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Keywords:

  • activated protein C;
  • aptamer;
  • enzyme-capture-assay;
  • oligonucleotide-based enzyme capture assay

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgement
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Summary.  Background: Human-activated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory and cytoprotective functions. This feature renders APC to be a promising vascular-inflammatory biomarker.Objective: The aim of the present study was the development and validation of a technique that allows the measurement of APC plasma levels under practical laboratory conditions.Methods/patients: Based on the APC-binding ssDNA aptamer HS02-52G we developed an oligonucleotide-based enzyme capture assay (OECA) that quantifies aptamer-captured APC through hydrolysis rates of a fluorogenic peptide substrate. After optimization of pre-analytical conditions, plasma APC levels were measured in healthy individuals and patients undergoing hip replacement surgery.Results and conclusion: A combination of APC–OECA with an aprotinin-based quenching strategy allowed APC analysis with a limit of detection as low as 0.022 ± 0.005 ng mL−1 (0.39 ± 0.10 pmol L−1) and a limit of quantification of 0.116 ± 0.055 ng mL−1 (2.06 ± 0.98 pmol L−1). While APC plasma levels in healthy individuals fell below the quantifiable range of the APC–OECA platform, levels substantially increased in patients undergoing hip replacement surgery reaching peak values of up to 12 ng mL−1 (214 pmol L−1). When normalized to the amount of thrombin generated, interindividual variabilities in the APC generating capacity were observed. In general, with a turn-around time from blood sampling to generation of test results of < 7 h, the APC–OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgement
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, anti-inflammatory and anti-apoptotic functions. The active protein is generated from its precursor zymogen protein C (PC) on the surface of endothelial cells by an activation complex formed by thrombin and thrombomodulin (TM) [1]. The endothelial cell protein C receptor (EPCR) augments this process [2]. APC released from the endothelial cell surface down-regulates thrombin formation by cleavage of the activated cofactors V and VIII [1,2], whereas APC still bound to the cell surface induces anti-inflammatory and cytoprotective signaling responses in endothelial cells [3–5].

A failure to generate sufficient amounts of APC is associated with a prothrombotic and a hyperinflammatory phenotype. The severity of the clinical symptoms depends on the residual APC activity. The prothrombotic phenotype is the leading symptom in milder forms of APC deficiency such as in heterozygous PC deficiency, whereas more severe forms of APC deficiency such as in homozygous PC deficiency are characterized by a thrombo-inflammatory phenotype [2]. Acquired APC dysfunction is critically involved in the pathogenesis of several thrombo-inflammatory diseases including severe sepsis [6].

In spite of the important physiological functions of APC and its crucial role in the pathogenesis of a variety of diseases, plasma levels of APC are not considered in diagnostic or therapeutic decision making. One reason for this is the lack of an appropriate method allowing the measurement of APC plasma levels under practical laboratory conditions.

In 1992, Gruber and Griffin [7] first reported the use of an enzyme-capture-assay (ECA) for the direct measurement of APC plasma levels through microtiter analysis. This assay is based on a monoclonal antibody used as capture ligand that recognizes the light chain of human PC. As this antibody does not discriminate between PC and APC, samples have to be extensively diluted to ascertain that binding sites are not completely blocked by PC as a result of the large excess of PC over APC in human plasma. As a consequence of the very low concentrations of captured APC, the chromogenic substrate used for the quantification of APC has to be incubated for several weeks to generate a signal strong enough for APC quantification [7]. Reduction of the substrate incubation times to 16–18 h was achieved by coupling a non-discriminating catching antibody to high-capacity agarose beads [8]. In 2003, Liaw et al. reported the generation of the monoclonal antibody HAPC 1555, which revealed a 10-fold higher binding affinity to APC as compared with PC in the presence of physiological calcium concentrations [9,10]. However, even with the use of this monoclonal antibody, detection of subnanogram concentrations of APC by a plate-based assay still requires incubation of the chromogenic substrate for at least 19 h rendering this assay hardly applicable for routine testing [9].

An alternative approach to direct APC measurement is to assess circulating levels of APC–protein C inhibitor (PCI) complexes. Stenflo et al. impelled this approach by employing an assay based on a monoclonal antibody (M36) that recognizes a neoepitope on both, cleaved/dissociated and APC-bound PCI [11,12]. However, plasma levels of APC–PCI complexes reflect that of freely circulating APC only to a certain extent [13]. For instance, it remains unclear how changes in levels of PCI affect the formation kinetics of APC–PCI in vivo. Furthermore, changes in the clearance rates of APC–PCI complexes might lead to the over- or underestimation of obtained APC levels. Moreover, although PCI is esteemed as a main physiologic inhibitor of APC [2,5], complex formation between APC and alpha-1-antitrypsin or alpha-2-macroglobulin takes place in healthy individuals and also plays a major role in various pathological conditions [14,15].

Based on APC–PCI determinations, a substractive approach has also been developed for APC determinations. Blood is collected into two separate tubes containing either heparin to augment APC–PCI complex formation or benzamidine to inhibit APC–PCI complex formation. Circulating concentrations of APC are calculated from the difference of the concentrations of APC–PCI complexes determined in the two samples [16]. Crucially, this approach requires multi-well analysis, close attention to heparin-triggered effects and also depends on plasma levels of APC inhibitors. Thus, direct measurement of enzyme plasma levels remains desirable to more accurately assess the impact of APC on health and disease.

The improvement of direct APC measurement requires a ligand that more specifically binds to APC. We have recently identified a ssDNA aptamer that binds APC with high affinity and specificity [17]. In the present study, we used this aptamer as a capture ligand and developed an oligonucleotide-based-enzyme-capture assay (OECA). This assay allows sensitive and fast measurement of plasma APC levels. The combination with a quenching strategy to prevent pre-analytical inactivation of APC established a diagnostic platform that allows expeditious measurement of plasma APC levels under routine clinical conditions. We were able to determine inter-individual differences in trauma-associated APC formation patterns by applying this platform to patients undergoing hip replacement surgery.

Patients, materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgement
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Patients and blood sampling

For measurement of APC and thrombin activation markers during major surgery, blood samples of five patients (three female, age 39–88) undergoing total hip replacement surgery were taken after anesthesia induction, after removal of the patient′s femoral head, after implantation of the artificial joint and after skin closure. This diagnostic study was approved by the local Institutional Review Board and Ethics committee with informed consent obtained in compliance with the declaration of Helsinki. For details on surgery procedures and blood sampling refer to the Supporting Information.

Materials and general methods

A complete list of used materials and descriptions of general methods are provided in Data S1.

Measurement of protein C, prothrombin activation fragments F1.2 and thrombin–antithrombin complexes

PC levels in human plasma were measured using a BCS XP coagulation analyzer and the Berichrom Protein C Kit according to the manufacturer’s instructions (Siemens Healthcare Diagnostics, Marburg, Germany). Plasma levels of thrombin–antithrombin (TAT) complexes and F1.2 were measured using the Enzygnost TAT micro and F1+2 (monoclonal) assays (Siemens Healthcare Diagnostics) according to the manufacturer’s instructions.

Oligonucleotide-based enzyme capture assay (OECA)

The OECA was performed in the microtiter plate format using white Maxisorp Fluoronunc microtiter modules (Nunc; Thermo Fisher Scientific, Roskilde, Denmark). During incubation, wells were generally sealed and stored at room temperature (RT) in the dark. For washing, if not otherwise stated, wells were rinsed three times with 300 μL of washing buffer using an automated plate washer (SLT Columbus, Tecan, Germany).

Microtiter modules were coated with 10 μg mL−1 bovine serum albumin (BSA)-biotin (100 μL well−1) in coating buffer (30 mmol L−1 Na2CO2, 200 mmol L−1 NaHCO3, pH 9.0) at 4 °C overnight. After washing with phosphate-buffered saline (PBS) containing 0.05% Tween 20, 10 μg mL−1 streptavidin in PBS containing 1 mg mL−1 BSA was added to the wells and incubated for 1 h at RT. After washing, the wells were blocked using 200 μL well−1 of blocking buffer (PBS, 20 mg mL−1 BSA, 0.05% Tween 20). After incubation for 2 h at RT, the remains were aspirated and aptamers loaded.

For loading of aptamers into the streptavidin-coated wells, 3′-biotinylated HS02-G52 molecules were diluted in TBS (pH 7.6, 1 mmol L−1 each MgCl2 and CaCl2, 0.05% Tween 20, 1 mg mL−1 BSA) and 100 μL of the solution added to the wells of streptavidin-primed modules and incubated at RT for 1 h. After incubation, the wells were washed with TBS washing buffer (TBS, pH 7.6, 1 mmol L−1 each MgCl2 and CaCl2, 0.05% Tween 20) and samples or calibrators directly added (100 μL well−1) or aptamer-loaded modules stored at 4 °C for up to 8 weeks until used.

Calibrators covering a ½-log10 concentration range from 0 to 10 ng mL−1 of rAPC (0–182 pmol L−1) or pAPC (0–178 pmol L−1) were prepared in the corresponding sample matrices. Thus pAPC-based calibrators for the measurement of plasma APC were prepared in normal pooled plasma (NPP) that was spiked with aprotinin to prevent the inactivation of APC over time (see Results section for details). Before analysis, plasma samples and calibrators were re-calcified by the addition of 1 mol L−1 CaCl2 to yield a final concentration of 7.5 mmol L−1 (Fig. S1), because binding of HS02-52 to APC has been shown to improve in the presence of calcium ions [17]. To prevent clotting of re-calcified plasma or unintended activation of PC by generated thrombin, the potent thrombin inhibitor hirudin was added to plasma samples and calibrators at a final concentration of 20 μg mL−1. Alternatively, hirudin (15 μg mL−1) was added to the anticoagulant buffer used in the blood collection tubes.

After incubation for 2 h at RT, samples and calibrators were removed from the wells using an eight-channel pipette and fresh tips for each column to prevent carry-over contamination during automated washing. Then, 250 μL of TBS-washing buffer were manually added to the wells and the modules washed using the standard TBS-washing procedure. Subsequently, 100 μL of fluorogenic substrate solution (300 μmol L−1 of Pefafluor PCa in TBS, pH 8.5, containing 4 mmol L−1 CaCl2) was added to the wells and baseline fluorescence intensities measured using a plate fluorescence reader (FLx-800; Bio-Tek, Bad Friedrichshall, Germany). Changes in fluorescence over a time period of 4 h were taken as the measure of APC captured in the wells. Data obtained from the calibrators were interpolated using four-parameter logistic curve fit and used to calculate APC concentrations in the samples.

Pre-analytical stability studies

To assess the quenching capacity of the aprotinin-citrate blood sampling buffer, whole blood was spiked with rAPC to achieve a final concentration of 1 ng mL−1. Subsequently, the preparation was split and stored at RT or on ice. Aliquots were removed over time, centrifuged and plasma obtained stored at −40 °C until assayed by OECA. To determine the half-life of APC in the absence of aprotinin, citrate-anticoagulated whole blood was spiked with 1 ng mL−1 rAPC and stored over time as described. Prior to centrifugation, aprotinin was added to sub-sampled aliquots to achieve final concentrations of 10 μmol L−1 aprotinin in order to stop rAPC inactivation. Regarding the half-life of APC in (aprotinin-primed) plasma, a comparable strategy, applying final concentrations of 20 μmol L−1 aprotinin, was followed.

APC and thrombin generation testing in vitro

Thrombin generation in normal and defibrinated human plasma in the presence of increasing concentrations of thrombomodulin was initiated by the addition of tissue factor at a final concentration of 1 pmol L−1 and monitored by calibrated automated thrombography (CAT) using standard reagents and equipment as described elsewhere [18] (Thrombinoscope B.V., Maastricht, The Netherlands).

For OECA-based monitoring of APC generation, a sub-sampling protocol based on the CAT assay reaction mixture was applied. For details see Supporting Information.

Statistical analyzes and validation of assay sensitivity

All measurements were at least performed in duplicate, and data are shown as mean values ± standard deviation (SD) if not otherwise stated. Standard curves for calculation of APC concentrations were interpolated using four-parameter logistic curve fit. Correlation of data sets was assessed by calculation of Pearson’s product-moment correlation coefficient (r). All calculations were done using SigmaPlot 9.0 (Systat, Erkrath, Germany) or Excel 2003 software.

For determination of the limit of detection (LOD) and the lower limit of quantification (LLOQ) of pAPC, hexaplicates of plasma-based calibrators were assayed by OECA and the LOD or LLOQ esteemed as the concentrations that correspond to change in fluorescence of 3 (LOD) or 9 times (LLOQ) the SD of the blanks. Determinations were repeated on different days and calculated mean values (±SD) defined as the LOD and LLOQ of the assay.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgement
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Detection of APC using the OECA configuration

In a first series of experiments, we evaluated basic assay conditions including the loading concentration of the aptamer. Using an APC concentration of 1 ng mL−1, stable hydrolysis rates of the fluorogenic substrate were achieved at aptamer loading concentrations above 2 nmol L−1 (Fig. 1A). Based on these results, an aptamer loading concentration of 5 nmol L−1 has been used in all further experiments. Applying sample and substrate incubation times of 2 and 4 h, respectively, rAPC and pAPC were detectable down to lower picomolar concentrations in buffer. The fluorescence signals obtained with rAPC were significantly higher than those obtained with pAPC (Fig. 1A,B). Plasma-derived APC yielded 45.2% ±4.7% of fluorescence read-out signals when compared with that of rAPC. This discrepancy remained stable at increasing concentrations of aptamers ruling out that this effect is caused by different binding kinetics of rAPC and pAPC to immobilized aptamers (Fig. 1A). To further investigate whether this effect can be explained by differences between both APC preparations we performed additional functional and antigenic analyses. The results summarized in Fig. S2 revealed a higher purity of the rAPC preparation and a higher content of the beta-form in the rAPC preparation indicating differences in the glycosylation pattern between rAPC and pAPC [19–21].

image

Figure 1.  Key data on initial assay development and preliminary validation. (A) Evaluation of aptamer loading concentrations. The biotinylated activated protein C (APC)-aptamer HS02-52G was incubated in the wells of streptavidin-coated microtiter modules at the indicated concentrations and unbound molecules removed. Recombinant APC (unfilled circles) or plasma-derived APC (filled circles) were added at final concentrations of 1 ng mL−1 in buffer and incubated at room temperature (RT). APC captured in the wells was measured through hydrolysis rates of the fluorogenic APC substrate Pyr-Pro-Arg-AMC. (B) A dose-response curve of the APC-oligonucleotide-based enzyme capture assay (OECA) at low APC input concentrations. Recombinant APC (unfilled circles) and plasma-based APC (filled circles) were diluted in buffer yielding the indicated concentrations and analyzed using the optimized APC-OECA. The inlet shows the full range results.

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Aprotinin prevents pre-analytical inactivation of APC in whole blood and plasma

To avoid pre-analytical inactivation of APC by endogenous inhibitors a quenching strategy using a reversible inhibitor is required. The addition of benzamidine to plasma spiked with 1 ng mL−1 pAPC or rAPC increased the recovery rates of APC with a maximum reached at ∼5 mmol L−1. At higher benzamidine concentrations APC detection rates decreased continuously (Fig. 2A). In the presence of aprotinin, maximal recovery rates were achieved at concentrations exceeding 10 μmol L−1. Compared with benzamidine, aprotinin revealed higher recovery rates that also remained stable up to a concentration of 60 μmol L−1 (Fig. 2A). More detailed analysis revealed hampered binding of APC to the immobilized aptamers in the presence of benzamidine but not in the presence of aprotinin (Fig. S3). Based on the results of these experiments, a final aprotinin concentration of 20 μmol L−1 was used for APC detection in plasma. When using whole blood, a final aprotinin concentration of 10 μmol L−1 was found to be sufficient (Fig. S4).

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Figure 2.  Stabilization of plasma activated protein C (APC) activity and evaluation of pre-analytical conditions. (A) Temporary quenching of APC. The reversible protease inhibitors benzamidine (triangles) or aprotinin (boxes) were added to human plasma at the indicated concentrations and mixtures subsequently spiked with plasma-derived APC to achieve final concentrations of 1 ng mL−1. The preservation of APC activity in the samples was assessed using the oligonucleotide-based enzyme capture assay (OECA). The inlet shows the results of an identical experiment performed with recombinant APC. (B) The influence of sample storage conditions on APC recovery rates. Recombinant APC was added to citrate-anticoagulated whole blood in the presence (triangles) or absence (circles) of aprotinin and samples stored at room temperature (RT) (filled symbols) or at 4 °C (unfilled symbols). At the indicated time points, APC plasma levels were measured using OECA.

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Testing of APC quenching efficiency in whole blood by the aprotinin-citrate buffer over time revealed that ∼95% of APC activity was preserved during a 4 h storage time on ice (Fig. 2B). Storage at RT decreased the recovery rate to ∼80%. Similar results were obtained in plasma (Fig. S5). Based on these results, blood to be analyzed for APC was collected into blood sampling tubes containing citrate–aprotinin–hirudin buffer, the tubes were immediately stored at 4 °C after blood sampling, centrifuged within 4 h and plasma samples stored at −40 °C for up to 6 months until assayed.

The plasma matrix only marginally influences the sensitivity of the APC–OECA

To analyze the influence of PC and other plasma components on the sensitivity of the APC–OECA, we compared recovery rates of APC in buffer and serially diluted NPP. In addition, the APC–OECA results were compared with the results obtained using an enzyme capture assay based on polyclonal PC antibodies as capture ligand. As shown in Fig. S6A,B, APC diluted in buffer was detectable at each dilution point independent of the capture ligand used. However, when analyzed in the plasma matrix the antibody-based enzyme capture assay failed to detect APC activity at each dilution point (Fig. S6B), whereas the APC–OECA results were nearly identical to the results obtained in the APC-buffer samples (Fig. S6A).

To further assess the influence of PC on APC–OECA results, plasma samples showing endogenous PC levels ranging from 39% to 150% were spiked with different concentrations of pAPC and assayed using the OECA. As shown in Fig. 3A, the recovery rates of pAPC, even at concentrations matching the LLOQ of the OECA, were only marginally influenced by the underlying PC concentrations.

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Figure 3.  Performance characteristics of the activated protein C (APC)–oligonucleotide-based enzyme capture assay (OECA) on the quantification of plasma APC. (A) The influence of pro-enzyme concentrations on assay outcome. To study the influence of protein C on OECA results, aprotinin-primed plasma samples containing the indicated concentrations of endogenous protein C were spiked with increasing concentrations of plasma-derived APC (closed circles: 0.1 ng mL−1, open circles: 0.5 ng mL−1, filled triangles: 8 ng mL−1) and recovery rates measured using the APC–OECA. The horizontal dashed and dotted lines represent the assay limit of detection (LOD), and the lower limit of quantification (LLOQ), respectively. (B) Sensitivity of the APC–OECA. The lower concentration range of a typical standard curve obtained with plasma APC calibrators is shown. The vertical dashed and dotted lines represent the LOD and the LLOQ of the assay, respectively. The inlet shows the full range of the standard curve. Data points were interpolated using a four-parameter logistic curve fit.

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To study to what extend the basal level of endogenous APC and PC influences APC–OECA results, APC recovery rates were analyzed in APC-/PC-depleted plasma (PCdp). As shown in Fig. S7A,B, NPP yielded slightly higher background values in comparison to buffer or PCdp at APC concentrations below 0.1 ng mL−1. On the basis of the PCdp- or buffer-derived data points, APC levels in the low (≤ 25) pg mL−1 range were calculated for NPP. Interestingly, extension of the substrate incubation times increased the hydrolysis rate of the APC substrate in the NPP samples to a higher extend than in the PCdp- or buffer-samples (Fig. S7C).

The APC–OECA allows sensitive and reproducible measurement of APC plasma levels

To assess the quantitative performance of the APC–OECA, we measured the limit of detection (LOD), the lower limit of quantification (LLOQ) and the reproducibility of the APC–OECA using dilution series of pAPC in NPP. At this, the APC–OECA showed a dynamic range from 0 to 10 ng mL−1. The LOD was determined as 22 ± 5.5 pg mL−1 (0.39 ± 0.10 pmol L−1) whereas the LLOQ was calculated as 116 ± 55 pg mL−1 (2.06 ± 0.98 pmol L−1) (Fig. 3B).

To assess the reproducibility of the APC–OECA, plasma samples containing pAPC at concentrations ranging from 0.5 to 5 ng mL−1 were assayed in triplicates in a single run repeated on 3 days. As shown in Table S1, within- and between-run coefficients of variation did not exceed 17% even at the lowest concentrations tested.

In vitro generation of APC starts during the initiation phase of thrombin generation

To study whether the APC–OECA sensitively reflects changes in plasma levels of APC, we next monitored APC generation in human plasma. Endogenous formation of APC was induced by TF-triggered blood coagulation in the presence of TM. The CAT results showed a concentration-dependent decrease in the maximal levels of thrombin generation at TM concentrations above 0.25 U mL−1, whereas lower TM concentrations did not significantly change the thrombin generation profiles (Fig. 4A).

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Figure 4. In vitro plasma thrombin and activated protein C (APC) generation profiles. (A) Thrombin generation profiles measured using calibrated automated thrombography (CAT). Normal pooled plasma (NPP) was spiked with the indicated concentrations of thrombomodulin and thrombin generation triggered by 1 pmol L−1 tissue factor. The inlet shows the results of an identical experiment performed with defibrinated plasma. (B) The measurement of plasma APC levels during tissue factor-driven thrombin generation. Defibrinated plasma was spiked with different concentrations of thrombomodulin (circles, 2 U mL−1; boxes, 0.125 U mL−1; diamonds, 0 U mL−1) and thrombin generation triggered by 1 pmol L−1 tissue factor. Reactions were manually stopped at the indicated time points and plasma APC levels measured using the oligonucleotide-based enzyme capture assay (OECA). The inlet shows the same data on the log scale, demonstrating the detection of early APC generation within the lag-time of CAT (A). The horizontal dashed and dotted lines represent the assay-specific limit of detection and the lower limit of quantification, respectively.

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Direct monitoring of APC formation using the APC–OECA revealed a TM-dependent increase in APC levels (Fig. 4B). In contrast to the CAT method, direct APC measurement identified APC levels even at low TM concentrations. Low levels of APC formation were detectable even in the absence of exogenously added TM. This APC formation might be triggered by soluble TM or by TM-independent cleavage of PC. In general, these results demonstrate the high sensitivity of the APC–OECA to measure plasma levels of APC.

Plasma levels of APC significantly increase during hip replacement surgery but show a high degree of interindividual variability

Among 20 healthy individuals tested, plasma levels of APC were detectable in five individuals but fell short of the LLOQ of the assay. In the remaining 15 individuals, plasma levels of APC were found to be below the LOD (Fig. 5A). Simultaneously, measurement of plasma levels of TAT complexes identified five patients with values above the LLOQ of the TAT-ELISA (Fig. 5B). Among them, three also showed enhanced values in the APC–OECA. The TAT levels of the remaining 15 individuals were below the LLOQ.

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Figure 5.  Plasma levels of activated protein C (APC) and thrombin–antithrombin (TAT) complexes in healthy individuals and patients undergoing hip-replacement surgery. Plasma samples were obtained from 20 healthy individuals as well as five patients undergoing hip-replacement surgery and analyzed for APC (A, C) and TAT complexes (B, D). During hip-replacement surgery, blood samples were taken after anesthesia induction, after removal of the patient′s femoral head, after implantation of the artificial joint and after skin closure. Determined values are shown in this order from the left to the right. The horizontal dashed, dotted and solid lines represent the limit of detection, the lower limits of quantification and the upper normal range, respectively.

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During hip-replacement surgery, plasma levels of TAT increased up to 200-fold when compared with the upper normal range of this parameter reaching peak values in blood samples obtained at the end of the operation (Fig. 5D). The time course of the APC plasma levels showed a nearly identical pattern with peak values of 12 ng mL−1, corresponding to ∼200 pmol L−1 (Fig. 5C).

There was only a moderate overall correlation (r = 0.575) between the APC levels and the TAT levels in spite of similar half-life times of these markers within the circulation (Fig. 6A). Studying the correlations on a more individualized level by grouping the data sets of individual patients, the patient-specific correlations increased and showed a range from 0.761 to 0.993, although the mean levels of APC and TAT confirmed the overall poor correlation between the amount of thrombin generated and the amount of APC generation (Fig. 6B). Similar results were obtained when comparing mean levels of APC with that of circulating prothrombin activation peptide F1.2 (Fig. S8).

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Figure 6.  Correlation between plasma levels of activated protein C (APC) and thrombin–antithrombin complexes (TAT). (A) The overall correlation between measured APC and TAT plasma levels. The dotted horizontal and vertical lines represent the lower limit of quantification of the APC and the TAT assay, respectively. (B) Means and correlations of TAT and APC levels determined in the plasma samples obtained during individual surgeries. The error bars represent the min–max range of quantifiable values whereas the ovals demarcate individual patient clusters only for illustration purposes without stressing any statistical interpretations. The mean correlation of intra-operative values (r = 0.877 ± 0.102) and the correlation of cluster means (r = 0.507) are also graphically shown in the inlet.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgement
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Using the recently identified and well-characterized APC-targeting ssDNA aptamer HS02-52G, we developed an enzyme capture assay that allows highly sensitive determination of APC in human blood. As binding of the aptamer does not block the active center of APC, aptamer-captured APC can be easily quantified by hydrolysis rates of a sensitive fluorogenic peptide substrate. The use of this substrate, in conjunction with the specific binding of HS02-52G to APC, accounts for the unrivaled sensitivity and speed of the APC–OECA when compared with previously published assays for direct APC measurement [7–9]. Total assay time for the detection of subnanogram concentrations, including all incubation steps, is now reduced to < 7 h.

Using filter retention analysis, we previously demonstrated that HS02-52G binds with high selectivity and similar affinity to rAPC and pAPC [17]. Thus, both forms of APC were used for initial validation of the APC–OECA. At identical concentrations of both APC species, consistently higher values were measured for rAPC probably as a result of its higher purity as well as its different glycosylation pattern. Thus, to avoid underestimation of APC plasma levels, we decided to use calibrators based on pAPC. Using these standards, the LOD and LLOQ of the assay were calculated to be ∼0.02 ng mL−1 (0.4 pmol L−1) and ∼0.12 ng mL−1 (2 pmol L−1), respectively. However, such assay parameters strongly depend on the specific activities of the APC-preparations used. For example, using rAPC (Xigris®, Eli Lilly, Indianapolis, IN, USA), the assay LOD and LLOQ were found to be ∼0.01 ng mL−1 (0.2 pmol L−1) and ∼0.03 ng mL−1 (0.6 pmol L−1), respectively (data not shown). This emphasizes the need for an APC-reference preparation to make APC testing similar between different methods and laboratories.

Although the plasma half-life of APC of ∼15–20 min is relatively long, endogenous inhibitors such as the serpin PCI continuously inactivate APC. A quenching strategy is therefore required that preserves APC plasma levels until analyzed. As inhibition by endogenous serpins depends on the amidolytic activity of their target enzymes [22], temporary quenching of APCs active site activity is a feasible strategy to preserve levels of circulating APC after blood sampling. The inhibitor to be used should not interfere with the binding of APC to the aptamer and should be completely dissociable after binding of APC to the aptamer. The broad-spectrum inhibitor benzamidine has been introduced as a quenching reagent in APC testing by Gruber et al. [7]. However, vigorous washing of wells is needed to efficiently restore the amidolytic activity of captured APC [7,9]. A possible alternative to benzamidine is aprotinin. This kunitz-type inhibitor has been shown to reversibly bind to APC thereby blocking the active site with a Ki of 1.35 μmol L−1 [23,24]. When comparing the efficacy of benzamidine with that of aprotinin, we found aprotinin superior in terms of recovery rates and the range of the optimal concentration.

To study, if the APC–OECA reflects changes in APC plasma levels, we measured APC generation over time in TF-activated plasma in the presence of TM. A correlation between the TM concentration and the amount of APC formed was observed. Using the APC–OECA, APC formation was measurable even at TM concentrations that revealed no effect on the CAT assay. Moreover, we were able to show that formation of APC becomes detectable early during the lag-phase of CAT, underlining the high sensitivity of the APC–OECA. Interestingly, APC formation was also measurable in the absence of exogenously added TM. Thus, although further studies are needed for confirmation, it is tempting to speculate that the measurement of in vitro APC generation might allow sensitive detection of functional active soluble TM.

In spite of the high sensitivity of the APC–OECA, we were not able to establish a reference range for APC in healthy individuals, as none of the plasma samples showed APC levels above the LLOQ and only five individuals showed plasma levels above the LOD of the assay. These data are in contrast to previous studies showing APC mean levels ranging from 1 to ∼3 ng mL−1 in healthy individuals [7–9,11]. Several factors and methodical aspects might explain these differences. For example, none of the two previously described plate-based enzyme-capture-assays were run with calibrators that consistently reflect the sample plasma-matrix at each dilution point, potentially leading to overestimation of APC levels as a result of unspecific conversion of substrate by accompanying plasma components (Fig. S7) [7,9]. In addition, these assays require unusual long substrate incubation times of more than 16 h. During such prolonged substrate incubation times, cleavage of the chromogenic substrate by proteases might induce a wrong positive assay result (Fig. S7C).

It should also be mentioned that the validation of none of the previously published assays for either direct [7–9] or indirect [11] APC plasma measurements included the determination of the LOD or LLOQ within the sample plasma matrix. Thus, based on our results, we conclude that APC circulates at concentrations around or even below 0.1 ng mL−1 (1.8 pmol L−1) under physiological conditions.

To study the correlation between thrombin formation and APC generation in vivo, we analyzed blood samples obtained from patients during hip-replacement surgery. Parallel to the increased thrombin formation, an increase in plasma levels of circulating APC was detectable reaching peak values of APC of 12 ng mL−1. However, when normalized to the amount of thrombin generated, there was a high degree of interindividual variability in the APC generating capacity. Although these results need to be confirmed in larger studies, they demonstrate that simultaneous measurements of a biomarker indicating thrombin formation such as TAT and APC plasma levels should enable the measurements of changes in the endothelial cell-determined anticoagulant phenotype.

Taken together, the APC–OECA platform allows quantification of APC under routine clinical conditions. Simultaneously quantification of thrombin formation will help to study patients’ APC generating capacity and may help quantifying endothelial cell dysfunction. Further clinical and diagnostic studies are required to understand the diagnostic impact of APC testing and to establish APC-based treatment regimens.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgement
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Contributions of the individual authors are as follows: J. Müller, G. Mayer, and B. Pötzsch designed the research and wrote the manuscript. J. Müller, M. Friedrich, T. Becher, J. Braunstein, F. Rohrbach and T. Kupper performed experiments and analyzed data; P. Berdel and S. Gravius took care of the patients, performed the operations and reviewed the manuscript. J. Oldenburg contributed to writing and reviewing of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgement
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgement
  9. Disclosure of Conflict of Interests
  10. References
  11. Supporting Information

Figure S1. Typical full MALDI-TOF    MS spectrum of peptide library substrate and products.

Figure S2. Fractional representation of peptide sequences in libraries.

Figure S3. Kinetic analysis of the effect of ethylene glycol on FIXa reactivity.

Figure S4. Fractional reactivities of sequences in response to ethylene glycol.

Figure S5. Fractional reactivities of sequences in response to Ca2+.

Figure S6. Fractional reactivities of sequences in response to LMWH.

Table S1. Predicted monoisotopic masses of peptides and hydrolysis products for the AT3.2-P3 library (Ac-XGRSL-Am).

Table S2. Predicted monoisotopic masses of peptides and hydrolysis products for the AT3.2-P2 library (Ac-AXRSL-Am).

Table S3. Predicted monoisotopic masses of peptides and hydrolysis products for the AT3.2-P1′ library (Ac-AGRXL-Am).

Table S4. Predicted monoisotopic masses of peptides and hydrolysis products for the AT3.2-P2′ library (Ac-AGRSX-Am).

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JTH_4623_sm_FigS3.pdf1236KSupporting info item
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