrations in our set-up i.e., final concentrations of 5–8 μmol/L has not been investigated before in the papers measuring EV-TF. This increased concentration only affected LT at very low TF concentrations (Fig 5)–at a TF activity of 10 fM the activity was overestimated 10–50% with PPL concentrations at 5–8 μM, but no overestimation at a TF activity of 30 fM or higher. Therefore, when measuring TF using EVs containing procoagulant phospholipids, a potential increase of PPL concentrations above the 4 μM in the reagent has a very small effect on the measurements, but very low concentrations of TF will probably be overestimated.

We hypothesized that addition of FVIIa might improve the signals because TF could immediately bind to this activated FVII, but it did not improve the method since LT after addition of aTF was also shortened, probably because FVIIa in combination with phospholipids can activate FXa, i.e. a TF-independent activation [5].

Vallier et al. [10] investigated the effect of various centrifugations to isolate EVs and based on this we chose 20,000*g for one hour as we have also used before [27]. At very low EV-TF concentrations a TF-independent TG was seen in the presence of aTF (Fig 4A). Noubouossie et al. [24] described recently that red blood cell EVs could activate the coagulation probably via an activation of prekallikrein and FIX and this activity was not inactivated by CTI but could be inhibited by soybean trypsin inhibitor [24]. However, addition of soybean trypsin inhibitor was not successful because SBTI inhibited this activity as well as the TF activity (S7 Fig). But the apparent TF concentration from this activity induced by EVs (i.e. when aTF was added) was subtracted from the activity read on the calibration curve for the sample in the absence of aTF, and it only reduced the TF concentration to a lesser extent at the low level and negligibly at the higher levels (Fig 4B). As mentioned, the premise for this subtraction is additivity of the TF-dependent and TF-independent activity of the EVs. This way of adjusting for the TF-independent activity may not be completely correct, and especially at low TF activities this may affect the result, whereas at higher TF concentrations it is less important. In combination with the potential effect of PPL from EVs when measuring low levels of TF, this reduces the accuracy of these measurements. The results indicate a very low level of TF activity and although the measurements are rather reproducible the exact activity has an inherent uncertainty. At higher levels as found in LPS stimulated samples the interference from these potential sources of error have virtually no importance for the result. Thus, when using the method to estimate the TF activity in procoagulant patients with an increased level of TF, the method is reliable.

The variation of the method calculated from the duplicates of calibrators was low: a very low CV_within, and a quite low CV_total. The total variation of measuring TF activity of samples based on CV calculated from duplicates was reasonable and comparable to the results of Hellum et al. [16]. The isolation procedure of EVs, however, has an inherent high degree of variation as shown by Vallier et al. [10] who recently published an improved version of a previous method to measure TF concentration based on FXa formation. They could detect TF in normal donors which had levels of 7 ± 4 fM, a similar or slightly higher level than our values. However, comparison is difficult when the calibrant differs. The CVs were not very different from the present method, probably slightly lower but it is difficult to compare CVs at different levels. A drawback of this method is the duration of the assay because of rather long incubation periods and the possibility of some TF-independent activity as stated by Østerud et al. [5]. The method recently published by Østerud et al. [5] is rapid but probably less sensitive since they found no activity of TF in normal controls. It had a lower CV than the present one and it is faster. The advantage of our method is that it is a one-stage method using commercially available reagents and the CAT system present in many research laboratories, and, therefore, simpler to establish.

In conclusion, we have established a method to detect TF activity based on thrombin generation with a high sensitivity and a reasonably low variation.

Supporting information

S1 Fig. Effect of aFXII and CTI to inhibit the contact activation.

Addition of a FXII increases LT by some minutes, but addition of CTI to the blood increases LT substantially. The bars show mean and SD of 4 plasma samples.

(TIF)

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S2 Fig. Effect of increasing the concentration of aTF.

The figure shows the ratio between LT in the presence and the absence of antibodies, when TG is activated by 0.05 and 0.1 pM TF. I.e., the more the antibodies inhibit TF the higher ratio will be expected. It appears that increasing aTF to higher concentrations do not increase the ratio indicating that 7.8 μg/mL is sufficient to inhibit the TF activity. The bars show mean and SD of 4 plasma samples.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S3 Fig. Comparison between 18.3 μg/mL and 36.6 μg/mL CTI.

Calibration curves (i.e. LT vs TF concentration) using 0.01–0.10 pM TF to plasma from blood samples where CTI was added in the two concentrations.

(TIF)

Click here for additional data file.^{(1.9MB, tif)}

S4 Fig. Effect of addition of aTFPI.

Addition of 100 μg/L or 150 μg/L aTFPI reduce LT in samples with 0.1, 0.2 or 0.3 pM TF considerably but no difference between 100 and 150 μg/mL. The bars show mean and SD of 4 plasma samples.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S5 Fig. Effect of addition of FVIIa.

Addition of FVIIa reduces LT but it also reduces LT in the same sample in the presence of aTF, i.e. that the effect is not a TF dependent activity.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S6 Fig. Measurements of TF in samples.

The same figure as Fig 4, but including all seven normal volunteers with TF activity of 1–6 pM. Below the figure is described LT in the presence and absence of aTF and the corresponding TF activity.

(TIF)

Click here for additional data file.^{(2.6MB, tif)}

S7 Fig. Effect of soybean trypsin inhibitor.

SBTI in various concentrations was added to a sample with 1 pM TF. It appears that the higher concentrations of SBTI inhibit the activity in samples without aTF as well as in the presence of aTF.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

Data Availability

All relevant data are within the paper.

Funding Statement

The authors received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0288918.r001

Decision Letter 0

Heather Faith Pidcoke

24 Feb 2023

PONE-D-23-00356

A sensitive tissue factor activity assay determined by an optimized thrombin generation method

PLOS ONE

Dear Dr. Kristensen,

Please submit your revised manuscript by Apr 10 2023 11:59PM. If you will need more time than this to complete your revisions, particularly if you chose to add the experiment suggested by Reviewer 1, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Additional Editor Comments:

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This editor and both reviewers are of the opinion that the statistical analysis requires improvement. Please consider requesting input from someone trained in statistical analysis of scientific data.

Reviewer 1 further makes the recommendation that the authors add a comparison to the Mackman lab in-house assay, published by Hisada et al. While this is sound advice and would strengthen this publication, it is not a requirement for resubmission of the manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors present a publication titled “A sensitive tissue factor activity assay determined by an optimized thrombin generation method”. The purpose of this publication as stated by the authors was to “establish a sensitive method to detect low levels of tissue factor (TF) based on the calibrated automated thrombogram (CAT) method”. This is important and relevant research to have simple measurement of the major initiator of the coagulation cascade. The study strengths are to use commercial grade reagents and a well-known method to rapidly determine tissue factor activity in plasma samples. This test would hopefully be an important step to bring measure of tissue factor from the research bench using chromatography assays to more of a commercial test. Despite these strengths, the paper is poorly written, organized, and fails to provide the data to be sufficient for publication in current form. Please see general comments and specific comments below:

The first main weakness in this paper is poor presentation to support their findings. Although the authors present graphical examples of the seven donors tested they fail to supply table listing the demographics of these donors (all men? how old?). Another table in the paper or supplemental data should list the computed CAT values and the CVs of each of the runs. This prevents a detailed review of the papers finding which is hard to get from the graphs. Without this date it is difficult to determine the CVs independently. Also help in understanding if additional donor testing is needed to reduce the CV to less than 10%, which is needed for high-quality research assay. Therefore, it is recommending the authors present this data and consider increasing the number of donors to reduce the CV value. As there are great number of tests out their increasing the number of donors documenting the sensitivity and specificity of the test is important. This is needed despite previously published low donor tested Hellum et al (only three donors) and Osterund et al (did not report how many). Furthermore, sex and age and other pertinent demographic variables are important for rigor and reproducibility in research. Although, Scully et al (J Thromb Thrombolysis, 2009) documented no difference in monocyte expression of TF between men and women. The NIH states that we should always consider sex, race, and other characteristics as biological variable. . Lastly, the authors should report sensitivity and specificity in predicting a TF expression in a donor using their test, which is necessary to compare tests.

Furthermore, as many studies have been published to measure tissue factor activity, it would be good for the authors to review the publications, cite from Mackman group, and discuss other methods in their paper including:

1. Mackman N, Sachetto ATA, Hisada Y. Measurement of tissue factor-positive extracellular vesicles in plasma: strengths and weaknesses of current methods. Curr Opin Hematol. 2022 Sep 1;29(5):266-274. doi: 10.1097/MOH.0000000000000730. Epub 2022 Jul 11. PMID: 35852819.

2. Hisada Y, Mackman N. Measurement of tissue factor activity in extracellular vesicles from human plasma samples. Res Pract Thromb Haemost. 2018 Nov 20;3(1):44-48. doi: 10.1002/rth2.12165. PMID: 30656275; PMCID: PMC6332748.

The first is excellent review that should be cite and evaluated in reporting a novel sensitive test. The second paper (Hisada et al) is the publication of the Mackman lab in-house assay which would strengthen this publication if the authors were to add a comparison to this test. This reviewer recommends repeating the study to compare these methods. Otherwise, this paper will not have the impact it needs to move toward a commercial test. Lastly, explain why an increased phospholipids would increase the lag time. As Mackman et al states above phospholipids that come from cellular activation (especially phosphatidylserine) activate cryptic TF to become active TF. This would lead to a decrease in lag time. Please explain why you found an increase in lagtime or was it not significant because of high CV.

Overall, figure legends should be at the end or connected to figures not in the text. All acronyms need to be spelled out. Overall, methods are very disorganized and large sections are missing compared to recent Osterund et al paper included NTA analysis and other details on the research process.

ABSTRACT:

Line 22: Tissue factor concentration is different than activity as you are measuring only activity it's important to mention that. You are not measuring the total amount of tissue factor, the active vs. non-active (aka cryptic).

Line 23: Are you determining the sensitivity and specificity of your assay or are you studying the CV?

Line 25: Please correc that the assay is based on “Thrombin generation”, it's based on :”lag time” to thrombin generation

Line 28: define lag time

Line 30: define calibrators

Line 31: This should be in methods: “Contact activation of the coagulation system was avoided using CTI plasma samples in Monovette tubes.”

Line 34: There is no mention on the methods of how you measured EV concentrations. Clarify EV up concentrated as this is not a standard term

Line 35: “However, the step with isolation of EVs have a higher inherent CV.” This is not appropriate for abstract but should be in the limitations.

Line 37: Precise would be all CV less than 10% for research-based assay and less than 5% for diagnostic assays. I would remove this word.

INTRODUCTION:

Line 42: Tissue factor is the PRIMARY coagulation factor that activates the coagulation system, please see papers on cell-based coagulation

Line 43: Do you mean increased expression of tissue factor?

Line 45: What do you mean present inside vascular walls, do you mean endothelial cell?

Line 48: What do you mean the measurement of TF, protein, or activity?

Line 61: Please discuss the Yohei paper as referenced above

Line 62: Please clarify the purpose of the hypothesis is it to determine the sensitivity of the test or accuracy (CV) and is it for protein expression or tissue factor activity

MATERIALS AND METHODS:

Line 67: Please make materials section into one paragraph

Line 81: Most TF activity assays discard the first three ml of blood draw, why was this not followed?

Line 95: Why did you do CAT measurements in duplicates? The thrombinoscope software recommends triplicates.

Line 99: Spell out all acronyms…ETP

Line 104: Please state the standard dilution of the MP reagent, not spelled out acronym

Line 107: Please clarify that the incubation with aTF was done before CAT or is it wrong section of the methods

Line 113: Why no sensitivity or specificity calculation as you did a test on TF EV from donor blood.

RESULT

Line 119: This is methods part and need to be moved up and explained.

Line 145: Watch out for acronyms like fig. please spell out

Line 127-129: This explanation needs to go to the methods.

Line 131 to 135 Please put into figure legend (put all figure descriptions into a legend)

Line 137 to 139: This is discussion and needs to be betterl explained

Line 149 to 151: Goes into methods

Line 151: How were EVs concentration measured, this needs to be put in methods

Line 153: Were not all samples treated the same? Why are higher ones achieved with LPS?

Line 153: we need a table describing the age, gender, and others to help repeat study

Line 165: Phospholipids usually activate TF, please explain in discussion why would lag time be prolonged with increased phospholipids

Line 175: a table needed to list all of this data, the CV and other can be in supplemental.

Line 179-181: As the variation is high it would be good to test vs. a published assay (Hisada et al) for reference.

For graphs and figures be sure your legends are present and spelled out in figures.

DISCUSSION:

Line 195: Can you comment on the CTI differences between 18.3 and 36.6 ug/mL as the data is not presented. Can it be a difference because the first 3 ML of blood was not discarded as performed in other studies.

Line 198-201: Very long sentence, please revise in two or three sentences.

Line 201-208: This is a list of papers with no relation of their findings with the results

Line 208-211: Rephrase to say we chose the previous method to include in our study as..

Line 221: Evs are made up of phospholipids. How do you distinguish that one of the donors EVS do not contribute the phospholipids to override the system. How many of the EVs were leukocytes vs. platelets? Platelet EVs do not contain TF but are more numerous that leukocyte

Line 224-225: Should this be iits own paragraph?

Line 230: Did you do test with soybean trypsin inhibitor? Nothing was reported in results. please include the data in supplemental.

Line 236-237: Please see line 224-225

Line 238: Please report your CVs for all values and then mention the ones for Vallier so we can know the actual difference. If your CVs are not less than 10% for lower TF, then how can you call this improved?

Line 243: Is it possible they found no TF in normal controls because they discarded the first 3 mL?

Line 246: This conclusion is too short and does not have a limitation section.

Reviewer #2: Summary: The authors present an interesting study detailing a novel method for determination of tissue factor (TF) concentration utilizing a commercially available thrombin generation assay (CAT; STAGO). Development of such an assay would make quantification of TF more readily available for both clinical and research investigations.

While the overarching objective of the study, to measure TF based on TG, is clear, the specific sub-aims/experimental procedures implemented are not clearly defined in the introduction or methods sections. There is no study hypothesis. Currently, the manuscript is missing a clear delineation of Methods/Results/Discussion as portions of each of these sections are presented in different areas/not clearly in a sequential order. The reader is left to go back and forth between sections for clarity at several points. It would be useful to have 1-2 sentences in the introduction clarifying how the authors planned to go about developing and evaluating the TG based TF detection method, and what conditions/variables will be assessed in this investigation – as this is not readily apparent. Further, the statistical methods are lacking. The sample sizes are not clearly reported, and only representative data displayed. It is not stated whether a power analysis was performed or whether the study was accurately powered to assess each of the outcome measures investigated. At various points throughout the manuscript the authors mentioned that they evaluated an experimental condition – but there is no data shown to support this. The manuscript could strongly benefit from a detailed Supplemental Methods and Supplemental Results document so that all data and experimental procedures referenced are accounted for and presented. It would be important to perform a comparative study to assess TF using the TG method developed in this study alongside other reference methods, prior to making claims that this is an “established” method.

Additional comments:

Methods:

How many healthy donors were included in the donor pool for preparation of the SP? Likely the experiment should be repeated additional times with SP prepared from separate donor pools. Likewise, may be important to repeat the study using SP prepared from patients with pathologies subject to TF investigation.

The method section could benefit from a table that indicates what additives /components were utilized in the sample mixtures for each of the experiments performed (ex. aTFPI, SP, aTF, EV suspension, CTI, etc.)

How was the concentration of CTI to be used decided upon? *Update - this is mentioned later in discussion but should be clarified in the methods earlier and cited appropriately. Were other concentrations tested by these authors? And where are those results reported?

Throughout methods and results it is difficult to ascertain how many times the experiments were repeated. The statistical analysis section in the methods states that representative data is presented, but throughout the manuscript the sample sizes are never clarified - it is only mentioned that "several" replicates were performed. The sample sizes should be clearly stated. Additionally, was a power analysis performed prior to the study? Or how were the sample sizes decided upon? Was the study accurately powered at each phase to draw the conclusions reported?

Results

Are there reference data or experimental data that could be added to a supplement to show the variability in LT when aTFPI was applied versus when it was not? How was the concentration of aTFPI decided on?

Results line 137-138 states, “Ten pM [FVIIa] had a small effect which increased at higher concentrations, but an effect was also seen in samples with aTF added….” – Where are these results? Could they be provided in a supplement? What did the authors define as “a small effect”/what was the outcome of interest here? LT? How many times was this experiment repeated?

In Fig 2 and 3, again it is not clearly stated how many replicates were performed to generate the TF concentration vs LT calibration curve; or how many replicates were performed when assessing linearity of the dilutions of TF in the LPS stimulated samples.

In Figure 4, thrombin generation in EV samples from 3 normal controls having 1-3 fM TF is shown – but earlier in the results it states that levels of TF in the normal controls ranged from 1-6 fM. Why are results for 4-6 fM not shown? Why do the authors speculate that LT was prolonged with escalating TF concentration in Fig 4A? What were the actual TF concentrations for EV5-8 in Fig 4B?

In Figure 5, the abbreviation “PPL” is used in the figure caption and manuscript, but the abbreviation “PL” is used on the figures themselves – this should be standardized.

Throughout the results, it may be useful to provide the numerical LTs – it is difficult to ascertain the LTs and variability in LT from the figures and CV alone, especially when no sample size is provided. The ETP/Peak are more clearly depicted in figure format; however, if LT is the sole outcome of interest it may be beneficial to report mean results in a table format with standard deviations and sample sizes. This information can be obtained in part from Figure 2A – however there are no error bars or samples sizes included in the figure to indicate how many replicates were performed to achieve a lagtime result at each TF concentration.

Discussion

The discussion mentions that additional CTI concentrations were evaluated – where are these results/could the be provided in supplement?

Lines 211-218 provide important information that should be explained in the methods section, rather than in discussion.

Line 224-225 Where are these results shown?

Line 230-233 Is this referring to the present study? If so, were these results reported?

Further discussion on how the presented novel method is potentially superior to currently available methods would be important. What are the next steps/future directions for this work?

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Reviewer #1: Yes: Andrew D. Meyer, MD, MS

Reviewer #2: No

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PLoS One. 2023 Jul 19;18(7):e0288918. doi: 10.1371/journal.pone.0288918.r002

Author response to Decision Letter 0

6 Apr 2023

We have uploaded a "Response to reviewers" with an answer to all the points raised

Attachment

Submitted filename: Response to reviewers.docx

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PLoS One. doi: 10.1371/journal.pone.0288918.r003

Decision Letter 1

Heather Faith Pidcoke

19 Jun 2023

PONE-D-23-00356R1A sensitive tissue factor activity assay determined by an optimized thrombin generation methodPLOS ONE

Dear Dr. Kristensen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please note that in response to your concerns about the reviewer comments regarding your original submission, we have requested the evaluation of a third reviewer whose paper you have cited in your submission. This reviewer (Reviewer #3) has provided comments regarding your work. Please address these comments in full prior to re-submitting your manuscript.

Please submit your revised manuscript by Aug 03 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Heather Faith Pidcoke, MD, MSCI, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: It would have been great to have a reliable sensitive and specific assay for the measurement of TF activity in blood/plasma. However, the present assay is not any easier and cheaper assay for TF activity than the present assays available today. It is rather complicated as the EVs must be isolated, and then they are added back to a pool of plasma from different individuals. To avoid any plasma contribution to thrombin generation, they are adding CTI to block the intrinsic pathway and aTFPI to avoid plasma inhibition of TF activity, both expensive reagents.

In the abstract, line 37, the authors state: “addition of PPL only increased lagtime slightly at very low concentrations of TF showing--” How can this be as they in the section 231-237, and Fig 5, shows that TG is enhanced with adding larger amounts of PPL, and LT is slightly shorter at a concentration of 10 fM TF. This does not make sense and should be corrected.

It looks like detection of TF activity in plasma of healthy individuals, is the major goal of this study. To detect the TF activity, they concentrate the EVs up to 25 times, compared to the level of EVs in the plasma. This gives rise to very high levels of PPL that will cause more non-specific TF activity. But what else can be associated with the large amounts of EVs? There are several other procoagulant activities associated with EVs as for example FVIIa, FVa, FIXa and FXIa, that may contribute to shorter LT, especially at low concentrations of TF. I do not believe that subtracting values obtained with aTF from results without aTF, is giving the right answer for the presence of TF activity or not. As the authors themselves states in lines 235-237: “Therefore, when measuring TF using EVs with procoagulant phospholipids, a potential increase of PPL concentrations has a very small effect of the measurement, but very low concentrations of TF may be overestimated”. I agree and believe that the detected TF activity in this assay system in EVs of healthy individuals, has nothing to do with TF activity. To disprove this, they must rule out the contribution of other procoagulant activities associated with highly concentrated EVs in the generation of thrombin.

FVIIa has different ways to mediate thrombin generation in plasma, and to assure that FVIIa is not involved in the generation of thrombin in the present assay in the absence of TF, they should use FVIIai to block the extrinsic pathway.

The authors are focusing on the detection of very low concentrations of TF in plasma to be used for analyzing samples from venous thrombosis patients. In my opinion, procoagulant EVs from activated cells expressing large amounts of phosphatidylserine, play a more important role in thrombin generation in venous thrombosis than the traces of TF detected. Only EVs from plasma of certain cancer patients may have TF levels that can be associated with their events of thrombosis.

Some minor points with strange sentences:

Line 127: “In seven normal volunteers TF activity was measured in plasma and in plasma from LPS-stimulated blood”. How do you define a normal volunteer? TF activity was not measured in plasma but in EVs isolated from plasma.

Line 208: The levels in 7 normal persons.

Line 214: “marked samples with a low TF activity which were concentrated quite much (i.e. a high amount of EVs in”

Line 221: “without aTF, so this non-TF activity did not interfere much with the result”

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PLoS One. 2023 Jul 19;18(7):e0288918. doi: 10.1371/journal.pone.0288918.r004

Author response to Decision Letter 1

6 Jul 2023

6. Review Comments to the Author

Reviewer #1: (No Response)

R: It is correct that it is not very simple or cheap – and this has certainly not been stated in the paper. EVs must be isolated as in other assays for TF activity, but the advantage is that you can use thrombin generation (or CAT) which many researchers have admittance to do. Unlike the two most used methods (as described in the Introduction) you do not have to buy several different reagents which is not always easy and uncomplicated (which I have learned from a long life in the lab). In this respect it is more straightforward to use the CAT technique with few modifications, and in this way it is an alternative to other methods. An optimized method for TF activity measurement based on CAT has not been described before.

R: We have now explicitly described the effect on the estimation of TF activity concentration

R: No, it is not very important to measure TF activity in normal, healthy individuals. As stated in the introduction it may be interesting to quantitate TF activity in cancer patients and other procoagulant patients. We have done this in some cancer patients previously and using the Mackman method it is not possible to detect an activity in all the patients. It could, therefore, be advantageously to measure lower concentrations. In their optimized method (Ref. #10 in the paper) they could actually measure TF activity in some of the healthy controls, and we therefore found it interesting that it was also possible with this method if we concentrated the EVs. But we believe that the main application of a method like this will be for patients who have a higher TF concentration. EVs were not concentrated 25 times because in the assay 20 µL of this solution is added to 60 µL SP, as described in Methods – we have now added this in the text. Thus it is concentrated about 6 times compared to normal plasma, and in the reaction it is further diluted to two thirds.

As we have described in the paper, high levels of PPL and FVIIa can activate FX, but this is the reason why we have tested the effect of increasing PPL in the assay which have a minor effect. What is “very high levels of PPL”? I do not think that we have very clear estimations of PPL concentrations of EVs. According to measurements with Zymuphene MP activity assay it should be at or below 10 nM PS equivalents. So even if the EVs are concentrated and only part of PPL in the reagent has PS activity, the amount of PPL from EVs cannot be more than the 4µM that we added.

But what else can be associated with the large amounts of EVs? There are several other procoagulant activities associated with EVs as for example FVIIa, FVa, FIXa and FXIa, that may contribute to shorter LT, especially at low concentrations of TF.

R: As stated, there is not a large amount of EVs in the assay, especially not when we measure higher concentrations of TF. There is no reference to these statements? We are not aware of papers that have estimated the concentration of these factors. It must be remembered that EVs are added to a standard plasma with normal concentrations of coagulation factors so this addition will probably be negligible. Even if it is correct, it will only be a problem at very low TF activities. Otherwise, it will also be a problem in other methods where EVs are added. Perhaps the reviewer means that the EVs have a procoagulant surface? That is exactly the reason why we tested the effect of adding phospholipids (fig 5). And as shown by this experiment, this is only a minor problem at low TF concentrations.

I do not believe that subtracting values obtained with aTF from results without aTF, is giving the right answer for the presence of TF activity or not.

R: This may be true, but it is a way to try to correct for it. We have now explicitly described the premise for this procedure, and also a possible uncertainty of the estimations because of this. In all the methods for TF activity there is some “blank value” which must be corrected for, and this way of correction may not be true in any of the methods. In any case, it only seems to be a potential interference at a very low TF activity as discussed explicitly in the paper now.

As the authors themselves states in lines 235-237: “Therefore, when measuring TF using EVs with procoagulant phospholipids, a potential increase of PPL concentrations has a very small effect of the measurement, but very low concentrations of TF may be overestimated”. I agree and believe that the detected TF activity in this assay system in EVs of healthy individuals, has nothing to do with TF activity. To disprove this, they must rule out the contribution of other procoagulant activities associated with highly concentrated EVs in the generation of thrombin.

R: Firstly, it is only when measuring TF activity in normal healthy persons that EVs are concentrated, and it is more relevant to measure higher concentrations in procoagulant persons. Secondly, we have participated in a trial of measurements of TF activity and the present method performed quite well. Making a measurement without and with aTF is usually what has been done to measure TF activity. Furthermore, I can tell that we have used the Mackman-method previously in samples from cancer patients – here we determine an activity with and without aTF. Frequently we got a higher activity with aTF included, i.e. we actually found a “negative concentration”. The present method seems to be much more reliable for lower concentrations.

I think it is a revealing statement that the measured TF activity has nothing to do with TF activity. We measure an activity which is inhibited by aTF. It may be slightly inaccurate but certainly not completely misleading – this is nonsense. How can aTF inhibit an activation which is not performed by TF?

R: As shown in fig 1B there is no thrombin generation in the absence of TF. We have actually tried to add FVIIa and concentrations up to 10 pM did not result in a shorter lagtime, and 100 pM only slightly. Why should there be a high concentration of FVIIa in the assay? Should it come from the EVs? I cannot find any references describing this. FVIIai will bind to TF and therefore inhibit this activity, but will it block all other reactions for the alleged FVIIa? I cannot find documentation for this. When we get a large difference between samples with and without aTF, it is most likely due to a TF activity

R: It is not correct that we focus on very low concentrations of TF. We agree that it is mainly interesting in certain cancer patients which we intend to investigate (which is also stated in the Introduction). That is the reason why we have established this method which we find more easy to use than the Mackman method.

We have now expressed that more clearly in the paper

Some minor points with strange sentences:

R: We have changed it to healthy volunteers. We have changed plasma to EVs.

Line 208: The levels in 7 normal persons.

R: We have changed it to healthy controls

Line 214: “marked samples with a low TF activity which were concentrated quite much (i.e. a high amount of EVs in”

R: It has been rephrased

Line 221: “without aTF, so this non-TF activity did not interfere much with the result”

R: We have changed non-TF activity to TF-independent activity

________________________________________

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PLoS One. doi: 10.1371/journal.pone.0288918.r005

Decision Letter 2

Heather Faith Pidcoke

7 Jul 2023

A sensitive tissue factor activity assay determined by an optimized thrombin generation method

PONE-D-23-00356R2

Dear Dr. Kristensen,

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Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0288918.r006

Acceptance letter

Heather Faith Pidcoke

11 Jul 2023

PONE-D-23-00356R2

A sensitive tissue factor activity assay determined by an optimized thrombin generation method

Dear Dr. Kristensen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Effect of aFXII and CTI to inhibit the contact activation.

Addition of a FXII increases LT by some minutes, but addition of CTI to the blood increases LT substantially. The bars show mean and SD of 4 plasma samples.

(TIF)

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S2 Fig. Effect of increasing the concentration of aTF.

(TIF)

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S3 Fig. Comparison between 18.3 μg/mL and 36.6 μg/mL CTI.

Calibration curves (i.e. LT vs TF concentration) using 0.01–0.10 pM TF to plasma from blood samples where CTI was added in the two concentrations.

(TIF)

Click here for additional data file.^{(1.9MB, tif)}

S4 Fig. Effect of addition of aTFPI.

Addition of 100 μg/L or 150 μg/L aTFPI reduce LT in samples with 0.1, 0.2 or 0.3 pM TF considerably but no difference between 100 and 150 μg/mL. The bars show mean and SD of 4 plasma samples.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S5 Fig. Effect of addition of FVIIa.

Addition of FVIIa reduces LT but it also reduces LT in the same sample in the presence of aTF, i.e. that the effect is not a TF dependent activity.

(TIF)

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S6 Fig. Measurements of TF in samples.

(TIF)

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S7 Fig. Effect of soybean trypsin inhibitor.

SBTI in various concentrations was added to a sample with 1 pM TF. It appears that the higher concentrations of SBTI inhibit the activity in samples without aTF as well as in the presence of aTF.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(41.6KB, docx)}

Attachment

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Data Availability Statement

All relevant data are within the paper.

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A sensitive tissue factor activity assay determined by an optimized thrombin generation method

Søren Risom Kristensen

Jette Nybo

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Materials

Experiments

Blood samples

Thrombin generation

Statistical analysis

Results

Fig 1. Thrombin generation in Vacutainer and Monovette tubes, and Monovette tubes after addition of CTI.

Fig 2. A calibration curve for TF concentration.

Fig 3.

Fig 4. Measurements of TF in samples.

Fig 5. Effect of an increased concentration of phospholipid.

Variation

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Heather Faith Pidcoke

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Author response to Decision Letter 0

Decision Letter 1

Heather Faith Pidcoke

Roles

Author response to Decision Letter 1

Decision Letter 2

Heather Faith Pidcoke

Roles

Acceptance letter

Heather Faith Pidcoke

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Associated Data

Supplementary Materials

Data Availability Statement

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