Monitoring of reversal agents for anticoagulants

Abstract

The invention relates to pharmaceutical agents in the field of hemostasis. The invention allows monitoring of anticoagulant reversal agents. The invention further provides for precise dosing of such reversal agents.

Description

[0001] Monitoring of reversal agents for anticoagulants

[0002] Field of the invention

[0003] The invention relates to pharmaceutical agents in the field of hemostasis. The invention allows monitoring of anticoagulant reversal agents. The invention further provides for precise dosing of such reversal agents.

[0004] Background art

[0005] Hemostasis is the biological process that prevents and repairs blood loss in a damaged blood vessel. This process involves various stages, including coagulation, when clot formation is stabilized by the formation of a fibrin network. Abnormalities in the coagulation process can lead to medical conditions such as hemophilia and thrombosis. In thrombosis, a blood vessel is occluded by a blood clot, potentially resulting in heart or brain infarctions. Patients who have suffered an infarction can be treated with direct oral anticoagulants (DOAC) to reduce blood clotting and prevent recurrence.

[0006] The use of DOACs can be accompanied with adverse effects, the most common of which is an increased risk of bleeding, comprising both nonmajor and major bleeding events. A patient under the effect of DOACs who requires direct management of bleeding can be administered so- called reversal agents, which act as antidotes for the DOACs. The product information for DOACs includes guidance on the management of bleeds and bleeding complications. Specific reversal agents are available for several DOACs such as for dabigatran (for example praxbind, also known as idarucizumab) and for apixaban and rivaroxaban (for example andexanet alfa).

[0007] Dosing of these reversal agents is rather coarse, where for instance idarucizumab is dosed at 5 gram and Andexanet alfa is dosed at “low” or “high” doses (see Ana Marco-Rico, in “anticoagulation - an update”, 2023, DOI: 10.5772 / intechopen.112079). It has been suggested that calibrated quantitative anti-Factor Xa (anti-FXa) assays may help to inform clinical decisions about use of apixaban, edoxaban, or rivaroxaban, for example in overdose and emergency surgery.

[0008] However, the general advice of official bodies, such as of the UK Medicines & Healthcare products Regulatory Agency, is to monitor reversal effects using clinical parameters. For example, it is stated that anti-FXa assays should not be used to measure the effectiveness of andexanet alfa as the results may not be reliable (see the “Drug Safety Update” pulished on 29 June 2020, available on the internet at www.gov.uk / drug-safety-update / direct-acting-oral-anticoagulants-doacs- reminder-of-bleeding-risk-including-availability-of-reversal-agents). It is recommended by such official bodies that treatment monitoring should be based mainly on clinical parameters indicative of appropriate response (achievement of hemostasis), lack of efficacy (re-bleeding), and adverse events (thromboembolic events).

[0009] It would be desirable for many clinical cases to be able to measure the efficiency of DOAC reversal agents with more precision, and to determine the most accurate dosage regimen for each patient. There is a need for reliable methods that allow the monitoring of DOAC reversal agents. There is a need for improved precision in determining the effect of reversal agents. There is a need for methods that improve the dosage precision of reversal agents. There is a need for improved monitoring of DOAC activity. There is a need for improved methods to measure DOAC reversal. There is a need for improved methods to monitor whether DOAC reversal occurred, and to what extent. There is a need for improved methods to measure DOAC reversal in a reliable manner.

[0010] Summary of the invention

[0011] Use and dosage of anticoagulants and their reversal agents is well known, although an improvement in hemostatic efficacy may be achieved by optimizing their dosing. Hitherto, the lack of reliable standardized or validated laboratory assays was an obstacle to this achievement. The invention for the first time allows the precise determination of reversal agent efficiency. It was surprisingly found that the described chemiluminescent assays are sufficiently sensitive at robustly useful concentration ranges, allowing the assays to be used for determining not only anticoagulant activity, but even reversal agent activity. This sensitivity is enabled in part by the good window of opportunity offered by the chemiluminescent assays, i.e. the broad range over which signals can be usefully measured (compare for instance Fig. 8A, a chromogenic assay where signal intensity is spread over about 30% of the signal intensity range, while the chemiluminescent assay of Fig. 8B has signal intensity with a variation that is spread over about 85% of the signal intensity range).

[0012] Accordingly the invention provides a method for quantifying the activity of a reversal agent in a sample, wherein the sample comprises an anticoagulant and a coagulation factor, the method comprising the steps of: a) contacting the sample with a composition comprising a chemiluminescent substrate for the coagulation factor to release aminoluciferin; b) contacting the aminoluciferin with luciferase; and c) determining the relative light intensity generated by the luciferase. Preferably the anticoagulant is a direct oral anticoagulant. Preferably the anticoagulant is hirudin, bivalirudin, desirudin, lepirudin, argatroban, dabigatran, efegatran, inogatran, melagatran, ximelagatran, apixaban, betrixaban, darexaban, edoxaban, otamixaban, or rivaroxaban, preferably it is rivaroxaban, apixaban, edoxaban, or dabigatran. Preferably the coagulation factor is factor Xa (FXa) or factor Ila (Fl la) . Preferably the reversal agent is andexanet alfa, idarucizumab, ciraparantag, VMX-C001 , prothrombin complex concentrate (PCC), or factor eight inhibitor bypassing activity (FEIBA), preferably it is andexanet alfa, idarucizumab, or FEIBA, more preferably it is andexanet alfa or idarucizumab. Preferably the sample is a blood sample, preferably wherein the blood sample is known or suspected of comprising a reversal agent, preferably wherein the blood sample has been previously obtained from a subject, highly preferably the blood sample is a plasma sample.

[0013] In preferred embodiments the reversal agent is andexanet alfa or VMX-C001 , and the anticoagulant is apixaban or edoxaban or rivaroxaban or betrixaban, and the coagulation factor is FXa; orthe reversal agent is idarucizumab, and the anticoagulant is dabigatran, and the coagulation factor is Fl la. Preferably the reversal agent is andexanet alfa and the anticoagulant is apixaban and the coagulation factor is FXa. In preferred embodiments the chemiluminescent substrate is a compound of general formula (1-3) or (1-4),

[0014] (1-3) (1-4) wherein r is 0, 1 , 2, or 3; r’ is 0, 1 , 2, or 3; d is 0, 1 , or 2; g is 0 or 1 ; g’ is 0 or 1 ; and X is a terminal moiety selected from NH2, OH, O(Ci-6alkyl), (OCH2CH2)I-6OH, (OCH2CH2)i-6O(Ci-6alkyl), NHC(=0)(0)o-i(Ci-6alkyl), NHC(=O)(O)0-i(Ci-6alkylene)(O-CH2CH2)i-6OH, NHC(=0)(0)o-i(Ci- 6alkylene)(OCH2CH2)i-6O(Ci-6alkyl), NHC(=0)(0)o-i(Ci-6alkylene)0(Ci-6alkyl), NHC(=0)(0)o-i(Ci- ealkylene)OH, and NP’ wherein P’ is an amine protecting group; or wherein the chemiluminescent substrate is pyroGlu-Phe-Lys-aminoluciferin; Gly-Gly-Arg-aminoluciferin; beta-Ala-Gly-Arg- aminoluciferin; Ala-Gly-Arg-aminoluciferin; or CH3O(CH2CH2O)n-acetyl-Gly-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 2; or CH3O(CH2CH2O)n-acetyl-beta- Ala-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 4; CH3O(CH2CH2O)n-acetyl-Ala-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 4; optionally wherein the aminoluciferin moiety is replaced by a different chemiluminescent amine; or a physiologically acceptable salt thereof.

[0015] Also provided is a method for determining a suitable dosage of reversal agent for a subject whose hemostasis is disrupted by an anticoagulant, the method comprising the steps of: i)contacting a blood sample that has been previously obtained from the subject with a composition comprising a chemiluminescent substrate for FXa or Flla to release aminoluciferin; ii) contacting the aminoluciferin with luciferase; iii) determining the relative light intensity generated by the luciferase; iv) comparing the determined relative light intensity to a reference value to determine the activity of the anticoagulant; v) determining a suitable dosage of reversal agent based on the determined activity of the anticoagulant. Preferably the reversal agent is andexanet alfa, and the anticoagulant is apixaban, wherein the suitable dosage of step v) has a stoichiometric ratio of andexanet alfa to apixaban in the range of 0.05-0.5 to 1 , preferably about 0.16 to 1 ; or the reversal agent is andexanet alfa, and the anticoagulant is edoxaban, wherein the suitable dosage of step v) has a stoichiometric ratio of andexanet alfa to edoxaban in the range of 1 .1 -2 to 1 , preferably about 1.56 to 1 ; or the reversal agent is idarucizumab, and the anticoagulant is dabigatran, wherein the suitable dosage of step v) has a stoichiometric ratio of idarucizumab to dabigatran in the range of 1.05-2 to 1 , preferably 1.17 to 1. Also provided is a reversal agent for use as a medicament to restore hemostasis, wherein the reversal agent is administered in a dosage as determined in the method described earlier herein. Also provided is a method for reversing the activity of an anticoagulant in a subject, the method comprising the steps of obtaining a blood sample from the subject, determining the activity of the anticoagulant in said sample by using a method as defined earlier herein, determining an effective dose of a reversal agent based on the determined activity of the anticoagulant, and administering said effective dose of the reversal agent to the subject.

[0016] Also provided is a kit of parts comprising a reversal agent and at least one further compound selected from the group consisting of luciferase, ATP, an Mg2+source, and a coagulation factor such as factor Xa. Also provided is a kit of parts comprising a reversal agent and a chemiluminescent substrate for a coagulation factor.

[0017] Description of embodiments

[0018] Use and dosage of anticoagulants and their reversal agents is well known, although an improvement in hemostatic efficacy may be achieved by optimizing their dosing. Hitherto, the lack of reliable standardized or validated laboratory assays was an obstacle to this achievement. The invention for the first time allows the precise determination of reversal agent efficiency. It was surprisingly found that the described chemiluminescent assays are sufficiently sensitive at roboustly useful concentration ranges, allowing the assays to be used for determining not only anticoagulant activity, but even reversal agent activity

[0019] Method

[0020] Accordingly the invention provides a method for quantifying the activity of a reversal agent in a sample, wherein the sample comprises an anticoagulant and a coagulation factor, the method comprising the steps of: a) contacting the sample with a composition comprising a chemiluminescent substrate for the coagulation factor to release aminoluciferin; b) contacting the aminoluciferin with luciferase; and c) determining the relative light intensity generated by the luciferase.

[0021] Such a method is referred to hereinafter as an assay according to the invention. In this context, quantification of the activity of a reversal agent can be understood as quantification of the coagulation factor itself. A skilled person will understand that when activity of a zymogen is determined, the activity of its corresponding enzyme is part of the assay.

[0022] The principle of chemiluminescence involving luciferase is well known by the skilled person. It typically uses luciferase, luciferin, ATP, and molecular oxygen for photon production; Mg2+is known to improve luminescence yield of the reaction. Luciferase catalyzes the conjugation of luciferin to ATP, and also the subsequent oxidation of the luciferyl-AMP intermediate. Ultimately, the luciferase provides an environment in which the oxidized luciferin intermediate rearranges to produce oxyluciferin and a single photon with high-quantum efficiency. Light intensity resulting from such luminescence is dependent on the concentrations of the components involved in the chemical or enzymatic conversion of the liberated luminescent molecule. By using an excess of such components, the luminescent signal of the method of the invention becomes dependent only on the generation of free luminescent molecules by cleavage of the substrate by the coagulation factor of interest, e.g., FXa or thrombin or another factor as later described herein. Under such circumstances, the light intensity thus is proportional to the generation of said coagulation factor, e.g., FXa generation. Thus the coagulation factor of interest can be quantified through design of the assay according to the invention, as its concentration is proportional to the light output of the assay. Along this principle, when the amount of coagulation factor is known, an amount of anticoagulant that inhibits the coagulation factor can be determined as well. Extending this principle, when the amount of coagulation factor is known, an amount of reversal agent can be determined.

[0023] The inventors found that by using compounds according to the invention, generation of hemostasis factor, e. g. FXa generation be it direct or indirect, or FXa concentration as such, can be measured continuously, semi-continuously, or in a direct way without requiring calculation of the first derivative as is required for chromogenic or fluorogenic method for measuring generation of blood clotting factors, or for determining anticoagulant activity or for determining activity of reversal agents. Typically, in a method according to the invention, upon cleavage of a substrate by a hemostasis factor of interest, a ‘luminescent molecule’ is liberated, generally being the aminoluciferin, which is prone to a subsequent chemical or enzymatic conversion that produces a detectable light signal (or “light quant” or “photon” or “light unit”). Since the light quant is produced in an irreversible step, there is no accumulation of output signal; the signal is a direct measure of coagulation factor activity (and thus possibly of the activity of a factor or inhibitor that is involved in factor activity). It is a significant advantage of the present method, as compared to existing methods employing fluorescent or chromogenic substrates, that a signal can be detected real-time that is directly proportional to the amount of the hemostasis factor activity present at any given time point; there is no need to calculate the first derivative of an accumulating optical signal. In the present method there is no interference with further production of light signals. In addition, no external light source and optical filters are required for measuring the signal. Thus, the method of the present invention is more convenient than the prior art methods.

[0024] A sample can be a sample from a subject, preferably it is a sample that has been previously obtained from a subject. A subject can be a human. A subject can be non-human. A sample is preferably a fluid. A preferred sample is blood or derived from blood. Suitable samples are whole blood and plasma such as platelet poor plasma or platelet rich plasma. A most preferred sample is platelet poor plasma, such as platelet poor plasma that has been previously obtained from a subject. In preferred embodiments the sample is a blood sample, preferably wherein the blood sample is known or suspected of comprising a reversal agent, preferably wherein the blood sample has been previously obtained from a subject. In some embodiments the blood sample has been processed, for instance to render it a plasma sample. Thus in some embodiments the blood sample is a plasma sample. Coagulation factors are well known in the art. In the context of the assay according to the invention, preferred coagulation factors are factor IX (FIX), factor IXa (FIXa), factor VIII (FVIII), factor Villa (FVIIIa), factor VII (FVII), factor Vila (FVIIa), factor XI (FXI), factor Xia (FXIa), Factor XII (FXI I), factor XII a (FXI la), factor X (FX), and factor Xa (FXa). A notation such as FX(a) references both or either of FX and FXa. A skilled person understands that coagulation factors, when cleaving substrates, are active coagulation factors, and as such active coagulation factors such as FXa and Fl la are preferred. Most preferably the coagulation factor is factor Xa (FXa) or factor Ila (Fl la) . In one embodiment the coagulation factor is FXa. In one embodiment the coagulation factor is Fl la.

[0025] A physiological role of coagulation factors is to ultimately enable thrombin generation. Thrombin (active Factor II, or Flla) is the most important constituent of the coagulation cascade in terms of its feedback activation roles. In humans parts of the process are generally as follows: FVIIa circulates in a higher amount than any other activated coagulation factor. Following damage to the blood vessel, FVII(a) can come into contact with tissue factor (TF), ultimately forming an activated complex (TF-FVIla). TF-FVIla activates FIX to form FIXa, and activates FX to form FXa. The activation of FX (to form FXa) by TF-FVIla is almost immediately inhibited by the TF (tissue factor) pathway inhibitor (TFPI) in the TF-TFPI-FXa complex. FXa and its co-factor FVa form the prothrombinase complex, which activates prothrombin to thrombin. Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which forms a complex with FIX), and activates and releases FVIII from being bound to vWF, forming FVIIIa. FVIIIa is the co-factor of FIXa, and together they form the "tenase" complex, which activates FX, continuing the cycle.

[0026] In preferred embodiments is provided the assay according to the invention, wherein the coagulation factor is selected from factor IX, factor IXa, factor VIII, factor Villa, factor VII, factor Vila, factor XI, factor Xia, Factor XII, factor Xlla, factor X, and factor Xa. The coagulation factor should preferably be FXa or should contribute to formation of FXa, or should be Flla or should contribute to formation of Flla. For example, when the coagulation factorto be assayed is FXa, then the coagulation factor is FXa. When the coagulation factor to be assayed is not FXa, then the coagulation factor should contribute to formation of FXa. For this, FX should be present.

[0027] In certain combinations the generation of FXa is proportional to the concentration of several coagulation factors like FVIII, FIX and FX in the tenase complex, or tissue factor or FVIIa in the TF- FVIla complex. When all factors but a missing one are present in excess, then the activity of the missing factor can be correlated to eventual FXa activity as determined via luminescence output. FX can be converted into FXa by FVIIa or by FVIIa in the presence of TF, so when FVIIa is to be assayed, the coagulation factor should be FX; this is because the FVIIa in the sample can then convert an excess of FX into FXa.

[0028] FX can also be converted into FXa by FIXa and FVIIIa, in a so-called tenase complex. A tenase complex consists of FIXa, FVIIIa, FX, anionic phospholipids, and calcium. Accordingly, when FIXa is to be assayed, the coagulation factor should be FX or FVIIIa, and preferably both FX and FVIIIa should be present, more preferably along with anionic phospholipids and calcium. Accordingly, when FVIIIa is to be assayed, the coagulation factor should be FX or FIXa, and preferably both FX and FIXa should be present, more preferably along with anionic phospholipids and calcium. In turn, FIX can be converted into FIXa by FXIa, so when FIX is to be assayed, FXIa is preferably present in addition to the conditions described for FIXa. A skilled person is aware of the various interactions in coagulation pathways and will be capable of selecting suitable coagulation factors to be present in an assay according to the invention, depending on which coagulation factor is to be assayed. In general, components of the coagulation pathway are to be present in excess, with the component to be assayed omitted. The coagulation factor to be assayed is then supplied via the sample, and because any of its substrates, cofactors, or other interactors are present in excess, the amount of coagulation factor to be assayed will directly correlate to the FXa activity that is generated and that is ultimately detected via the compounds according to the invention. For this, required factors can be added to for instance a blood sample.

[0029] A skilled person will understand that detection of a zymogen will generally involve detection of the corresponding enzyme, and that a detection of the zymogen thus amounts to detection of both the zymogen and the corresponding active enzyme. For example, detection of FVII results in simultaneous detection of FVII and FVIIa.

[0030] In one class of preferred embodiments, all components of the coagulation pathway are present. Such an assay is referred to herein as a global assay, and it is preferably preformed using the coagulation factors provided by the sample itself. Accordingly, in a global assay, preferably the components of the coagulation pathway are present in ratios that resemble the ratios found in physiological systems. Preferred samples for a global assay are whole blood and plasma such as platelet rich plasma, or platelet poor plasma, more preferably plasma, most preferably platelet poor plasma. A global hemostasis assay is preferably initiated by addition of active coagulation factors such as FXIIa, FXIa, or FIXa, of tissue factor (TF) and / or of calcium to plasma, more preferably of TF and / or of calcium, most preferably of both TF and calcium; this leads to FXa generation followed by thrombin generation and subsequent clot formation. Following initiation, thrombin is required for propagation and termination of the cascade. Measuring for instance the FXa concentration in such a global design gives information about the sample’s capacity of the coagulation cascade. The impact of an anticoagulant on the hemostasis of a sample can be determined this way, as can the effect of a reversal agent.

[0031] Step a) - release of aminoluciferin

[0032] In step a) the sample is contacted with a composition comprising a chemiluminescent substrate for the coagulation factor to release aminoluciferin. Depending on the substrate, a factor such as FXa is able to recognize the peptide comprised in the substrate, and cleaves it from the luminescent moiety to release aminoluciferin. In highly preferred embodiments, calcium and phospholipids are present during step a). These can be natively present in the sample when the sample is a blood sample. The calcium and phospholipids should be suitable for formation of a tenase complex. These phospholipids are preferably anionic phospholipids. Conditions for coagulation factor activity are known in the art, and it is under these circumstances that the contacting is preferably done. An example is the use of a physiologically acceptable buffer, for example Tris buffer optionally comprising 1 % serum albumin such as BSA. An example of a suitable Tris buffer is Tris buffered saline (TBS; 50 mM Tris-HCI, 150 mM NaCI; pH 7.4). The concentration of the substrate according to the invention is preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 pM or more, more preferably at least 20 or 30 pM such as at least 30 pM. The concentration of the substrate according to the invention is preferably at most 5000, 4000, 3000, 2000, 1750, 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60 pM or lower, more preferably at most 2500 or 200 pM or 1750 pM or 1500 pM or 1250 pM or 1000 pM or 900 pM or 800 pM or 700 pM or 600 pM or 500 pM or lower, even more preferably at most 2000 or 750 pM or lower, such as at most 750 pM.

[0033] This step is to correlate the presence of coagulation factor to be assayed to a luminescent output, and accordingly a substrate for luciferase is released in this step. In this context, release is to be interpreted as a hydrolysis of for example the aminoluciferin moiety as shown in general formula II-4 wherein the carboxylic acid is S. Depending on the substrate, other luminescent moieties can also be released. The release is to make the chemiluminescent substrate available for subsequent conversion in step b), as the chemiluminescent substrate is not available for further conversion when it is comprised in a compound according to the invention.

[0034] Step b) - oxidation of aminoluciferin by luciferase

[0035] In step b) the aminoluciferin is contacted with luciferase. The purpose of this contacting is to generate a light quant from the chemiluminescent molecule that was released in step a) such as from the released aminoluciferin. Methods for converting a chemiluminescent substrate to produce a light quant are established in the art, and the luciferase, preferably firefly luciferase, can become more functional when further substances are also present during this contacting. Accordingly, in preferred embodiments, step b) further comprises contacting the released chemiluminescent molecule with ATP. In preferred embodiments, step b) further comprises contacting the released chemiluminescent molecule with Mg2+. In highly preferred embodiments, step b) further comprises contacting the released chemiluminescent molecule with ATP and Mg2+. When ATP is also present during step b), it can be present at a final concentration of about 50-1000 pM, preferably at a final concentration of about 250-500 pM, more preferably of about 300-400 pM such as about 333 pM. When Mg2+ is present during step b), it can be present at a final concentration of about 1 -30 pM, it is preferably present at a final concentration of about 4-12 mM, more preferably of about 6-10 mM, such as about 8.3 mM. Luciferase is preferably present at about 0.05-50 mg / mL, more preferably at about 0.1-10 mg / mL, even more preferably at about 0.5 to 5 mg / mL such as at about 0.9 mg / mL.

[0036] Step a) and step b) can be performed simultaneously and / or in the same reaction volume. In preferred embodiments, step a) and step b) are performed in the same reaction volume, which preferably simultaneously comprises all reagents required for both steps a) and b). This allows a released chemiluminescent molecule to be converted by luciferase without delay. Step c) - determining the relative light intensity

[0037] In step c) the luminescent signal is determined. This can be done in any way that is known in the art, for example using a luminometer. The determined light intensity is used as a basis for guantifying the coagulation factor to be assayed. This is because, as described herein, the concentration of the coagulation factor correlates to the relative light intensity, preferably expressed as relative light units (RLU). In some embodiments, the relative light intensity is compared to a reference value or to a calibration curve. Such a calibration curve has preferably been prepared using known amounts of the coagulation factor to be assayed, for example as demonstrated in the examples. A reference value can be a set value such as a predetermined value, or it can be the assay result from a control sample. In this context a control sample is preferably a sample that is known to meet certain specifications, or a sample (previously) obtained from a healthy subject, or it is normal pooled plasma.

[0038] In some embodiments, the relative light intensity is compared to a reference value or to a calibration curve wherein the calibration curve has preferably been prepared using known amounts of anticoagulant in blood, for example as demonstrated in the examples. A reference value can be a set value such as a predetermined value, or it can be the assay result from a control sample. In this context a control sample is preferably a sample that comprises a known amount of anticoagulant.

[0039] The relative light intensity directly represents the actual luciferase activity, which in turn directly represents the actual amount of luminescent substrate being released, which in turn directly represents coagulation factor activity. The relative light intensity thus provides direct information about the amount of coagulation factor to be assayed, owing to the fixed kaof the enzymes and substrates involved. This is in contrast to accumulating assays such as fluorogenic assays or chromogenic assays. For the latter two assays the slope must be calculated to determine conversion rate. In luminescent assays the flat output (for example in RLU) directly indicates the conversion rate. In preferred embodiments, step c) does not comprise determining a derivative of any signal determined in step c). In more preferred embodiments, step c) does not comprise determining the first derivative of the relative light intensity generated by the luciferase. In this context, light intensity generated by the luciferase relates to the relative light intensity resulting from the luminescent molecule such as aminoluciferin being released from the substrate. Luminescence can be measured at wavelengths according to methods known in the art. Examples of suitable wavelengths are wavelengths between 360-630 nm.

[0040] The relative light intensity as determined at a point in time thus directly provides relevant information about coagulation factor activity. When compared to a reference value such as normal pooled plasma, this provides information about the activity of anticoagulant. When compared to a reference value such as blood comprising a known amount of anticoagulant, this provides information about the activity of reversal agent. Determination of relative light intensity during a set duration of time, or until a certain condition is met, can provide relevant information about the dynamics of an assay. This is particularly useful for a global assay as described above, wherein the output will generally be a bellshaped curve. Accordingly, in preferred embodiments, step c) comprises determining the relative light intensity generated by the luciferase over a period of time. This period of time is preferably at most 150, 120, 90, 80, 70 , 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute or shorter, more preferably at most 35, 30, 15, or 10 minutes or shorter. A period of time is preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds, more preferably at least 10, 20, or 30 seconds such as at least 30 seconds.

[0041] Relevant to the invention is an assay to allow quantification of anticoagulants, such as a method for quantifying an anticoagulant in a sample, the method comprising the steps of: a) contacting the sample with a composition comprising a coagulation factor and a chemiluminescent substrate to release aminoluciferin; b) contacting the aminoluciferin with luciferase; and c) determining the relative light intensity generated by the luciferase.

[0042] This method is a method that quantifies inhibition of coagulation factor activity, and it is referred to hereinafter as an inhibition assay according to the invention. In this method a factor such as FXa is provided in step a). A result of this is that a known amount of luminescent signal will be generated when no inhibitor of the factor is present, as a result of factor activity releasing aminoluciferin from the compounds according to the invention. Therefore any reduction in luminescent output can be ascribed to factor inhibition, such as when an anticoagulant is present.

[0043] In preferred embodiments, the invention provides an assay or an inhibition assay according to the invention, wherein step c) comprises determining the relative light intensity generated by the luciferase over a period of time, and / or wherein the assay or inhibition assay does not comprise determining the first derivative of the relative light intensity generated by the luciferase.

[0044] Anticoagulants

[0045] Preferred anticoagulants to be assayed are direct oral anticoagulants (DOACs), heparin, and heparinoids. Preferably, anticoagulants used in the invention are direct oral anticoagulants. Preferred anticoagulants are hirudin, bivalirudin, desirudin, lepirudin, argatroban, dabigatran, efegatran, inogatran, melagatran, ximelagatran, apixaban, betrixaban, darexaban, edoxaban, otamixaban, or rivaroxaban, preferably it is rivaroxaban, apixaban, edoxaban, or dabigatran.

[0046] Examples of preferred DOACs are against FXa are rivaroxaban (CAS 366789-02-8), apixaban (CAS 503612-47-3), betrixaban (CAS 330942-05-7), and edoxaban (CAS 480449-70-5). Further DOACs against FXa are darexaban and otamixaban. Good assay results were obtained with rivaroxaban, apixaban, and edoxaban. Examples of preferred DOACs are against Flla are hirudin (CAS 8001-27-2), bivalirudin (CAS 128270-60-0), desirudin (CAS 120993-53-5), lepirudin (CAS 138068-37-8), argatroban (CAS 74863-84-6), efegatran (CAS 105806-65-3), inogatran (CAS 155415-08-0), melagatran (CAS 159776-70-2), and ximelagatran (192939-46-1). Good assay results were obtained with dabigatran.

[0047] Anticoagulants can be spiked in a sample, for instance to create a calibration curve. Anticoagulants can be present in a sample, for instance when the sample is a blood sample of a subject whose hemostasis is disrupted by the anticoagulant. Use and dosage of anticoagulants is well known.

[0048] Reversal agents

[0049] Reversal agents are antidotes for anticoagulants, in that they can reverse the disruptive effect that an anticoagulant has on hemostasis. As an example, idarucizumab is a non-competitive inhibitor that forms complexes with dabigatran to counteract its anticoagulant effect within minutes of administration. It binds to dabigatran that is free or bound to thrombin, as well as dabigatran's active metabolites. Idarucizumab is specific to dabigatran and has an affinity that is around 350 times stronger compared to thrombin.

[0050] Another example is andexanet alfa, a recombinant modified version of human FXa. Andexanet alfa differs from native FXa due to the removal of a 34 residue fragment that contains the Gia domain, which reduces andexanet alfa's anticoagulant potential. Additionally, a serine to alanine (S419A) mutation in the active site eliminates its activity as a prothrombin to thrombin catalyst, but still allows the molecule to bind to FXa inhibitors. Thus FXa inhibitors bind to andexanet alfa with the same affinity as to natural FXa. As a consequence in the presence of andexanet alfa natural FXa is partially freed, which can lead to effective hemostasis. Andexanet alfa reverses effect of all anticoagulants that act directly through FXa. Similarly, VMX-C001 is a modified human FX zymogen that is insensitive to DOACs.

[0051] Prothrombin complex concentrate (CAS 37224-63-8) is a combination medication made up of blood clotting factors II, IX, and X, which can reverse the effect of anticoagulants. Factor eight inhibitor bypass activity (FEIBA) comprises nonactivated factors II, IX, X, and activated VII, which means that it is similar to prothrombin complex concentrate and recombinant factor Vila combined.

[0052] In preferred embodiments the reversal agent is andexanet alfa, idarucizumab, ciraparantag (CAS 1438492-26-2), VMX-C001 , prothrombin complex concentrate (PCC), or factor eight inhibitor bypassing activity (FEIBA), preferably it is andexanet alfa, idarucizumab, or FEIBA, more preferably it is andexanet alfa or idarucizumab. Use and dosage of anticoagulants is well known.

[0053] As the activity of individual reversal agents and individual anticoagulants can be related, various combinations are preferred. For instance, in preferred embodiments the reversal agent is andexanet alfa or VMX-C001 , and the anticoagulant is apixaban or edoxaban or rivaroxaban or betrixaban, and the coagulation factor is FXa; or the reversal agent is idarucizumab, and the anticoagulant is dabigatran, and the coagulation factor is Flla. In more preferred embodiments the reversal agent is andexanet alfa, and the anticoagulant is apixaban or edoxaban or rivaroxaban or betrixaban, and the coagulation factor is FXa. Alternatively the reversal agent is andexanet alfa, and the anticoagulant is apixaban, and the coagulation factor is FXa. Alternatively the reversal agent is andexanet alfa, and the anticoagulant is edoxaban, and the coagulation factor is FXa. Alternatively the reversal agent is andexanet alfa, and the anticoagulant is rivaroxaban, and the coagulation factor is FXa. Alternatively the reversal agent is idarucizumab, and the anticoagulant is dabigatran, and the coagulation factor is Fl la.

[0054] Chemiluminescent substrate

[0055] Based on which coagulation factor is related to the reversal agent to be quantified, a suitable substrate is to be selected by the skilled person. In preferred embodiments the chemiluminescent substrate is a compound of general formula (I-3) or (I-4),

[0056] (I-3) (I-4) wherein r is 0, 1 , 2, or 3; r’ is 0, 1 , 2, or 3; d is 0, 1 , or 2; g is 0 or 1 ; g’ is 0 or 1 ; and

[0057] X is a terminal moiety selected from NH2, OH, O(Ci-6alkyl), (OCH2CH2)i-eOH, (OCH2CH2)I- 6O(Ci-6alkyl), NHC(=0)(0)o-i(Ci-6alkyl), NHC(=0)(0)o-i(Ci.6alkylene)(0-CH2CH2)i-60H,

[0058] NHC(=0)(0)o-i(Ci-6alkylene)(OCH2CH2)i-60(Ci-6alkyl), NHC(=0)(0)o-i(Ci.6alkylene)0(Ci. salkyl), NHC(=0)(0)o-i(Ci-6alkylene)OH, and NP’ wherein P’ is an amine protecting group; or wherein the chemiluminescent substrate is pyroGlu-Phe-Lys-aminoluciferin; Gly-Gly-Arg-aminoluciferin; beta-Ala-Gly-Arg- aminoluciferin; Ala-Gly-Arg-aminoluciferin; or

[0059] CH3O(CH2CH2O)n-acetyl-Gly-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 2; or

[0060] CH3O(CH2CH2O)n-acetyl-beta-Ala-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 4; CH3O(CH2CH2O)n-acetyl-Ala-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 4; optionally wherein the aminoluciferin moiety is replaced by a different chemiluminescent amine; or a physiologically acceptable salt thereof. Peferably the aminoluciferin is not replaced by a different chemiluminescent amine. The compounds of general formula (I-3) or (I-4) are preferred when the coagulation factor is FXa, and these substrates and their preparation are known from W02020079155. The other compounds listed above are preferred when the coagulation factor is Flla, and these substrates and their preparation are known from WO2012096566. Particularly preferred are substrates selected from MePEG2-IEGR, MePEG2-IDGR, MePEG3-IEGR, MePEG3-

[0061] In more preferred embodiments the substrate is selected from MePEG2-IEGR, MePEG2- IDGR, MePEG3-IEGR, MePEG3-IDGR, MePEG2-RGR, and MePEG3-RGR. In even more preferred embodiments the substrate is selected from MePEG2-IEGR, MePEG2-IDGR, MePEG3- IEGR, and MePEG3-IDGR. In most preferred embodiments the substrate is selected from MePEG2-IEGR and MePEG2-IDGR, preferably it is MePEG2-IEGR; more preferably a physiologically acceptable salt thereof, such as a TFA salt. This is preferred when FXa is involved. In other preferred embodiments the substrate is beta-Ala-Gly-Arg-aminoluciferin (shown below for reference); more preferably a physiologically acceptable salt thereof, such as a TFA salt. This is preferred when Fl la is involved.

[0062] Medical applications

[0063] The invention also provides a method for determining a suitable dosage of reversal agent for a subject whose hemostasis is disrupted by an anticoagulant, the method comprising the steps of: i) contacting a blood sample that has been previously obtained from the subject with a composition comprising a chemiluminescent substrate for FXa or Fl la to release aminoluciferin; ii) contacting the aminoluciferin with luciferase; iii) determining the relative light intensity generated by the luciferase; iv) comparing the determined relative light intensity to a reference value to determine the activity of the anticoagulant; v) determining a suitable dosage of reversal agent based on the determined activity of the anticoagulant.

[0064] Dosing of reversal agents is now generally performed by administering one-size-fits-all doses that comprise a large excess of reversal agent. This can be unattractive for it may exacerbate side effects of these agents. Additionally, these agents are often costly and labour intensive to manufacture, further indicating a need for their reduced consumption. Up until now the required dosage of reversal agent could not be reliably determined because assays for anticoagulant and reversal agent activity were not sufficiently robust. By determining the activities using a method as provided herein, a more tailored dosage of reversal agent can be determined.

[0065] The subject is preferably a mammal, more preferably a primate, most preferably a human. Preferred subjects are humans above the age of 50, more preferably above the age of 60. Preferred subjects are humans who are undergoing concomitant treatment with an anticoagulant, preferably with a DOAC. The subjects have hemostasis that is disrupted by an anticoagulant, preferably the subjects are in need of restoration of hemostasis. Preferably the subject is bleeding or is at risk of bleeding.

[0066] In step i) a blood sample, which can be a plasma sample, previously obtained from the subject is contacted with a chemiluminescent substrate as described for step a) earlier herein. Steps ii) and iii) have features and definitions as defined for steps b) and c) as described earlier herein.

[0067] The light intensity generated in step iii) is a measure for the activity of FXa or Fl la depending on which substrate was used. In step iv) this intensity is compared to a reference value to determine the activity of the anticoagulant. This can mean that the intensity is compared to a calibration curve, or to a preciously determined reference value. Such a calibration curve has preferably been prepared using known amounts of the coagulation factor to be assayed, for example as demonstrated in the examples. A reference value can be a set value such as a predetermined value, or it can be the assay result from a control sample such as a stored sample from the subject where no anticoagulant is present. In this context a control sample is preferably a sample that is known to meet certain specifications, or a sample (previously) obtained from a healthy subject, or it is normal pooled plasma.

[0068] As demonstrated in the examples, the activity of anticoagulant is related to the amount of anticoagulant. When the amount of anticoagulant is determined, a suitable dosage of reversal agent is determined in step v) based on the determined activity of the anticoagulant. The suitable dosage of reversal agent can also be based on a relevant reference value, or on a relevant calibration curve.

[0069] As demonstrated in the examples, certain ratios of reversal agent to DOAC were identified as being suitable for achieving complete reversal of DOAC activity. Accordingly, the method is provided wherein the reversal agent is andexanet alfa, and the anticoagulant is apixaban, wherein the suitable dosage of step v) has a stoichiometric ratio of andexanet alfa to apixaban in the range of 0.05-0.5 to 1 , preferably about 0.16 to 1 ; or the reversal agent is andexanet alfa, and the anticoagulant is edoxaban, wherein the suitable dosage of step v) has a stoichiometric ratio of andexanet alfa to edoxaban in the range of 1 .1 -2 to 1 , preferably about 1 .56 to 1 ; or the reversal agent is idarucizumab, and the anticoagulant is dabigatran, wherein the suitable dosage of step v) has a stoichiometric ratio of idarucizumab to dabigatran in the range of 1 .05-2 to 1 , preferably 1 .17 to 1 .

[0070] In preferred embodiments the reversal agent is andexanet alfa, and the anticoagulant is apixaban, wherein the suitable dosage of step v) has a stoichiometric ratio of andexanet alfa to apixaban in the range of 0.1 -0.4 to 1 , preferably 0.12-0.3 to 1 , more preferably 0.13-0.2 to 1 , most preferably 0.14-0.18 to 1 , such as about 0.16 to 1 .

[0071] In preferred embodiments the reversal agent is andexanet alfa, and the anticoagulant is edoxaban, wherein the suitable dosage of step v) has a stoichiometric ratio of andexanet alfa to edoxaban in the range of 1 .1-2 to 1 , preferably 1 .2-1 .9 to 1 , more preferably 1 .3-1 .8 to 1 , still more preferably 1 .4-1 .7 to 1 , even more preferably 1 .5-1 .6 to one, such as about 1 .56 to 1 .

[0072] In preferred embodiments the reversal agent is idarucizumab, and the anticoagulant is dabigatran, wherein the suitable dosage of step v) has a stoichiometric ratio of idarucizumab to dabigatran in the range of 1 .05-2 to 1 , preferably 1 .07-1 .9 to 1 , more preferably 1 .09-1 .75 to 1 , still more preferably 1.1-1 .6 to 1 , still more preferably 1 .12-1 .5 to 1 , even more preferably 1 .14-1 .4 to 1 , even more preferably 1.15-1 .3 to 1 , most preferably 1.16-1 .2 to 1 , such as about 1 .17 to 1 .

[0073] Use and dosage of anticoagulants is well known, although an improvement in hemostatic efficacy may be achieved by optimizing the dosing of these reversal agents. As described above, the lack of standardized and validated laboratory assays is an obstacle to this achievement, while the invention allows the precise determination of a suitable dosage. Accordingly the invention provides a reversal agent for use as a medicament to restore hemostasis, wherein the reversal agent is administered in a dosage as determined in a method as described above.

[0074] Preferably the reversal agent is administered in a dosage that has a given stoichiometric ratio to the amount of anticoagulant as determined in step iv). Examples of these ratios are as described above.

[0075] In some embodiments is provided andexanet alfa, wherein the andexanet alfa is administered to a subject who is taking concomitant apixaban, wherein the activity of apixaban in the subject has been determined, wherein the dosage of andexanet alfa has a stoichiometric ratio of andexanet alfa to apixaban in the range of 0.1 -0.4 to 1 , preferably 0.12-0.3 to 1 , more preferably 0.13-0.2 to 1 , most preferably 0.14-0.18 to 1 , such as about 0.16 to 1. The activity of apixaban in the subject can be determined using methods as described herein.

[0076] In some embodiments is provided andexanet alfa, wherein the andexanet alfa is administered to a subject who is taking concomitant edoxaban, wherein the activity of edoxaban in the subject has been determined, wherein the dosage of andexanet alfa has a stoichiometric ratio of andexanet alfa to edoxaban in the range of 1.1 -2 to 1 , preferably 1.2-1 .9 to 1 , more preferably 1 .3-1 .8 to 1 , still more preferably 1 .4-1 .7 to 1 , even more preferably 1 .5-1 .6 to one, such as about 1 .56 to 1 . The activity of edoxaban in the subject can be determined using methods as described herein. In some embodiments is provided idarucizumab, wherein the idarucizumab is administered to a subject who is taking concomitant dabigatran, wherein the activity of dabigatran in the subject has been determined, wherein the dosage of idarucizumab has a stoichiometric ratio of idarucizumab to dabigatran in the range of 1 .05-2 to 1 , preferably 1 .07-1 .9 to 1 , more preferably 1.09-1.75 to 1 , still more preferably 1.1 -1.6 to 1 , still more preferably 1.12-1.5 to 1 , even more preferably 1 .14-1 .4 to 1 , even more preferably 1 .15-1 .3 to 1 , most preferably 1 .16-1 .2 to 1 , such as about 1 .17 to 1. The activity of dabigatran in the subject can be determined using methods as described herein.

[0077] These products for use can be used in a method for reversing the activity of an anticoagulant in a subject, the method comprising the steps of obtaining a blood sample from the subject, determining the activity of the anticoagulant in said sample by using a method as defined above, determining an effective dose of a reversal agent based on the determined activity of the anticoagulant, and administering said effective dose of the reversal agent to the subject.

[0078] Combinations and kits

[0079] The invention provides a kit of parts comprising a reversal agent and at least one further compound selected from the group consisting of luciferase, ATP, an Mg2+source, and a coagulation factor such as factor Xa or factor Ila. The invention also provides a combination of a reversal agent and at least one further compound selected from the group consisting of luciferase, ATP, an Mg2+source, and a coagulation factor such as factor Xa or factor Ila. Thus the invention provides a combination of a reversal agent and luciferase, or a combination of a reversal agent and ATP, or a combination of a reversal agent and an Mg2+source, or a combination of a reversal agent and a coagulation factor such as factorXa or factor Ila. Also provided is a kit of parts comprising a reversal agent and a chemiluminescent substrate for a coagulation factor. The substrate is preferably as defined above.

[0080] Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence using luciferin as a substrate. Luciferases do not require an external light source, but do require luciferin and O2, and often also ATP. Mg2+is known to increase luminescent yield of luciferases. Luciferases and their assays are known in the art, and a skilled person can select a suitable luciferase for combination with a luciferin substrate as comprised in a compound according to the invention. The luciferase should be capable of converting the aminoluciferin comprised in the compound according to the invention into its oxidated analogue after the aminoluciferin has been liberated, under emission of a light quant. Suitable luciferases are firefly luciferase (EC 1.13.12.7), Renilla-luciferin 2-monooxygenase (EC 1.13.12.5), and Metridia luciferase (MetLuc). When the compound according to the invention comprises a 2-(6-amino-1 ,3-benzothiazol-2-yl)-4,5- dihydrothiazole-(4 / 5)-carboxylic acid moiety, the luciferase is preferably firefly luciferase. The luciferase may be any luciferase known in the art or yet to be discovered or engineered. Many luciferases are known in the art. They can be commercially obtained from manufacturers such as Promega, Sigma, and the like. The luciferase may be a native, a recombinant or a mutant luciferase. Said mutant luciferase may be a modified luciferase comprising one or more amino acid substitutions, amino acid deletions, or amino acid insertions, as long as it retains its luciferase activity, preferably at least 25%, 50%, 75% of the luciferase activity of the native (recombinant) luciferase. It may be derived from any organism, as long as it has luciferase activity. In preferred embodiments, the luciferase is a fast acting luciferase; this is particularly suitable for assays wherein the luciferase should provide quantitative information, because fast acting of luciferase reduces interference that might be caused by luciferase being saturated or outside its linear range.

[0081] ATP is required by luciferase and is preferably present in the combination according to the invention in an amount suitable for enabling luciferase activity, or in a stock solution in an amount suitable for preparing dilutions that enable luciferase activity. A suitable ATP stock solution is a 1 mM solution in distilled water, but it can be any stock solution in the range of 200 pM to 10 mM in any physiologically acceptable solvent system.

[0082] The combinations or kits according to the invention may further comprise magnesium ions as it was found that these magnify the luminescent signal generated by luciferase. However, it is also possible to achieve this effect with other divalent cations such as Mn2+(Rodionova and Petushkov, J. Photochem. Photobiol B., DOI: 10.1016 / j.jphotobiol.2005.12.014). An Mg2+source is a source of magnesium ions, which enhances luciferase functioning. Preferred sources of Mg2+are magnesium salts such as magnesium citrate, MgSO4, MgCOs, MgO, MgCh, MgF2, Mgh, MgBr2, and hydrates thereof. Magnesium halides are more preferred, being MgCh, MgF2, Mgh, MgBr2, and hydrates thereof. A most preferred Mg2+source is MgCh or a hydrate thereof.

[0083] The combination or kits according to the invention can further comprise a coagulation factor. Coagulation factors are sometimes referred to as hemostasis factors and are well-known in the art. They can be as defined above. Examples of suitable coagulation factors are the group of serine proteases, in particular serine endopeptidases (EC 3.4.21), preferably selected form the group consisting of thrombin, FXa, plasmin, factor Vila, factor IXa, plasma kallikrein, factor Xlla, factor Xia, tissue-type plasminogen activator (tPA) (preferably two-chain tPA (tc-tPA)), activated Protein C, and urokinase (uPA) (preferably tc-uPA). Zymogens of serine proteases are also encompassed, such as prothrombin, FX, FVII, FIX, prekallikrein, FXII, FXI, sc-tPA (single-chain tPA), protein C, and sc-uPA. In a highly preferred embodiment, the hemostasis factor is FXa or FX. Preferably, coagulation factors are present in such a combination that FXa can be generated by the factors present, or that FXa can be generated when the combination according to the invention is contacted with a sample comprising a further coagulation factor. In such a case, the sample provides the coagulation factor missing from the cascade to generate FXa, and contact with the sample allows FXa generation. Preferably, all factors present in such a cascade that misses only a single factor are present in excess relative to the expected concentration of the missing factor. This allows the factor from the sample to generate FXa as a function of its concentration, after which FXa can generate a luminescent signal proportional to its concentration, and thus proportional to the concentration of factor in the sample. The section on methods has more details on how such compositions can be constituted. In some embodiments the substances of the combination are comprised in a single composition. Such a composition may comprise further substances such as excipients. Water such as distilled water is a suitable excipient. Other suitable excipients are buffer salts such as Tris (tris(hydroxymethyl)aminomethane). In other, preferred, embodiments, the substances of the combination are comprised in distinct compositions. This can be convenient for the provision of kits of parts. In preferred embodiments, the invention provides a kit of parts comprising a reversal agent in a first container, and at least one further compound as defined above in a second container.

[0084] General definitions

[0085] In preferred embodiments, compounds and compositions according to the invention are for use in methods according to the invention, or are for use according to the invention. Each embodiment as identified herein may be combined together unless otherwise indicated. When a structural formula or chemical name is understood by the skilled person to have chiral centers, yet no chirality is indicated, for each chiral center individual reference is made to all three of either the racemic mixture (having any enantiomeric excess), the pure R enantiomer, and the pure S enantiomer.

[0086] Whenever a parameter of a substance is discussed in the context of this invention, it is assumed that unless otherwise specified, the parameter is determined, measured, or manifested under physiological conditions. Physiological conditions are known to a person skilled in the art, and comprise aqueous solvent systems, atmospheric pressure, pH-values between 6 and 8, a temperature ranging from room temperature to about 37 °C (from about 20 °C to about 40 °C), and a suitable concentration of buffer salts or other components. It is understood that charge is often associated with equilibrium. A moiety that is said to carry or bear a charge is a moiety that will be found in a state where it bears or carries such a charge more often than that it does not bear or carry such a charge. As such, an atom that is indicated in this disclosure to be charged could be non-charged under specific conditions, and a neutral moiety could be charged under specific conditions, as is understood by a person skilled in the art.

[0087] In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter.

[0088] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. “Hemostasis” and “Haemostasis” can be used interchangeably herein. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 1 % of the value. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a composition of the invention may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristics of the invention.

[0089] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0090] In the context of this invention, a cell or a sample can be a cell or a sample from a sample obtained from a subject. Such an obtained sample can be a sample that has been previously obtained from a subject. Such a sample can be obtained from a human subject. Such a sample can be obtained from a non-human subject.

[0091] The following are sequences referred to in this invention:

[0092] SEQ ID NO: 1 IEGR SEQ ID NO: 2 IDGR

[0093] SEQ ID NO: 3 IEGK SEQ ID NO: 4 IDGK

[0094] Description of the figures

[0095] Fig. 1 - The Coagulation cascade (extrinsic pathway) is initiated by the TF-FVIla complex, hereby thrombin is formed and the propagation phase will start where the production of thrombin will be enhanced. When the coagulation is performed it will be terminated by fibrinolysis. The coagulation is regulated by Tissue Factor Pathway Inhibitor (TFPI), Antithrombin, Activated Protein C (APC) and Thrombin-activatable fibrinolysis inhibitor (TAFI).

[0096] Fig. 2 - Example of a thrombin generation assay (TGA) graph, which contains different parameters such as lag time (min), time to peak (min) and peak height (nM or RLU).

[0097] Fig. 3A - The mean thrombin generation curves of three different concentration (0, 350 and 700 ng / ml) DOAC spiked NPP measured in triplicates in the TGAIum. Panel A shows the effect of Apixaban on the thrombin generation.

[0098] Fig. 3B - Same as in Fig. 3A, but for the effect of Edoxaban on the thrombin generation.

[0099] Fig. 3C - Same as in Fig. 3A and 3B, but for the effect of Dabigatran on the thrombin generation.

[0100] Fig. 4A - The mean results of the TGA parameters of the FXa DOAC reversal Andexanet alfa (Aa) measured in the TGAium. The different samples used to see the DOAC reversal effect of Andexanet alfa are: NPP without FXa-DOAC and Andexanet alfa, 700 ng / ml FXa-DOAC spiked NPP and 700 ng / ml FXa-DOAC spiked NPP with the addition of 4 concentration, 1 , 5, 10 and 80 pg / ml, of Andexanet alfa. Each sample was measured in duplicates. Panel A shows the TPH (Maximum RLU).

[0101] Fig. 4B - Same experiment as in Fig. 4A, but showing the LT (minutes).

[0102] Fig. 4C - Same experiment as in Fig. 4A and 4B, but showing the AUC (RLU).

[0103] Fig. 4D - Same experiment as in Fig. 4A-4C, but showing the velocity index (RLU / min).

[0104] Fig. 4E - TGAium curves of 700 ng / ml Apixaban reversal with 4 concentrations (1 , 5, 10, 80 pg / ml) of Andexanet alfa. iq. 5A - The mean results of the reversal effect of Idarucizumab on Dabigatran and coagulation. The samples that were measured in duplicates using TGAium: NPP without Dabigatran and Idarucizumab, 700 ng / ml Dabigatran spiked NPP, and 700 ng / ml Dabigatran spiked NPP supplemented with four concentrations (30, 40, 50, and 60 ng / ml) of Idarucizumab. In panel A, TPH is depicted, showing an increase with rising Idarucizumab concentrations.

[0105] Fiq. 5B - Same experiment as in Fig. 5A, but showing the LT (minutes). In the condition wherein 30 pg / ml Dabigatran is used, the lag-time could not be determined as there was no signal.

[0106] Fiq. 5C - Same experiment as in Fig. 5A and B, but showing the AUC (RLU).

[0107] Fiq. 5D - Same experiment as in Fig. 5A-C, but showing the VI (RLU / min).

[0108] Fiq. 6A - The reversal effect of Prothromplex on DOACs. The measurement was performed in duplicates. The addition of 0.5 lU / ml Anti-heparin to all DOAC concentration with 1 lU / ml Prothromplex was made to intervene the anti-coagulant effect of heparin in Prothromplex. Panel A shows the mean results of the TPH.

[0109] Fiq. 6B - Same experiment as in Fig. 6A, but showing the mean effect of PT on the LT (minutes).

[0110] Fiq. 6C - Same experiment as in Fig. 6A and B, but showing the mean of the overall formed thrombin over time (AUC (RLU)).

[0111] Fiq. 6D - Same experiment as in Fig. 6A-C, but showing the mean VI (RLU / min).

[0112] Fiq. 7A - The reversal effect of FEIBA on 350 and 700 ng / ml DOACs measured in duplicates. Panel A shows the mean results of the TPH.

[0113] Fiq. 7B - Same experiment as in Fig. 7A, but showing the mean effect of PT on the LT (minutes). In the condition wherein 350 and 700 ng / ml Dabigatran is used in absence of FEIBA, the lag-time could not be determined as there was no signal.

[0114] Fiq. 7C - Same experiment as in Fig. 7A and B, but showing the mean of the overall formed thrombin over time (AUC (RLU)).

[0115] Fiq. 7D - Same experiment as in Fig. 7A-C, but showing the mean VI (RLU / min).

[0116] Fiq. 8A - Calibration line of the chromogenic Dabigatran assay. Three concentrations of 50, 250 and 500 ng / ml Dabigatran spiked NPP were measured. The calibration line describes a linear function with y = -0.0009x + 1 .4496 with a R2of 0.984.

[0117] Fiq. 8B - Luminescent Dabigatran assay is used to measure the concentration Dabigatran in a plasma sample. The calibration curve contains 4 concentrations of Dabigatran measured in duplicates: 0, 50, 250 and 500 ng / ml. The calibration curve describes an exponential function with y = 278020e-°OO43xand a R2of 0.996.

[0118] Fiq. 9A - The luminescent anti-FXa assay is employed for the quantitative measurement of Apixaban, Edoxaban and Rivaroxaban concentration in plasma samples. Panel A shows the calibration curve of Apixaban, comprising four concentrations: 0, 158, 317, and 632 ng / ml of Apixaban. The calibration curve follows an exponential function y = 910663e_°OO243xwith an R2of 0.9673.

[0119] Fiq. 9B - Same as in Fig. 9A, but showing the calibration curve of four concentrations of 0, 125, 251 , and 500 ng / ml Edoxaban, which follows an exponential decline with a function y = 114651e-°00232xand R2of 0.9732.

[0120] Fig. 9C - Same as in Fig. 9A, but showing the calibration curve of 0, 125, 251 , and 513 ng / ml Rivaroxaban in the luminescent anti-FXa assay. The function of the calibration curve is described by y = 1148409e(_°'OO4x)with an R2of 0.9718.

[0121] Fig. 10 - Reversal of FXa-DOACs with Andexanet alfa using the luminescent anti-FXa assay. The FXa-DOAC concentrations are guantitatively measured with the function of the calibration curve of the assay. The FXa-DOAC concentrations are guantitatively measured using the calibration curve of the assay. Following the addition of 80 pg / ml Andexanet alfa, the FXa-DOAC concentrations at both lower (317 ng / ml Apixaban, 250 ng / ml Edoxaban, and 250 ng / ml Rivaroxaban) and higher concentrations (632 ng / ml Apixaban, 500 ng / ml Edoxaban, and 513 ng / ml Rivaroxaban) are determined. The assay can measure that Andexanet alfa effectively reduces the concentration of FXa-DOAC, and to which level.

[0122] Examples

[0123] Example 1 - Introduction

[0124] In pursuit of advancing the field of hemostasis diagnostics, we developed a diagnostic point-of-care device. It offers accurate and real-time assessments of patients’ hemostasis status by employing the FVIIlium assay and luminescent Thrombin Generation Assay (TGAium) within a microfluidic cartridge.

[0125] Coagulation: coagulation is part of the secondary hemostasis and includes two pathways, namely the intrinsic and extrinsic pathways. Various clotting factors play a role in promoting blood clot formation in these processes. The intrinsic pathway is a more extensive pathway in the coagulation process, activated by contact with damaged tissues. In this pathway, activation of factor XII (FXIIa) initiates a series of events culminating in the activation of factor IX, which subseguently binds to factor VIII to form the tenase complex. The extrinsic pathway (Fig. 1), is initiated by the release of Tissue Factor (TF) by injured subendothelial cells, this leads to the formation of the TF-Factor Vila (FVIIa) complex. These two pathways are brought together into the common pathway where the activation of Factor X (FX) takes place, which in complex with activated Factor V (Fva) converts prothrombin (Fll) into thrombin (Flla). Thrombin is an important enzyme that converts fibrinogen into fibrin and it also results in more thrombin generation which is also called the propagation phase. Coagulation is important to stabilize blood platelets by forming the fibrin network, which will eventually be terminated by fibrinolysis. The process is regulated at multiple points in the pathway to prevent uncontrolled blood clotting. Abnormalities in the coagulation process lead to medical conditions such as hemophilia and thrombosis.

[0126] Table 1. Summary of the structure, mechanism of action, treatment, dosing (adjustment), pharmacodynamics and kinetics, clearance and most common side effect of Apixaban, Edoxaban, Rivaroxaban and, Dabigatran

[0127] Direct oral ant-coagulants (DOACs): to prevent diseases such as thrombosis, patients are treated with anticoagulants. Traditionally, anticoagulants like vitamin K antagonists (VKA) and unfractionated heparin were employed for blood clot management. However, these treatments come with certain limitations, such as the need for frequent monitoring, variability in dosage adjustments, interindividual variation in anticoagulant effects, and drug-food interactions. Over the years, the use of DOACs has increased, as they address these limitations. DOACs inhibit blood coagulation by binding to Fl la or Fxa. In the Netherlands, four registered DOACs are currently used for blood clot management, including three Fxa inhibitors: Apixaban (Eliquis), Edoxaban (Lixiana), Rivaroxaban (Xarelto), and a Flla inhibitor Dabigatran (Pradaxa). Apixaban, Edoxaban, and Rivaroxaban specifically inhibit both bound and unbound Fxa generated through either of the two pathways. Similarly, Dabigatran targets and inhibits both bound and unbound Flla.

[0128] Food intake does not affect DOAC concentration and the risk of extended bleeding is the most common side effect of all DOACs. All DOACs are commonly used in the treatment of various cardiovascular conditions, with one of the most prevalent being atrial fibrillation, particularly non- valvular atrial fibrillation (NVAF). NVAF refers to atrial fibrillation occurring independently of mitral stenosis orthe presence of valvular prostheses, which increases the risk of stroke, systemic emboli, and death. Studies have consistently shown that treating NVAF with DOACs can be a preferable therapy compared to vitamin K antagonists. Additionally, DOACs have demonstrated effectiveness in the treatment of Venous Thromboembolism (VTE), which can be a complication of NVAF. VTE is caused by the formation of blood clots in the veins. VTE includes Deep Venous Thrombosis (DVT), which occurs when a blood clot forms in the deep veins. DVT is the third leading cause of cardiovascular disease-related mortality. Leaving blood clots untreated may result in their rupture, leading to serious complications such as pulmonary embolism (PE), which is a blood clot in the lungs. Overall, DOACs play a crucial role in the management of NVAF and VTE, offering advantages over traditional therapies in terms of efficacy and safety. The dosage, pharmocodynamica and kinetica features as well as the structures of the DOACs are shown in Table 1 .

[0129] DOAC antidotes: over the years, various DOAC antidotes have been developed to facilitate the monitoring and control of DOAC usage. These antidotes play an important role in mitigating the anticoagulant effects, particularly during moments such as surgical interventions. These DOAC antidotes can be categorized in different classes, including anti-Fxa inhibitors like Andaxanet alfa, anti-Flla inhibitors such as Idarucizumab, and procoagulant factor concentrates like prothrombin complex concentrates.

[0130] Andexanet alfa (Anexxa / Ondexxya) is an anti-Fxa inhibitor designed to neutralize the anticoagulant effects of direct Fxa DOACs Apixaban and Rivaroxaban. There is not enough evidence for the reversal of Edoxaban. It is a modified recombinant human Fxa protein that functions as a decoy protein. Unlike human FX, Andexanet Alfa lacks the GLA domain, preventing it from binding to phospholipids and calcium ions, thereby avoiding pro- or anticoagulant activity. Individually, Andexanet alfa is also catalytically inactive due to the presence of a distinct catalytic domain (A419), divergent from the S419 catalytic domain of Fxa. However, it retains the capacity to bind to and neutralize Fxa inhibitors. This mechanism ultimately restores the activity of human Fxa. Clinical trials of healthy volunteers pretreated with apixaban or rivaroxaban shows that Andexanet alfa restored the thrombin generation within 2 to 5 minutes. The administration dosage is contingent upon the prescribed dose of Apixaban and Rivaroxaban. In cases where a low dose of DOAC is utilized (<10 mg), a corresponding administration of 480 mg is employed. In instances where a higher dosage exceeding 10 mg is prescribed, an administration of 960 mg will be implemented. The elimination half-life is 5 to 7 hours after injection.

[0131] Idarucizumab (Praxbind) functions as a monoclonal antibody which neutralize the inhibitory impact of Dabigatran on thrombin. This specific binding forms a high-affinity complex, Idarucizumab binds to dabigatran with an affinity which is 350 times higher than dabigatran’s affinity for Flla, swiftly mitigating the anticoagulant effects and restoring the balance of coagulation. Patients are administered with 5 grams Idarucizumab, and a second dose of 5 grams is used if prolonged clotting times are observed. It has a rapid onset of action, the peak plasma is reached within minutes with a half-life of 10 hours. Elimination is through renal clearance and endogenous proteases (protein catabolism).

[0132] In contrast to the direct DOAC inhibitor, an alternative approach involves the strategic use of prothrombin complex concentrate (PCC). Unlike the DOAC inhibitors, PCC does not directly interfere with the active sites of the DOAC rather, it employs a competitive inhibition strategy to counteract the anticoagulant effects induced by DOACs. PCC contains factors II, VII, IX, and X, with an overall concentration which is 25 times higher than in normal plasma. It also contains heparin to prevent activation of the coagulation factors. When administered PCC competes for the active binding sites on coagulation factors, particularly factors II and X. By competitively binding to these active sites, PCC effectively reduces the availability of binding sites for direct inhibition by DOACs. As a result of this competitive inhibition and factor replenishment, PCC counteracts the anticoagulant effects induced by DOACs. The increased concentration of coagulation factors facilitates an enhanced thrombin generation. Factor eight inhibitor bypassing activity (FEIBA), comprising both activated and non-activated factors of PCC, is commonly utilized in Hemophilia A therapy. However, due to its composition of the same 4-factor concentrate as PCC, it will be used in this project to explore the potential for DOAC reversal.

[0133] Monitoring assays: the variability in inter- and interlevel of DOACs poses challenges in accurately measuring DOAC plasma levels. To gain insights into the coagulation status and DOAC concentration, the TGA, Anti-Fxa, and Anti-Fl I a assays were employed.

[0134] The TGA is a global hemostasis assay that measures thrombin generation in plasma in real time, contrasting with traditional tests that measure the endpoint. This assay provides a comprehensive view of thrombin generation over time. It operates on the principle of initiating the extrinsic coagulation pathway, utilizing components such as TF, phospholipids, and Normal Pooled Plasma (NPP). We utilized a luminescent variant of TGA, employing a luminescent thrombin substrate. When this reacts with thrombin, D-amino luciferin is formed, which is converted into oxyluciferin and light by firefly luciferase. In Fig. 2, a general graph of the Thrombin Generation Assay is presented, this includes parameters such as lag-time, peak height, and time to peak. Lag- time (LT) represents the duration between the initiation of coagulation and the first-formed thrombin. Thrombin peak height (TPH) indicates the maximum thrombin concentration reached during the assay. The time to peak (TTP) correspond to the duration from initiation to reaching the peak thrombin concentration.

[0135] The Dabigatran assay utilizes a luminescent Flla substrate to measure the impact of Dabigatran on Flla (anti-FII a assay). The assay is based on the following principle:

[0136] [Dabigatran] + [Flla (excess)] — [Flla-Dabigatran] + [Flla residual]

[0137] [Flla (residual)] + Flla luminescent substrate — D-aminoluciferin + luciferase +MgCl2 + ATP + O2 — Oxyluceferin + light

[0138] This mechanism involves a DOAC inhibiting excess Flla, forming a complex. Residual Flla reacts with a luminescent substrate, generating D-amino luciferin. In the presence of luciferase, magnesium chloride, ATP, and oxygen, D-amino luciferin undergoes enzymatic reactions, resulting in the production of oxyluciferin and light emission.

[0139] Similarly, the luminescent anti-Factor Xa (Fxa) assay operates on the same principle, with a luminescent Fxa substrate employed to determine the anticoagulant effect of Apixaban, Edoxaban and Rivaroxaban on Fxa.

[0140] [Fxa DOAC] + [Fxa (excess)] [Fxa-Fxa DOAC] + [Fxa residual]

[0141] [Fxa (residual)] + Fxa luminescent substrate — D-aminoluciferin + luciferase + MgC + ATP + O2 — Oxyluceferin + light

[0142] Aim: in contemporary medical practice, the administration of direct oral anticoagulants (DOACs) is not routinely accompanied by systematic monitoring of their predictable effects. However, it is imperative to underscore the necessity of monitoring in specific demographics, including the elderly and individuals afflicted with renal impairment or related conditions, to tailor the treatment to the unique requirements of such patients. To regulate the effects of DOACs, this project at employs DOAC antidotes such as Andexanet alfa, Idarucizumab, and prothrombin complex concentrate (PCC). Additionally, the impact of FEIBA on DOACs is being analyzed. The aim of this study was to develop a DOAC (-inhibitor) monitoring plan. This plan involves the measurement of the effects and concentrations of both DOACs and various antidotes using the luminescent Thrombin Generation Assay (TGA), anti-FI la, and anti-Fxa assays.

[0143] Example 2 - Materials and Methods

[0144] For monitoring and regulating the utilization of DOACs and their antidotes, a 60 pL luminescent thrombin generation assay was employed, allowing the measurement of the general coagulation process. To determine the concentrations of DOACs, the Anti-Dabigatran and Anti-Fxa assays were utilized.

[0145] Reagents: Bovine serum album (BSA), calcium chloride (CaCL), citric acid, 4-(2-hydroxyethyl)-1- piperazine-ethane-sulfonic acid (HEPES), magnesium chloride (MgCL) sodium chloride (NaCI), were obtained from Sigma Aldrich (USA). The assay buffer consists of 25 mM HEPES, 0,5% BSA, and 125 mM NaCI. Adenosine 5’triphosphate (ATP) was provided by Merck (Germany), the phospholipids were obtained from Rossix AB (Molndal, Sweden), TF was obtained from Siemens Healthineers (Germany), and tPA was provided by Radboud Pharmacy (Nijmegen, the Netherlands). Both Luminescent beta-AGR-aminoluciferin thrombin substrate (referred to as S12) and the FXa substrate MePEG2-IEGR as shown earlier herein (referred to as S30) were obtained from Symeres (Nijmegen, the Netherlands). The human thrombin substrate of the Direct Thrombin Inhibtor kit provided by Hyphen BioMed (France) was used in the chromogenic Dabigatran assay. The DOACs Apixaban (C2109) and Dabigatran (C1586), were obtained from Alsachim (France), Rivaroxaban (FA140518) from Hyphen BioMed (France) and Edoxaban was provided by Technoclone (Austria). The DOAC reversal agents Prothromplex (PCC) and FEIBA were provided by Takeda (Zurich, Switzerland), Idarucizumab was obtained from Boehringer Ingelheim and Andexanet alfa was provided by AstraZeneca AB (Sweden). Normal pooled plasma was obtained from Radboud UMC (Nijmegen, the Netherlands). The lyophilized Fxa-DOACs spiked plasmas, which were used in the luminescent anti-Fxa assay, with predetermined concentrations of Fxa DOAC were established and provided by Hyphen BioMed (France). Thrombin-ctM is obtained from Stago (Canada) and human Fxa is provided by CoaChrom Diagnostica GmbH (Maria Enzersdorf, Austria). Quantilum® Recombinant firefly (Photinus pyralis) luciferase and Ultra-Gio™ recombinant firefly (Photuris pennsylvanica) luciferase were provided by Promega (Leiden, the Netherlands).

[0146] Luminescent thrombin generation assay (TGAium): to perform the 60 pL luminescent thrombin generation assay, a component mix was first prepared, consisting of 8.3 pM phospholipids, 1 mM MgCI2, and 0.75 mg / mL Quantilum® (a luciferase expressed from a cloned gene from the North American firefly Photinus pyralis) in assay buffer. Subsequently, the start reagent was prepared, comprising of 830 pM S12 Beta-ARG-amino luciferin thrombin substrate, 16.7 mM CaCI2, 1.1 mM ATP, 0.6 pg / mL tPA, and 1 pM tissue factor. The component mix, start reagent, and normal pooled plasma were incubated for 1 minute at 37°C in a water bath (Memmert). Following this, 40 pL NPP, 6.5 pL component mix and 4 pL of different end concentrations of DOAC and / or DOAC antidote were pipetted into the pre-incubated (37°C) half area opaque assay plate (Greiner Bio-One). I specific concentrations of the DOACs and antidotes are shown in the tables below. The plate was incubated for one minute at 37 degrees Celsius and 550 rpm in the ThermoMixerOC (Eppendorf). To initiate thrombin generation, 9.5 pL start reagent were added to the wells, and the luminescence was kinetically measured every 30 seconds for 30 minutes using the Flexstation®3 (a multi-mode microplate reader that measures absorbance, fluorescence intensity, fluorescence polarization, luminescence, and time-resolved fluorescence, Molecular devices) at 37°C.

[0147] Table 2. DOAC concentrations that were used in TGAium.

[0148] Table 3. DOAC antidotes concentrations that were used in the TGAium.

[0149] Dabigatran assay: The determination of Dabigatran concentration in plasma is achieved through the Dabigatran assay. To perform the chromogenic method the Dabigatran-spiked plasma was initially diluted 10-fold in assay buffer. Subsequently, in a pre-incubated (37°C) 96-well EIA / RIA flatbottom plate (Corning), 50 pL of the diluted Dabigatran-spiked plasma was combined with 50 pL of Thrombin substrate (Hyphen). This mixture was then incubated for 2 minutes ThermoMixer®C at 37°C and 550 rpm. Following the initial incubation, 50 pL of pre-incubated (37°C) 20 nM thrombin (Hyphen) was added to the plasma and incubated in the ThermoMixer®C for an additional 2 minutes at 37°C and 550 rpm. The reaction was halted by adding 100 pL of 2% citric acid, and the optical density (CD) was measured at 37°C and 405 nm using the Flexstation®3 (Molecular Devices).

[0150] To convert the chromogenic assay into a luminescent assay, a Lucimix solution was first prepared comprising 1 mM MgCI2, 830 pM S12 luminescent thrombin substrate, 1 mM ATP, and 0.75 mg / ml Quantilum® in assay buffer. Four concentrations of Dabigatran-spiked plasma (0, 50, 250, and 500 ng / ml) were diluted 5-fold in assay buffer. Subsequently, 50 pL of the diluted Dabigatran-spiked plasma and 50 pL of the Lucimix were added into a pre-incubated (37°C) 96 wells half-area opaque assay plate. The plate was then incubated ThermoMixerOC for three minutes at 37°C and 550 rpm. To initiate the reaction, 50 pL of 100 nM thrombin-a2M was added, and luminescence was measured for 30 minutes at 37°C using the Flexstation®3.

[0151] Luminescent anti-Fxa assay: Like the luminescent anti-Dabigatran assay the anti Fxa assay determines the concentration of the Fxa DOACs in plasma. To perform the assay, a lucimix was prepared, comprising 1 mM S30 Fxa substrate, 1 mM MgCI2, 1 mM ATP, and 0.7 mg / ml Ultragio in HEPES, NaCI, and BSA buffer. The four predetermined concentrations of the lyophilized Fxa DOACs (Apixaban, Edoxaban and Rivaroxaban) are shown in the table. The Fxa DOAC spiked plasma was diluted 16 times in HEPES, NaCI, and BSA buffer. Next, 5 pL of the diluted Fxa DOAC spiked plasma and 20 pL of HEPES, NaCI, BSA buffer were pipetted into pre-incubated (37°C) wells of a 96-well half-area opaque assay plate. The plate was then incubated in a ThermoMixer®C for 1 .5 minutes at 37°C and 550 rpm. Subsequently, 10 pL of 10 nM preheated human Fxa and 10 pL of the lucimix were added to the diluted plasma. Luminescence was measured for 10 minutes at 37°C using the Flexstation®3.

[0152] Data analysis: The TGAium data collected from the Flexstation®3 in a Microsoft Excel (Microsoft 365, USA) file were transferred into Graphpad prism 10 (Dotmatics, United Kingdom) to obtain the TGAium graphs. The various parameters, including thrombin peak height (TPH), lag time (LT), area under the curve, and velocity index of the different TGAium samples, were calculated using Microsoft Excel. TPH was determined by identifying the point of maximum measured thrombin within 30 minutes, and the time taken to reach this point was defined as the time to peak (TTP). The Microsoft Excel function (=MAX) was utilized for the calculation of the TPH. Lag time values were obtained from the TGAium curves by identifying the moment where the curve first bends beyond three standard deviations of its initial average value. To calculate the area under the curve (AUC), the sum of all thrombin formed in relative light units (RLU) over 30 minutes was computed.

[0153] AUC = Relative light units of 1 (spiked)plasma sample

[0154] The velocity index was calculated using the formula below for each measured plasma sample: Velocity index = TPH / (TTP - LT)

[0155] To create the bar charts depicting various parameters of the TGAium, data obtained in Microsoft Excel was transferred to GraphPad Prism 10.

[0156] For the luminescent Dabigatran assay, the maximum luminescent signal value was calculated in Microsoft Excel to generate the calibration curve in GraphPad Prism 10. Additionally, the analysis function was utilized to conduct an exponential growth analysis, resulting in an exponential function representing the calibration curve along with its corresponding R-squared value. Similarly, calibration curves for the luminescent anti-Fxa assay were constructed using the same approach. The Fxa-DOAC concentration of different Fxa-DOAC spiked plasma samples with the addition of 80 pg / ml Andexanet alfa were calculated in Microsoft Excel with the function of the calibration curve.

[0157] Example 3 - Results

[0158] Effect of DO AC on coagulation: to analyze the global effect of the DOACs on the coagulation the TGAium was utilized. Fig. 3 illustrates the mean thrombin generation curves resulting from various concentrations of the DOACs, Apixaban, Edoxaban and Dabigatran, utilized in TGAium. Three data points were acquired per sample of DOAC-spiked NPP. Notably, the NPP samples across all three TGAium assays exhibited luminescence levels ranging from 1 to 1 .2 million relative light units (RLU). Among the three DOACs investigated, Apixaban demonstrated the least inhibitory effect on thrombin generation (fig. 3A). Specifically, the mean thrombin peak (TPH) height for Apixaban at a concentration of 350 ng / ml showed a reduction of 30%, along with an extension of 1 minute in lag- time (LT) and 1.5 minutes in time to peak (TTP) compared to the baseline coagulation profile observed in NPP without Apixaban. Upon increasing the concentration of Apixaban to 700 ng / ml, the anticoagulant effect intensified. This was evidenced by a 70% decrease in TPH compared to the NPP sample, accompanied by further extensions in both LT and TTP. The anticoagulant effect of Edoxaban (Fig. 3B) appears more pronounced than that of Apixaban, despite both being Factor Xa inhibitors. At a concentration of 350 ng / ml, Edoxaban reduces TPH by 75%, accompanied by a notable extension in lag time, ranging from 2.5 to 3 minutes. In comparison to the NPP samples spiked with 350 ng / ml Edoxaban, the higher concentration of 700 ng / ml Edoxaban spiked NPP samples do not fully suppress coagulation, with only marginal differences observed in TPH, LT, and TTP between the two concentrations. In the TGAium, both concentrations of 350 and 700 ng / ml Dabigatran-spiked NPP effectively inhibit thrombin generation, as depicted in Fig. 3C.

[0159] Hence, Fig. 3 shows that as the concentration of Apixaban increases from 0 to 350 and 700 ng / ml, coagulation is reduced by 30 to 70%, accompanied by prolonged lag times, indicating that thrombin formation takes longer compared to the baseline situation. Similarly, the addition of 350 and 700 ng / ml Edoxaban to NPP leads to a 70 to 80% inhibition of coagulation, along with extended lag times. Both concentrations of 350 and 700 ng / ml Dabigatran entirely suppress thrombin generation.

[0160] Fxa-DOAC reversal with Andexanet alfa: The reversal of Fxa DOACs, namely Apixaban and Edoxaban, with Andexanet alfa, was also assessed using the TGAium. Mean results of thrombin peak TPH, LT, AUC, and VI are depicted in Fig. 4. Notably, in the Apixaban measurement, the baseline NPP sample without Fxa-DOAC and Andexanet alfa exhibited lower values in TPH, AUC, and VI compared to the Edoxaban measurement, as shown in Fig. 4A, C and D. Additionally, a slight extension in LT of 0.5 minutes was observed, as depicted in fig. 4B.

[0161] Both 700 ng / ml Fxa-DOAC spiked NPP samples demonstrated an equivalent decrease in TPH signal to 290,000 RLU, as shown in fig. 4A. Similarly, the AUC and VI values were approximately consistent across both 700 ng / ml Fxa-DOAC spiked NPP samples. In the case of 700 ng / ml Apixaban spiked NPP samples, there was a LT extension of 1.5 minutes compared to NPP samples without Fxa DOAC and Andexanet alfa. Conversely, for the 700 ng / ml Edoxaban spiked NPP plasma, the difference in lag time compared to NPP samples without Fxa-DOAC was 2 minutes compared to the baseline situation, as shown in fig. 4B.

[0162] The impact of Andexanet alfa on TPH in the presence of 700 ng / ml Apixaban is shown in Fig. 4A. The luminescent signal of TPH exhibits an increase with increasing concentrations of Andexanet alfa. Specifically, when 5 to 10 pg / ml Andexanet alfa was introduced to 700 ng / ml Apixaban-spiked NPP, the anticoagulant effect of Apixaban was entirely neutralized. Consequently, TPH and AUC become comparable to the baseline levels observed in NPP, as illustrated in Fig. 4A and C. Remarkably, compared to the baseline measurement, the LT and TTP was further shortened to 0.5 minutes upon supplementation with 10 and 80 pg / ml Andexanet alfa to 700 ng / ml Apixaban spiked NPP, as shown in Fig. 4B and Fig. 4E. Moreover, the VI demonstrates an increase compared to NPP samples, beginning at an additive concentration of 5 pg / ml Andexanet alfa (Fig. 4D). Beyond a concentration range of 10 to 80 pg / ml Andexanet alfa, a plateau was reached wherein TPH, AUC, and VI did not further increase, and the lag time remains unaltered, fig. 4A and B also demonstrated that TPH and AUC increased with increasing concentrations of Andexanet alfa in 700 ng / ml Edoxaban-spiked NPP. The baseline condition was achieved only with the addition of 80 pg / ml Andexanet alfa, where TPH, LT, and AUC were approximately the same as the baseline. Additionally, the VI of 80 pg / ml Andexanet alfa in 700 ng / ml Edoxaban falls within the range of the NPP sample, as shown in fig. 4D.

[0163] Thus, the addition of Andexanet alfa to Fxa-DOAC spiked plasma effectively reverses the anticoagulant effect of the DOACs, as shown in Fig. 4. For Apixaban-spiked plasma, all parameters are comparable to the baseline situation from 10 pg / ml Andexanet alfa and higher. The shortened LT and TTP observed when 10 pg / ml Andexanet alfa is added to 700 ng / ml Apixaban spiked plasma indicate the inhibitory effect of Andexanet alfa on TFPI. Conversely, for 700 ng / ml (1.523 pM) Apixaban, complete reversal is achieved with only 10 pg / ml (0.249 pM) Andexanet alfa, corresponding to a stoichiometric Apixaban to Andexanet alfa ratio of 1 :0.16. The pro-coagulant effect of Andexanet alfa was less pronounced at lower concentrations of Edoxaban, until the addition of 80 pg / ml (1.995 pM) Andexanet to 700 ng / ml (1.277 pM) Edoxaban, which leads to complete reversal of the anticoagulant effect of Edoxaban. Because Edoxaban exhibited a stronger anticoagulant effect compared to Apixaban in the TGAium assay, a higher concentration of Andexanet alfa was required, with a stoichiometric Edoxaban to Andexanet alfa ratio of 1 :1.56 in this case. Hence, this indicates that a lower concentration of Andexanet alfa is sufficient for complete neutralization with Apixaban, in contrast to Edoxaban, where a higher concentration is necessary for neutralization.

[0164] Flla-DOAC reversal with Idarucizumab: The reversal of Dabigatran with Idarucizumab was assessed using the TGAium, and the TGA parameters are shown in Fig. 5. The TPH (fig. 5A) increased as the concentration of Idarucizumab rises, with a complete reversal to baseline conditions observed when 60 pg / ml Idarucizumab is added to 700 ng / ml Dabigatran-spiked NPP. This effect is similarly observed in LT, AUC and VI, as shown in fig. 5B, C and D. The same trend was evident with the addition of 50 pg / ml Idarucizumab, although the condition was restored to only 80%, along with an extended LT of 4 to 5 minutes. There was a minimal reversal in TPH and AUC observed with 30 and 40 pg / ml Idarucizumab (Fig. 5A and 5C), but this concentration was insufficient to reach baseline conditions when looked at the LT and VI (Fig. 5B and Fig. 5D).

[0165] Therefore, with the addition of varying concentrations of Idarucizumab, the observed parameters returned toward baseline levels as the Idarucizumab concentration increased. Complete reversal of the anticoagulant effect of 700 ng / ml (1.115 pM) Dabigatran was achieved at 60 pg / ml (1.25 pM) Idarucizumab, suggesting a stoichiometric ratio of Dabigatran to Idarucizumab of approximately 1 :1 .17 for neutralizing the anticoagulant effect. Furthermore, from 30 pg / ml (0.625 pM) Idarucizumab onwards, the reversal of anticoagulants was starting to emerge, indicating that a stoichiometric ratio of Dabigatran to Idarucizumab of 1 :0.585 can already be beneficial.

[0166] DOAC reversal with PCC: To assess the reversal of DOACs using competitive inhibition, Prothromplex (PT) was utilized. Baseline measurements with NPP without DOAC and PT consistently yielded similar TPH, LT, AUC, and approximately equivalent VI across all three measurements.

[0167] Spiking 350 ng / ml of Apixaban into NPP resulted in minimal coagulation inhibition, as indicated by TPH; however, at a higher concentration of 700 ng / ml, Apixaban reduced TPH by 40%, as depicted in fig. 6A. LT was extended with both concentrations, reaching 1 minute with 350 ng / ml and 1 .5 minutes with 700 ng / ml Apixaban, as shown in fig. 6B. Edoxaban at 350 ng / ml caused 70% inhibition, while 700 ng / ml led to 80% inhibition of TPH. Both concentrations prolonged LT to 2.5 minutes at 350 ng / ml and 3.5 minutes at 700 ng / ml Edoxaban, as illustrated in fig. 6B. Both concentrations of Dabigatran completely halted coagulation.

[0168] Adding 1 lU / ml PT to 350 ng / ml Apixaban-spiked NPP amplified thrombin generation, resulting in a TPH of 1 .7 million RLU (Fig. 6A), though it did not alter LT (Fig. 6B). Moreover, adding 1 lU / ml PT to 350 ng / ml Apixaban-spiked plasma doubled the total thrombin formed compared to NPP samples, as shown in Fig. 6C. Addition of 1 lU / ml PT to 700 ng / ml Apixaban-spiked NPP reversed the condition to that of NPP in terms of TPH, and it led to a slightly lower VI and a slight increase in AUC. The LT of 700 ng / ml Apixaban-spiked NPP remained unchanged after PT addition.

[0169] The effect of adding 1 lU / ml PT to 350 ng / ml Edoxaban-spiked NPP was less pronounced than that with Apixaban-spiked NPP. PT addition restored coagulation by 50% in terms of TPH for 350 ng / ml Edoxaban, while for 700 ng / ml Edoxaban, TPH increased from 183,000 to 390,000 RLU. Additionally, PT addition did not shorten LT at either concentration of Edoxaban-spiked NPP; LT increased from 2.5 to 3 minutes with 350 ng / ml Edoxaban and remained at 3.5 minutes with 700 ng / ml Edoxaban. For both concentrations of Dabigatran-spiked NPP, the addition of 1 lU / ml PT did not reverse coagulation. DOAC reversal with activated PCC: Similar to PCC, the activated form was also used to assess the reversal effect. Baseline measurements with NPP without DOAC and PT consistently yielded similar TPH, LT, AUC, and VI across all three measurements, as depicted in Fig. 7. Spiking 350 ng / ml of Apixaban into NPP resulted in a 50% reduction in TPH, while 700 ng / ml led to a 70% reduction, extending the LT to 3 minutes. Both concentrations of Edoxaban exhibited stronger inhibition in thrombin generation, with 350 ng / ml causing a 75% reduction in TPH, and 700 ng / ml resulting in a 90% reduction, as shown in fig. 7A. Additionally, the LT was extended to 3-5 minutes. In this measurement as well, coagulation was completely inhibited by both concentrations of Dabigatran.

[0170] The addition of 1 lU / ml FEIBA induces prothrombotic effects in both concentrations of Apixaban-spiked NPP, with TPH (Fig. 7A) values exceeding 2 million RLU and total RLU ranging from 40 to 60 million when considering AUC, as depicted in Fig. 7C. Additionally, it leads to a shortened LT of 0.5 minutes in both concentrations of Apixaban-spiked NPP. Furthermore, there is a significant increase in thrombin formation per minute, as illustrated in Fig. 7D.

[0171] In fig. 7A, it is observed that the addition of FEIBA to 350 ng / ml Edoxaban-spiked plasma results in a reversal of the anticoagulant effect of Edoxaban. Additionally, the LT returns to the condition observed in the NPP samples, with an LT of 1 minute. However, the rate of thrombin formation per minute is slower than the baseline situation of the NPP samples, as depicted in fig. 7D. The total thrombin formed is also lower than the baseline situation in NPP (Fig. 7C). In this measurement the addition of 1 lU / ml FEIBA to 700 ng / ml Edoxaban spiked plasma causes prothrombotic events.

[0172] The addition of 1 lU / ml FEIBA to 350 ng / ml Dabigatran-spiked NPP results in an increase in TPH to 1.5 million RLU. However, in 700 ng / ml Dabigatran-spiked NPP, FEIBA is not potent enough to neutralize the anticoagulant effect of Dabigatran. Additionally, the total thrombin formed is higher in 350 ng / ml Dabigatran-spiked NPP with the addition of 1 lU / ml FEIBA. FEIBA also leads to an increase in VI in 350 ng / ml Dabigatran-spiked NPP, as depicted in fig. 7D. In 350 ng / ml Dabigatran, 1 lU / ml FEIBA prolongs LT by 9 minutes, and this extends to 25 minutes in 700 ng / ml Dabigatran with 1 lU / ml FEIBA, as shown in fig. 7B.

[0173] Development of the luminescent Dabigatran assay: To conduct the luminescent Dabigatran assay, the initial step involved utilizing the chromogenic method to ascertain the suitability of the measurement range for determining Dabigatran concentration. Fig. 8A illustrates the calibration curve generated from the chromogenic assay. Three concentrations 50, 250, and 500 ng / ml of Dabigatran-spiked plasma samples were employed to establish this calibration curve. Concentrations below 250 ng / ml of Dabigatran were selected to align with practical dosing levels used in clinical practice, while 500 ng / ml represents a higher concentration to achieve a wide window of opportunity for accurate measurement of Dabigatran concentration. In the chromogenic assay a narrow range was observed between the samples, wherein the optimal density (OD) transitioned from 1 .4 for the 50 ng / ml Dabigatran-spiked plasma sample to an OD of 1 .0 for the 500 ng / ml Dabigatran-spiked sample. The calibration line is described by a linear function of y = - 0.0009* + 1.4496 with an R2of 0,984. The y-axes represent the OD and the x-axes represent the Dabigatran concentration (ng / ml).

[0174] Fig. 8B shows calibration curve of the luminescent method of the Dabigatran assay. The luminescent method was assessed with the aim to obtain a wider range in between samples to measure the concentration Dabigatran more precisely. The calibration curve is constructed using the peak luminescence corresponding to each individual spiked plasma concentration of Dabigatran. The luminescent calibration curve shows an exponential decline in luminescence (RLU) as the concentration of Dabigatran increases. The exponential curve is described by the function of y = 278020e_°OO43xwith a R2of 0,996. In Fig. 8B, y represents luminescence (RLU) and x represents the Dabigatran concentration (ng / ml). Surprisingly, the assay exhibits a wide window of opportunity, particularly between 0 and 250 ng / ml Dabigatran, enabling the quantitative measurement of Dabigatran concentrations in plasma samples. Therefore, utilizing this calibration curve enables the accurate quantitative determination of unknown Dabigatran concentrations in plasma inside the range of 0 to 500 ng / ml Dabigatran.

[0175] Luminescent Anti-Fxa assay: The luminescent anti-Fxa assay is employed to measure the concentration of Fxa DOACs in plasma, fig. 9A depicts the calibration curve of the anti-Fxa assay using Apixaban-spiked plasma samples. Concentrations ranging from 0 to 317 ng / ml were selected to align with practical Apixaban dosing levels. Additionally, a higher concentration of 632 ng / ml Apixaban was included to establish a calibration curve, with a wide window of opportunity, capable of quantitatively measuring Apixaban concentrations spanning from 0 to 632 ng / ml. This curve is constructed based on the luminescent signal of the various concentrations of Apixaban spiked in plasma, measured at t = 1 minute. Due to the decrease in signal over time, in the measurement it was not possible to construct the calibration curve with the maximum luminescence. The curve exhibits an exponential decrease in luminescence with increasing concentrations of Apixaban, described by the function y = 910663ef_O OO2',3xjwith an R2of 0.9673. The y-axes represents the luminescent signal (RLU) and the x-axes represent the Fxa-DOAC concentration (ng / ml).

[0176] The luminescent anti-Fxa assay is also conducted using four concentrations of 0, 125, 251 and 500 ng / ml Edoxaban. The concentrations between 0 and 251 ng / ml of Edoxaban are reflective of the dosages commonly used in clinical practice, while 500 ng / ml represents a higher concentration intended to assess a broader window of opportunity spanning from 0 to 500 ng / ml of Edoxaban. The calibration curve, depicted in fig. 9B, is established based on the luminescent signal at t = 1 of the individual curves. Demonstrating an exponential decrease in luminescence as Edoxaban concentration rises, the curve is defined by the function y = 114651ef_(l (l(l232xjwith an R2of 0.9732.

[0177] Finally, the luminescent anti-Fxa assay is conducted using four concentrations 0, 125, 251 and 513 ng / ml Rivaroxaban, as depicted in Fig. 9C. The concentrations between 0 and 251 ng / ml Rivaroxaban are comparable to the dosages used in clinical practice, while 500 ng / ml represents a higher concentration intended to assess a broader window of opportunity spanning from 0 to 500 ng / ml of Rivaroxaban. Luminescent signals at t=1 for the Rivaroxaban concentrations are utilized to establish the calibration curve. Fig. 16C shows an exponential decline in signal with increasing Rivaroxaban concentration. The curve’s function is described by y = 1148409ef_o'oo',XJwith an R2of 0.9718.

[0178] Hence, the luminescent anti-Fxa assay can be used for the quantitative measurements of Apixaban, Edoxaban and Rivaroxaban concentrations. The calibration curves of all Fxa-DOACs have the same trend with an exponential decline in luminescent signal when the Fxa-DOAC concentration increases, with a wide window of opportunity in between the different concentrations Fxa-DOAC which enables the quantitative measurement of unknown Fxa-DOAC concentrations in plasma.

[0179] Fxa-DOAC reversal with Andexanet alfa measured with the Luminescent anti-Fxa assay: The calibration curves of Apixaban, Edoxaban, and Rivaroxaban enable the quantitative measurement of Fxa-DOAC samples, facilitated by the addition of 80 pg / ml Andexanet alfa, which demonstrates complete reversal of Fxa-DOAC activity within the TGAium. This concentration of Andexanet alfa is added to known concentrations of 317 and 632 ng / ml Apixaban, 251 and 500 ng / ml Edoxaban, and 251 and 513 ng / ml Rivaroxaban. Subsequently, the Fxa-DOAC concentration is quantitatively determined using the calibration curve functions, as depicted in Fig. 10.

[0180] The calibration curve enabled the quantitative measurement of two Apixaban-spiked plasma samples, with concentrations of 317 and 632 ng / ml Apixaban, respectively, along with 80 pg / ml Andexanet alfa. The addition of 80 pg / ml Andexanet alfa to the 317 ng / ml Apixaban sample resulted in an 88% concentration reduction to 38.77 ng / ml Apixaban. In the Apixaban-spiked plasma sample containing 632 ng / ml Apixaban and 80 pg / ml Andexanet alfa, the luminescent signal corresponded to 145.08 ng / ml Apixaban according to the calibration curve function. Therefore, the addition of 80 pg / ml Andexanet alfa to the 632 ng / ml Apixaban sample leads to a 77% reduction in Apixaban concentration, as shown in Fig. 10.

[0181] Fig. 10 illustrates that the addition of 80 pg / ml Andexanet to 251 ng / ml of Edoxaban resulted in a reduction in Edoxaban concentration of 71 % to 72.98 ng / ml Edoxaban. Similarly, the Edoxaban- spiked plasma sample of 500 ng / ml with the addition of 80 pg / ml Andexanet alfa exhibited a Edoxaban concentration reduction to 169.77 ng / ml Edoxaban according to the calibration curve function. This demonstrates a reduction of 66.1 % in Edoxaban concentration compared to the sample with 500 ng / ml Edoxaban without Andexanet alfa.

[0182] The addition of 80 pg / ml Andexanet alfa to 251 ng / ml Rivaroxaban reduced the Rivaroxaban concentration to 30.21 ng / ml, as shown in Fig. 10. This represents an 88% reduction in Rivaroxaban concentration compared to 251 ng / ml Rivaroxaban without Andexanet alfa. Similarly, supplementing 80 pg / ml Andexanet alfa to the 513 ng / ml Rivaroxaban-spiked plasma sample reduced the Rivaroxaban concentration to 58.96 ng / ml. This demonstrates an 88.5% reduction in Rivaroxaban concentration compared to 513 ng / ml Rivaroxaban-spiked plasma without Andexanet alfa. Example 4 - Discussion

[0183] In current medical practice, the administration of DOACs is not routinely accompanied by systematic monitoring because of their predictable effects (Pfreppr, C. et al., 2020). However, it may be imperative to underscore the necessity of monitoring in specific target groups, including the elderly and individuals afflicted with renal impairment or related conditions, to tailor the treatment to the unique requirements of such patients (Ahuja, T., 2023). To regulate the effects of DOACs, this project employed DOAC antidotes such as Andexanet alfa, Idarucizumab, PCC. Additionally the effect of FEIBA was also analyzed as a potential DOAC antidote. The aim of this study was to develop a means to efficiently monitor DOAC (-inhibitor) levels. This involves the measurement of the global effects of both DOACs and various antidotes using the TGAium and the quantitative measurement of the DOAC concentrations using the luminescent Dabigatran and luminescent anti- FXa assays.

[0184] Effect of DOAC in coagulation: The global impact of varying concentrations of Apixaban, Edoxaban, and Dabigatran was assessed using the TGAium assay. Among the FXa-DOACs, concentrations of 350 and 700 ng / ml Apixaban inhibited coagulation by 30 to 70%, resulting in prolonged LT, TTP, and a decrease in AUC and VI. Concentrations of 350 and 700 ng / ml Edoxaban exhibited a more pronounced anticoagulant effect, inhibiting coagulation by 70 to 80%, accompanied by extended LT and TTP compared to Apixaban. Dabigatran, which inhibits coagulation by binding to Flla, completely halted the coagulation process. Therefore, Edoxaban demonstrates a greater anticoagulant effect than Apixaban, while Dabigatran effectively prevents the coagulation cascade. However, the TGAium with Dabigatran reveal a more pronounced inhibitory effect, whereas the TGAium with Apixaban demonstrates a less inhibitory effect compared to what has been observed in the study of Shaw, J. R., et al. (2022). Hence, the TGAium can be employed in practical scenarios to monitor the global coagulation status in plasma samples of patients treated with DOACs.

[0185] FXa-DOAC reversal with Andexanet alfa: The reversal of FXa DOACs is achieved through the administration of Andexanet alfa, which directly binds to Apixaban and Edoxaban. Concentrations of 10 and 80 pg / ml (0.249 and 1 .995 pM) Andexanet are found to be sufficiently potent in restoring the anticoagulant effect of 700 ng / ml (1.523 pM) Apixaban to baseline conditions. Likewise, the addition of 100 pg / ml Andexanet alfa can fully reverse the effects of 1000 ng / ml FXa-DOACs in the TGA. The complete reversal of the effect of 700 ng / ml (1 .523 pM) Apixaban by lower concentrations of 10 pg / ml (0.249 pM) Andexanet alfa was unexpected, with a stoichiometric Apixaban to Andexanet alfa ratio of 1 :0,16. Especially when compared to the findings of Siddiqui, F. et al. (2019), Lu, G. et al. (2020), and Kaatz, S. et al. (2017), where the addition of Andexanet alfa at a 1 :1 stoichiometric ratio was required for complete neutralization.

[0186] Additionally, the observed reduction in TTP and LT is attributed to the inhibitory effect of Andexanet alfa on Tissue Factor Pathway Inhibitor (TFPI). A study indicates that Andexanet alfa therapy leads to a decrease in TFPI activity, a key inhibitor of the TF-FVIla complex. Furthermore, Andexanet alfa competes with Fxa for binding to antithrombin, which primarily inhibits both Fxa and Flla. The binding of Andexanet alfa to TFPI may result in an increase in prothrombin fragments 1 and 2, elevated concentrations of D-dimer, and formation of thrombin-antithrombin complexes. These alterations can lead to the observed shortening of LT and TTP when 10 and 80 pg / ml Andexanet alfa are added to 700 ng / ml Apixaban.

[0187] The pro-coagulant effect of Andexanet alfa in Edoxaban is less pronounced at lower concentrations of Andexanet alfa, until the addition of 80 pg / ml (1 .995 pM) Andexanet to 700 ng / ml (1.277 pM) Edoxaban, which leads to complete reversal of the anticoagulant effect of Edoxaban. Because Edoxaban exhibits a stronger anticoagulant effect compared to Apixaban in the TGAium assay, a higher concentration of Andexanet alfa is required, with a stoichiometric Edoxaban to Andexanet alfa ratio of 1 :1 .56 in this case. This aligns closely with expectations, as previous studies have demonstrated that a 1 :1 ratio was sufficient to neutralize the anticoagulant effects of Fxa- DOACs.

[0188] Thus, the global coagulation effect can also be evaluated using TGAium with Fxa-DOAC spiked plasma treated with Andexanet alfa, where a concentration of 80 pg / ml Andexanet alfa is sufficient to reverse the effects of 700 ng / ml Apixaban and Edoxaban.

[0189] Dabigatran reversal with Idarucizumab: The neutralization of Dabigatran using the specific antidote Idarucizumab is assessed using the TGAium assay. In plasma samples without Idarucizumab, 700 ng / ml (1.115 pM) Dabigatran completely inhibits coagulation. Upon the addition of various concentrations of Idarucizumab, it is observed that the parameters return towards baseline levels as the concentration of Idarucizumab is increased, with complete reversal of the anticoagulant effect of Dabigatran achieved at 60 pg / ml (1 .25 pM) Idarucizumab. This suggests that a stoichiometric ratio of Dabigatran to Idarucizumab of 1 :1.17 is required to neutralize the anticoagulant effect of Dabigatran. These findings are consistent with the study by Mojca Mijovski et al. (2021), where the concentration 125 pg / ml Idarucizumab effectively neutralized the anticoagulant effect of up to 1500 ng / ml Dabigatran.

[0190] DOAC reversal with PCC and FEIBA: The competitive reversal of DOACs using both PT (PCC) and FEIBA was examined via the TGAium assay. Addition of 1 lU / ml PT to 350 ng / ml Apixaban demonstrated a prothrombotic effect, while at 350 ng / ml Edoxaban, it exhibited a less pronounced reversal effect, restoring only 50% of coagulation compared to baseline. PT failed to neutralize the anticoagulant effect of Dabigatran. Coagulation was partially restored by adding 1 lU / ml PT to 700 ng / ml Apixaban without inducing a prothrombotic state, albeit with an observed longer lag time. For 700 ng / ml Edoxaban, similarto the 350 ng / ml concentration, 1 lU / ml PT was insufficient for reversal. Moreover, at 700 ng / ml Dabigatran, the addition of 1 lU / ml PT did not effectively neutralize the anticoagulant effect. In contrast, the addition of 1 lU / ml FEIBA to both 350 and 700 ng / ml Apixaban induces prothrombotic situations. Notably, adding 1 lU / ml FEIBA to 350 ng / ml Edoxaban achieves complete neutralization of the anticoagulant effect, whereas in 700 ng / ml Edoxaban, it results in a prothrombotic effect in these findings. This discrepancy may arise from errors in the handling performed during the analysis. At 350 ng / ml Dabigatran, the addition of 1 lU / ml FEIBA restores the Time to Peak Hemoglobin (TPH) but with a lag time of 9 minutes. However, 1 lU / ml FEIBA demonstrates no reversal effect at 700 ng / ml Dabigatran.

[0191] Comparatively, FEIBA not only facilitates the reversal of DOACs but also induces prothrombotic effects. This stronger effect of FEIBA may be attributed to its composition containing activated coagulation factors, unlike PT, which lacks activated coagulation factors, potentially resulting in a less effective neutralization effect. Therefore, FEIBA cannot be utilized as a DOAC reversal agent due to its association with prothrombotic events.

[0192] The development of the luminescent Dabigatran assay: To assess Dabigatran concentration in plasma samples, the luminescent Dabigatran assay is employed. Initially, the chromogenic method is utilized to measure Dabigatran concentration and evaluate the feasibility of accurately measuring its levels. A linear relationship is observed, represented by the equation y = -0.0009x + 1.4496 with an R2of 0,984. However, it is noted that the window of opportunity within the chromogenic method is relatively narrow, as shown in Fig. 9, limiting the precision of Dabigatran concentration measurements.

[0193] In contrast, the luminescent method yields a calibration curve characterized by an exponential decline, described by the function y = 278020e_°OO43xwith a R2of 0,996. This method enables precise measurement of Dabigatran concentration, owing to the wider window of opportunity spanning from 0 to 250 ng / ml Dabigatran due to the addition of 100 nM thrombin-c^M. The addition of thrombin- c^M also results in a stable signal over time, unlike normal human thrombin. This broad range is advantageous in practical applications to measure Dabigatran concentrations, as it accommodates variations in daily dosage within this range. Therefore, the luminescent Dabigatran assay provides improved accuracy and reliability, making it an optimal method for practical applications that demand precise measurements of Dabigatran concentrations.

[0194] Luminescent anti-Fxa assay: The luminescent anti-Fxa assay was conducted to determine the concentrations of Fxa-DOACs. In the case of Apixaban measurements, the calibration curve exhibits an exponential decline, represented by the y = 114651ef_(l (l(l232xjwith an R2of 0.9732. The lower R-squared value is attributed to the plateau reached in inhibition beyond 300 ng / ml Apixaban, resulting in a narrow range of signal between 317 and 632 ng / ml Apixaban. Nevertheless, this method was able to measure the concentrations of two Apixaban samples, 317 and 632 ng / ml, in the presence of 80 pg / ml Andexanet alfa, resulting in a reduction of 80-90% to concentrations from 317 ng / ml to 38.77 ng / ml and 632 ng / ml to 144.08 ng / ml Apixaban.

[0195] The calibration curve of the Edoxaban measurement exhibits an exponential decline, represented by the y = 114651ef_(l (l(l232xjwith an R2of 0.9732. The lower R-squared value is attributed to the limited range of signal between 251 and 500 ng / ml Edoxaban. Nevertheless, this method measured the concentrations of two Edoxaban samples, 251 and 500 ng / ml, in the presence of 80 pg / ml Andexanet alfa, resulting in a reduction of 65-70% to concentrations from 251 ng / ml to 72.98 ng / ml and 500 ng / ml to 169.77 ng / ml Edoxaban.

[0196] The calibration curve of the Rivaroxaban measurement exhibits an exponential decline, represented by the y = 1148409ef_o'oo',XJwith an R2of 0.9718. The lower R-squared value is attributed to the limited range of signal between 251 and 513 ng / ml Rivaroxaban. Nevertheless, this method measured the concentrations of two Rivaroxaban samples, 251 and 513 ng / ml, in the presence of 80 pg / ml Andexanet alfa, resulting in a reduction of 88% to concentrations from 251 ng / ml to 30.21 ng / ml and 513 ng / ml to 58.96 ng / ml Rivaroxaban.

[0197] In all measurements involving Apixaban, Edoxaban, and Rivaroxaban, the signal exhibits instability over time. Therefore, for future studies, it may be advantageous to consider replacing human Fxa with Fxa-a2M. Given that a2M functions as a cage around Fxa, only the luminescent Fxa substrate can bind to Fxa-a2M. This approach could potentially result in more stable signals, improved window of opportunity, and consequently, more accurate measurement of Fxa-DOAC concentrations. However, the luminescent anti-Fxa assay can be utilized to quantitatively measure the concentration of FXa-DOACs in patient plasma samples, including those treated with the Fxa- DOAC reversal agent Andexanet alfa.

[0198] References

[0199] Ahuja, T., Raco, V., Bhardwaj, S., and Green, D., “To Measure or Not to Measure: Direct Oral Anticoagulant Laboratory Assay Monitoring in Clinical Practice,” Adv Hematol, vol. 2023, 2023, doi: 10.1 155 / 2023 / 9511499.

[0200] Kaatz, S., Bhansali, H., Gibbs, J., Lavender, R., Mahan, C.E., and Paje, D.G., “Reversing factor Xa inhibitors - clinical utility of andexanet alfa,” J Blood Med, vol. 8, p. 141 , Sep. 2017, doi: 10.2147 / JBM.S121550.

[0201] Lu, G., Lin, J., Bui, K., Curnutte, J.T., and Conley, P.B., “Andexanet versus prothrombin complex concentrates: Differences in reversal of factor Xa inhibitors in in vitro thrombin generation,” Res Pract Thromb Haemost, vol. 4, no. 8, p. 1282, Nov. 2020, doi: 10.1002 / RTH2.12418.

[0202] Lu, G., De Guzman F.R., Hollenbach S.J., et al. A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa. Nat Med. 2013;19(4):446-451 Pfrepper, C.,Metze, M., Siegemund, A., Kloter, T., Siegemund, T.„ and Petros, S., “Direct oral anticoagulant plasma levels and thrombin generation on ST Genesia system,” Res Pract Thromb Haemost, vol. 4, no. 4, p. 619, May 2020, doi: 10.1002 / RTH2.12340.

[0203] Shaw, J.R., Castellucci, L.A., Siegal, D., and Carrier, M., “DOAC-associated bleeding, hemostatic strategies, and thrombin generation assays - a review of the literature,” Journal of Thrombosis and Haemostasis, vol. 21 , no. 3, pp. 433-452, Mar. 2023, doi: 10.1016 / J.JTHA.2022.11 .029.

[0204] Siddiqui, F. et al., “Reversal of Factor Xa Inhibitors by Andexanet Alfa May Increase Thrombogenesis Compared to Pretreatment Values,” Clinical and Applied Thrombosis / Hemostasis, vol. 25, Jul. 2019, doi: 10.1177 / 1076029619863493.

Claims

1. Claims1. Method for quantifying the activity of a reversal agent in a sample, wherein the sample comprises an anticoagulant and a coagulation factor, the method comprising the steps of: a) contacting the sample with a composition comprising a chemiluminescent substrate for the coagulation factor to release aminoluciferin; b) contacting the aminoluciferin with luciferase; and c) determining the relative light intensity generated by the luciferase.

2. The method according to claim 1 , wherein the anticoagulant is a direct oral anticoagulant.

3. The method according to claim 1 or 2, wherein the anticoagulant is hirudin, bivalirudin, desirudin, lepirudin, argatroban, dabigatran, efegatran, inogatran, melagatran, ximelagatran, apixaban, betrixaban, darexaban, edoxaban, otamixaban, or rivaroxaban, preferably it is rivaroxaban, apixaban, edoxaban, or dabigatran.

4. The method according to any one of claims 1 -3, wherein the coagulation factor is factor Xa (FXa) or factor Ila (Fl la).

5. The method according to any one of claims 1 -4, wherein the reversal agent is andexanet alfa, idarucizumab, ciraparantag, VMX-C001 , prothrombin complex concentrate (PCC), or factor eight inhibitor bypassing activity (FEIBA), preferably it is andexanet alfa, idarucizumab, or FEIBA, more preferably it is andexanet alfa or idarucizumab.

6. The method according to any one of claims 1 -5, wherein the sample is a blood sample, preferably wherein the blood sample is known or suspected of comprising a reversal agent, preferably wherein the blood sample has been previously obtained from a subject.

7. The method according to any one of claims 1 -6, wherein the reversal agent is andexanet alfa or VMX-C001 , and the anticoagulant is apixaban or edoxaban or rivaroxaban or betrixaban, and the coagulation factor is FXa; or the reversal agent is idarucizumab, and the anticoagulant is dabigatran, and the coagulation factor is Fl la.

8. The method according to claim 7, wherein the reversal agent is andexanet alfa and the anticoagulant is apixaban and the coagulation factor is FXa.

9. The method according to any one of claims 1-8, wherein the chemiluminescent substrate is a compound of general formula (I-3) or (I-4),(1-3) (1-4) wherein r is 0, 1 , 2, or 3; r’ is 0, 1 , 2, or 3; d is 0, 1 , or 2; g is 0 or 1 ; g’ is 0 or 1 ; andX is a terminal moiety selected from NH2, OH, O(Ci-ealkyl), (OCH2CH2)i-eOH, (OCH2CH2)I- 6O(Ci-6alkyl), NHC(=0)(0)o-i(Ci-6alkyl), NHC(=0)(0)o-i(Ci-6alkylene)(0-CH2CH2)i-60H,NHC(=0)(0)o-i(Ci-6alkylene)(OCH2CH2)i-60(Ci-6alkyl), NHC(=0)(0)o-i(Ci-6alkylene)0(Ci- salkyl), NHC(=0)(0)o-i(Ci-6alkylene)OH, and NP’ wherein P’ is an amine protecting group; or wherein the chemiluminescent substrate is pyroGlu-Phe-Lys-aminoluciferin; Gly-Gly-Arg-aminoluciferin; beta-Ala-Gly-Arg- aminoluciferin; Ala-Gly-Arg-aminoluciferin; orCH3O(CH2CH2O)n-acetyl-Gly-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 2; orCH3O(CH2CH2O)n-acetyl-beta-Ala-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 4;CH3O(CH2CH2O)n-acetyl-Ala-Gly-Arg-aminoluciferin, wherein n is 0, 1 , 2, 3, or 4, preferably 2 or 4, more preferably 4; optionally wherein the aminoluciferin moiety is replaced by a different chemiluminescent amine; or a physiologically acceptable salt thereof.

10. Method for determining a suitable dosage of reversal agent for a subject whose hemostasis is disrupted by an anticoagulant, the method comprising the steps of: i) contacting a blood sample that has been previously obtained from the subject with a composition comprising a chemiluminescent substrate for FXa or Fl la to release aminoluciferin; ii) contacting the aminoluciferin with luciferase; iii) determining the relative light intensity generated by the luciferase;iv) comparing the determined relative light intensity to a reference value to determine the activity of the anticoagulant; v) determining a suitable dosage of reversal agent based on the determined activity of the anticoagulant.11 . The method according to claim 10, wherein the reversal agent is andexanet alfa, and the anticoagulant is apixaban, wherein the suitable dosage of step v) has a stoichiometric ratio of andexanet alfa to apixaban in the range of 0.05-0.5 to 1 , preferably about 0.16 to 1 ; or the reversal agent is andexanet alfa, and the anticoagulant is edoxaban, wherein the suitable dosage of step v) has a stoichiometric ratio of andexanet alfa to edoxaban in the range of 1 .1 -2 to 1 , preferably about 1 .56 to 1 ; or the reversal agent is idarucizumab, and the anticoagulant is dabigatran, wherein the suitable dosage of step v) has a stoichiometric ratio of idarucizumab to dabigatran in the range of 1 .05-2 to 1 , preferably 1 .17 to 1 .

12. Reversal agent for use as a medicament to restore hemostasis, wherein the reversal agent is administered in a dosage as determined in method 10 or 11 .

13. Kit of parts comprising a reversal agent and at least one further compound selected from the group consisting of luciferase, ATP, an Mg2+source, and a coagulation factor such as factor Xa.

14. Kit of parts comprising a reversal agent and a chemiluminescent substrate for a coagulation factor.

15. Method for reversing the activity of an anticoagulant in a subject, the method comprising the steps of obtaining a blood sample from the subject, determining the activity of the anticoagulant in said sample by using a method as defined in any one of claims 1-9, determining an effective dose of a reversal agent based on the determined activity of the anticoagulant, and administering said effective dose of the reversal agent to the subject.

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