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. Author manuscript; available in PMC: 2024 Jan 1.
Published in final edited form as: Methods Mol Biol. 2023;2709:319–332. doi: 10.1007/978-1-0716-3417-2_23

Anticoagulant Activity of Nucleic Acid Nanoparticles (NANPs) Assessed by Thrombin Generation Dynamics on a Fully Automated System

Renata de Freitas Saito 1, Bárbara Gomes Barion 2, Tania Rubia Flores da Rocha 2, Alex Rolband 3, Kirill A Afonin 3, Roger Chammas 4
PMCID: PMC10482313  NIHMSID: NIHMS1925783  PMID: 37572292

Abstract

Rapidly reversible anticoagulant agents have great clinical potential. Oligonucleotide-based anticoagulant agents are uniquely positioned to fill this clinical niche, as they are able to be deactivated through the introduction of the reverse complement oligo. Once the therapeutic and the antidote oligos meet in solution, they are able to undergo isothermal reassociation to form short, inactive, duplexes that are rapidly secreted via filtration by the kidneys. The formation of the duplexes interrupts the structure of the anticoagulant oligo, allowing normal coagulation to be restored. To effectively assess these new anticoagulants, a variety of methods may be employed. The measurement of thrombin generation (TG) reflects the overall capacity of plasma to produce active thrombin and provides a strong contribution to identifying new anticoagulant drugs, including DNA/RNA thrombin binding aptamer carrying fibers which are used through this chapter as an example. Here we describe the TG assessed by Calibrated Automated Thrombogram (CAT) assay in a fully automated system. This method is based on the detection of TG in plasma samples by measuring fluorescent signals released from a quenched fluorogenic thrombin substrate and the subsequent conversion of these signals in TG curves.

Keywords: Thrombin, Thrombin generation, Nucleic acid nanoparticles, Aptamer, Fiber, Blood coagulation tests, ST Genesia

1. Introduction

For more than 50 years, heparin and vitamin K antagonists (VKAs) were the only anticoagulant therapeutic option to treat and prevent thrombotic diseases [1]. Although effective, these drugs have a variable anticoagulant response and limitations, including multiple drug and food interactions. The low therapeutic index of these drugs, which have multiple targets in the coagulation pathway, prompted the development of a new oral anticoagulant with selective targets, the direct oral anticoagulants (DOACs) (Fig. 1) [2]. They include synthetic small molecules which selectively inhibit the enzymatic activity of factors IIa or Xa, with predictable pharmacokinetic/pharmacodynamic profiles, limited drug or food interactions, and lower risk of bleeding [3]. Although clinical evidence demonstrates that DOACs are at least as effective and safe as the long-standing standards of anticoagulant drugs, and in some cases, even more, effective and safer, there are still some limitations implicated in their clinical use [3]. Certain patient-specific comorbidities, including renal insufficiency, hepatic impairment, and extreme body weight, affect the appropriate efficacy and safety of DOACs. Different from VKA which requires continuous laboratory monitoring to prevent adverse outcomes and allows dose adjustment, DOACs are used in a fixed dose without plasma level assessment. Although it represents a reduction in laboratory monitoring tests, DOACs are substantially more expensive than VKA. While DOACs have changed the paradigm for the treatment of venous thromboembolism (VTE), they should be used with caution in patients with comorbidities listed above, and the lack of specific antidotes makes challenging the reversal of bleeding complications [4]. As such, the development of new and safer anticoagulant strategies is of mounting importance.

Fig. 1.

Fig. 1

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In vitro coagulation cascade: pathways, targets of anticoagulant drugs, and coagulations tests. Prothrombin Time (PT), Activated Partial Thromboplastin Time (aPTT), Thrombin Time (TT), thrombin generation (TG), LMWH (Lower Molecular Weight Heparin) and direct oral anticoagulants (DOACs). Created with BioRender.com

Nucleic acid nanoparticles (NANPs) have emerged as a promising approach to designing new anticoagulants. Several aptamers, single-stranded oligonucleotides selected in vitro for high affinity binding to target molecules, have been developed to target coagulation cascade proteins [5]. These aptamers can be conditionally deactivated by complementary sequences, producing low-molecular-weight assemblies that undergo rapid renal excretion [6]. Despite encouraging preclinical results, no aptamer targeting blood coagulation factors have been approved for clinical use mainly due to their short post-infusion half-lives. To address this limitation, we designed a dynamic platform based on RNA/DNA fibers containing multiple thrombin-binding aptamers as a strategy to increase their molecular weight, prolong blood stability, and increase retention time in vivo [6]. One of the key issues with ARC183 and an adapted formulation, NU172, has been the rapid removal from circulation through the urinary system [7, 8]. The RNA/DNA fibers used as the example in this text were designed to carry multiple copies of NU172, which was added as an extension to the constituent DNA oligos entering the structure of RNA/DNA fibers [9-12]. Thrombin is a particularly relevant target for anticoagulants, as both the intrinsic and extrinsic coagulation pathways converge to thrombin, which serves as the start of the common pathway [13]. ARC183, also referred to as HD1, inhibits thrombin activity by binding to exosite 1 on its surface, which is responsible for thrombin’s recognition of fibrinogen [10, 13]. Attaching the aptamers to a fibrous scaffold provides several advantages. Through the increased size and molecular weight of the fiber NANPs, the circulation time is increased. The facile assembly of the fibers, as well as their self-assembly through canonical Watson-Crick base pairing, offers excellent batch-to-batch consistency. The fibers also maintain the ability to be readily deactivated by introducing a reverse complement structure that hybridizes with the anticoagulant fiber [11].

We demonstrated the anticoagulant effect of these NANPs in vitro by tests routinely used in the clinic to evaluate hemostasis, such as Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), and Thrombin Time (TT) [14]. These clinical tests of coagulation evaluate the rate of clot formation from the time that the coagulation cascade is activated through increased turbidity or decreased optical clarity, based on the instrumentation used. In the PT test, calcium chloride is used as initiating coagulation substance to restore the calcium required for coagulation and effectively replaces the calcium that was bound by sodium citrate present in the blood collector tube. Partial thromboplastin, which is a contact activator, and a platelet phospholipid substitute are the APTT coagulation initiation reagents. While in PT and APTT tests plasma is exposed to substances initiating coagulation via the extrinsic and intrinsic clotting pathway, respectively, the TT test monitors clot formation of plasma in the presence of dilute thrombin to measure the conversion of fibrinogen to fibrin, the final common pathway of the coagulation cascade. These methods are indirect forms to measure thrombin activity which rely on the static measurements of single end-point values. The monitoring of thrombin generation (TG) is a direct manner to address the targeting of anti-thrombin aptamers. Additionally, TG assays based on fluorogenic substrates overcome absorbance interference caused by the formation of fibrin clots, allowing for the detection of thrombin activity [15].

Here, we describe a methodology for monitoring the effect of anti-thrombin NANPs on thrombin generation (TG) performed by a fully automated analyzer. This method is based on calibrated automated thrombography (CAT), a global coagulation assay that measures thrombin generation in plasma [16]. The methodological principle encompasses recording fluorescent signals as a function of time, resulting from the cleavage of a quenched fluorogenic substrate by thrombin, after adding a standard amount of human recombinant tissue factor and synthetic phospholipids to the test plasma (Fig. 3). Thrombin amount is estimated from the change in fluorescence signal in the test plasma compared to the same plasma containing a known concentration of thrombin.

Fig. 3.

Fig. 3

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Measurement principle of TG generation by a fluorimetric assay. To initiate TG a mixture of recombinant tissue factor (TF) and procoagulant phospholipids (PPL) are added to the plasma sample. After incubation at 37 °C, a fluorogenic substrate (Z-Gly-Gly-Arg-AMC) is added along with calcium chloride (CaCl2) to the plasma (Fig. 3). The molecule Z-Gly-Gly-Arg-AMC contains a highly fluorescent 7-amino-4-methyl coumarin (AMC) group that is quenched by the amide bond formed between its amino group and the carboxyl group of the Arg residue. Thrombin is capable of cleaving this amide bond, and free AMC is excited at 377 nm light producing measured fluorescence at approximately 440 nm. AMC generation is monitored every 15–20 seconds and mirrors the kinetic of thrombin generation. All measurements are carried out in duplicate, and final results are reported as the mean of the two duplicate measures. Created with BioRender.com

2. Materials

All reagents are commercially available by Stago, and the entire study needs to be performed with the same lot of reagents.

2.1. Anticoagulant and Antidote Fiber NANP Preparation

  1. Chemically synthesized oligonucleotides.

  2. Endotoxin-free (ET-free) ultra-pure water.

  3. Hybridization buffer (89 mM Tris, 80 mM boric acid (pH 8.2), 2 mM magnesium chloride, 2 mM potassium chloride).

  4. Heat bath set to 95 °C.

  5. Pipettes ranging from 0.5 μL to 1 mL.

  6. Sterile, RNAse- and DNAse-free pipette tips.

  7. Vortexer.

  8. Microcentrifuge.

  9. 1.5 mL microcentrifuge tubes.

2.2. Blood Collection and Plasma Preparation

  1. Vacuum blood collection tubes filled with a buffered sodium citrate solution (3.2%), Light Blue-Top (see Notes 1 and 2).

  2. Safety needles (22 g or less) and butterfly needles (21 g or less).

  3. Tourniquets, antiseptic, gauze, and bandages or tape.

  4. Sharps disposal container.

  5. Micropipettes, tips, and 15 mL tubes.

  6. Microcentrifuge.

2.3. Calibration

  1. STG®-ThrombiCal. Reconstitute 1 vial with 2 mL of distilled water, and allow the reagent to stand at room temperature (18–25 °C) for 30 min before use. Then, swirl and invert the vial properly to obtain a homogeneous solution.

  2. STG®-FluoStart. Allow the reagent to stand at room temperature (18–25 °C) for 30 min before use (see Note 3).

  3. STG®-FluoSet. Allow the reagent to stand at room temperature (18–25 °C) for 30 min before use (see Note 3).

  4. STG®-ThrombiClean. Ready to use (see Note 4).

2.4. Quality Controls

  1. STG®-RefPlasma DS. Reconstitute 1 vial with 1 mL of distilled water, and allow the reagent to stand at room temperature (18–25 °C) for 30 min before use (see Note 5).

  2. STG®-QualiTest Norm DS. Reconstitute 1 vial with 1 mL of distilled water, and allow the reagent to stand at room temperature (18–25 °C) for 30 min before use (see Note 5).

  3. STG®-QualiTest Low DS. Reconstitute 1 vial with 1 mL of distilled water, and allow the reagent to stand at room temperature (18–25 °C) for 30 min before use (see Note 4).

  4. STG®-DrugScreen (see Notes 3 and 5).

2.5. End-of-Day Procedure

  1. STG®-ThrombiClean. Ready to use.

3. Methods

3.1. Anticoagulant and Antidote Fiber Preparation

  1. Mix the monomer DNA and RNA units that are appropriate for the structure being assembled at their appropriate concentrations with hybridization buffer in a 1.5 mL microcentrifuge tube.

  2. Thoroughly vortex the solution.

  3. Centrifuge briefly to collect the liquid at the bottom of the tube.

  4. Heat the solution to 95 °C for 5 min.

  5. Remove the solution and allow it to cool to room temperature for a minimum of 20 min.

  6. Store the solution at 4 °C until just prior to use.

3.2. Blood Collection and Plasma Preparation

  1. Draw blood by direct venipuncture into a citrate anticoagulated tube from at least ten health volunteers after full informed consent in accordance with the declaration of Helsinki.

  2. Tubes should be mixed by gentle inversion immediately after drawing to assure that the sodium citrate is homogeneously dispersed.

  3. Centrifuge citrated tubes at 3000 × g for 20 min at room temperature to obtain the platelet-poor plasma (PPP) (see Note 6).

  4. As soon as centrifugation is complete, collect the supernatant avoiding the platelets at the top surface and those at the buffy coat. This is a critical preanalytical step; thus, samples should be processed as quickly as possible, ideally within 1 h of collection.

  5. Pool together the plasma, make aliquots, and immediately store them at −80 °C until use. Coagulation proteins are stable for a minimum of 2 years at −80 °C.

3.3. Turning the Analyzer on and Priming the System at Startup

  1. Turn on the ST Genesia® and activate the ST Genesia® interface (Fig. 2).

  2. Access the Service Maintenance screen.

  3. Select “Arm1” and then “Global” and “Needle purge.” The procedure lasts approximately 2 min.

  4. After completion, restart the system to quit the Service Maintenance mode (see Note 7).

  5. Load cuvettes.

Fig. 2.

Fig. 2

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Equipment home screen. Circles mean the tube’s position in each tray (S1,S2,R1, and R3). The upper right lists the number of cuvettes inserted in the equipment, and the lower right shows the cuvette bin. The lower left indicates the level of cleaning solution in the tank, and the upper left shows the number of totals, running, and remaining tests

3.4. Calibration

  1. Scan reagents’ barcodes in the “QC/Calibration” menu.

  2. Load STG®-FluoStart and in R3.

  3. When the STG®-ThrombiCal is stabilized, load it and the STG®-ThrombiClean in R1 or R2 and press Go.

  4. The calibration is run automatically and lasts STG®-FluoSet approximately 30 min.

  5. When the calibration is completed, you must check if all parameters are in the expected ranges (see Note 8) (Table 1).

Table 1.

Composition of commercially available STG®-Genesia kits and their clinical use

Kits/reagents Type of
samples		 Composition Concentration Clinical use
STG®- Cal & flu – STG®-FluoStart
STG®-FluoSet
STG®-ThrombiCal		 Fixed amount of thrombin
Fluorogenic substrate+ Ca2+
Fixed amount of AMC		 Bleeding risk evaluation
Thrombotic risk evaluation
Effect of antithrombotic drugs
STG®-ThrombiClean – STG®-ThrombiClean Descontamination solution –
STG®-BleedScreen PPP Tissue factor phospholipids Low concentration of TF Severe haemophiliacsA and B Haemorrhage risk evaluation
STG®-ThromboScreen PPP Tissue factor phospholipids +/− Thrombomodulin Intermediate concentration of TF Thrombotic risk evaluation
STG®-DrugScreen PPP Tissue factor phospholipids High concentration of TF Effect of antithrombotic drugs (NOACs/DOACs, LMWH, UFH or VKA)
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3.5. Quality Controls

  1. When the reagents are stabilized, load them in R1 or R2.

  2. Press Go. The quality controls and reference plasma are run automatically if the calibration of the day is valid.

  3. When QCs and reference plasma are completed, check if the parameters are in the expected ranges (see Note 9).

3.6. Samples

  1. Pipette the plasma in 1.5 mL tubes, and add the antithrombin nanoparticles (NU172 fiber) or nanoparticle control in different concentrations (for antithrombin nanoparticles’ synthesis, see Chap. 20). Ensure your samples contain a minimum of 500 μL of material to be tested (see Notes 9 and 10).

  2. Load the 1.5 mL tubes to be tested on rack S1 or S2 (see Note 11).

  3. Identify the sample on the software.

  4. Press “Add/Delete,” check the “DrugScreen” test, and press “Save.”

  5. All samples are measured in duplicates, and the fluorogenic assay requires between 50 and 120 min be completed.

  6. After the raw data are collected, the TG parameters are normalized based on reference plasma (STG®-RefPlasma DS), and mathematical calculations convert these data into thrombograms (see Note 12).

  7. Thrombin generation parameters obtained from the thrombogram are lag time (initiation phase of coagulation), endogenous thrombin potential (ETP; area under the curve), peak height (PH; maximum level of thrombin generated), time to peak, and velocity index (rate of thrombin generation until the PH) (Figs. 4 and 5).

Fig. 4.

Fig. 4

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Thrombogram outlining major parameters of the reaction curve. By taking the integral (dx/dt) of the relative change in fluorescence emitted by AMC, it is possible to determine the Endogenous Thrombin Potential or ETP (nMxmin), which corresponds to the area under the curve (AUC). From the resulting curve, it is possible to determine lag time (min), the time it takes for fibrin clot formation; time to peak (min), the amount of time it takes from the start of the assay to reach the maximal rate of thrombin generation; the velocity index (nM/min), the speed to reach the peak; and peak height (nM), the maximal amount of thrombin generated. Created with BioRender.com

Fig. 5.

Fig. 5

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Thrombin generation curves in the presence of anti-thrombin nanoparticles (NU172 aptamer)

3.7. End-of-Day Procedure

  1. Load the STG®-ThrombiClean in R1 or R2.

  2. Access the Service Maintenance screen.

  3. Go to “Robotic” and select “Needle decontamination.”

  4. Wait until the completion of the cleaning procedure, which lasts approximately 15 min.

  5. After completion, shut down the system.

4. Notes

  1. Tubes with different additives are used for collecting blood for specific types of tests. For coagulation studies, blood should be collected in tubes containing sodium citrate 3.2%, which acts as an anticoagulant by chelating calcium and confers more stability for factors V and VIII. To avoid preanalytical variability, blood collection needs to be standardized by always using identical tubes for brand and draw volume [17].

  2. As with all hemostasis tests, thrombin generation assay is highly vulnerable to preanalytical variables, which may ultimately influence the final quality of test results. Although TG fully automated devices reduce variability by offering thermostability, such devices do not overcome variability caused by preanalytical steps. Thus, it is critical to follow the recommendations of the Scientific and Standardization Committee (SSC) of the International Society of Thrombosis and Hemostasis (ISTH) regarding preanalytical variables such as the procedures used for drawing blood and for transportation of samples and their preparation and storage to reduce assay imprecision [18].

  3. 1 vial of STG®-FluoStart, STG®-FluoSet, and STG®-DrugScreen can be used for a series of 8 samples, but if you pool together 2 vials, it can be used for a series of up to 20 samples.

  4. ThrombiClean after opening is stable for 4 h on ST Genesia® analyzer.

  5. Once reconstituted, STG®-DrugScreen is stable for 8 h on ST Genesia® analyzer, while STG®-RefPlasma DS, STG®-QualiTest Norm DS, and STG®-QualiTest Low DS are stable for 4 h.

  6. TG is analyzed in STG®-Genesia using three different sets of reagents balanced for sensitivity to procoagulant and anticoagulant protein deficiencies (STG-ThromboScreen, STG-BleedScreen, and STG-DrugScreen) (Table 1). For assessing the effect of anticoagulants, the STG-DrugScreen is the adequate set of reagents.

  7. Approximately 4 mL of PPP can be obtained from 10 mL of venous blood.

  8. When the equipment is not used on a day-to-day basis, it is highly recommended to perform the priming procedure at start-up to prevent any air bubbles in the line.

  9. Calibration is performed by measuring the fluorescence derived from a known concentration of thrombin (STG®-ThrombiCal) and fluorogenic substrate (STG®-FluoSet) in a buffer. A known concentration of thrombin solution (STG-ThrombiCal) is divided into two subsamples: to one subsample, fluorogenic substrate (STG-FluoStart) is added and fluorescence is measured in time until all substrate is converted, and to the other subsample, a fixed amount of AMC (STG-FluoSet) is added, and fluorescence is measured for a few minutes. The thrombin generation in the test sample will be generated as a function of time by comparing the fluorescent signal from the thrombin-generating test sample to that from a known stable concentration of thrombin activity measured simultaneously in parallel. Both data are combined by the software of ST Genesia and through a mathematical calculation; the thrombin concentration is displayed in real time during the experiment. This measurement of thrombin solution is performed only once before the start of the measurements in the plasma and allows the correction of the inner filter effect and substrate consumption. As the concentration of fluorophore increases, there is a nonlinear increase in the fluorescence quenching, which occurs when the fluorophore absorbs light generated from fluorescence. In parallel, the substrate concentration is continuously decreasing as it is being consumed by the thrombin generated in the PPP. These two factors combine to cause a rapid decrease in the rate of change (dF/dt) in the overall fluorescence after the initial “burst” of fluorescence [16].

  10. As an internal quality control, two quality controls, one for low (STG®-QualiTest Low DS) and the other for normal (STG®-QualiTest Norm) TG potential, are used to evaluate the precision and accuracy of the reagent-analyzer combination.

  11. Frozen plasma should be thawed at 37 °C before testing. It is not recommended to use plasma that has been freeze-thawed more than once.

  12. Samples with hemolysis, icteric indices, and/or lipemia should not be used because they may impair the quality and reliability of test results. Hemolysis can be avoided at the time of blood collection by using the widest needle available, but icteric indices and lipemia are characteristics found in some blood samples. This can be observed after centrifugation, and if detected, the samples must be discarded. The remaining platelets and/or white cells may lead to an incorrect estimation of TG [19].

  13. For TG by chromogenic detection, blood samples must be cleared of cells and then defibrinated to reduce the background absorption, compromising the physiological status of these samples. The use of florigenic substrates to TG overcomes this limitation, and samples do not need to be pretreated to make them suitable for analysis. This method can be used to analyze thrombin generation in fibrin-containing lyophilized plasma, platelet-poor plasma, platelet-rich plasma, and even whole blood.

  14. The cap of the 1.5 mL tubes should be cut before being loaded into the rack.

  15. Each TG parameter is normalized based on reference plasma for each test to reduce the interlaboratory variability as well as inter- and intra-assay variability.

  16. A2-macroglobulin competes with anti-thrombin to halt the thrombin generation but still allows thrombin to cleave the florigenic substrate. This means that during the reaction the substrate conversion will continue although all thrombin went back to zero. The correction of α2-macroglobulin activity is automatically subtracted by the software [20, 21].

Acknowledgments

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM139587 (to K.A.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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