EP4087939A1 - Combination of clot-based fibrinogen test and enzyme-based fibrinogen test - Google Patents

Combination of clot-based fibrinogen test and enzyme-based fibrinogen test

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Publication number
EP4087939A1
EP4087939A1 EP21701051.1A EP21701051A EP4087939A1 EP 4087939 A1 EP4087939 A1 EP 4087939A1 EP 21701051 A EP21701051 A EP 21701051A EP 4087939 A1 EP4087939 A1 EP 4087939A1
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Prior art keywords
fibrinogen
test
clot
sample
enzyme
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EP21701051.1A
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German (de)
French (fr)
Inventor
Christian Müller
San Pun
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PENTAPHARMA AG
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DSM IP Assets BV
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Publication of EP4087939A1 publication Critical patent/EP4087939A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen

Definitions

  • the present invention is related to an in vitro method of measuring the clottability of a sample, and also to an in vitro method of checking the health status of an individual via measurement of the blood or plasma clottability.
  • the normal range of fibrinogen that is predominantly synthesized by the liver, is between 1.5 to 3 mg/ ml plasma. A lot of health issues are associated with deviations from such normal fibrinogen level. Infectious diseases, cancer, diabetes mellitus and other diseases are for example associated with low fibrinogen levels, whereas some chronic diseases might be associated with high fibrinogen levels in the blood. Normal physiological processes like pregnancy are known to have higher fibrinogen expression. Additionally, the contraceptive pills are inducing higher fibrinogen expression.
  • Fibrinogen is part of the clot formation occurring in bleeding disorders and thrombogenesis. Under normal conditions, fibrinogen-formation is activated by the action of thrombin (factor I la), leadingto formation of fibrin monomers that upon interaction are transformed into fibrin polymers, which under the influence of factor XI I la, are crosslinked to form cross-linked fibrin polymers also known as clot formation. In massively bleeding patients such as traumatic or perioperative settings fibrinogen is the first and key coagulation factor to reach critical levels.
  • Inflammatory events in the body including e.g. infectious disease induced disseminated intravascular coagulation with massive thrombosis and fibrinolyis co-existing at the same and at different time points might be very challenging for assessment of the actual fibrinogen level.
  • Various clot-based fibrinogen assays are known, including PT-derived fibrinogen assay, thrombin time or the so-called “Clauss-assay” or “Clauss-test” (as illustrated by Mackie et a l, Thromb Haemost. 2002 J u n;87(6):997-1005), the latter being the state-of-the-art test used for fibrinogen measurement, i.e. measurement of the time needed for a fibrin clot to form upon excess of thrombin.
  • the present invention is directed to a method of measuring clottability of a sample, comprising a combination of a clot-based fibrinogen test and enzyme-based fibrinogen test.
  • enzyme-based fibrinogen test preferably means a clot-independent enzyme-based fibrinogen test.
  • the method preferably is an in vitro method.
  • said clot-independent enzyme-based fibrinogen test refers to a competitive peptide substrate-based fibrinogen test.
  • the expression "clot-independent enzyme-based fibrinogen test” and the expression “competitive peptide substrate-based fibrinogen test” are used interchangeably herein.
  • POCT point-of-care-testing
  • an aspect of the present invention is directed to a method, preferably an in vitro method, of measuring the clottability of a sample, preferably the clottability of blood or plasma sample, comprising a combination of a clot- based fibrinogen test and a clot-independent competitive peptide substrate- based fibrinogen test.
  • a further aspect of the present invention is to provide an in vitro method of assessingthe health status of an individual, such as e.g. the thrombotic risk in an individual, said method comprising the application of both a clot-based and a clotting-independent competitive peptide substrate-based test method, preferably comprising the application of the Clauss-test and a clot-independent competitive peptide substrate-based fibrinogen test as disclosed above.
  • the present invention is directed to an in vitro method of measuring the thrombotic risk of an individual, particularly with measurement of real-time fibrinogen activation and generation of fibrin.
  • the presently disclosed method enables measuring the clotting of blood or plasma independent of the presence of compounds such as fibrin-derived species like soluble fibrin, fibrin monomer complex and a series of other degradation products which might be present in the blood or plasma and which could negatively influence the measurement.
  • the presently disclosed method is particularly useful for individuals with critically low fibrinogen level.
  • the presently disclosed method is particularly useful as screening method/test to check the risk for an individual of developing thrombosis, haemophilia and the like.
  • the presently disclosed method can be particularly useful for individuals suffering infections disease induced disseminated intravascular coagulation, where massive thrombosis and fibrinolysis could co-exist at the same and different time points.
  • the presently disclosed method is useful in individuals suffering massive fibrinolysis.
  • the presently disclosed method is useful in individuals undergoing thrombolytic treatment, including intake of (direct) (oral) anti coagulation medication.
  • the present invention is directed to an in vitro method for measuring non-activated fibrinogen levels independently of any pro- and/or anti coagulating factors present in the sample.
  • clottability can be measured even in patients suffering from chronic diseases, i.e. wherein the level of pro- coagulating factors are chronically present in the blood.
  • the presently disclosed method can measure the clottability in a sample selected from blood or plasma, including assessing whether the individual is suffering from hyper- or hypo-coagulability of the blood, wherein the hyper- or hypo-coagulability is associated with different health issues.
  • hypo-coagulability or “hyper-clottability” might be associated with increased thrombotic risk, i.e. the blood has the tendency to form fibrin-clots which is higher than the normal (healthy) range.
  • hypo-coagulability or “hypo-clottability” might be associated with increased fibrinolysis, i.e. the blood has the tendency to form fibrin-clots which is lower than the normal (healthy) range.
  • the clot-based fibrinogen test might be selected from Clauss-test (CTT), PT (Prothrombin-Time)-derived fibrinogen test or PTT (determination of partial thromboplastin time), or any known clot-based test. More preferably, the clot-based fibrinogen test is the Clauss-test.
  • the Clauss-test is based on thrombin clotting time
  • PT test is based on the change in light scattering or optical density during the prothrombin time, wherein the source and composition of reagents as well as assay protocol can be variable.
  • Said Clauss-test and PT test can be performed as described by Mackie et al, Thromb Haemost. 2002 Jun;87(6):997-1005.
  • said any known clot-based test can refer to a batroxobin-based test, wherein said batroxobin is optionally a recombinant batroxobin, and wherein the source and composition of reagents as well as assay protocol can be variable.
  • Said batroxobin-based test can be performed as described by Park R, Song J. Performance Evaluation of a New Fibrinogen Assay Reagent Using Recombinant batroxobin [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1), or can be performed as described by Ha J, Park R, Yang M-, Kim S-, Kim H-,
  • the presently disclosed method is particularly useful to measure the clottability/coagulability of an individual suffering from e.g. cardiovascular disease, diabetes mellitus, cancer, neurodegenerative diseases, autoimmune diseases like inflammatory bowel disease, antiphospholipid syndrome or other chronic disease. And by doing so, one can determine if this individual at the time of blood sampling suffers from hyper-coagulability, namely increased thrombotic risk, or other coagulability and/or fibrinogen level issues, e.g. reduced clottability due to fibrinolysis.
  • cardiovascular disease e.g. cardiovascular disease, diabetes mellitus, cancer, neurodegenerative diseases, autoimmune diseases like inflammatory bowel disease, antiphospholipid syndrome or other chronic disease.
  • hyper-coagulability namely increased thrombotic risk, or other coagulability and/or fibrinogen level issues, e.g. reduced clottability due to fibrinolysis.
  • the presently disclosed method can be used as screening test in individuals such as e.g. at the beginning of potential thrombosis on either a short-term or as a long-term measurement, wherein the measurement might be performed every day either via a physician in a hospital or surgery or by the individual itself, i.e. without the help of a physician or other person.
  • the presently disclosed method could be used in drug screening assays or clinical studies that involve activation and inhibition of immune and coagulation systems of individuals, including human or other animal species.
  • the coagulation system can entail hemostasis and anti coagulation pathways including those mediated by protein C and fibrinolysis.
  • the presently disclosed method can be capable of predicting the risk of thrombosis, or fibrinolysis, or is useful as screening test such as daily measurement of real-time blood coagulability, wherein a clot-based and an enzyme based measurement is combined with a comparison of the result from the clot-based test (A), e.g. Clauss-test, with the result from the clot- independent competitive peptide substrate-based test (B), as disclosed above.
  • A e.g. Clauss-test
  • B competitive peptide substrate-based test
  • the present invention is directed to an in vitro method of checking the health status of an individual, particularly for checking the risk for developing thrombosis, comprising:
  • the present invention is directed to an in vitro method of checking the health status of an individual, particularly for checking the risk for developing thrombosis, comprising: (l) providing a sample from blood or plasma obtained from a subject;
  • the present invention is directed to an in vitro method of checking the health status of an individual, particularly for checking the risk for developing thrombosis, comprising:
  • a still further aspect of the present invention is to provide an in vitro method of diagnosing early fibrinolysis in an individual, comprising: (l) providing a sample from blood or plasma obtained from a subject;
  • the novel diagnostic method comprising measurement of fibrinogen, wherein the results of the clot-based method (l), such as e.g. the Clauss-test, is compared with the results of the clot-independent competitive peptide substrate- based method (2), i.e. the clot-independent competitive peptide substrate- based fibrinogen test as disclosed above with both measurements being in the same range or being different, i.e. wherein the result of (l) is higher or lower than the result of (2), can be used for prediction of the risk of developingthrombosis as well as of the development of fibrinolysis in an individual.
  • the diagnosis is dependent on the ratio /difference between fibrinogen measurement according to method (1) and (2). With the assessment of both measurements (1) and (2) the adequate treatment can be provided.
  • the presently disclosed method comprising the clot-based measurement, particularly Clauss-test, and the clot-independent competitive peptide substrate- based fibrinogen test as disclosed above might be performed in one single device or in two different devices, including but not limited to any device described for such measurement, such as e.g.
  • the present invention is directed to an in vitro method of measuring the clottability of the blood or plasma in an individual, comprising:
  • Also disclosed is a method and diagnostic test used for assessing the health status of an individual comprising measuring the blood clottability with a combination of a clot-based and enzyme-based fibrinogen test method.
  • the blood coagulation cascade is inhibited, more preferably inhibition of both intrinsic and extrinsic pathways.
  • said clot-independent competitive peptide substrate-based fibrinogen test comprises an enzymatic cleavage reaction, wherein the enzymatic cleavage of fibrinogen present in the sample competes with enzymatic cleavage of a detection substrate added to the test medium.
  • said clot-independent competitive peptide substrate-based fibrinogen test comprises the use of a serine endopeptidase for enzymatic cleavage of fibrinogen.
  • the speed of enzymatic cleavage depends on the fibrinogen level in the sample.
  • said clot-independent competitive peptide substrate-based fibrinogen test comprises measuring the proteolytic activity of a serine endopeptidase in sense of converting a peptide substrate, which is inversely proportional to the fibrinogen level in said sample.
  • said clot-independent competitive peptide substrate-based fibrinogen test is performed in the absence of CaC and/or in the absence of thrombin activity.
  • said clot-independent competitive peptide substrate-based fibrinogen test comprises the presence of protease inhibitors, more preferably inhibitors of fibrin polymerization, even more preferably thrombin inhibitors.
  • said clot-independent competitive peptide substrate-based fibrinogen test does not include the generation of a calibration curve and/or the generation/presence of a fibrinogen standard.
  • clottability and "coagulability” is used interchangeably herein. It is defined as the process of the tendency and capability of the plasma or blood to clot, clot-formation or coagulation process, i.e. transformation of fibrinogen into fibrin that are available as soluble oligomeric (fibrin) aggregates, i.e. the development and accumulation of soluble oligomeric fibrin aggregates that can lead to thrombus formation as seen e.g. in pulmonary embolism, cerebral stroke or heart attack. Clot-formation can be influenced by many factors including pro-coagulating and anti-coagulating factors.
  • clottability occurs in 3 different levels, i.e. normal, higher than normal or lower than normal, which can be illustrated in the schematic diagram ( Figure 4).
  • a "normal clottability" is defined as small net-contribution of pro- and anti-coagulable factors, i.e. a non-significant contribution as in healthy individuals. This normal clottability is shown in Figure 4 as hollow circles, where they are exhibiting similar or equal values, estimated by these 2 different methods, Clauss-test vs clot-independent competitive peptide substrate-based fibrinogen test as disclosed above.
  • high than normal clottability or “hyper-clottability” is used interchangeably herein and is defined as a status of significantly more pro- coagulable factors than anti-coagulable factors or the net-effects of pro- and anti-coagulable factors are favoring clotting in the blood, as represented by the black circles in Figure 4, where they are exhibiting very different values from each other estimated by these 2 different methods, specifically, values obtained with Clauss-test are greater than values obtained with clot-independent competitive peptide substrate-based fibrinogen test.
  • a "lower than normal clottability" or “hypo-clottability” is defined as a status of significantly more anti-coagulable factors than pro- coagulable factors or the net-effects of pro- and anti-coagulable factors are not favoring clotting in the blood, as represented by the grey circles in Figure 4, where they are exhibiting very different values from each other estimated by these 2 different methods, specifically, values obtained with Clauss-test are smaller than values obtained with clot-independent competitive peptide substrate-based fibrinogen test.
  • clotting rate is the time needed to reach a defined threshold in a machine that detects clot-formation.
  • pro-coagulating factors or “pro-coagulable factors” as interchangeably used herein, differentiate themselves from fibrinogen and include but are not limited to soluble fibrin monomers, soluble fibrin monomer complex, soluble oligomeric fibrin aggregates, chemically modified fibrinogen/fibrin monomers, soluble fibrin complexes, soluble oligomeric fibrin aggregates, chemically modified fibrinogen/fibrin molecules and any derivatives of fibrinogen via enzyme- and/or non-enzyme-mediated processes, and/or factors of non-fibrinogen origin which are capable of increasing/enhancing the aggregation dynamics of clotting measured by the method according to the present invention.
  • a situation of increased level of pro-coagulating factors is defined herein as "hyper-clottability", i.e. the ability to form clots is increased as compared to healthy individuals, with at the same starting unmodified fibrinogen levels within normal ranges.
  • hyper-clottability i.e. the ability to form clots is increased as compared to healthy individuals, with at the same starting unmodified fibrinogen levels within normal ranges.
  • the significant effects of such factors can be seen in the black circles of Figure 4. If 50% of fibrinogen has been activated to form pro-coagulating factors, the clot-independent competitive peptide substrate-based fibrinogen test will show 50% remaining fibrinogen, but the Clauss-test will show >150 or 300% "fibrinogen" level.
  • anti-coagulable factors include but are not limited to fibrin degradation products (FDPs), fibrinogen-degradation product, chemically modified fibrinogen/fibrin molecules with anti-coagulating activity but excluding fibrinogen and other factors of non-fibrinogen origin which are capable of slowing or decreasing or inhibitingthe aggregation dynamics of clotting measured by the method according to the present invention.
  • a situation of increased level of anti-coagulable factors is defined herein as "hypo- clottability", i.e. the ability to form clots is reduced as compared to healthy individuals, with the same starting unmodified fibrinogen levels within normal ranges. Such individuals may have compromised ability to form clot due to these factors, as compared to situations without these factors.
  • hyper-clottability situation means a level of clottability which is higher compared to the normal or healthy situation.
  • hypo-clottability situation is defined as a level of clottability which is lower compared to the normal or healthy situation.
  • higher means that the fraction of pro-coagulable factors exists in significant amount than the background level.
  • lower means that the fraction of anti-coagulable factors exists in significant amount than the background level.
  • thrombotic risk or "risk for developing thrombosis” are used interchangeably herein and are defined as tendency to develop thrombus as compared to healthy individuals. Thrombus is produced when coagulation cascade is activated or over-activated. Two individuals may have the same normal fibrinogen level, but one of them has uncontrolled or massive activation of the coagulation system, and this individual has a higher thrombotic risk.
  • a further aspect of the present invention is to provide a kit for use in any method disclosed above, wherein said kit comprises a serine endopeptidase, optionally also comprises further essential substance(s) for the tests used.
  • said essential substance(s) refer to the reagents in addition to (and including) said serine endopeptidase which are essential for performing the desired clot-based fibrinogen test as well as clot independent competitive peptide substrate-based fibrinogen test.
  • said essential substance comprises one or more reagents selected from the non-limiting group as described in table 1 below.
  • said kit also comprises a detection substrate which is in combination with said serine endopeptidase.
  • a detection substrate which is in combination with said serine endopeptidase.
  • Table 1 Examples of essential substances to be useful in connection with the Clauss-test (CCT) or the clot-independent competitive peptide substrate-based fibrinogen test (TFT. Figures
  • Figure 1 fibrinogen plasma level measurement with 3 different assays, i.e. CCT, ELISA and TFT according to W02019/068940, plotted on the x-axis and the fibrinogen level in g/l plotted on the y-axis.
  • Fig. 1A shows the average values from citrated plasma samples are plotted according to the indicated method (X- axis) and the corresponding fibrinogen values (Y-axis). The line in the middle of each data set represents the average of each assay.
  • Fig. 1B shows a typical standard curve of TFT with different known fibrinogen calibrators indicated by the filled dot along the curve, with dotted area representing the 95% confidence interval created using GraphPad Prism. For more details, see text.
  • Figure 2 distribution of the average CRP values in ng/ml sample plotted on the Y-axis of individuals plotted on the X-axis. The wide spread of CRP values is reflecting the health status of the individual and are a strong indication of heterogeneity of health status from these volunteers. For more details, see text.
  • Figure 3 fibrinogen plasma level measurement using CCT and TFT (Fig.3A), with side-by-side comparison of individual data points derived from Fig. 3A in the presence of high CRP (Fig.3B) or without high CRP (Fig.3C). For more details see Figure 1A or text.
  • Figure 4 A schematic graph depicting the effects of pro- and anti-coagulable factors on the real-time clottability/coagulability, using 2 different fibrinogen assays as indicated.
  • the fibrinogen levels are 2—4 g/L, but are not limited by these range.
  • FIG. 5 A TFT performed in the presence of plasma and various concentrations of FDPs.
  • Plasma of various well-defined concentration of FDPs were subjected to standard TFT in the absence (blue bracket) and presence of fibrin polymerization inhibitor (green bracket, Pefabloc ® FG). Each condition was duplicated in measurement, and error bar represents SD, and the mean value is plotted.
  • FDP concentrations indicated in the legend are determined by the supplier based on their certificate of analysis. The final concentrations of FDPs are 1 ⁇ 2 of those indicated at the legend.
  • the tracing of both kinds of reactions can be clearly visible: 1. clotting events, including the release of para- nitroaniline (pNA), and 2. the release of pNA only event, both catalyzed by batroxobin.
  • pNA para- nitroaniline
  • FIG. 6 (A) Fibrinogen plasma level measurement with CCT and TFT according to W02019/068940, distribution plotted each on the outer side on the x-axis, the fibrinogen level in g/l plotted on the y-axis. The different fibrinogen levels determined with both methods are connected with red lines on the inner side of the x-axis to show the shift for each individual sample. (B) Difference calculated from the fibrinogen plasma level in g/l of individual samples measurement with CCT and TF, distribution plotted on the y-axis. Samples resulting in a difference of higher than the upper threshold of +0.5 g/l are categorized as hyper- clottable. Samples resulting in a difference of less than the lower threshold of - 0.5 g/l are categorized as hypo-clottable. Examples
  • CCT Clauss-test
  • TFT clot independent competitive peptide substrate-based fibrinogen test
  • CCT was carried out according to the standardized procedure of Clauss fibrinogen test in accordance to Multifibren U (Siemens) and measurement was performed on a GMP-validated standard coagulometer BCS-XP (Siemens). Measurements of ELISA and TFT were both performed on GLP-validated plate reader ClarioStar (BMG Labtech), and ELISA was performed according to the instruction of the kitZymutest Fibrinogen #RK024A (Hyphen BioMed). Each sample was measured twice to obtain average value from each method.
  • the typical signal-dose response curve between optical density (OD) and fibrinogen concentration (g/L) covers the range between ⁇ 0.5 and > 4.0 g/L ( Figure 1).
  • Option 2 values of CCT are higher/much higher than TFT. This situation has been measured with an individual suffering chronic illness associated with high CRP values. The CCT values fluctuated at highly abnormal levels of 4.4 and 4.9 at different occasions, but the TFT values were quite constant at 3.3 and 3.4, respectively. This indicates quite a significant increase in the pro-coagulable factors in this individual at different time points.
  • Option 3 values of CCT are lower/much lower than TFT (see Example 2).
  • Example 2 Measuring the clottability activity from healthy individuals Determination of the clotting activity was performed with 3 different samples from the blood donation center of Switzerland, including measurement of CCT and TFT. Plasma from frozen aliquots was thawed and measurement performed as described in Example 1. The results are shown in Table 2 giving both values for CCT and TFT.
  • S4 and S5 show similar values obtained from CCT and TFT, which correspond to the clear or hollow circles in Figure 4.
  • S7 was showing hyper-clottability, which correspond to one of the black circles in Figure 4.
  • three individuals with plasmas SI, S2 and S3 were found to show hypo-clottability, namely the prominent inhibitory effects of anti- coagulable factors in the clotting process in CCT.
  • clear evidence of hypo- clottability (grey circles in Figure 4) is shown in this example.
  • the currently available fibrinogen assays have some limitations. Being the most used method, the Clauss fibrinogen assay and other clot-based methods are influenced positively and negatively by fibrin-derived intermediates due to the activities of thrombin and plasmin. These fibrin-derivatives are able to participate in clotting itself, by being part of the components for a clot.
  • a kind of fibrin-derived intermediates which is exemplified by fibrin degradation products (FDPs), is well-known to inhibit clotting and interfere with clot-based fibrinogen measurement.
  • FDPs fibrin degradation products
  • the existing literature shows a strong need for a fibrinogen assay that is not affected by the presence of FDPs.
  • the invention of a unique fibrinogen test called TFT according to
  • W02019/068940 is able to overcome the analytical problems faced by the current state of the art fibrinogen test, due to its assay principle, which is not clot-dependent. Additionally, experiments were carried out to proof the assay (W02019/068940) is not affected by FDPs ( Figure 5). These FDPs, which exist in many different molecular forms and at different time of the molecular life cycle, are exhibiting anti-clotting effects, hence they are some of the most significant anti-coagulable factors known to interfere with clotting which is a key process in bleeding prevention.
  • Example 5 This specificity is further validated in Example 5 using a bigger set of plasma samples from healthy donors, tested with CCT and TFT. Within this Example 5, many individuals showed significantly under-estimated fibrinogen levels using CCT, but not the very specific TFT. This is consistent with the molecular mechanism illustrated in this Example 3 and many reports regarding the clot- inhibitory activities of FDPs or other anti-coagulable factors. The opposite is also true for pro-coagulable factors, which are exemplified by many individuals with significantly over-estimated CCT, but not the very specific TFT (Example 5). The very similar observation was obtained from different set of individuals at different time in Example 2.
  • this TFT according to W02019/068940 is able to solve the specificity issue faced by the most widely used state of art fibrinogen test based on clotting.
  • the combination of CCT and TFT is able to indicate the clottability status of that sample at the time of sampling.
  • Example 4 Real world clinical samples of various pathological indications
  • DD means D-dimer.
  • Liver has a key and vital function in hemostasis, namely in the synthesis and production of many coagulation factors, including fibrinogen.
  • Chronic inflammatory liver disease such as liver cirrhosis is well recognized to manifest both increased risk of thrombosis and bleeding.
  • This group of samples were assayed for a few biomarkers such as coagulation factor V (to classify % of liver function), D-dimer (DD, a fibrinolysis marker due to thrombin-activation) and fibrinogen.
  • DD is one of many molecular species of fibrin-degradation products and hence the precursors of DD are FDPs, and the precursors of FDPs are soluble fibrin monomer complexes or fibrin.
  • FDPs FDPs
  • FDPs soluble fibrin monomer complexes or fibrin.
  • fibrinolysis Much slower proteolytic process called fibrinolysis.
  • DD may exist at different levels at different time points, with over 500 signifies high levels.
  • DD is mainly used to determine or confirm if thrombosis did occur, when combined with visual diagnosis of big-enough-clots.
  • DD is high after the clot or thrombosis has happened and the slow fibrinolysis action has started for some time.
  • DD is a marker indicating the activities of thrombin.
  • DD levels demonstrate chronic thrombin generation or activation, and such thrombin activity has left its footprint on fibrin or fibrin-derived molecules which are pro-coagulable or facilitating clotting in nature. Based on the clottability scheme illustrated in Figure 4, most of these samples are therefore showing hyper-clottability, which is in line with the reported observation and DD levels. Patients (ID 5 and 7) were not showing hyper-clottability as in majority of the examples.
  • Table 5 Comparison of CCT and TFT in a set of plasma samples collected from patients needing anti-thrombotic treatment 1. This set of plasmas was collected from patients receiving direct oral anticoagulants (DOACs), targeting at the level of coagulation Factor Xa, to reduce the risk of thrombosis. From the left to the right column of the table, one can see a list of sample ID, the drug plasma concentrations are indicated next to the type of DOACs, followed by the CCT and TFT fibrinogen levels, and the corresponding DD levels.
  • DOACs direct oral anticoagulants
  • the DD is a biomarker, explained previously at Table 4. Just basing on the DD levels, some patients were still not sufficiently anti coagulated.
  • This set of plasmas was collected from patients receiving direct oral anticoagulants (DOACs), targeting at the level of coagulation Factor I la (thrombin), to reduce the risk of thrombosis. From the left to the right column of the table, one can see a list of sample ID, the drug plasma concentrations are indicated next to the type of DOACs, followed by the CCT and TFT fibrinogen levels, and the corresponding DD levels, respectively.
  • the DD is a biomarker, explained previously at Table 4. Just basing on the DD levels, some patients were still not sufficiently anti-coagulated by these anti-thrombin inhibitors. The majority of these samples showed hyper-clottability, except plasma with ID 15, where slightly more anti-coagulable factors were exerting their effects on clotting.
  • the clottability is a dynamic manifestation of physiological levels of both anti- and pro-coagulable factors, which is a unique feature of individual under specific treatments or indications along a period of time.
  • This example illustrates a small representation of a population suffering from chronic diseases. Majority of individuals in this example displayed hyper- clottability states, and these snap shots are captured by this method, which could explain the observations of individuals under anti-coagulant treatments could still suffer from thrombosis, though with reduced risks.
  • Example 5 Real world clinical samples of self-proclaimed healthy individuals
  • self-proclaimed healthy donors were recruited to provide their plasmas for testing, similar to the example shown in Table 1, but with much larger sample size. Beside sample size difference, Example 5 and 2 are representing different sampling times of a local healthy population.

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Abstract

The present invention is related to an in vitro method of measuring the clottability of a sample and also to an in vitro method of checking the health status of an individual via measurement of the blood or plasma clottability.

Description

COMBINATION OF CLOT-BASED FIBRINOGEN TEST AND ENZYME-BASED
FIBRINOGEN TEST
The present invention is related to an in vitro method of measuring the clottability of a sample, and also to an in vitro method of checking the health status of an individual via measurement of the blood or plasma clottability. The normal range of fibrinogen, that is predominantly synthesized by the liver, is between 1.5 to 3 mg/ ml plasma. A lot of health issues are associated with deviations from such normal fibrinogen level. Infectious diseases, cancer, diabetes mellitus and other diseases are for example associated with low fibrinogen levels, whereas some chronic diseases might be associated with high fibrinogen levels in the blood. Normal physiological processes like pregnancy are known to have higher fibrinogen expression. Additionally, the contraceptive pills are inducing higher fibrinogen expression.
Fibrinogen is part of the clot formation occurring in bleeding disorders and thrombogenesis. Under normal conditions, fibrinogen-formation is activated by the action of thrombin (factor I la), leadingto formation of fibrin monomers that upon interaction are transformed into fibrin polymers, which under the influence of factor XI I la, are crosslinked to form cross-linked fibrin polymers also known as clot formation. In massively bleeding patients such as traumatic or perioperative settings fibrinogen is the first and key coagulation factor to reach critical levels.
Thus, the determination of the actual fibrinogen level is very crucial.
Inflammatory events in the body including e.g. infectious disease induced disseminated intravascular coagulation with massive thrombosis and fibrinolyis co-existing at the same and at different time points might be very challenging for assessment of the actual fibrinogen level. Various clot-based fibrinogen assays are known, including PT-derived fibrinogen assay, thrombin time or the so-called "Clauss-assay" or "Clauss-test" (as illustrated by Mackie et a l, Thromb Haemost. 2002 J u n;87(6):997-1005), the latter being the state-of-the-art test used for fibrinogen measurement, i.e. measurement of the time needed for a fibrin clot to form upon excess of thrombin.
Unfortunately, the known clot-based tests are not suitable for measuring the true fibrinogen level: besides fibrinogen, other fibrin-derived species like soluble fibrin, fibrin monomer complexes and various degradation products present in the blood might influence the results, i.e. the time needed for a fibrin clot formation. Thus, other test methods are needed to circumvent this disadvantage. One way is the use of immune-based ELISA with polyclonal antibodies raised against human fibrinogen, detecting all kinds of fibrinogen- or fibrin-derived molecular species of full-length or degraded products. However, said method is highly error-prone, tedious, and needs to be performed by a specialized laboratory. Furthermore, it typically requires 4 hours of laboratory time, thus not applicable in emergency situations. Disadvantageous to ELISA and immunoturbidimetry is that no differentiation of fragmented fibrinogen- or fibrin-related product from intact fibrinogen or fibrin is possible. Therefore, as described in W02019/068940 other clot-independent enzyme- based methods have been developed wherein the true fibrinogen level can be determined in a competitive reaction involving catalytic cleavage by snake venom serine endopeptidase. In this regard, said clot-independent enzyme- based method refers to a competitive peptide substrate-based fibrinogen test. Until now there is no test method available giving a reliable information on the clottability, i.e. to determine the ability of the blood to form fibrin-clots, with measurement of non-activated fibrinogen level. This information would be extremely helpful in assessing the health status of an individual based on fibrinogen measurement. Surprisingly, we now found a novel and inventive way to assess the health status of an individual, particularly with regards to thrombotic risk, via measuring blood clottability, particularly using a combination of a clot-based and an enzyme-based fibrinogen test. In a general aspect, the present invention is directed to a method of measuring clottability of a sample, comprising a combination of a clot-based fibrinogen test and enzyme-based fibrinogen test.
As defined herein, the term "enzyme-based fibrinogen test" preferably means a clot-independent enzyme-based fibrinogen test. The method preferably is an in vitro method.
In the present invention, said clot-independent enzyme-based fibrinogen test refers to a competitive peptide substrate-based fibrinogen test. The expression "clot-independent enzyme-based fibrinogen test" and the expression "competitive peptide substrate-based fibrinogen test" are used interchangeably herein.
The presently disclosed method is particularly useful in point-of-care-testing (POCT) such as e.g. used in hospitals or surgeries but also to be used at home. Additionally, the new method and diagnostic test can be easily implemented in centralized lab-based solutions.
Thus, an aspect of the present invention is directed to a method, preferably an in vitro method, of measuring the clottability of a sample, preferably the clottability of blood or plasma sample, comprising a combination of a clot- based fibrinogen test and a clot-independent competitive peptide substrate- based fibrinogen test.
Said clot-independent competitive peptide substrate-based fibrinogen test is disclosed in W02019/068940.
A further aspect of the present invention is to provide an in vitro method of assessingthe health status of an individual, such as e.g. the thrombotic risk in an individual, said method comprising the application of both a clot-based and a clotting-independent competitive peptide substrate-based test method, preferably comprising the application of the Clauss-test and a clot-independent competitive peptide substrate-based fibrinogen test as disclosed above.
Preferably, the present invention is directed to an in vitro method of measuring the thrombotic risk of an individual, particularly with measurement of real-time fibrinogen activation and generation of fibrin.
The presently disclosed method enables measuring the clotting of blood or plasma independent of the presence of compounds such as fibrin-derived species like soluble fibrin, fibrin monomer complex and a series of other degradation products which might be present in the blood or plasma and which could negatively influence the measurement.
The presently disclosed method is particularly useful for individuals with critically low fibrinogen level. The presently disclosed method is particularly useful as screening method/test to check the risk for an individual of developing thrombosis, haemophilia and the like.
The presently disclosed method can be particularly useful for individuals suffering infections disease induced disseminated intravascular coagulation, where massive thrombosis and fibrinolysis could co-exist at the same and different time points.
In one embodiment, the presently disclosed method is useful in individuals suffering massive fibrinolysis.
In another embodiment, the presently disclosed method is useful in individuals undergoing thrombolytic treatment, including intake of (direct) (oral) anti coagulation medication.
Preferably, the present invention is directed to an in vitro method for measuring non-activated fibrinogen levels independently of any pro- and/or anti coagulating factors present in the sample. Thus, clottability can be measured even in patients suffering from chronic diseases, i.e. wherein the level of pro- coagulating factors are chronically present in the blood.
Preferably, the presently disclosed method can measure the clottability in a sample selected from blood or plasma, including assessing whether the individual is suffering from hyper- or hypo-coagulability of the blood, wherein the hyper- or hypo-coagulability is associated with different health issues.
As used herein, the term "hyper-coagulability" or "hyper-clottability" might be associated with increased thrombotic risk, i.e. the blood has the tendency to form fibrin-clots which is higher than the normal (healthy) range.
As used herein, the term "hypo-coagulability" or "hypo-clottability" might be associated with increased fibrinolysis, i.e. the blood has the tendency to form fibrin-clots which is lower than the normal (healthy) range.
Preferably, according to the present invention, the clot-based fibrinogen test might be selected from Clauss-test (CTT), PT (Prothrombin-Time)-derived fibrinogen test or PTT (determination of partial thromboplastin time), or any known clot-based test. More preferably, the clot-based fibrinogen test is the Clauss-test.
In this regard, as well known by the skilled person in the art, the Clauss-test is based on thrombin clotting time, and PT test is based on the change in light scattering or optical density during the prothrombin time, wherein the source and composition of reagents as well as assay protocol can be variable. Said Clauss-test and PT test can be performed as described by Mackie et al, Thromb Haemost. 2002 Jun;87(6):997-1005.
In addition, said any known clot-based test can refer to a batroxobin-based test, wherein said batroxobin is optionally a recombinant batroxobin, and wherein the source and composition of reagents as well as assay protocol can be variable. Said batroxobin-based test can be performed as described by Park R, Song J. Performance Evaluation of a New Fibrinogen Assay Reagent Using Recombinant batroxobin [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1), or can be performed as described by Ha J, Park R, Yang M-, Kim S-, Kim H-,
Song Y, Song J. Batroxobin Based Method to Measure Fibrinogen to Overcome Interfering Effects of New Anticoagulant Agents Targeting Thrombin [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1).
In one embodiment, the presently disclosed method is particularly useful to measure the clottability/coagulability of an individual suffering from e.g. cardiovascular disease, diabetes mellitus, cancer, neurodegenerative diseases, autoimmune diseases like inflammatory bowel disease, antiphospholipid syndrome or other chronic disease. And by doing so, one can determine if this individual at the time of blood sampling suffers from hyper-coagulability, namely increased thrombotic risk, or other coagulability and/or fibrinogen level issues, e.g. reduced clottability due to fibrinolysis.
In one particular embodiment, the presently disclosed method can be used as screening test in individuals such as e.g. at the beginning of potential thrombosis on either a short-term or as a long-term measurement, wherein the measurement might be performed every day either via a physician in a hospital or surgery or by the individual itself, i.e. without the help of a physician or other person.
In one embodiment, the presently disclosed method could be used in drug screening assays or clinical studies that involve activation and inhibition of immune and coagulation systems of individuals, including human or other animal species. The coagulation system can entail hemostasis and anti coagulation pathways including those mediated by protein C and fibrinolysis.
The presently disclosed method can be capable of predicting the risk of thrombosis, or fibrinolysis, or is useful as screening test such as daily measurement of real-time blood coagulability, wherein a clot-based and an enzyme based measurement is combined with a comparison of the result from the clot-based test (A), e.g. Clauss-test, with the result from the clot- independent competitive peptide substrate-based test (B), as disclosed above.
In one embodiment, the present invention is directed to an in vitro method of checking the health status of an individual, particularly for checking the risk for developing thrombosis, comprising:
(1) providing a sample from blood or plasma obtained from a subject;
(2) measuring the fibrinogen level via clot formation in the sample, particularly via application of the Clauss-test; (3) measuring the true fibrinogen level in the sample, via the clot- independent competitive peptide substrate-based fibrinogen test as disclosed above;
(4) comparing the result of the measurement of (2) and (3); wherein in the case of both measurements are in about a range of 1.5 to 3 mg fibrinogen per ml plasma, the individual has low or no risk of developing thrombosis.
In a further embodiment, the present invention is directed to an in vitro method of checking the health status of an individual, particularly for checking the risk for developing thrombosis, comprising: (l) providing a sample from blood or plasma obtained from a subject;
(2) measuring the time for clot formation in the sample, particularly via application of the Clauss-test;
(3) measuring the true fibrinogen level in the sample, via the clot- independent competitive peptide substrate-based fibrinogen test as disclosed above;
(4) comparing the result of the measurement of (2) and (3); wherein in the case of both measurements are not in the same range of 1.5 to 3 mg fibrinogen per ml plasma and the result of (2) is higher than the result of (3), the individual has a risk of developing thrombosis. In even a further embodiment, the present invention is directed to an in vitro method of checking the health status of an individual, particularly for checking the risk for developing thrombosis, comprising:
(1) providing a sample from blood or plasma obtained from a subject;
(2) measuring the time for clot formation in the sample, particularly via application of the Clauss-test; (3) measuring the true fibrinogen level in the sample, via the clot- independent competitive peptide substrate-based fibrinogen test as disclosed above;
(4) comparing the result of the measurement of (2) and (3); wherein in the case of both measurements are not in the same range of 1.5 to 3 mg fibrinogen per ml plasma and the result of (2) is lower than the result of (3), the individual the individual is goingthrough early fibrinolysis, dependent on the degree of fibrinolysis and fibrinogen level.
A still further aspect of the present invention is to provide an in vitro method of diagnosing early fibrinolysis in an individual, comprising: (l) providing a sample from blood or plasma obtained from a subject;
(2) measuring the time for clot formation in the sample, particularly via application of the Clauss-test;
(3) measuring the true fibrinogen level in the sample, via the clot- independent competitive peptide substrate-based fibrinogen test as disclosed above; wherein the fibrinogen level according to measurement (2) is lower than the level according to measurement (3).
The novel diagnostic method comprising measurement of fibrinogen, wherein the results of the clot-based method (l), such as e.g. the Clauss-test, is compared with the results of the clot-independent competitive peptide substrate- based method (2), i.e. the clot-independent competitive peptide substrate- based fibrinogen test as disclosed above with both measurements being in the same range or being different, i.e. wherein the result of (l) is higher or lower than the result of (2), can be used for prediction of the risk of developingthrombosis as well as of the development of fibrinolysis in an individual. The diagnosis is dependent on the ratio /difference between fibrinogen measurement according to method (1) and (2). With the assessment of both measurements (1) and (2) the adequate treatment can be provided.
The presently disclosed method comprising the clot-based measurement, particularly Clauss-test, and the clot-independent competitive peptide substrate- based fibrinogen test as disclosed above might be performed in one single device or in two different devices, including but not limited to any device described for such measurement, such as e.g. Xprecia Stride (Siemens Healthcare), CoaguChek® (Roche Diagnostics), i-Stat® systems (Abbott), ESR or qLabs systems (Operon Biotech & Healthcare), Alere INRatio® systems (Alere™), LabPad® (Avalun®), microlNR (i Line® Microsystems), Mission® PT (Aeon®), Cobas series (Roche Diagnostics), ACL Top series (I L), BCS series and Atellica series (Siemens Healthcare), CS-series (Sysmex), STA series (Stago), Accriva/IL's Hemochron or VerifyNow, LABGEO (Samsung), or other systems used or known in the art for lab-based tests or POCT in the field of blood analysis. Preferably, the present invention is directed to an in vitro method of measuring the clottability of the blood or plasma in an individual, comprising:
(a) providing a sample from whole blood or plasma obtained from a subject,
(b) applying a clot-based test, particularly the Clauss-test,
(c) applying a clot-independent competitive peptide substrate-based fibrinogen test as explained above,
(d) comparing the results from (b) and (c), and
(e) making a decision of whether to take anti-coagulation drugs or providing fibrinogen or further drugs like e.g. tranexamic acid or provide plasma exchange.
Also disclosed is a method and diagnostic test used for assessing the health status of an individual comprising measuring the blood clottability with a combination of a clot-based and enzyme-based fibrinogen test method.
Preferably, during the clot-independent competitive peptide substrate-based fibrinogen test, the blood coagulation cascade is inhibited, more preferably inhibition of both intrinsic and extrinsic pathways. Preferably, said clot-independent competitive peptide substrate-based fibrinogen test comprises an enzymatic cleavage reaction, wherein the enzymatic cleavage of fibrinogen present in the sample competes with enzymatic cleavage of a detection substrate added to the test medium.
Preferably, said clot-independent competitive peptide substrate-based fibrinogen test comprises the use of a serine endopeptidase for enzymatic cleavage of fibrinogen.
Preferably, in said clot-independent competitive peptide substrate-based fibrinogen test, the speed of enzymatic cleavage depends on the fibrinogen level in the sample. Preferably, said clot-independent competitive peptide substrate-based fibrinogen test comprises measuring the proteolytic activity of a serine endopeptidase in sense of converting a peptide substrate, which is inversely proportional to the fibrinogen level in said sample. Preferably, said clot-independent competitive peptide substrate-based fibrinogen test is performed in the absence of CaC and/or in the absence of thrombin activity.
Preferably, said clot-independent competitive peptide substrate-based fibrinogen test comprises the presence of protease inhibitors, more preferably inhibitors of fibrin polymerization, even more preferably thrombin inhibitors.
Preferably, said clot-independent competitive peptide substrate-based fibrinogen test does not include the generation of a calibration curve and/or the generation/presence of a fibrinogen standard.
As used herein, the term "clottability" and "coagulability" is used interchangeably herein. It is defined as the process of the tendency and capability of the plasma or blood to clot, clot-formation or coagulation process, i.e. transformation of fibrinogen into fibrin that are available as soluble oligomeric (fibrin) aggregates, i.e. the development and accumulation of soluble oligomeric fibrin aggregates that can lead to thrombus formation as seen e.g. in pulmonary embolism, cerebral stroke or heart attack. Clot-formation can be influenced by many factors including pro-coagulating and anti-coagulating factors. The more clottable fibrino- or fibrin-derived products are available, the faster the clotting rate, and it is the clotting rate that the instrument derives the fibrinogen level from. Generally, clottability occurs in 3 different levels, i.e. normal, higher than normal or lower than normal, which can be illustrated in the schematic diagram (Figure 4).
As used herein, a "normal clottability" is defined as small net-contribution of pro- and anti-coagulable factors, i.e. a non-significant contribution as in healthy individuals. This normal clottability is shown in Figure 4 as hollow circles, where they are exhibiting similar or equal values, estimated by these 2 different methods, Clauss-test vs clot-independent competitive peptide substrate-based fibrinogen test as disclosed above.
The term "higher than normal clottability" or "hyper-clottability" is used interchangeably herein and is defined as a status of significantly more pro- coagulable factors than anti-coagulable factors or the net-effects of pro- and anti-coagulable factors are favoring clotting in the blood, as represented by the black circles in Figure 4, where they are exhibiting very different values from each other estimated by these 2 different methods, specifically, values obtained with Clauss-test are greater than values obtained with clot-independent competitive peptide substrate-based fibrinogen test.
As used herein, a "lower than normal clottability" or "hypo-clottability" is defined as a status of significantly more anti-coagulable factors than pro- coagulable factors or the net-effects of pro- and anti-coagulable factors are not favoring clotting in the blood, as represented by the grey circles in Figure 4, where they are exhibiting very different values from each other estimated by these 2 different methods, specifically, values obtained with Clauss-test are smaller than values obtained with clot-independent competitive peptide substrate-based fibrinogen test.
As used herein, "clotting rate" is the time needed to reach a defined threshold in a machine that detects clot-formation.
As used herein, "pro-coagulating factors" or "pro-coagulable factors" as interchangeably used herein, differentiate themselves from fibrinogen and include but are not limited to soluble fibrin monomers, soluble fibrin monomer complex, soluble oligomeric fibrin aggregates, chemically modified fibrinogen/fibrin monomers, soluble fibrin complexes, soluble oligomeric fibrin aggregates, chemically modified fibrinogen/fibrin molecules and any derivatives of fibrinogen via enzyme- and/or non-enzyme-mediated processes, and/or factors of non-fibrinogen origin which are capable of increasing/enhancing the aggregation dynamics of clotting measured by the method according to the present invention. A situation of increased level of pro-coagulating factors is defined herein as "hyper-clottability", i.e. the ability to form clots is increased as compared to healthy individuals, with at the same starting unmodified fibrinogen levels within normal ranges. The significant effects of such factors can be seen in the black circles of Figure 4. If 50% of fibrinogen has been activated to form pro-coagulating factors, the clot-independent competitive peptide substrate-based fibrinogen test will show 50% remaining fibrinogen, but the Clauss-test will show >150 or 300% "fibrinogen" level.
As used herein, "anti-coagulable factors" include but are not limited to fibrin degradation products (FDPs), fibrinogen-degradation product, chemically modified fibrinogen/fibrin molecules with anti-coagulating activity but excluding fibrinogen and other factors of non-fibrinogen origin which are capable of slowing or decreasing or inhibitingthe aggregation dynamics of clotting measured by the method according to the present invention. A situation of increased level of anti-coagulable factors is defined herein as "hypo- clottability", i.e. the ability to form clots is reduced as compared to healthy individuals, with the same starting unmodified fibrinogen levels within normal ranges. Such individuals may have compromised ability to form clot due to these factors, as compared to situations without these factors.
According to the present invention, hyper-clottability situation means a level of clottability which is higher compared to the normal or healthy situation. Meanwhile, the term hypo-clottability situation is defined as a level of clottability which is lower compared to the normal or healthy situation. As used herein, "higher" means that the fraction of pro-coagulable factors exists in significant amount than the background level. As used herein, "lower" means that the fraction of anti-coagulable factors exists in significant amount than the background level.
As used herein, the terms "thrombotic risk" or "risk for developing thrombosis" are used interchangeably herein and are defined as tendency to develop thrombus as compared to healthy individuals. Thrombus is produced when coagulation cascade is activated or over-activated. Two individuals may have the same normal fibrinogen level, but one of them has uncontrolled or massive activation of the coagulation system, and this individual has a higher thrombotic risk.
A further aspect of the present invention is to provide a kit for use in any method disclosed above, wherein said kit comprises a serine endopeptidase, optionally also comprises further essential substance(s) for the tests used. In this regard, said essential substance(s) refer to the reagents in addition to (and including) said serine endopeptidase which are essential for performing the desired clot-based fibrinogen test as well as clot independent competitive peptide substrate-based fibrinogen test. Preferably said essential substance comprises one or more reagents selected from the non-limiting group as described in table 1 below.
Preferably, said kit also comprises a detection substrate which is in combination with said serine endopeptidase. Table 1: Examples of essential substances to be useful in connection with the Clauss-test (CCT) or the clot-independent competitive peptide substrate-based fibrinogen test (TFT. Figures
Figure 1: fibrinogen plasma level measurement with 3 different assays, i.e. CCT, ELISA and TFT according to W02019/068940, plotted on the x-axis and the fibrinogen level in g/l plotted on the y-axis. Fig. 1A shows the average values from citrated plasma samples are plotted according to the indicated method (X- axis) and the corresponding fibrinogen values (Y-axis). The line in the middle of each data set represents the average of each assay. Fig. 1B shows a typical standard curve of TFT with different known fibrinogen calibrators indicated by the filled dot along the curve, with dotted area representing the 95% confidence interval created using GraphPad Prism. For more details, see text. Figure 2: distribution of the average CRP values in ng/ml sample plotted on the Y-axis of individuals plotted on the X-axis. The wide spread of CRP values is reflecting the health status of the individual and are a strong indication of heterogeneity of health status from these volunteers. For more details, see text. Figure 3: fibrinogen plasma level measurement using CCT and TFT (Fig.3A), with side-by-side comparison of individual data points derived from Fig. 3A in the presence of high CRP (Fig.3B) or without high CRP (Fig.3C). For more details see Figure 1A or text.
Figure 4: A schematic graph depicting the effects of pro- and anti-coagulable factors on the real-time clottability/coagulability, using 2 different fibrinogen assays as indicated. For simplicity sake, the fibrinogen levels are 2—4 g/L, but are not limited by these range. For more details, see text.
Figure 5: A TFT performed in the presence of plasma and various concentrations of FDPs. Plasma of various well-defined concentration of FDPs were subjected to standard TFT in the absence (blue bracket) and presence of fibrin polymerization inhibitor (green bracket, Pefabloc® FG). Each condition was duplicated in measurement, and error bar represents SD, and the mean value is plotted. FDP concentrations indicated in the legend are determined by the supplier based on their certificate of analysis. The final concentrations of FDPs are ½ of those indicated at the legend. Within this graph, the tracing of both kinds of reactions can be clearly visible: 1. clotting events, including the release of para- nitroaniline (pNA), and 2. the release of pNA only event, both catalyzed by batroxobin. The clotting was inhibited when the FDPs were at 57 pg/mL, which is consistent with previous reported findings. Whereas in a typical TFT, which are represented by the green-bracketed tracings, FDPs at any tested concentrations demonstrated no interference.
Figure 6: (A) Fibrinogen plasma level measurement with CCT and TFT according to W02019/068940, distribution plotted each on the outer side on the x-axis, the fibrinogen level in g/l plotted on the y-axis. The different fibrinogen levels determined with both methods are connected with red lines on the inner side of the x-axis to show the shift for each individual sample. (B) Difference calculated from the fibrinogen plasma level in g/l of individual samples measurement with CCT and TF, distribution plotted on the y-axis. Samples resulting in a difference of higher than the upper threshold of +0.5 g/l are categorized as hyper- clottable. Samples resulting in a difference of less than the lower threshold of - 0.5 g/l are categorized as hypo-clottable. Examples
Example 1: Fibrinogen level assessment under influence of inflammation markers
In a first step, 3 different test methods including the Clauss-test (CCT), ELISA- based test (ELISA) and clot independent competitive peptide substrate-based fibrinogen test (TFT) which is also described in W02019/068940 were used to evaluate the fibrinogen level in plasma samples from volunteers and commercial sources (n=25). This population is seen as "healthy" individuals. Citrated plasmas were collected and prepared according to standard protocol as recommended by the supplier of Multifibren U (Siemens). Samples when not used on the same day were stored frozen according to standard protocol at - 80°C to maintain biological functions required by the assays. CCT was carried out according to the standardized procedure of Clauss fibrinogen test in accordance to Multifibren U (Siemens) and measurement was performed on a GMP-validated standard coagulometer BCS-XP (Siemens). Measurements of ELISA and TFT were both performed on GLP-validated plate reader ClarioStar (BMG Labtech), and ELISA was performed according to the instruction of the kitZymutest Fibrinogen #RK024A (Hyphen BioMed). Each sample was measured twice to obtain average value from each method. The typical signal-dose response curve between optical density (OD) and fibrinogen concentration (g/L) covers the range between <0.5 and > 4.0 g/L (Figure 1). All these 3 fibrinogen assays have been calibrated against known calibrators to create standard curves of their own so that the amount of fibrinogen in g/L can be estimated from the samples. As shown in Figure 1A, the average values fall within the measurable ranges covered by each method. Notably, CCT and ELISA were similar in their wide spread of values, whereas TFT exhibited much narrower spread for the same plasma sample set. Importantly, when we followed the data of the individuals, more comparable values of fibrinogen were found to be the case between CCT and ELISA for the same sample, whether the values are low or high. On the contrary to CCT and ELISA, the data set from TFT exhibited narrower spread from its set average. When the values from TFT were compared with CCT, we saw a subset of values having comparable values between TFT and CCT, while another subset displayed huge difference between TFT and CCT values. In a next step, health status of the samples with known inflammatory marker C- reactive protein (CRP) was tested via ELISA (commercially available from Hyphen BioMed), as it is well known that CRP can induce release of tissue factor from immune cells, which can further activate coagulation cascade. As shown in Figure 2, the CRP values were also wide spread in values. Remarkably, the CRP- values correspond to the values obtained via CCT, i.e. high CCT-value corresponds to high CRP-value.
In a next step, the correlation between CCT and TFT was evaluated at individual level, which is meaningful when the fibrinogen value range extent beyond the value < 2.3 for CCT-fibri nogen (Figure 1), preferably approaching 1. The results are shown in Figure 3. We observed much better correlation between CCT and TFT when only relatively much healthier plasmas were included into the comparison, as shown by the basic R2 calculation performed by Microsoft Excel and its improvement (see Fig. 3C). In comparing the values of CCT and TFT, 3 different possible situations can be detected:
Option 1: values of CCT and TFT were about in the same range in real healthy individuals, with the correlation indicator R2 being improved to 0.91, with 95% confidence interval of slope between 0.8 and 1.
Option 2: values of CCT are higher/much higher than TFT. This situation has been measured with an individual suffering chronic illness associated with high CRP values. The CCT values fluctuated at highly abnormal levels of 4.4 and 4.9 at different occasions, but the TFT values were quite constant at 3.3 and 3.4, respectively. This indicates quite a significant increase in the pro-coagulable factors in this individual at different time points. Option 3: values of CCT are lower/much lower than TFT (see Example 2).
From the set of data evaluated here, we have confirmed that the CCT will reflect true fibrinogen level only in very healthy individuals, with the values for CCT and TFT being more or less in the same range. Since the health situation of an individual is not always known, it is recommended to use a combination of both CCT and TFT to check the extent of pro- or anti-coagulable factors in the individual and decide on further investigations, before more critical situation is developing.
Example 2: Measuring the clottability activity from healthy individuals Determination of the clotting activity was performed with 3 different samples from the blood donation center of Switzerland, including measurement of CCT and TFT. Plasma from frozen aliquots was thawed and measurement performed as described in Example 1. The results are shown in Table 2 giving both values for CCT and TFT.
Table 2: Comparison of CCT and TFT with healthy blood samples.
With plasma (1) and (2) we realized that the CCT should be approximately doubled as compared to values obtained with TFT. In contrast to plasma (3), where the CCT-value is far too high as compared to the TFT, indicating that both a high amount of fibrinogen has been converted to fibrin/fibrin-related products and the presence of abundant pro-coagulable factors.
In addition, determination of the clotting activity was performed with 6 different random samples from the blood donation center of Switzerland, which cover a wide range of fibrinogen level determined by CCT, to further substantiate the previous example. These plasmas came from a completely different source and population. Plasma from frozen aliquots was thawed and measurement performed as described in Example 1. These plasmas were measured by CCT and TFT. The results are shown in Table 3 giving both values for CCT and TFT.
Table 3: Comparison of CCT and TFT with healthy blood samples.
From this table, very consistent observation as illustrated in Example 1 is demonstrable in S4, S5 and S7. S4 and S5 show similar values obtained from CCT and TFT, which correspond to the clear or hollow circles in Figure 4. S7 was showing hyper-clottability, which correspond to one of the black circles in Figure 4. In this example, three individuals with plasmas SI, S2 and S3 were found to show hypo-clottability, namely the prominent inhibitory effects of anti- coagulable factors in the clotting process in CCT. Hence, clear evidence of hypo- clottability (grey circles in Figure 4) is shown in this example.
Example 3: Unresolved analytical issues of the state of the art fibrinogen tests, and how TFT solves them
The currently available fibrinogen assays have some limitations. Being the most used method, the Clauss fibrinogen assay and other clot-based methods are influenced positively and negatively by fibrin-derived intermediates due to the activities of thrombin and plasmin. These fibrin-derivatives are able to participate in clotting itself, by being part of the components for a clot. A kind of fibrin-derived intermediates, which is exemplified by fibrin degradation products (FDPs), is well-known to inhibit clotting and interfere with clot-based fibrinogen measurement. The existing literature shows a strong need for a fibrinogen assay that is not affected by the presence of FDPs. The invention of a unique fibrinogen test called TFT, according to
W02019/068940, is able to overcome the analytical problems faced by the current state of the art fibrinogen test, due to its assay principle, which is not clot-dependent. Additionally, experiments were carried out to proof the assay (W02019/068940) is not affected by FDPs (Figure 5). These FDPs, which exist in many different molecular forms and at different time of the molecular life cycle, are exhibiting anti-clotting effects, hence they are some of the most significant anti-coagulable factors known to interfere with clotting which is a key process in bleeding prevention. The effects of such anti-coagulable factors include reduced clotting propensity or efficiency, hence the term hypo-clottability (Figure 4), when compared with plasma conditions of similar fibrinogen levels but in the absence of inhibitory effect from anti-coagulable factors like FDPs. In this example, measurement of TFT reactions were carried out in 2 different groups from a same set of plasma samples containing different FDP concentrations (Figure 5). These 2 groups differ in only one component in their reactions, namely Pefabloc FG. Pefabloc FG, an artificial clot inhibitor, is added by default in one group, as in a typical TFT reaction, to stop clot formation. In the other group without Pefabloc FG, thrombin-like-enzyme-induced clot formation was observed and can be measured in the spectrophotometer (see Figure 5 legend for details). Evidently, the FDPs were inhibiting clotting and the inhibitions were concentration-dependent (Figure 5). Similar clot-inhibitory effects of Pefabloc FG were evident in TFT, and the effect of FDPs did not interfere with the enzyme kinetics in TFT (Figure 5). Additionally, these fibrin-derivatives, as exemplified here with FDPs, have neither negative nor positive influences on the TFT reaction (Figure 5). These fibrin-derivatives, though all originated from fibrinogen, are not exerting any influence in such TFT, which is not the case for thrombin and thrombin-induced clotting like CCT. It was reported that such fibrin-derivatives were able to inhibit clotting and the enzyme activity of thrombin. This is another strong argument that such assay design as TFT is able to improve the specificity for fibrinogen that is not seen in any state of the arts.
This specificity is further validated in Example 5 using a bigger set of plasma samples from healthy donors, tested with CCT and TFT. Within this Example 5, many individuals showed significantly under-estimated fibrinogen levels using CCT, but not the very specific TFT. This is consistent with the molecular mechanism illustrated in this Example 3 and many reports regarding the clot- inhibitory activities of FDPs or other anti-coagulable factors. The opposite is also true for pro-coagulable factors, which are exemplified by many individuals with significantly over-estimated CCT, but not the very specific TFT (Example 5). The very similar observation was obtained from different set of individuals at different time in Example 2.
Therefore, this TFT according to W02019/068940 is able to solve the specificity issue faced by the most widely used state of art fibrinogen test based on clotting. At the same time, the combination of CCT and TFT is able to indicate the clottability status of that sample at the time of sampling.
Example 4: Real world clinical samples of various pathological indications
In this example, specific clinical samples, obtained from local university hospital, with defined clinical indications were tested with 2 different fibrinogen measurement methods, CCT and TFT, to further demonstrate the specificity of TFT and the advantage of the combination of CCT an TFT.
Table 4: Comparison of CCT and TFT in a set of plasmas with liver indications. "DD" means D-dimer. Liver has a key and vital function in hemostasis, namely in the synthesis and production of many coagulation factors, including fibrinogen. Chronic inflammatory liver disease such as liver cirrhosis is well recognized to manifest both increased risk of thrombosis and bleeding. This group of samples were assayed for a few biomarkers such as coagulation factor V (to classify % of liver function), D-dimer (DD, a fibrinolysis marker due to thrombin-activation) and fibrinogen. Briefly, DD is one of many molecular species of fibrin-degradation products and hence the precursors of DD are FDPs, and the precursors of FDPs are soluble fibrin monomer complexes or fibrin. This is the life cycle of fibrinogen started when fibrinogen is proteolytically modified by thrombin either through activation of intrinsic or extrinsic coagulation pathway, followed by much slower proteolytic process called fibrinolysis. Depending on the plasma-sampling time, DD may exist at different levels at different time points, with over 500 signifies high levels. DD is mainly used to determine or confirm if thrombosis did occur, when combined with visual diagnosis of big-enough-clots. DD is high after the clot or thrombosis has happened and the slow fibrinolysis action has started for some time. Here, DD is a marker indicating the activities of thrombin. Hence, in this group of liver disease patients, DD levels demonstrate chronic thrombin generation or activation, and such thrombin activity has left its footprint on fibrin or fibrin-derived molecules which are pro-coagulable or facilitating clotting in nature. Based on the clottability scheme illustrated in Figure 4, most of these samples are therefore showing hyper-clottability, which is in line with the reported observation and DD levels. Patients (ID 5 and 7) were not showing hyper-clottability as in majority of the examples. This apparent normal clottability could be due to the negligible effects from both anti- and pro-coagulable factors at the time of sampling. In another word, both anti- and pro-coagulable factors could exist to neutralize each other's effect on clottability. When these 2 patients were sampled at different times, the contributions from both anti- and pro-coagulable factors might be very different.
Table 5: Comparison of CCT and TFT in a set of plasma samples collected from patients needing anti-thrombotic treatment 1. This set of plasmas was collected from patients receiving direct oral anticoagulants (DOACs), targeting at the level of coagulation Factor Xa, to reduce the risk of thrombosis. From the left to the right column of the table, one can see a list of sample ID, the drug plasma concentrations are indicated next to the type of DOACs, followed by the CCT and TFT fibrinogen levels, and the corresponding DD levels. The DD is a biomarker, explained previously at Table 4. Just basing on the DD levels, some patients were still not sufficiently anti coagulated. The majority of these samples showed hyper-clottability, except plasmas with ID 12 and 29, where more anti-coagulable factors were exerting their effects on clotting. Hence, the clottability is a dynamic manifestation of physiological levels of both anti- and pro-coagulable factors, which is a unique feature of individual under specific treatments or indications along a period of time. Table 6: Comparison of CCT and TFT in a set of plasma samples collected from patients needing anti-thrombotic treatment 2.
This set of plasmas was collected from patients receiving direct oral anticoagulants (DOACs), targeting at the level of coagulation Factor I la (thrombin), to reduce the risk of thrombosis. From the left to the right column of the table, one can see a list of sample ID, the drug plasma concentrations are indicated next to the type of DOACs, followed by the CCT and TFT fibrinogen levels, and the corresponding DD levels, respectively. The DD is a biomarker, explained previously at Table 4. Just basing on the DD levels, some patients were still not sufficiently anti-coagulated by these anti-thrombin inhibitors. The majority of these samples showed hyper-clottability, except plasma with ID 15, where slightly more anti-coagulable factors were exerting their effects on clotting. Hence, the clottability is a dynamic manifestation of physiological levels of both anti- and pro-coagulable factors, which is a unique feature of individual under specific treatments or indications along a period of time. Currently, there is no such state of the art available in the market.
This example illustrates a small representation of a population suffering from chronic diseases. Majority of individuals in this example displayed hyper- clottability states, and these snap shots are captured by this method, which could explain the observations of individuals under anti-coagulant treatments could still suffer from thrombosis, though with reduced risks.
Example 5: Real world clinical samples of self-proclaimed healthy individuals In this example, self-proclaimed healthy donors were recruited to provide their plasmas for testing, similar to the example shown in Table 1, but with much larger sample size. Beside sample size difference, Example 5 and 2 are representing different sampling times of a local healthy population.
Similar to the observation shown in Example 2, many more individuals can be categorized according to their own CCTs and the corresponding TFTs. Similarly, these individuals are either hyper- or normal or hypo-clottability. The averages of the 2 measurements, CCT and TFT, are similar, but the spreads of the values of CCT and TFT are very different (Figure 6A). This pattern is also seen in another clinical sample set illustrated in Example 1, Figure 1A. In these 2 examples, CCT values are consistently showing wider range and TFT values. Hence, the wider ranges seen in CCT are the results of low specificity for fibrinogen discussed in the main text and examples. The same low specificity is also illustrated by the method based on antibodies, which by themselves not able to differentiate molecular species from fibrinogen and fibrin-derivatives (Example 1, Figure 1A). If the thresholds of 0.5 and -0.5 g/L are applied to categorize the clottability of these individuals, about 50% are within the normal clottability and about 50% are either hypo- or hyper-clottable (Figure 6B). The strong fibrinolytic activities are notable in this population, as these activities are manifested in hypo- clottable states, which is rarely observed in the population described in Example 4. This is in line with the numerous reports and well-known pathway in fibrinolysis inhibition, even though DD levels indicated fibrinolysis did occur. Hence, it is possible to monitor the fibrinolysis pathway within an individual over a period of time to see if fibrinolysis pathway is sufficiently activated.
Hence, again in this example, as in other previous examples, the presence of anti- and pro-coagulable factors are present physiologically, and they influence the clottability of the plasma. Through this clottability, this method reveals the thrombin activities that are well-known to be activated when immune system and coagulation pathways, which are tightly connected, are active.

Claims

Claims
1. A method of measuring clottability of a sample, comprising a combination of a clot-based fibrinogen test and an enzyme-based fibrinogen test.
2. The method accordingto claim 1, which is an in vitro method and/or wherein the enzyme-based fibrinogen test is a clot-independent enzyme-based fibrinogen test.
3. The method accordingto claim 1 or 2, wherein the clot-based fibrinogen test is selected from determination of the prothrombin time (PT), determination of partial thromboplastin time (PTT), and Clauss-test.
4. The method accordingto any one of claims 1 to 3, wherein the enzyme- based fibrinogen test involves catalytic cleavage by a serine endopeptidase.
5. The method accordingto any one of claims 1 to 4, wherein the enzyme- based fibrinogen test involves catalytic cleavage of fibrinogen by snake venom serine endopeptidase, preferably by venombin A.
6. The method accordingto any one of claims 1 to 5, comprising measuring the proteolytic activity of a serine endopeptidase which is inversely proportional to the fibrinogen level in said sample.
7. The method accordingto any one of claims 1 to 6, wherein the sample is blood or plasma.
8. The method accordingto any one of claims 1 to 7, comprising:
(a) providing a sample selected from whole blood or plasma obtained from a subject,
(b) applying the clot-based test, preferably the Clauss-test,
(c) applying the enzyme-based fibrinogen test, (d) comparing the results from (b) and (c), and
(e) making a decision of whether to take anti-coagulation drugs or providing fibrinogen or further drugs or provide plasma exchange.
9. A method of assessing the health status of an individual, comprising a combination of the clot-based fibrinogen test and the enzyme-based fibrinogen test of any one of claims 1-7.
10. The method of assessing the health status of an individual accordingto claim 8, which is an in vitro method and/or is comprising:
(l) providing a sample from blood or plasma obtained from a subject, (2) measuring the time for clot formation in the sample, preferably via application of the Clauss-test;
(3) measuring the true fibrinogen level in the sample, via the enzyme-based fibrinogen test; (4) comparing the result of the measurement of (2) and (3), wherein in the case of both measurements are in about a range of 1.5 to 3 mg fibrinogen per ml plasma, the individual has low or no risk of developing thrombosis, or wherein in the case of both measurements are not in the same range of 1.5 to 3 mg fibrinogen per ml plasma and the result of (1) is higher than the result of (2), the individual has a risk of developing thrombosis, or wherein in the case of both measurements are not in the same range of 1.5 to 3 mg fibrinogen per ml plasma and the result of (1) is lower than the result of (2), the individual the individual is goingthrough early fibrinolysis.
11. An in vitro method of diagnosing early fibrinolysis in an individual, comprising a combination of the clot-based fibrinogen test and the enzyme- based fibrinogen test of any one of claims 1-7.
12. The in vitro method of diagnosing early fibrinolysis in an individual accordingto claim 11, comprising: (l) providing a sample from blood or plasma obtained from a subject,
(2) measuring the time for clot formation in the sample, preferably via application of the Clauss-test;
(3) measuring the true fibrinogen level in the sample, via the enzyme-based fibrinogen test; wherein the fibrinogen level accordingto measurement (1) is lower than the level accordingto measurement (2).
13. The method according to any one of claims 1 to 12 which is used in centralized haematological or clinical laboratories, emergency rooms, emergency situations occurring even outside hospitals, medical practices, private home, paddocks, barns, or point-of-care testing (POCT) environment.
14. A kit for use in the method of any one of claims 1 to 13, comprising a serine endopeptidase.
15. The kit accordingto claim 14, comprising a detection substrate.
EP21701051.1A 2020-01-08 2021-01-08 Combination of clot-based fibrinogen test and enzyme-based fibrinogen test Pending EP4087939A1 (en)

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