WO2025175397A1 - Assay to measure antifibrinolytic agents - Google Patents

Abstract

The present disclosure relates to methods for determining concentration of a lysine analog antifibrinolytic agent in a sample. Also provided are kits comprising (a) a composition comprising a fluorescently labeled inactive plasminogen variant and a fluorescence quencher labeled fibrin-degradation product (FDP) and (b) a lysine analog antifibrinolytic agent standard and methods of use thereof.

Description

ASSAY TO MEASURE ANTIFIBRINOLYTIC AGENTS

RELATED APPLICATION

[0001] This application claims benefit of US Provisional Application serial no. 63/556,471 filed on February 22, 2024, incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates to an assay for quantifying levels of a lysine analog antifibrinolytic agent in a sample.

BACKGROUND

[0003] T ranexamic acid (TXA) is the most used drug for the treatment of bleeding, a class of drugs often referred to as antifibrinolytics. TXA is often used to manage bleeds in mild hemophilia, von Willebrand Disease (vWD), rare platelet disorders, menorrhagia, and trauma. In addition, antifibrinolytics are used during invasive surgical procedures at high doses [1], whereby the doses are based on the patient’s risk of bleeding and renal impairment [1 ,2], Currently, there are no readily available methods that can accurately measure TXA levels with relative ease and accuracy.

[0004] Previous studies have suggested plasma TXA concentration of ~10 mg/L as the threshold required to attain sufficient inhibition of fibrinolysis [3], However, patient plasma TXA concentrations have been described ranging from approximately 10 mg/L to over 100 mg/L, depending on the dosing regimen used [3-6], Because there are no readily available methods to measure TXA, it is challenging to ensure that a patient is given the appropriate dose - high enough to prevent bleeding while not excessive to minimize the risks of complications such as seizure [7], The challenge of determining the appropriate TXA dosage is made even more complex by individual factors, such as renal function/clearance, who would require adjusted dosage to minimize the risk of overdose [8,9], Current methods to quantify TXA levels in plasma include liquid chromatography and mass spectrometry. However, these methods require specialized laboratory settings and techniques, thus rendering them not suitable for point-of-care testing (POCT) in a clinical setting [10], [0005] The background herein is included solely to explain the context of the disclosure. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.

SUMMARY

[0006] Provided herein is a novel method to measure concentration of a lysine analog antifibrinolytic agent accurately and rapidly in a sample. The reaction mixture of a plasminogen variant labeled with a fluorescent probe and a fibrin degradation product (FDP) labeled with a fluorescence quencher measures the lysine analog antifibrinolytic agent concentration by measuring the relative fluorescence. The method demonstrated herein has clear viability to become a rapid point-of-care testing for clinical applications.

[0007] Accordingly, an aspect of the disclosure is a method for determining concentration of a lysine analogue antifibrinolytic agent in a liquid sample, the method comprising: a. contacting the liquid sample with a mixture of: i. a fluorescently labeled inactive plasminogen variant, and ii. a fluorescence quencher labeled fibrin-degradation product (FDP) b. measuring a relative fluorescence signal, wherein the relative fluorescence signal is calculated by subtracting the fluorescence signal generated in the absence of the lysine analog antifibrinolytic agent from the fluorescence signal generated in the presence of the lysine analog antifibrinolytic agent; wherein in the absence of the lysine analog antifibrinolytic agent, the fluorescence quencher labeled FDP quenches the fluorescence generated by the fluorescently labeled plasminogen thereby dampening the fluorescence signal and the presence of the lysine analog antifibrinolytic agent disrupts the interaction of the fluorescently labeled plasminogen and fluorescence quencher labeled FDP thereby generating a higher fluorescence signal; and c. determining the concentration of the lysine analog antifibrinolytic agent in the liquid sample, wherein the fluorescence signal is indicative of the concentration of the lysine analog antifibrinolytic agent.

[0008] In some embodiments, determining the concentration comprises reference to a fluorescence signal standard curve generated with known amounts of the lysine analog antifibrinolytic agent; and wherein the concentration of the lysine analog antifibrinolytic agent is proportional to the fluorescence signal.

[0009] In some embodiments, the liquid sample comprises plasma, serum, and/or blood.

[0010] In some embodiments, the fluorescently labeled inactive plasminogen variant is a recombinant full length Glu-plasminogen with a single mutation in the latent active site, Lys-plasminogen with a mutation in the latent active site, mini-plasminogen with a single mutation in the latent active site, or any plasminogen variant that contains a lysine-binding Kringle domain and a mutation in the latent active site.

[0011] In some embodiments, the recombinant full length Glu- plasminogen is a human plasminogen, optionally encoded by the nucleotide sequence and/or the amino acid sequence as shown in Gene ID: 5340. In some embodiments, the amino acid sequence of human plasminogen as set forth in UniProt ID: P00747 or NCBI Reference Sequence: NP_000292.1.

[0012] In some embodiments, the single mutation in the latent active site is serine to a cysteine at position 741 (S741 C), also known as [Glu1 ]Plg(S741 C).

[0013] In some embodiments, the fluorescently labeled inactive plasminogen variant is labeled with a fluorescence probe on a free cysteine, such as C741.

[0014] In some embodiments, the fluorescence probe comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide. [0015] In some embodiments, the fluorescence probe is 5- iodoacetamidofluorescein (5IAF), fluorescein-5-maleimide, Alexa Fluor™ 488 C5 maleimide, DyLight™ 488 Maleimide, BDP™-FL Maleimide.

[0016] In some embodiments, the fluorescence probe is 5IAF.

[0017] In some embodiments, the method further comprises first generating the fluorescence quencher labeled FDP by: i) reducing purified fibrinogen with a reducing agent; ii) labelling the purified fibrinogen with the fluorescence quencher to generate fluorescence quencher-fibrinogen; iii) contacting the fluorescence quencher-fibrinogen with thrombin to generate a fluorescence quencher-insoluble fibrin clot; iv) contacting the fluorescence quencher-insoluble fibrin clot with plasmin to degrade the insoluble fibrin clot; and v) inhibiting the thrombin and plasmin to generate the fluorescence quencher labeled FDP.

[0018] In some embodiments, the fluorescence quencher comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

[0019] In some embodiments, the fluorescence quencher is QSY™ 9 Cs- maleimide (QSY), QSY™ 7 C5-Maleimide, or Tide Quencher™ 3 Maleimide (TQ3 Maleimide).

[0020] In some embodiments, wherein the fluorescence quencher is QSY.

[0021] In some embodiments, the reducing agent comprises betamercaptoethanol, (2-carboxyethyl) phosphine hydrochloride (TCEP) or dithiothreitol (DTT).

[0022] In some embodiments, the reducing agent comprises betamercaptoethanol. [0023] In some embodiments, inhibiting the thrombin comprises adding phe-pro-arg-chloromethylketone (FPR-ck), Lepirudin, Desirudin, Argatroban, Bivalirudin, Hirudin, or Dabigatran.

[0024] In some embodiments, inhibiting plasmin comprises adding val- phe-lys-chloromethylketone (VFK-ck), O2-antiplasmin, or aprotinin.

[0025] In some embodiments, the lysine analog antifibrinolytic agent is tranexamic acid (TXA) or £-aminocaproic acid (sACA).

[0026] In some embodiments, the lysine analog antifibrinolytic agent is TXA.

[0027] In some embodiments, the lower limit of detection of TXA is about 0.28 pM

[0028] In some embodiments, the lower limit of detection of TXA is about 0.044 mg/L.

[0029] In some embodiments, in step a), the mixture is a pre-mixed mixture.

[0030] In some embodiments, the pre-mixed mixture is mixed up to 6 months prior to use.

[0031] In some embodiments, the pre-mixed mixture is stored at about - 20°C to about -80°C, about -20°C, or about -80°C.

[0032] In some embodiments, the fluorescently labeled inactive plasminogen variant is at a concentration of about 25 nM to about 1000 nM, optionally about 200 nM.

[0033] In some embodiments, the fluorescence quencher labeled fibrindegradation product is at a concentration of about 0.1 pM to about 10 pM, optionally about 1 pM.

[0034] In some embodiments, the liquid sample is provided from a patient being treated with the lysine analog antifibrinolytic agent, optionally the patient is a human patient. [0035] In some embodiments, the liquid sample is provided from a patient being treated with the lysine analog antifibrinolytic agent that is undergoing a surgical procedure.

[0036] In some embodiments, the method further comprises repeating steps a)-c) on one or more subsequent liquid samples from the same patient obtained at one or more subsequent time points.

[0037] Another aspect of the disclosure is a kit comprising (a) a composition comprising a fluorescently labeled inactive plasminogen variant and a fluorescence quencher labeled fibrin-degradation product (FDP); and (b) a lysine analog antifibrinolytic agent standard.

[0038] In some embodiments, the fluorescently labeled inactive plasminogen variant is a recombinant full length Glu-plasminogen with a single mutation of the latent active site, Lys-plasminogen with a mutation in the latent active site, mini-plasminogen with a single mutation in the latent active site, or any plasminogen variant that contains a lysine-binding Kringle domain and a mutation in the latent active site.

[0039] In some embodiments, the recombinant full length Glu- plasminogen is a human plasminogen, optionally encoded by the nucleotide sequence and/or the amino acid sequence as shown in Gene ID: 5340. In some embodiments, the amino acid sequence of human plasminogen as set forth in in UniProt ID: P00747 or NCBI Reference Sequence: NP_000292.1.

[0040] In some embodiments, the single mutation of the latent active site is serine to a cysteine at position 741 (S741 C), also known as [Glu1]Plg(S741C).

[0041] In some embodiments, the fluorescently labeled inactive plasminogen variant is labeled with a fluorescence probe on a free cysteine, such as C741.

[0042] In some embodiments, the fluorescence probe comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide. [0043] In some embodiments, the fluorescence probe is 5- iodoacetamidofluorescein (5IAF), fluorescein-5-maleimide, Alexa Fluor™ 488 C5 maleimide, DyLight™ 488 Maleimide, BDP™-FL Maleimide.

[0044] In some embodiments, the fluorescence probe is 5IAF.

[0045] In some embodiments, the fluorescence quencher labeled FDP comprises was generated by: i) reducing purified fibrinogen with a reducing agent; ii) labelling the purified fibrinogen with the fluorescence quencher to generate fluorescence quencher-fibrinogen; iii) contacting the fluorescence quencher-fibrinogen with thrombin to generate a fluorescence quencher-insoluble fibrin clot; iv) contacting the fluorescence quencher-insoluble fibrin clot with plasmin to degrade the insoluble fibrin clot; and v) inhibiting the thrombin and plasmin to generate the fluorescence quencher labeled FDP.

[0046] In some embodiments, the fluorescence quencher comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

[0047] In some embodiments, the fluorescence quencher is QSY™ 9 Cs- maleimide (QSY), QSY™ 7 C5-Maleimide, or Tide Quencher™ 3 Maleimide (TQ3 Maleimide).

[0048] In some embodiments, the fluorescence quencher is QSY.

[0049] In some embodiments, the reducing agent comprises betamercaptoethanol, (2-carboxyethyl) phosphine hydrochloride (TCEP) or dithiothreitol (DTT).

[0050] In some embodiments, the reducing agent comprises betamercaptoethanol. [0051] In some embodiments, inhibiting the thrombin comprises adding phe-pro-arg-chloromethylketone (FPR-ck), Lepirudin, Desirudin, Argatroban, Bivalirudin, Hirudin, or Dabigatran.

[0052] In some embodiments, inhibiting plasmin comprises adding val- phe-lys-chloromethylketone (VFK-ck), O2-antiplasmin, or aprotinin.

[0053] In some embodiments, the composition is stable for up to 6 months.

[0054] In some embodiments, the composition is stored at about -20°C to about -80°C, about -20°C or about -80°C.

[0055] In some embodiments, the fluorescently labeled inactive plasminogen variant is at a concentration of about 25 nM to about 1000 nM. In some embodiments, the fluorescently labeled inactive plasminogen variant is at a concentration of about 200 nM.

[0056] In some embodiments, the fluorescence quencher-FDP is at a concentration of about 0.1 pM to about 10 pM. In some embodiments, the fluorescence quencher labeled FDP is at a concentration of about 1 pM.

[0057] In some embodiments, the lysine analog antifibrinolytic agent standard is tranexamic acid (TXA) and/or £-aminocaproic acid (sACA).

[0058] In some embodiments, the lysine analog antifibrinolytic agent standard is TXA.

[0059] In some embodiments, the kit is for use in performing the methods described herein.

[0060] In another aspect, there is provided an assay for determining concentration of antifibrinolytic agents in a liquid sample, comprising: a) providing a liquid sample b) adding to the sample: i. a fluorescently labeled mutant variant of plasminogen (5IAF-Pg), ii. a modified fibrin FDP selectively labeled with a fluorescence quencher (QSY-FDP). c) measuring the change in fluorescence signal value. d) determining the concentration of the antifibrinolytic agent in the unknown sample using a standard curve generated with known levels of the said antifibrinolytic agent, whereby the change in fluorescence signal detected in c) is proportional to the concentration of the antifibrinolytic agent present.

[0061] In an embodiment, the liquid sample comprises of biological material including plasma and blood.

[0062] In an embodiment, the mutant plasminogen does not generate an active end product that can affect the positive cycle of fibrinolysis.

[0063] In an embodiment, the mutant plasminogen is a recombinant full length Glu-plasminogen with a single mutation of the latent active site serine to a cysteine (S741C).

[0064] In an embodiment, the mutant plasminogen is labeled with a fluorescence probe.

[0065] In an embodiment, the fluorescence probe is 5- iodoacetamidofluorescein.

[0066] In an embodiment, the FDP is labeled with a fluorescence quencher.

[0067] In an embodiment, the fluorescence quencher is QSY 9 C5- maleimide (QSY).

[0068] In an embodiment, the antifibrinolytic agent disrupts plasminogen and FDP interaction.

[0069] In an embodiment, the antifibrinolytics are lysine analog antifibrinolytic agents.

[0070] In an embodiment, the lysine analog antifibrinolytic agents are selected from the group consisting of tranexamic acid (TXA) and £-aminocaproic acid (eACA). [0071] In an embodiment, the assay is not sensitive to other fibrinolysis inhibitors.

[0072] In an embodiment, the other fibrinolysis inhibitors comprise other proteases and class of direct protease inhibitors also known as serine protease inhibitors that affect clot breakdown.

[0073] In an embodiment, the fibrinolysis inhibiting proteases that affect clot breakdown include activated thrombin-activatable fibrinolysis inhibitor (TAFIa).

[0074] In an embodiment, the serine protease inhibitors include plasminogen activator inhibitor 1 (PAI-1) and O2-antiplasmin.

[0075] In an embodiment, the 5IAF-Pg/QSY-FDP mixture concentration can be varied to measure different amounts of antifibrinolytic agents in a liquid sample.

[0076] In an embodiment, higher concentrations of the reaction mixture can measure a higher quantity of inhibitor and lower concentrations of the reaction mixture can measure a lower quantity of inhibitor.

[0077] In an embodiment, the limit of detection of antifibrinolytic concentration present in a plasma sample is 0.28 pM or 0.044 mg/L.

[0078] In an embodiment, the concentration of the antifibrinolytic agent is less than 100 pM or 15.7 mg/L, whereby anything greater would require dilution.

[0079] In a further aspect provided is a method of testing antifibrinolytic agent concentration in a liquid sample that is periodically collected from a patient using the methods described herein.

[0080] In an embodiment, the patient is being monitored in real time during surgical procedures.

[0081] In an embodiment, patients that are prescribed antifibrinolytic agents.

[0082] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.

DRAWINGS

[0083] Certain embodiments of the disclosure will now be described in greater detail with reference to the attached drawings in which:

[0084] FIG. 1 shows the increased fluorescence signal from dissociation of QSY-FDP from 5IAF-Pg as a function of increasing concentration of TXA in exemplary embodiments of the disclosure. The standard curve was generated from four independent experiments, each measurement performed in triplicates. The data represents mean ± standard error of the mean.

[0085] FIG. 2 shows the measured levels of TXA in healthy plasma samples that were added with known levels of TXA plotted against expected levels of TXA present in the said plasma samples in exemplary embodiments of the disclosure. The samples were aliquoted and frozen and stored at -80°C. The samples were measured at four independent days. Symbols indicate mean ± standard error of the mean.

[0086] FIG. 3 shows the stability of the pre-made QSY-FDP/5IAF-Pg mixture in buffer with no loss of quantitation ability up to 28 days when stored in -80°C in exemplary embodiments of the disclosure. The intention was to perform the assay as a single step addition from a pre-mixed lot.

[0087] FIG. 4 shows measurement of D-dimers and TXA in plasma samples from five patients undergoing cardiac pulmonary bypass (CPB) surgery in exemplary embodiments of the disclosure. Pre - before surgery; Post - post surgery after heparin reversal with protamine sulfate.

[0088] FIG. 5 shows plasma samples obtained from patients undergoing CPB surgery in exemplary embodiments of the disclosure. T1 - before surgery; T2 - post-CPB after heparin reversal with protamine sulfate; T3 - at the time of chest closure or 60 min after heparin reversal. [0089] FIG. 6 shows a standard curve generation using s-aminocaproic acid (sACA), another lysine analog that is clinically used as an antifibrinolytic in exemplary embodiments of the disclosure.

[0090] FIG. 7 shows stability of the premixed reaction mixture at -80°C in exemplary embodiments of the disclosure. 5IAF-Pg and QSY-FDPs were premixed with the intention of performing the assay as a single step addition, aliquoted and frozen. Over the first 26 weeks, the reaction mixture showed excellent stability while stored at -80°C. From left to right, the bars represent relative fluorescence unit (RFU) of premixed reaction mixture measured at week 0, week 2, week 4, week 8, week 13, week 18, week 26, and week 39. Weeks 0, 2, and 4 are the same as those shown in FIG 3.

[0091] FIG. 8 shows stability of the premixed reaction mixture at -20°C in exemplary embodiments of the disclosure. 5IAF-Pg and QSY-FDPs were premixed with the intention of performing the assay as a single step addition, aliquoted and frozen. Over the first 26 weeks, the reaction mixture showed excellent stability while stored at -20°C. From left to right, the bars represent RFU of premixed reaction mixture measured at week 0, week 2, week 4, week 8, week 13, week 18, and week 26.

DETAILED DESCRIPTION

[0092] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure.

[0093] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature described herein may be combined with any other feature or features described herein.

I. Definitions

[0094] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0095] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.

[0096] The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps. [0097] Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

[0098] All numerical values herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art.

[0099] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated.

[00100] As used in this disclosure, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds.

[00101] In embodiments comprising an “additional” or “second” component, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[00102] The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

[00103] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of" or, when used in the claims, "consisting of' will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[00104] The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[00105] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5).

[00106] The term “plasminogen” as used herein refers to the zymogen, also known as the inactive precursor, of the enzyme plasmin and includes, without limitation, human plasminogen. Gene ID 5340 provides exemplary nucleotide and amino acid sequences of human plasminogen. The amino acid sequence of human plasminogen may be found with reference to UniProt ID: P00747 or NCBI Reference Sequence: NP_000292.1.

[00107] The term “Lys-plasminogen” as used herein refers to an intermediate zymogen created in the conversion of Glu-plasminogen to plasmin. Lys-plasminogen is a truncated form of Glu-plasminogen in which Glu- plasminogen is cleaved at the N-terminus, exposing a lysine residue. Optionally, Glu-plasminogen is cleaved between Lys77 and Lys78. The main consequence is that the loss of the 77-residue N-terminal peptide leads to a conformation change: closed form of Glu-plasminogen to an open form of Lys- plasminogen.

[00108] The term “mini-plasminogen” as used herein refers to a shorter zymogen that is a truncated plasminogen fragment lacking the N-terminal peptide along with the Kringle domains 1 through 4. Mini-plasminogen consists of Kringle domain 5 and the protease domain of plasminogen. Miniplasminogen is formed when plasminogen is digested by leukocyte elastase.

[00109] The term “fibrinolysis” as used herein refers to the enzymatic degradation of insoluble fibrin during the breakdown of a blood clot. Fibrinolysis is an enzymatic reaction that is driven by plasmin or plasmin-like activity. This can be achieved by 1) plasminogen being enzymatically converted to plasmin by plasminogen activators, or 2) plasminogen being bound by other activators such as streptokinase, which alters the structure of plasminogen to be plasmin-like while having plasmin-like activity. Plasmin, a serine protease, cleaves and solubilizes fibrin into fibrin degradation products to break up the blood clot.

[00110] The term “blood clot” or “clot” as used herein refers to a blood coagulation product. The term blood clot may include both thrombus or embolus. A thrombus refers to a stationary blood clot that forms in a blood vessel. An embolus refers to a blood clot that moves through the bloodstream. For example, an embolus may result from a piece of a thrombus breaking off.

[00111] The term “antifibrinolytic” or “antifibrinolytic agent” as used herein refers to a class of drugs that inhibit fibrinolysis. These drugs hinder blood clot breakdown by binding plasminogen.

[00112] The term “lysine analog antifibrinolytic agent” as used herein refers to a class of drugs that are synthetic derivatives of the amino acid lysine. These drugs disrupt the interaction between plasmin(ogen) and fibrin by binding to the lysine-binding sites on plasmin(ogen), thereby inhibiting the conversion of plasminogen to plasmin as well as plasmin activity towards fibrin. Examples of clinically used lysine analog antifibrinolytic agents include tranexamic acid (TXA) and £-aminocaproic acid (sACA).

[00113] The term “fluorescently labelled inactive plasminogen variant” as used herein refers to a mutant variant of plasminogen that has no enzyme activity even if it were cleaved by plasminogen activators, as the plasminogen has a mutation in its active site, for example mutation of serine at position 741 to a cysteine. This free cysteine may be labeled with a fluorescent probe. [00114] The term “fluorescence quencher labeled fibrin-degradation product” or “fluorescence quencher labeled-FDP” as used herein refers to fibrin degradation products (FDPs) that are labeled with a fluorescence quencher [12], Fluorescence quenchers as used herein refer to molecules which decrease the fluorescence intensity of a fluorophore (quenching). For example, quenching can occur when the fluorescence quencher and fluorophore interact or are placed in physical proximity to each other.

[00115] The term “fibrin degradation product” or “FDP” as used herein refers to fragments produced from the breakdown of fibrin during fibrinolysis. FDPs are commonly used as biomarkers of clot breakdown and include, for example, D- dimer.

[00116] The term “patient” as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Optionally, the term “patient” includes mammals that are undergoing a surgical procedure, will undergo a surgical procedure and/or have undergone a surgical procedure. The term “patient” may also include mammals that are taking, could benefit from taking or have taken a lysine analog antifibrinolytic agent. It will be understood that any component defined herein as being included may be explicitly excluded by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein.

[00117] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[00118] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.

II. Methods

[00119] Demonstrated herein is a method for determining concentration of a lysine analog antifibrinolytic agent in a liquid sample, the method comprising: a. contacting the liquid sample with a mixture of: i. a fluorescently labeled inactive plasminogen variant, and ii. a fluorescence quencher labeled fibrin-degradation product (FDP); b. measuring a relative fluorescence signal, wherein the relative fluorescence signal is calculated by subtracting the fluorescence signal generated in the absence of the lysine analog antifibrinolytic agent from the fluorescence signal generated in the presence of the lysine analog antifibrinolytic agent; wherein in the absence of the lysine analog antifibrinolytic agent, the fluorescence quencher labeled FDP quenches the fluorescence generated by the fluorescently labeled plasminogen thereby dampening the fluorescence signal and the presence of the lysine analog antifibrinolytic agent disrupts the interaction of the fluorescently labeled plasminogen and fluorescence quencher labeled FDP thereby generating a higher fluorescence signal; and c. determining the concentration of the lysine analog antifibrinolytic agent in the liquid sample, wherein the fluorescence signal is indicative of the concentration of the lysine analog antifibrinolytic agent.

[00120] In some embodiments, determining the concentration comprises reference to a fluorescence signal standard curve generated with known amounts of the lysine analog antifibrinolytic agent; and wherein the concentration of the lysine analog antifibrinolytic agent is proportional to the fluorescence signal.

[00121] In some embodiments, the liquid sample comprises plasma, serum, and/or blood.

[00122] In some embodiments, the fluorescently labeled inactive plasminogen variant is a recombinant full length Glu-plasminogen with a single mutation of the latent active site, Lys-plasminogen with a mutation in the latent active site, mini-plasminogen with a single mutation in the latent active site, or any plasminogen variant that contains a lysine-binding Kringle domain and a mutation in the latent active site.

[00123] In some embodiments, the recombinant full length Glu- plasminogen is a human plasminogen, optionally encoded by the nucleotide sequence and/or the amino acid sequence as shown in Gene ID: 5340 or the amino acid sequence of human plasminogen as set forth in UniProt ID: P00747 or NCBI Reference Sequence: NP_000292.1.

[00124] In some embodiments, the single mutation of the latent active site is serine to a cysteine at position 741 (S741 C), also known as [Glu1 ]Plg(S741 C).

[00125] In some embodiments, the fluorescently labeled inactive plasminogen variant is labeled with a fluorescence probe on a free cysteine, such as C741.

[00126] Methods of fluorescently labelling proteins are known to those skilled in the art. For example, fluorescently labelling a free cysteine can be achieved by incubating the protein of interest with a fluorescent probe containing a sulfhydryl reactive group. Examples of sulfhydryl reactive groups include, without limitation, maleimide, iodoacetamide, and disulfide containing probes.

[00127] In some embodiments, the fluorescence probe comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

[00128] In some embodiments, the fluorescence probe is 5- iodoacetamidofluorescein (5IAF), Alexa Fluor™ -488, fluorescein-5-maleimide, Alexa Fluor™ 488 C5 maleimide, DyLight™ 488 Maleimide, or BDP™-FL Maleimide.

[00129] In one embodiment, the fluorescence probe is 5- iodoacetamidofluorescein (5IAF).

[00130] In some embodiments, the method further comprises first generating the fluorescence quencher labeled FDP by: i) reducing purified fibrinogen with a reducing agent; ii) labelling the purified fibrinogen with the fluorescence quencher to generate fluorescence quencher-fibrinogen; iii) contacting the fluorescence quencher-fibrinogen with thrombin to generate a fluorescence quencher-insoluble fibrin clot; iv) contacting the fluorescence quencher-insoluble fibrin clot with plasmin to degrade the insoluble fibrin clot; and v) inhibiting the thrombin and plasmin to generate the fluorescence quencher labeled FDP.

[00131] In some embodiments, the fluorescence quencher comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

[00132] In some embodiments, the fluorescence quencher is QSY™ 9 Cs- maleimide (QSY), QSY™ 7 C5-Maleimide, or Tide Quencher™ 3 Maleimide (TQ3 Maleimide).

[00133] In one embodiment, the fluorescence quencher is QSY™ 9 Cs- maleimide (QSY).

[00134] In one embodiment, the fluorescence probe is 5- iodoacetamidofluorescein (5IAF) and the fluorescence quencher is QSY™ 9 Cs-maleimide (QSY).

[00135] The skilled person can readily select a compatible fluorescence probe and fluorescence quencher. For example, the emission spectrum of the fluorescence probe and the absorption spectrum of the fluorescence quencher can be used to assess compatibility.

[00136] In some embodiments, quenching of the fluorescence probe is accomplished by Forster Resonance Energy Transfer (FRET), which requires the fluorescence probe and fluorescence quencher to be in physical proximity to one another. Briefly, the fluorescence probe acts as a donor and the fluorescence quencher acts as an acceptor. When the fluorescence probe (donor) is excited by a wavelength of light (e.g. 488 nm) in the proximity of the fluorescence quencher (acceptor), the excited donor energy is transferred to the acceptor without photon emission thereby preventing fluorescence. In some embodiments, quenching occurs when the fluorescence probe and fluorescence quencher are less than 10 nm apart [15],

[00137] In some embodiments, the reducing agent is a disulfide reducing agent. In some embodiments, the reducing agent comprises betamercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) or dithiothreitol (DTT).

[00138] In one embodiment, the reducing agent comprises betamercaptoethanol.

[00139] In some embodiments, inhibiting thrombin comprises adding a thrombin inhibitor. Examples include phe-pro-arg-chloromethylketone (FPR- ck), Lepirudin, Desirudin, Argatroban, Bivalirudin, Hirudin, or Dabigatran.

[00140] In one embodiment, inhibiting the thrombin comprises adding FPR-ck.

[00141] In some embodiments, inhibiting the plasmin comprises adding a plasmin inhibitor. Examples include val-phe-lys-chloromethylketone (VFK-ck), 02-antiplasmin, or aprotinin.

[00142] In one embodiment, inhibiting the plasmin comprises adding VFK- ck.

[00143] In some embodiments, the lysine analog antifibrinolytic agent is tranexamic acid (TXA) or £-aminocaproic acid (sACA).

[00144] In some embodiments, the lysine analog antifibrinolytic agent is TXA.

[00145] In some embodiments, the method does not detect non-lysine analog antifibrinolytic agents, optionally the non-lysine analog antifibrinolytic agents comprise fibrinolysis inhibiting proteases and fibrinolysis inhibiting protease inhibitors.

[00146] In some embodiments, the fibrinolysis inhibiting proteases comprise activated thrombin-activatable fibrinolysis inhibitor (TAFIa). [00147] In some embodiments, the fibrinolysis inhibiting protease inhibitors comprises plasminogen activator inhibitor 1 (PAI-1 ) or O2-antiplasmin.

[00148] In some embodiments, the lower limit of detection of TXA is about

0.28 pM.

[00149] In some embodiments, the lower limit of detection of TXA is about

0.044 mg/mL.

[00150] In some embodiments, the upper limit of detection of TXA is about 100 pM. In some embodiments, the upper limit of detection of TXA is about 15.7 mg/L. The skilled person will appreciate that liquid samples exceeding the upper limit of detection can be diluted, thereby facilitating detection of any concentration of TXA in the liquid sample.

[00151] In some embodiments, the limit of detection of sACA is about 10 pM to about 500 pM.

[00152] In some embodiments, in step a), the mixture is a pre-mixed mixture.

[00153] In some embodiments, the pre-mixed mixture is mixed up to 6 months prior to use.

[00154] In some embodiments, the pre-mixed mixture is stored at about - 20°C to about -80°C, about -20°C, or about -80°C.

[00155] In some embodiments, the fluorescently labeled inactive plasminogen variant is at a concentration of about 25 nM to about 1000 nM. In one embodiment, the fluorescently labeled inactive plasminogen variant is at a concentration of about 200 nM.

[00156] In some embodiments, the fluorescence quencher-FDP is at a concentration of about 0.1 pM to about 10 pM. In some embodiments, the fluorescence quencher-FDP is at a concentration of about 1 pM.

[00157] In some embodiments, the liquid sample is provided from a patient being treated with the lysine analog antifibrinolytic agent, optionally the patient is a human patient. [00158] In some embodiments, the liquid sample is provided from a patient being treated with the lysine analog antifibrinolytic agent and undergoing a surgical procedure.

[00159] In some embodiments, the liquid sample is provided from a patient being treated with the lysine analog antifibrinolytic agent from and has heavy bleeding. In some embodiments, the heavy bleeding is caused by trauma, hemorrhage, post-partum hemorrhage, menorrhagia or surgery.

[00160] In some embodiments, the method further comprises use or administration of TXA in the patient if the concentration of TXA as determined in step c) is less than about 10 mg/L.

[00161] In some embodiments, the method further comprises repeating steps a)-c) on one or more subsequent liquid samples from the same patient provided at one or more subsequent time points.

[00162] In some embodiments, the one or more subsequent liquid samples are provided about 2 hours from the previously provided liquid sample.

[00163] In some embodiments, the liquid sample is deposited into the wells of a plate or in at least one test tube. In some embodiments, the plate is a multiwell plate, for example a 96-well plate or a 384-well plate.

[00164] In some embodiments, measuring the fluorescence signal comprises measuring the fluorescence signal using a spectrometer, such as a fluorescence plate reader.

[00165] In some embodiments, the fluorescence signal is measured using a 480 nm excitation wavelength and a 520 nm emission wavelength. The skilled person can readily determine the required and optimal excitation and emission wavelengths.

[00166] Another aspect of the disclosure is a method of testing lysine analog antifibrinolytic agent concentration of a patient comprising periodically collecting a liquid sample from the patient and subjecting the liquid sample to the methods described herein. [00167] In some embodiments, the patient is being monitored in real time during surgical procedures.

III. Kits

[00168] A further aspect of the disclosure is a kit comprising (a) a composition comprising a fluorescently labeled inactive plasminogen variant and a fluorescence quencher labeled fibrin-degradation product (FDP) and (b) a lysine analog antifibrinolytic agent standard.

[00169] In some embodiments, the kit further comprises instructions for use.

[00170] In some embodiments, the fluorescently labeled inactive plasminogen variant is a recombinant full length Glu-plasminogen with a single mutation of the latent active site, Lys-plasminogen with a mutation in the latent active site, mini-plasminogen with a single mutation in the latent active site, or any plasminogen variant that contains a lysine-binding Kringle domain and a mutation in the latent active site.

[00171] In some embodiments, the recombinant full length Glu- plasminogen is a human plasminogen, optionally encoded by the nucleotide sequence and/or the amino acid sequence as shown in Gene ID: 5340. In some embodiments, the amino acid sequence of human plasminogen as set forth in UniProt ID: P00747 or NCBI Reference Sequence: NP_000292.1.

[00172] In some embodiments, the single mutation of the latent active site is serine to a cysteine at position 741 (S741 C), also known as [Glu1]Plg(S741C).

[00173] In some embodiments, the fluorescently labeled plasminogen variant is labeled with a fluorescence probe on a free cysteine, such as C741 .

[00174] In some embodiments, the fluorescence probe comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

[00175] In some embodiments, the fluorescence probe is 5- iodoacetamidofluorescein (5IAF), Alexa Fluor™ -488, fluorescein-5-maleimide, Alexa Fluor™ 488 C5 maleimide, DyLight™ 488 Maleimide, BDP™-FL Maleimide.

[00176] In one embodiment, the fluorescence probe is 5- iodoacetamidofluorescein (5IAF).

[00177] In some embodiments, the fluorescence quencher labeled FDP is generated by: i) reducing purified fibrinogen with a reducing agent; ii) labelling the purified fibrinogen with the fluorescence quencher to generate fluorescence quencher-fibrinogen; iii) contacting the fluorescence quencher-fibrinogen with thrombin to generate a fluorescence quencher-insoluble fibrin clot; iv) contacting the fluorescence quencher-insoluble fibrin clot with plasmin to degrade the insoluble fibrin clot; and v) inhibiting the thrombin and plasmin to generate the fluorescence quencher labeled FDP.

[00178] In some embodiments, the fluorescence quencher comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

[00179] In some embodiments, the fluorescence quencher is QSY™ 9 Cs- maleimide (QSY), QSY™ 7 C5-Maleimide, or Tide Quencher™ 3 Maleimide (TQ3 Maleimide).

[00180] In one embodiment, the fluorescence quencher is QSY™ 9 Cs- maleimide (QSY).

[00181] In one embodiment, the fluorescence probe is 5- iodoacetamidofluorescein (5IAF) and the fluorescence quencher is QSY™ 9 Cs-maleimide (QSY).

[00182] In some embodiments, the reducing agent is a disulfide reducing agent. In some embodiments, the reducing agent comprises beta- mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) or dithiothreitol (DTT).

[00183] In one embodiment, the reducing agent comprises betamercaptoethanol.

[00184] In some embodiments, inhibiting thrombin comprises adding a thrombin inhibitor. Examples include phe-pro-arg-chloromethylketone (FPR- ck), Lepirudin, Desirudin, Argatroban, Bivalirudin, Hirudin, or Dabigatran.

[00185] In one embodiment, inhibiting the thrombin comprises adding phe-pro-arg-chloromethylketone (FPR-ck).

[00186] In some embodiments, inhibiting the plasmin comprises adding a plasmin inhibitor. Examples include val-phe-lys-chloromethylketone (VFK-ck), 02-antiplasmin, or aprotinin.

[00187] In one embodiment, inhibiting the plasmin comprises adding VFK- ck.

[00188] In some embodiments, the composition is stable for up to 6 months.

[00189] In some embodiments, the composition is stored at about -20°C to about -80°C, at about -20°C, or at about -80°C.

[00190] In some embodiments, the fluorescently labeled inactive plasminogen variant is at a concentration of about 25 nM to about 1000 nM. In one embodiment, the fluorescently labeled inactive plasminogen variant is at a concentration of about 200 nM.

[00191] In some embodiments, the fluorescence quencher-FDP is at a concentration of about 0.1 pM to about 10 pM. In some embodiments, the fluorescence quencher-FDP is at a concentration of about 1 pM.

[00192] In some embodiments, the composition further comprises a diluent.

[00193] In some embodiments, the diluent is a buffered salt solution, optionally buffered saline, optionally the buffered saline comprises hepes buffered saline, phosphate buffered saline, Hanks’ balanced salt solution. [00194] In some embodiments, the diluent further comprises a non-ionic detergent, optionally Tween-80.

[00195] In some embodiments, the lysine analog antifibrinolytic agent standard is for use to generate a serial dilution in a diluent.

[00196] Methods of creating a serial dilution are known to those skilled in the art. For example, the lysine analog antifibrinolytic agent standard may be serially diluted from the standard to concentrations of 0 pM, 1 pM, 2 pM, 5 pM, 10 pM, 20 pM, 100 pM, 1 ,000 pM, and 10,000 pM.

[00197] The skilled person can readily select a suitable diluent. For example, the diluent can be the same as the liquid sample, such as plasma, serum or blood.

[00198] In some embodiments, the lysine analog antifibrinolytic agent standard is tranexamic acid (TXA) or £-aminocaproic acid (sACA).

[00199] In some embodiments, the lysine analog antifibrinolytic agent standard is TXA.

[00200] In some embodiments, the kit further comprises one or more plates, optionally a multi-well plate. In some embodiments, the kit further comprises one or more test tubes.

[00201] In some embodiments, the kit further comprises a positive control. In some embodiments, the positive control is a liquid sample containing a known concentration of the lysine analog antifibrinolytic agent.

[00202] In some embodiments, the kit is for use in performing the methods described herein.

[00203] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

EXAMPLES

[00204] The following non-limiting examples are illustrative of the present disclosure:

Example 1 : Materials and Methods

Materials

[00205] Gibco® Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM F-12), newborn calf serum (NCS), Opti-MEM I media, and Pen Strep were purchased from Life Technologies Corporation (Grand Island, NY). ZnCI2 was purchased from Acros Organics (Fair Lawn, NJ). Methotrexate was purchased from Cayman Chemical Company (Ann Arbor, Ml). 0.25% EDTA was purchased from Biomatik (Kitchener, ON). Glu-Gly-Arg-chloromethylketone (GGA-ck), FPR-ck, and VFK-ck were purchased from Molecular Innovations (Novi, Ml). 5IAF was purchased from Thermo Scientific (Rockford, IL). Lysine Sepharose beads were generated in-house as described previously.[13] DEAE Sepharose Fast Flow resin was purchased from GE Healthcare (Uppsala, Sweden). QSY 9 C5-maleimide (QSY) was purchased from Invitrogen (Waltham, MA). PD-10 columns were purchased from GE Healthcare (Chicago, IL). TXA was purchased from TCI (Portland, OR). Normal human plasma was prepared as previously described. [14] Glutathione and sACA were purchased from Sigma (Oakville, ON).

Methods

[00206] Generation of a standard curve for measuring TXA and/or sACA levels in plasma - Experiments were performed in white 96-well microtiter plates pretreated with HBS with 1 % Tween-80, and rinsed thoroughly prior to use. Each well contained 80 pL of a reaction mixture containing 5IAF-Pg (200 nM final) and QSY-FDP (1 pM final) in HBS with 0.01 % Tween-80 (HBST). Once the fluorescent signal stabilized, 20 pL of the TXA and/or sACA standards (0, 1 , 2, 5, 10, 20, 100, 1 ,000, and 10,000 pM in NHP diluted 1 :5 with HBST) were added to the wells and mixed by pipetting. Fluorescence intensities were measured on a fluorescence plate reader (Spectra Max M2, Molecular Devices) with 480 nm excitation and 520 nm emission wavelengths, with a 495 nm emission cut-off filter. A positive control standard with 20 mM sACA was also prepared. Each assay was performed in triplicate on four separate days. A standard curve was generated by plotting the relative fluorescence unit (RFU) signal change at equilibrium as a function of TXA and/or sACA concentration.

[00207] To test whether a pre-mixed QSY-FDP/5IAF-Pg solution would be stable while stored in -80°C, a batch of the mixture including HBST was generated, aliquoted and frozen and stored at -80°C. Similarly, a large batch of TXA standards in plasma were generated, aliquoted, and also stored at -80°C. Standard curves were generated at 0, 14, and 28 days after the reaction mixture was made.

Example 2. Detection of increasing TXA concentration in plasma samples to generate a standard curve

[00208] To demonstrate that TXA can be measured in human blood plasma samples, the present inventors have shown that TXA added to normal human plasma can be calculated using relative fluorescence. After the fluorescence signal equilibrated, the RFU values were estimated by averaging the first ten minutes. The RFU determined with no TXA were subtracted from the remaining data set as the background signal. The change in the RFU values estimated were then plotted as a function of TXA concentration (FIG. 1). The data were fit to a 3-parameter logistic sigmoidal function y = — — using

(¹⁺ )

SigmaPlot (v11.0, SPSS Inc). The assay showed a TXA concentrationdependent RFU change between 1 pM and 100 pM TXA. Minimal signal change above 100 pM TXA was observed, suggesting full dissociation between QSY-FDP and 5IAF-Pg (i.e. no more signal increase can be obtained). This was confirmed with the use of 20 mM sACA, which showed RFU values comparable to 100 pM TXA (not shown).

[00209] To determine the reproducibility of the TXA assay, the intraassay variability and interassay variability were determined. The standard curves spanning the range between 0 and 100 pM TXA in diluted normal human plasma (NHP) were generated over four separate days, with each experiment being performed in triplicates. The mean RFUs ± SD of three measurements each day over 4 separate days are shown in Table 1. The intraassay variability and interassay variability were determined to be 5.7% and 3.0%, respectively.

Table 1

Relative Fluorescence Units (RFUs)

[TXA] (pM) Day 1 Day 2 Day 3 Day 4

1 100.34 ± 105.12 ± 98.32 ± 5.29 97.21 ±

13.48 13.34 12.65

2 181.44 ± 175.21 ± 182.80 ± 188.02 ±

8.21 48.86 10.65 10.59

5 365.54 ± 376.98 ± 362.23 ± 393.40 ±

13.79 27.04 34.32 3.03

10 575.61 ± 600.70 ± 591 .46 ± 597.34 ±

23.26 11.56 37.37 5.07

20 774.97 ± 803.37 ± 798.52 ± 834.08 ±

24.05 25.04 8.22 6.61

100 1121.07 ± 1167.47 ± 1190.19 ± 1193.41 ±

23.40 40.48 4.64 10.22

Known amounts of tranexamic acid (TXA) were added to diluted normal human pooled plasma as indicated. The intraassay variability and the interassay variability were determined to be 5.7% and 3.0%, respectively. All data are presented as means ± SD of four separate experiments.

[00210] Because the fluorescence change is indicative of dissociation of 5IAF-Pg (derived from a point mutation of the full-length Glu-plasminogen) from QSY-FDP, other derivative or truncated (Lys-plasminogen, mini-plasminogen) forms of plasminogen that (a) contain kringle domains, and/or (b) has a point mutation S741C, could generate similar fluorescence changes observed. Furthermore, the assay is not necessarily limited to using 5IAF as the probe to provide a signal change, although the labeling efficiency appears to be better for 5IAF compared with similar probes such as Alexa-488. 5IAF is expected to have 50% to 100% greater labeling efficiency when compared to Alexa-488. Example 3. Measuring TXA levels in NHP samples.

[00211] To demonstrate the accuracy of the TXA assay, the present inventors used the TXA standard curve to calculate known amounts of TXA in normal human plasma. NHP plasma samples that were pre-treated with 150, 80, and 15 pM TXA were subjected to quantitation across four separate days with each day being measured as five replicates. When averaged and plotted as a function of the expected values (FIG. 2), their ratio as determined by the slope of the line of best fit was 1.0151.

Example 4. Pre-mixed QSY-FDP/5IAF-Pg reaction mixture was stable for up to39 weeks.

[00212] To assess the stability of pre-mixed QSY-FDP and 5IAF-Pg, the present inventors demonstrated that storing the reaction mixture does not result in a significant loss in TXA quantitation. A premade mixture containing QSY- FDP, 5IAF-Pg, and buffer was tested for its stability at days 0, 14, and 28, when stored at -80°C. There was little to no discernable differences in the change in fluorescence observed after 28 days (FIG. 3). Further testing showed stability after 39 weeks when stored at -80°C (FIG. 7) and after 28 weeks when stored at -20°C (FIG. 8), demonstrating its storage capacity and stability (FIG. 3).

Example 5: TXA assay can quantify TXA concentration during cardiac pulmonary bypass surgery

[00213] The concentration of TXA was quantified in plasma samples obtained from five patients undergoing cardiac pulmonary bypass surgery. Samples were obtained before surgery, post surgery after heparin reversal (FIG. 4) and 60 minutes after heparin reversal (FIG. 5). This data demonstrates the clinical applicability of the TXA assay.

Example 6: Measurement of EACA in NHP

[00214] Similar to TXA as described in Example 3, the assay was able to demonstrate dose-dependence to sACA (FIG. 6), albeit higher concentrations of sACA are needed. This was consistent with lower binding affinity of sACA with lysine-binding proteins such as plasminogen when compared with TXA. [00215] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

[00216] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present disclosure is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

CITATIONS

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2 Karkouti K, Ho LTS. Preventing and managing catastrophic bleeding during extracorporeal circulation. Hematol Am Soc Hematol Educ Progr United States; 2018; 2018: 522-9.

3 Picetti R, Shakur-Still H, Medcalf RL, Standing JF, Roberts I. What concentration of tranexamic acid is needed to inhibit fibrinolysis? A systematic review of pharmacodynamics studies. Blood Coagul Fibrinolysis 2019; 30: 1-10.

4 Fiechtner BK, Nuttall GA, Johnson ME, Dong Y, Sujirattanawimol N, Oliver WC, Sarpal RS, Oyen LJ, Ereth MH. Plasma tranexamic acid concentrations during cardiopulmonary bypass. Anesth Analg 2001 ; 92: 1131-6.

5 Ng W, Jerath A, Wasowicz M. Tranexamic acid: A clinical review. Anaesthesiol Intensive Ther 2015; 47: 339-50.

6 Sharma V, Fan J, Jerath A, Pang KS, Bojko B, Pawliszyn J, Karski JM, Yau T, McCluskey S, Wgsowicz M. Pharmacokinetics of tranexamic acid in patients undergoing cardiac surgery with use of cardiopulmonary bypass. Anaesthesia 2012; 67: 1242-50.

7 Lecker I, Wang DS, Whissell PD, Avramescu S, Mazer CD, Orser BA. Tranexamic acid-associated seizures: Causes and treatment. Ann Neurol 2016; 79: 18-26.

8 Jerath A, Yang AJ, Pang KS, Looby N, Reyes-Garces N, Vasiljevic T, Bojko B, Pawliszyn J, Wijeysundera D, Beattie WS, Yau TM, Wasowicz M. Tranexamic acid dosing for cardiac surgical patients with chronic renal dysfunction: A new dosing regimen. Anesth Analg 2018; 127: 1323-32.

9 Relke N, Chornenki NLJ, Sholzberg M. Tranexamic acid evidence and controversies: An illustrated review. Res Pract Thromb Haemost John Wiley and Sons Inc.; 2021 ; 5: e12546-e 12546.

10 Silva EMP, Barreiros L, Sa P, Afonso C, Kozek-LangeneckerS, Segundo MA. Analytical methods for quantification of tranexamic acid in biological fluids: A review. Microchem J 2017; 134: 333-42. Horrevoets AJG, Smilde AE, Fredenburgh JC, Pannekoek H, Nesheim ME. The activation-resistant conformation of recombinant human plasminogen is stabilized by basic residues in the amino-terminal hinge region. J Biol Chem 1995; 270: 15770-6. Kim PY, Foley J, Hsu G, Kim PY, Nesheim ME. An assay for measuring functional activated thrombin-activatable fibrinolysis inhibitor in plasma. Anal Biochem 2008; 372: 32-40. Horrevoets AJ, Pannekoek H, Nesheim ME. Production and characterization of recombinant human plasminogen(S741C- fluorescein). A novel approach to study zymogen activation without generation of active protease. J Biol Chem Departments of Biochemistry and Medicine, Queen’s University, Kingston, Ontario, Canada, K7L 3N6; 1997; 272: 2176-82. Kim PY, Stewart RJ, Lipson SM, Nesheim ME. The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular disease. J Thromb Haemost 2007; 5: 1250-6. Toseland CP. Fluorescent labeling and modification of proteins. J Chem Biol. 2013 Apr 13;6(3):85-95. doi: 10.1007/s12154-013-0094-5. PMID: 24432126; PMCID: PMC3691395. Kim PY, Vong M, Lee D, Wu C. Development of an assay to quantify tranexamic acid levels in plasma. Anal Biochem. 2025 Feb;697:115714. doi: 10.1016/j.ab.2024.115714. Epub 2024 Nov 8. PMID: 39521358.

Claims

Claims:

1 . A method for determining concentration of a lysine analogue antifibrinolytic agent in a liquid sample, the method comprising: a. contacting the liquid sample with a mixture of: iii. a fluorescently labeled inactive plasminogen variant, and iv. a fluorescence quencher labeled fibrin-degradation product

(FDP) b. measuring a relative fluorescence signal, wherein the relative fluorescence signal is calculated by subtracting the fluorescence signal generated in the absence of the lysine analog antifibrinolytic agent from the fluorescence signal generated in the presence of the lysine analog antifibrinolytic agent; wherein in the absence of the lysine analog antifibrinolytic agent, the fluorescence quencher labeled FDP quenches the fluorescence generated by the fluorescently labeled plasminogen thereby dampening the fluorescence signal and the presence of the lysine analog antifibrinolytic agent disrupts the interaction of the fluorescently labeled plasminogen and fluorescence quencher labeled FDP thereby generating a higher fluorescence signal; and c. determining the concentration of the lysine analog antifibrinolytic agent in the liquid sample, wherein the fluorescence signal is indicative of the concentration of the lysine analog antifibrinolytic agent.

2. The method of claim 1 , wherein the determining the concentration comprises reference to a fluorescence signal standard curve generated with known amounts of the lysine analog antifibrinolytic agent; and wherein the concentration of the lysine analog antifibrinolytic agent is proportional to the fluorescence signal.

3. The method of claim 1 or 2 wherein the liquid sample comprises plasma, serum, and/or blood.

4. The method according to any one of claims 1 to 3, wherein the fluorescently labeled inactive plasminogen variant is a recombinant full length Glu-plasminogen with a single mutation in the latent active site, Lys-plasminogen with a mutation in the latent active site, miniplasminogen with a single mutation in the latent active site, or any plasminogen variant that contains a lysine-binding Kringle domain and a mutation in the latent active site.

5. The method according to claim 4, wherein the recombinant full length Glu-plasminogen is a human plasminogen, optionally encoded by the nucleotide sequence and/or the amino acid sequence as shown in Gene ID: 5340, or the amino acid sequence of human plasminogen as set forth in in UniProt ID: P00747 or NCBI Reference Sequence: NP_000292.1.

6. The method according to claim 4 or 5, wherein the single mutation in the latent active site is serine to a cysteine at position 741 (S741C), known as [Glu1]Plg(S741C).

7. The method according to claim 6, wherein the fluorescently labeled inactive plasminogen variant is labeled with a fluorescence probe on a free cysteine, such as C741 .

8. The method according to claim 7, wherein the fluorescence probe comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

9. The method according to claim 7 or 8, wherein the fluorescence probe is 5-iodoacetamidofluorescein (5IAF), fluorescein-5-maleimide, Alexa Fluor™ 488 C5 maleimide, DyLight™ 488 Maleimide, BDP™-FL Maleimide.

10. The method according to any one of claims 7-9, wherein the fluorescence probe is 5IAF.

11 . The method according to any one of claims 1 to 10, further comprising first generating the fluorescence quencher labeled FDP by: i) reducing purified fibrinogen with a reducing agent; ii) labelling the purified fibrinogen with the fluorescence quencher to generate fluorescence quencher-fibrinogen; iii) contacting the fluorescence quencher-fibrinogen with thrombin to generate a fluorescence quencher-insoluble fibrin clot; iv) contacting the fluorescence quencher-insoluble fibrin clot with plasmin to degrade the insoluble fibrin clot; and v) inhibiting the thrombin and plasmin to generate the fluorescence quencher labeled FDP.

12. The method according to any one of claims 1-11 , wherein the fluorescence quencher comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

13. The method according to claim 11 or 12, wherein the fluorescence quencher is QSY™ 9 Cs-maleimide (QSY), QSY™ 7 C5-Maleimide, or Tide Quencher™ 3 Maleimide (TQ3 Maleimide).

14. The method according to any one of claims 11 to 13, wherein the fluorescence quencher is QSY.

15. The method of any one of claims 11 to 14, wherein the reducing agent comprises beta-mercaptoethanol, (2-carboxyethyl) phosphine hydrochloride (TCEP) or dithiothreitol (DTT).

16. The method of any one of claims 11 to 15, wherein the reducing agent comprises beta-mercaptoethanol.

17. The method of any one of claims 11 to 16, wherein inhibiting the thrombin comprises adding phe-pro-arg-chloromethylketone (FPR-ck), Lepirudin, Desirudin, Argatroban, Bivalirudin, Hirudin, or Dabigatran.

18. The method of any one of claims 11 to 17, wherein inhibiting plasmin comprises adding val-phe-lys-chloromethylketone (VFK-ck), ci2- antiplasmin, or aprotinin.

19. The method according to any one of claims 1 -18, wherein the lysine analog antifibrinolytic agent is tranexamic acid (TXA) or £-aminocaproic acid (sACA).

20. The method according to any one of claims 1 -19, wherein the lysine analog antifibrinolytic agent is TXA.

21 . The method according to claim 19 or 20, wherein the lower limit of detection of TXA is about 0.28 pM

22. The method according to claim 19 or 20, wherein the lower limit of detection of TXA is about 0.044 mg/L.

23. The method of any one of claims 1-22, wherein in step a), the mixture is a pre-mixed mixture.

24. The method of claim 23, wherein the pre-mixed mixture is mixed up to 6 months prior to use.

25. The method of claim 23 or 24, wherein the pre-mixed mixture is stored at about -20°C to about -80°C, about -20°C, or about -80°C.

26. The method of any one of claims 1-25, wherein the fluorescently labeled inactive plasminogen variant is at a concentration of about 25 nM to about 1000 nM, optionally about 200 nM.

27. The method of any one of claims 1-26, wherein the fluorescence quencher labeled fibrin-degradation product is at a concentration of about 0.1 pM to about 10 pM, optionally about 1 pM.

28. The method of any one of claims 1-27, wherein the liquid sample is provided from a patient being treated with the lysine analog antifibrinolytic agent, optionally the patient is a human patient.

29. The method of any one of claims 1-28, wherein the liquid sample is provided from a patient being treated with the lysine analog antifibrinolytic agent that is undergoing a surgical procedure.

30. The method of any one of claims 1-29, further comprising repeating steps a)-c) on one or more subsequent liquid samples from the same patient obtained at one or more subsequent time points.

31 . A kit comprising (a) a composition comprising a fluorescently labeled inactive plasminogen variant and a fluorescence quencher labeled fibrin-degradation product (FDP); and (b) a lysine analog antifibrinolytic agent standard.

32. The kit of claim 31 , wherein the fluorescently labeled inactive plasminogen variant is a recombinant full length Glu-plasminogen with a single mutation of the latent active site, Lys-plasminogen with a mutation in the latent active site, mini-plasminogen with a single mutation in the latent active site, or any plasminogen variant that contains a lysine-binding Kringle domain and a mutation in the latent active site.

33. The kit according to claim 32, wherein the recombinant full length Glu- plasminogen is a human plasminogen, optionally encoded by the nucleotide sequence and/or the amino acid sequence as shown in Gene ID: 5340, or the amino acid sequence of human plasminogen as set forth in in UniProt ID: P00747 or NCBI Reference Sequence: NP-000292.1.

34. The kit according to claim 32 or 33, wherein the single mutation of the latent active site is serine to a cysteine at position 741 (S741C), known as [Glu1]Plg(S741 C).

35. The kit according to claim 34, wherein the fluorescently labeled inactive plasminogen variant is labeled with a fluorescence probe on a free cysteine, such as C741 .

36. The kit according to claim 35, wherein the fluorescence probe comprises a functional group reactive with sulfhydryl groups, optionally the functional group comprises maleimide or iodoacetamide.

37. The kit according to claim 35 or 36, wherein the fluorescence probe is 5-iodoacetamidofluorescein (5IAF), fluorescein-5-maleimide, Alexa Fluor™ 488 C5 maleimide, DyLight™ 488 Maleimide, BDP™-FL Maleimide.

38. The kit according to any one of claims 35 to 37, wherein the fluorescence probe is 5IAF.

39. The kit according to any one of claims 31 to 38, wherein the fluorescence quencher labeled FDP comprises was generated by: i) reducing purified fibrinogen with a reducing agent; ii) labelling the purified fibrinogen with the fluorescence quencher to generate fluorescence quencher-fibrinogen; iii) contacting the fluorescence quencher-fibrinogen with thrombin to generate a fluorescence quencher-insoluble fibrin clot; iv) contacting the fluorescence quencher-insoluble fibrin clot with plasmin to degrade the insoluble fibrin clot; and v) inhibiting the thrombin and plasmin to generate the fluorescence quencher labeled FDP.

40. The kit according to any one of claims 31-39, wherein the fluorescence quencher comprises a functional group reactive with sulfhydryl groups, optionally the functional grouo comorises maleimide or iodoacetamide.

41 . The kit according to claim 39 or 40, wherein the fluorescence quencher is QSY™ 9 Cs-maleimide (QSY), QSY™ 7 C5-Maleimide, or Tide Quencher™ 3 Maleimide (TQ3 Maleimide).

42. The kit according to any one of claims 39 to 41 , wherein the fluorescence quencher is QSY.

43. The kit according to any one of claims 39 to 42, wherein the reducing agent comprises beta-mercaptoethanol, (2-carboxyethyl) phosphine hydrochloride (TCEP) or dithiothreitol (DTT).

44. The kit of any one of claims 39 to 43, wherein the reducing agent comprises beta-mercaptoethanol.

45. The kit of any one of claims 39 to 44, wherein inhibiting the thrombin comprises adding phe-pro-arg-chloromethylketone (FPR-ck), Lepirudin, Desirudin, Argatroban, Bivalirudin, Hirudin, or Dabigatran.

46. The kit of any one of claims 39-45, wherein inhibiting plasmin comprises adding val-phe-lys-chloromethylketone (VFK-ck), ci2- antiplasmin, or aprotinin.

47. The kit of any one of claims 31-46, wherein the composition is stable for up to 6 months.

48. The kit of any one of claims 31-47, wherein the composition is stored at about -20°C to about -80°C, about -20°C or about -80°C.

49. The kit of any one of claims 31 -48, wherein the fluorescently labeled inactive plasminogen variant is at a concentration of about 25 nM to about 1000 nM, optionally about 200 nM.

50. The kit of any one of claims 31-49, wherein the fluorescence quencher labeled FDP is at a concentration of about 0.1 pM to about 10 pM, optionally about 1 pM.

51 . The kit of any one of claims 31 -50, wherein the lysine analog antifibrinolytic agent standard is tranexamic acid (TXA) and/or e- aminocaproic acid (sACA).

52. The kit of any one of claims 31 -51 , wherein the lysine analog antifibrinolytic agent standard is TXA.

53. The kit of any one of claims 31-52, for use in performing the method of any one of claims 1-30.