METHODS AND SYSTEMS FOR DETECTING AND QUANTIFYING INDIRECT

THROMBIN INHIBITORS

[001] FIELD OF THE INVENTION

[002] The present invention relates to methods and systems for detecting and/or quantifying indirect thrombin inhibitors such as heparin in a sample containing at least one blood component, e.g., in a blood or plasma sample. The methods and systems employ prothrombin activators that cause the production of thrombin that is susceptible to action by such indirect thrombin inhibitors.

[003] BACKGROUND OF THE INVENTION

[004] The process of blood coagulation involves a complex series of interactions known as the coagulation cascade in which a succession of proteolytic actions leads to coagulation. Proteolysis is balanced by actions inhibiting coagulation. Coagulation factors, which are subject to proteolysis, start generally out as inactive zymogens which upon proteolysis turn into active proteases that activate the next zymogen in the cascade. Activated coagulation factors are generally labeled with a subscript "a."

[005] The activation of coagulation is often described as following one of two pathways: (a) the extrinsic pathway and (b) the intrinsic pathway. These two pathways converge at one point to form a common pathway leading to clot formation. In the extrinsic pathway, damaged tissue exposes tissue factor which activates factor VII (FVII) to its activated form FVII ₃ . Tissue factor and FVII ₃ form a complex which activates FX which initiates the common pathway of the extrinsic and intrinsic pathways. The intrinsic pathway is initiated by contact activation on an artificial surface, e.g., when blood is exposed to surfaces or during extracorporeal blood circulation such as during heart surgery or hemodialysis or when biomaterials such as catheters are inserted into the blood

circulation. In the intrinsic pathway, FXII is activated to FXiI ₃ which activates factor FXI to FXI _a which in turn activates FIX to FIX ₃ eventually leading to the common pathway. In the common pathway, prothrombin is cleaved by activated factor FXa, which eventually leads to active thrombin. Thrombin in turn cleaves fibrinogen resulting in fibrin which polymerizes to form a blood clot.

[006] Coagulation is, as mentioned above, balanced by a series of inhibitory actions. For example, antithrombin, in conjunction with heparin cofactor Il inactivates thrombin. Protein C, when activated by thrombin to form its activated form, APC, acts in conjunction with a cofactor, protein S, to inactivate Factors FVIIIg and FV _a . Tissue factor pathway inhibitor (TFPI) inhibits FX _a and the tissue factor- FVIIa complex in a FX ₃ dependent fashion.

[007] in sum, a wide variety of factors are required in the bloodstream to achieve a balanced coagulation leading to clotting at the right time, place and in the right amount. In certain conditions, e.g., hemophilia or lupus, essential factors are missing or anticoagulants are present which inactivate such essential factors. Some snakes produce venoms that simulate certain coagulation factors, leading to coagulation and clotting of the blood of their prey. In particular, some snake venoms simulate factor FX ₃ and/or factor FV ₃ , thus activating prothrombin. These snake venoms can be used in vitro anticoagulation assays.

[008] The publications and other materials, including patents and patent applications, used herein to illustrate the invention and, in particular, to provide additional details respecting the practice are incorporated herein by reference.

[009] It is often necessary to treat blood with substances having a coagulating effect, e.g., when the blood is deficient of certain factors, or, more often, with substances having anticoagulating effect, e.g., in the course of a surgical intervention. As a consequence, blood anticoagulants are among the most commonly used drugs in modern medicine, being used prophylactically as well

as for the treatment of thromboembolic diseases. Long-term blood anticoagulation can be achieved with vitamin K antagonists such as warfarin and other coumarin derived oral anticoagulants. These substances need several days until a stable anticoagulation is achieved. However, in many situations rapid coagulation is required. In those situations direct or indirect inhibition of activated coagulation factors is often employed. The most commonly used coagulation factor inhibitor is heparin, a mixture of polysulfated glycoaminoglycans, which promotes the binding of antithrombin to factors FXIIa, FXIa, FXa and thrombin and which acts essentially instantly.

[0010] In the course of a number of invasive procedures blood is brought into contact with foreign surfaces. For example, during cardiopulmonary bypass (CPB) for open-heart surgery, the patient's blood is channeled into an extracorporeal circuit. Foreign surfaces generally exhibit a strong procoagulant effect on the circulating blood, thus promoting the formation of blood clots. Blood clots can induce thrombosis or embolism, resulting in severe harm to the patient. To offset blood cloth formation, anticoagulants, in particular, heparin, an indirect thrombin inhibitor, are routinely administered. Direct thrombin inhibitors such as hirudin can also be employed.

[0011] It is important to closely monitor an anticoagulant's concentration in a patient's blood as well as its activity since its effect not only differs from patient to patient, but since it is often metabolized during surgery.

[0012] One method which aims at determining the concentration and activity of direct thrombin inhibitors such as hirudin is described in International Patent Publication WO00004662. In this method snake venom, such as ecarin, is added to a body fluid and converts prothrombin into atypical intermediates such as meizothrombin. These intermediates are selectively inactivated by direct thrombin inhibitors. Certain chromogenic or fluorogenic substances, which are known to be cleaved by thrombin, are also cleaved by these atypical

intermediates. The concentration as well as the activity of the direct thrombin inhibitor can be determined by following the reduction of light absorption- or emission resulting from the cleavage of the chromogenic or fluorogenic substances.

[0013] The indirect thrombin inhibitor heparin binds to the endogenous coagulation inhibitor antithrombin and significantly enhances the ability of antithrombin to bind and inactivate activated clotting factors. The biological action of heparin is believed to be mainly mediated by the inhibition of thrombin.

[0014] Heparin is metabolized during surgery and the anticoagulation effect of heparin varies from patient to patient. Thus, to maintain an adequate level of anticoagulation during, e.g., CPB surgery, heparin is generally administered both before and during surgery in amounts tailored to the specific patient. At the end of the invasive procedure, the exposition to the foreign surface and thus the need for anticoagulation ceases. Thus, agents such as protamine are administered, which block the activity of heparin by forming a complex with it.

[0015] A number of assays are currently available to quantify heparin activity. The more popular ones include the so called "activated partial thromboplasmin time" (aPTT) assay and the "activated clotting time" (ACT). The aPTT assay is able to quantify heparin concentrations up to 0.75U heparin/ml. Only ACT is able to quantify high levels of anticoagulants/anticoagulation.

[0016] The ACT assay (United States Patent 3,492,096) involves adding, e.g., a patient's blood sample to a defined amount of activators of coagulation that simulate a foreign surface, such as celite (diatomaceous earth), kaolin, or glass particles. These activate the intrinsic pathway of coagulation, causing, after running through the coagulation cascade, the blood to clot (Fig. 1). A measurement of the time interval between adding an activator to a blood sample and the detection of clotting is taken. This measurement represents the ACT.

The method has been simplified by a high degree of automation. The ACT is, in the absence of heparin, typically about 120 seconds. An ACT of more than 480 seconds, and the respective heparin concentration, is by many clinicians considered the minimum ACT/ heparin concentration required for, e.g., CPB surgery. The ACT test is aimed at determining whether adequate amounts of heparin have been administered to avoid the formation of blood clots during a surgical procedure. The main advantage of the ACT test over other available coagulation tests is that it can quantify heparin at relative high concentrations. However, presently there are a number of limitations to the ACT test such as poor correlation of the ACT to the heparin concentration in the sample (Leyvi et al., An investigation of a new activated clotting time "MAX-ACT" in patients undergoing extracorporeal circulation, Anesth Analg 92(3):578-83 (2001)), limited standardization (Murray et al, Heparin detection by the activated coagulation time: a comparison of the sensitivity of coagulation tests and heparin assays, J Cardiothorac Vase Anesth 11 : 24-8 (1997)) and distortion of measurements due to the presence of drugs such as aprotinin and/or due to hemodilution (i.e., dilution of the blood sample). In addition, the adaptation of the ACT test for the use with test cartridges such as the ones described in United States Patents 4,756,884; 5,110,727 and 6,060,323 has been problematic due to the method's lack of adaptability to smaller sample volumes.

[0017] While some progress has been made in recent years to overcome some of the limitations of the traditional ACT (United States Patent 6,417,004; Leyvi et al, 2001; United States Patent Application Publication 20020127730), there remains a need for alternative methods for detecting and quantifying and monitoring the activity of indirect thrombin inhibitors such as heparin in a sample containing at least one blood component. There is in particular a need for methods and/or systems that can detect and/or quantify high amounts of indirect thrombin inhibitors, e.g., as high as about 10 U/ml unfractionated heparin, as often needed in clinical practice. More in particular, there is a need for a method and/or system that can detect and/or quantify such high amounts of indirect

thrombin inhibitors but also low amounts of indirect thrombin inhibitor, such as about 0.75 U/ml of unfractionated heparin. The latter method is particularly needed in clinical practice where it is generally necessary to quantify the indirect thrombin inhibitor after its administration when high amounts generally need to be detected/quantified as well as after reversal of its effect when very low residual amounts need to be detected/quantified. Compared to using two different methods/systems for the high and low measurements, such a dual function method/system may cut down on time spent and reduce subsequent analysis costs.

[0018] Summary of the Invention

[0019] The present invention is directed at a method for detecting the presence and/or quantifying of at least one indirect thrombin inhibitor in a sample containing at least one blood component. The method comprises

- adding at least one prothrombin activator to the sample to create a sample mixture, wherein said at least one prothrombin activator causes prothrombin in the sample mixture to be processed to thrombin that is susceptible to inhibition via the at least one indirect thrombin inhibitor; and

- measuring at least one parameter indicative of thrombin activation and/or activity in the sample mixture.

[0020] The present invention is also directed to a system for detecting the presence or quantifying at least one indirect thrombin inhibitor in a sample containing at least one blood component. The system comprises (a) a receptacle or material for receiving, absorbing or adsorbing:

(1) the sample, and

(2) at least one prothrombin activator for processing prothrombin in the sample to thrombin that is susceptible to inhibition via the at least one indirect thrombin inhibitor; and

(b) at least one detector for directly or indirectly detecting a presence or quantity of thrombin formed, wherein a reading provided by the detector is correlatable to the presence or quantity of the at least one indirect thrombin inhibitor in the sample.

[0021] The present invention is also directed towards a method for determining antagonisation of an indirect thrombin inhibitor in a sample containing at least one blood component. This method comprises

- adding at least one prothrombin activator to the sample to create a sample mixture, wherein said at least one prothrombin activator causes prothrombin in the sample mixture to be processed to thrombin that is susceptible to inhibition via the at least one indirect thrombin inhibitor;

- adding at least one antagonist of the indirect thrombin inhibitor; and

- measuring at least one parameter indicative of thrombin activation and/or activity in the sample mixture.

[0022] Brief Description of the Drawings

[0023] Fig. 1 indicates the parts of the coagulation cascade that will be passed through in the ACT assay prior to reaching thrombin, the target of an indirect thrombin inhibitor.

[0024] Fig. 2 depicts the direct prothrombin activation according to the present invention.

[0025] Fig. 3 shows the detection of moderate amounts of heparin using textarin.

[0026] Fig. 4 shows the detection of higher amounts of heparin using noscarin in combination with the factor V activator RW-V (Russell's Viper Venom- factor V).

[0027] Fig. 5 shows the detection of high and low dosages of heparin using different combinations of the prothrombin activators textarin and noscarin.

[0028] Fig. 6 indicates the precision of repeated measurement of heparin concentrations using the method described (20 determinations using one plasma sample spiked with rising heparin concentrations).

[0029] Fig. 7 shows the effect of rising amounts of protamine. The results of 6 samples from patients undergoing open heart surgery are depicted. The amount of protamine required could be quantified within 3 minutes.

[0030] Fig. 8 depicts the detection of heparin using a solution rich in tissue factor as, e.g., applied for the determination of the prothrombin time (rombs) or a contact activator solution as , e.g., used for the determination of the aPTT (circles) as the trigger for clotting. As shown, the addition of rising amounts of heparin to the sample leads to an exponential rise of the clotting times, which limits the amount of heparin which can be quantified by this method.

[0031] Fig. 9 shows the detection of different concentrations of unfractionated heparin (UFH) in whole blood using a single reagent (Experiment 6) with a ROTEM-system.

[0032] Fig. 10 shows the consistency of results obtained with a reagent comprising noscarin 80 U/ml + textarin 480 U/ml + RW-V 240 U/ml + CaCI ₂ 0.06 mmol/ml in HEPES 50 mM 0.5% BSA pH 7.4 that was, freshly prepared (t=0), 1.5 hours old (t=1.5) or 3.5 hours old (t=3.5).

[0033] Detailed Description of Various and Preferred Embodiments of the Invention

[0034] Definitions

[0035] An "indirect thrombin inhibitor," according to the present invention, temporarily or permanently associates, for example, by binding, with a substance that directly interacts with thrombin and exerts a thrombin inhibitory activity via said association. Generally, an indirect thrombin inhibitor contains at least one domain that allows for such temporary or permanent association with the substance that directly interacts with thrombin. An indirect thrombin inhibitor, according to the present invention may, in particular, activate or augment the activity of an inhibitor of thrombin that interacts directly with thrombin or may deactivate or suppress the activity of an activator of thrombin that interacts directly with thrombin. For example, the indirect thrombin inhibitor heparin exerts its inhibitory activity on thrombin primarily by augmenting the activity of the endogenous coagulation inhibitor antithrombin.

[0036] In the context of the present invention, a substance, e.g., a prothrombin activator "derived from" a composition, e.g., snake venom when the substance originates from said composition.

[0037] A measurement or quantification is, according to the present invention, "substantially unaffected" by, e.g., a drug, if the measurement or quantification can be used in medical praxis, despite the presence of the drug, without introduction of a correcting factor.

[0038] A reagent, mixture or other composition is, according to the present invention, "substantially free" of a substance, if any amount of the substance that is present does not negatively affect a physical property, such as stability, of the reagent, mixture or other composition.

[0039] "Thrombin activation" describes, in the context of the present invention, the time when thrombin becomes effective and shows its effect.

[0040] "Thrombin activity" is, in the context of the present invention, used as a term that describes the effects of thrombin in quantitatively.

[0041] The "heparin" and "heparin derivatives" according to the present invention may be of any origin. For example, they may be isolated from a natural source or they may be synthesized by different methods. "Heparin" in a more technical sense includes, for example, UFH (unfractionated heparin) which is, e.g., available under the trade names LIQUEMIN, THROMBOPHOB, CALCIPARIN and HEPARIN, and which form part of the present invention. However, the term "heparin," as the person skilled in the art will appreciate, is used subsequently herein for the most part more generically to refer to any "heparin" or "heparin derivative" that may be employed in the context described.

[0042] "Heparin derivatives," according to the present invention, include any substance that comprises at least the heparin pentasaccharide, is able to augment the activity of antithrombin via association with the same and that does not correspond to UFH as defined above. These substances include, preferably, at least 15 oligosaccharides and more preferably at least 18 oligosaccharides (see, ,,The New Heparins", The Ochsner Journal: Vol. 4, No. 1 , pp. 41-47 (2002)). "Heparin derivatives" include, e.g., fractionated heparin (FH) and low molecular weight heparin (LMWH) such as Certoparin, Dalteparin, Enoxparin, Nadroparin, Reviparin, Tinzaparin, which are also available under the trade names MONO-EMBOLEX, FRAGMIN, CLEXANE, FRAXIPARIN, CLIVARIN and INNOHEP as well as dermatan sulfate and other substances that comprise, optionally sulfated, L-iduronate and GalNAc-4-sulfate as repeating disaccharide units and is able to augment the activity of antithrombin via association with the same. "Heparin pentasaccharide" refers to a structural unit of heparin having

three D-glucosamine and two uronic acid residues. The central D-glucosamine residue contains a unique 3-0-sulfate moiety:

(See also U.S. Patent Publications 2002/01069143 and 2004/0038932). Also included in this definition are so called heparinoids. The term heparinoids describes a group of substances with a heparin-like effect. These include, for example, sulfated vegetable oligo- and polysaccharides, e.g. polysulfates prepared from alginic acid, pectins, xylans, starches and dextrans, or sulfated animal glycosaminoglycans. Particular mention should be made of pentosan polysulfates, e.g. sodium pentosansulfonate, xylan sulfate, e.g. .beta.-1 ,4-D- xylan 2,3-bis(hydrogen sulfate), xylan poly(hydrogen sulfate) and sodium salts thereof, such as dextran sulfates, chitin sulfates, chondroitin polysulfates, as well as so called mucopolysaccharide polysulfates, polyvinylsulfonic acids, also called polyethylenesulfonic acids, e.g. sodium apolate, polygalacturonic acid sulfate (methyl ester methyl glucoside), alginate sulfates, e.g. sodium alginate sulfate and polymannuronic acid sulfate (e.g., Danaparoid-Natrium which is available under the trade name ORAGAN; see also U.S. Patent Application 2003/0161884).

[0043] A "prothrombin activator" according to the present invention is a prothrombin activator that acts directly on prothrombin. Prothrombin activators include substances that require, e.g., cofactors or other substances that are not part of the coagulation cascade as well as substances that are, e.g., augmented in their activity by other substances including certain coagulation factors. The term includes any substance that acts directly on prothrombin and converts prothrombin to a form of thrombin which is susceptible to inhibition via an indirect thrombin inhibitor. A prothrombin activator may be, but is not limited to,

substances derived from snake venom such as textarin, oscutarin, noscarin, pseutarin, trocarin, notecarin or hopsarin. Many snake venoms are group C and D prothrombin activators and show sequence homologies to mammalian FX _a . Accordingly, a prothrombin activator may be a "derivative of FX ₃ ." This includes human or non-human animal FX _a as well as synthetic FX ₃ that have been mutated according to methods well known in the art to exchange, delete etc., for example, single amino acids, but substantially retain their functionality as well as functional parts of naturally occurring or synthetic FX _a s, which are optionally mutated, and retain the functionality of FX ₃ . Mutated FX _a s may, for example, have sequence identities of about 90%, 95%, 98% or 99% with such human, non-human animal, synthetic FX _a s or fragements thereof. Under Swiss Prot No. P00742 the sequence of the unprocessed 488 amino acid sequence of the human coagulation factor X precursor is disclosed, (see also, e.g., Leytus, S. et al. "Gene for Human Factor X: A Blood Coagulation Factor Whose Gene Organization is Essentially Identical with that of Factor IX and Protein C." Biochem., 1986, pp. 5098-5102, vol. 25.; or, e.g., U.S. Patent 6,905,846, in particular, Fig. 1).

[0044] The term "protamine" defines a group of simple proteins found in fish sperm that are strongly basic, are soluble in water, and are not coagulated by heat, and yield chiefly arginine upon hydrolysis. Protamines are capable of inactivating indirect prothrombin activators, in particular, by binding to them. The term "protamine derivates" include substances such as protamine sulfate and other protamine-salts such as protamine chloride and protamine hydrochloride, which bind to and inactive heparin and other glycosaminoglycans.

[0045] An "antagonist" according to the present invention is any molecule or substance that can counteract the effects of an indirect thrombin inhibitor. Such an antagonist might exert its effect directly, for example, by neutralizing the indirect thrombin inhibitor through complex formation, but might also exert its effect indirectly. Those antagonists are often arginine-rich compounds or low

molecular weight compounds which exhibit a protamine-like effect on indirect prothrombin activators.

[0046] An indirect thrombin inhibitor is "effectively" antagonized if the anticoagulant effects of the indirect thrombin inhibitor are counteracted to a degree that is considered in clinical practice sufficient to be safe for a patient to whom the thrombin inhibitor had been administered, that is, sufficient to ensure that the patient is not exposed to the risk of bleeding resulting from the administration of the indirect thrombin inhibitor.

[0047] The present invention provides methods and systems for detecting and quantifying indirect thrombin inhibitors. Indirect thrombin inhibitors inhibit the coagulation cascade at the thrombin level, thereby interfering with the clotting of blood. To determine the presence and/or quantity of an indirect thrombin inhibitor in, e.g., a blood sample, the thrombin activation and/or activity in the sample is measured using an appropriate parameter. To that end the coagulation cascade is artificially triggered. The present invention provides for triggering the coagulation cascade at a late stage, preferably at the prothrombin stage of the cascade. In a preferred embodiment, a prothrombin activator,, preferably a reagent containing such a prothrombin activator, is added to a sample and causes the prothrombin in the sample to be processed to thrombin. In this embodiment, additional prothrombin might be added to avoid the prothrombin innate to the sample to be reaction limiting. Such additional prothrombin might be provided as part of an additional reagent and is preferably mixed with either the prothrombin activator containing reagent or with the sample at or around the time the latter two are combined or shortly after a mixture of the two has been produced. The inhibition of the thrombin formed via any indirect thrombin inhibitor in the sample is then assessed by measuring the parameters indicative of thrombin activity and/or activation. In contrast to the present invention, many detection and quantification methods for direct as well as indirect thrombin inhibitors, such as the ACT assay (Fig. 1), trigger the coagulation

cascade at an early stage. This renders these methods susceptible to substances that interfere with many factors and steps in the coagulation cascade between the point at which the coagulation cascade is triggered and thrombin. By triggering the coagulation cascade at a later stage, errors resulting from those interferences are reduced if not eliminated. In particular interferences with factors such a FVIII, FV or FXII and/or other steps in the coagulation cascade that precede prothrombin can be completely or substantially avoided. For example, kallikrein inhibitors which are often used during procedures such as open heart surgery, interfere with steps in the coagulation cascade that precede prothrombin. Aprotinin, for example, interferes with the activation of FXII. If a method such as the ACT assay is used this interference can lead to erroneous readings. Also, certain conditions or drugs result in a deficiency or super abundance of coagulation factor(s) that precede prothrombin in the coagulation cascade leading to Factor X _a . This again can lead in the ACT and other assays that rely on initiating the coagulation cascade at an early stage to erroneous readings. For example, in response to inflammation some patients develop a so called "acute phase reaction," which leads to high levels of the coagulation factor VIII (FVIII) in the sample. The superabundance of FVIII leads, when ACT is used to determine the quantity of heparin present, to short baseline ACT values, since high levels of factor VIII shorten the time interval from the activation of the ACT via the contact phase to the detection of clotting via the thrombin activity. This is turn may result in an underestimation of the quantity of heparin present in the sample as, in such a case, a short clotting time does not reflect low heparin activity, but rather a high level of FVIII in the sample. Such a superabundance of FVIII does, however, in many embodiments, not affect the systems and methods of the present invention since the coagulation cascade is triggered further downstream. Thus, the methods and systems of the present invention are, in many embodiments, substantially unaffected by those deficiencies and/or super abundances.

[0048] The activity of the prothrombin activator of the present invention processes prothrombin into thrombin which is susceptible to indirect thrombin inhibitors. The inhibition of the thrombin so produced can be monitored and its presence and/or quantity can be assessed (Fig. 2).

[0049] In certain embodiments, the present invention allows for a quantification of low and/or high concentration of indirect thrombin inhibitors. As will be shown in the following, a prothrombin inhibitor can be chosen so that the indirect thrombin inhibitor can be detected at low doses or high doses. For example, Fig. 3 shows prothrombin activation with a prothrombin activator such as textarin. This set-up allows for the detection of moderate amounts of heparin (see Example 1). Fig. 4 shows that with certain prothrombin activators such as noscarin the quantification of fairly high concentrations of heparin is possible. However, here the sensitivity with regard to lower concentrations of heparin may be reduced.

[0050] In certain embodiments the prothrombin inhibitor can be chosen so that both low and high concentrations of indirect prothrombin inhibitor can be detected and/or quantified. Preferably, a combination of prothrombin inhibitors is chosen to achieve this goal. Fig. 5 shows the results obtained with a combination of noscarin and textarin. As can be seen from this Figure, the concentrations of noscarin and textarin employed determine the shape of the dose-response curve, which can accordingly be adjusted. For example, a higher concentration of noscarin leads to a flatter dose-response curve at high heparin concentrations and therefore enhances the measuring range of the assay. Various combinations of two or more prothrombin activators, such as textarin and noscarin, at differing concentrations can therefore be used to increase the sensitivity of the methods and systems of the present invention and adjust the time required for testing.

[0051] As can be seen from, e.g., Fig. 5, the dose-response curve of the methods and systems of the present invention may be non-linear: First, there

may be a steep increase of the clotting time at low heparin concentrations, leading to a flatter part of the curve. Thus, already small amounts of heparin are reliably detected due to the steep increase of the clotting time at low concentrations. This allows for a very low lower level of quantification, e.g., in the range of 0.2 U of unfractionated heparin/ml which is, for example much lower than the lower level of quantification possible with methods such as the ACT method, which is only sensitive to heparin concentrations higher than 0.5 U/ml. On the other hand the flatter extended clotting times at higher heparin concentrations (less steep dose-response curve) limits the time which is required for the determination of high heparin concentrations. If the dose-response curve was linear throughout the heparin concentration range, either very long clotting times would be required to detect high heparin dosages or a low sensitivity in the low heparin concentration range would ensue. For example, assuming a test method/system has a linear dose-response curve and that a prolongation of the clotting time for at least 20 seconds is required to reliably detect heparin activity (due to a certain variation in the clotting times of subjects without heparin treatment), then methods/systems with a steep dose-response curve will render a low level of anticoagulant sufficient to prolong the clotting time by 20 seconds. However a very steep dose-response relationship between the anticoagulant and the clotting time, that is, a very steep dose-response curve, will also lead to very long clotting times at higher anticoagulant concentrations. For example, if 0.1 LJ of heparin prolongs the clotting time by 10 seconds, than 10 U of heparin will prolong the clotting time by 1000 seconds. If, however, 0.1 U of heparin prolong the clotting time only by 2 seconds, then 10 U of heparin will prolong the clotting time only by 200 seconds, but 1 U of heparin will be required to detect the anticoagulant activity. The methods and systems according to the present invention that provide a steep dose-response curve at low dosages of the anticoagulant and a less steep dose-response curve at high dosages of anticoagulant allow for the quantification of both high and low dosages of anticoagulant at comparatively short detection times.

[0052] Prior to an operation in which an indirect thrombin inhibitor is to be used to avoid the patient's blood to clot, it may be desired to test the sensitivity of the

patient's coagulation system towards the action of the indirect thrombin inhibitor. Accordingly, in one embodiment of the present invention, rising amounts of heparin are added to a patient's blood sample in order to predict the sensitivity of the patient's coagulation system to heparin and thus to allow one to select an appropriate dosage of the indirect thrombin inhibitor.

[0053] Similarly, at the end of a procedure that required an indirect thrombin inhibitor, it is generally desirable to reverse the effect of the indirect thrombin inhibitor. Thus, in another embodiment of the present invention an analysis is performed in which rising amounts of antagonists of indirect thrombin inhibitors such as protamine are added in order to quantify the amount of antagonist required to reverse or effectively reverse the impact of the indirect thrombin inhibitor in the sample. In Fig. 7 the results of the analysis of 6 samples of UFH treated patients undergoing open heart surgery are shown, assayed with the addition of rising amounts of protamine. Depending on the amount of heparin present in the sample different amounts of protamine are required to antagonize the heparin. Using the method of the present invention the amount of protamine required for reversing the effect of heparin in the patient could be quantified within about 3 minutes. However, the present invention includes embodiments in which such an amount can be quantified within about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes or about 10 minutes. However, as the person skilled in the art will appreciate, these times vary widely depending, among others, on the indirect thrombin inhibitor and the respective antagonist used.

[0054] Sometimes it is desired to reduce the risk of thrombosis in a patient, while this patient is still healthy or prior to a low risk medical procedure that somewhat increases the risk of thrombosis. In such a case moderate amounts of the indirect thrombin inhibitor are sufficient to prevent the development of a thrombosis. Therefore a "prophylactic dose" of an indirect thrombin inhibitor refers to an amount/concentration of an indirect thrombin inhibitor which is sufficient to prevent the formation of a thrombosis, but is not sufficient to treat a

thrombosis which is already present. For the treatment of an existing thrombosis or for impeding thrombosis in situations where there is a particularly high risk of formation of a thrombosis, such as when blood is circulated in a cardiopulmonary bypass device, significantly higher dosages, namely "therapeutic doses," of the indirect thrombin inhibitor are generally administered. In the case of unfractionated heparin, 0.75 U or less of heparin/ ml is considered a prophylactic dose, while doses higher than 0.75 U of heparin/ ml are considered a therapeutic dose. However, as the person skilled in the art will appreciate the doses of indirect prothrombin inhibitor used for prophylactic and treatment purposes vary widely from one indirect prothrombin inhibitor to the next. However, the person skilled in the art will be able to readily ascertain the required doses for the indirect prothrombin inhibitor of interest. In certain embodiments of the invention, the methods and systems can be employed to measure amounts/concentrations of indirect thrombin inhibitor used in prophylaxis or in treatment. In a preferred embodiment of the invention, the method and systems are capable of measuring both, amounts/concentrations of indirect thrombin inhibitor used in prophylaxis ("prophylactic doses") as well as those used in treatment ("therapeutic doses").

[0055] In certain embodiments of the present invention time intervals between adding a prothrombin activator to a sample and the commencement of, e.g., clotting or thrombin activity as measured via indirect parameters such as color change due to thrombin's effect on a chromogenic thrombin substrate, are determined. Several direct and indirect parameters may also be measured over a certain time interval or at predetermined times subsequent to addition of prothrombin. The length of those intervals/the set predetermined times again vary from one indirect thrombin inhibitor to the next. However, such time intervals/predetermined times are generally less than about 600 seconds, preferably less than about 500 seconds, more preferably less than about 400 seconds or less than about 300 seconds, but usually more than about 30 seconds or about 60 seconds.

[0056] A wide variety of prothrombin activators can be used in the context of the present invention. In a preferred embodiment, a prothrombin activator is used

which is derived from a snake venom. Noscarin, oscuratin, textarin, pseυtarin, trocarin, notecarin and hopsarin are just a few of these snake venoms. In another embodiment, the prothrombin activator may be a "derivative of FX ₃ " as defined herein. This term includes peptides exhibiting at least about 30% identity with the corresponding naturally-occurring protein or a functional part thereof, usually peptides exhibiting at least about 70% identity, more usually at least about 80% identity, preferably at least about 90% identity, and more preferably at least about 95% identity which maintain their ability to convert prothrombin to a form of thrombin which is susceptible to inhibition via an indirect thrombin inhibitor. However, as the person skilled in the art will appreciate, any substance that converts prothrombin to a form of thrombin which is susceptible to inhibition via an indirect thrombin inhibitor can be adapted for use in the inventive method/system. Typically those substances can be identified by their ability to clot blood or plasma. For example, a substance ability to clot plasma deficient in FVII and / or FXII shows that it is not activating clotting via the pathways of contact activation or tissue factor activation. By using various concentrations of an identified prothrombin activator, the clotting times can then be adapted. A rising concentration of the prothrombin activator will typically lead to shortened clotting times for samples with and without an indirect thrombin inhibitor. Shortened clotting times will typically requires higher concentrations of indirect thrombin inhibitor for reliable quantification during a fixed detection period. On the other hand, lower concentration of the prothrombin activator will lead to longer clotting times for samples with and without an indirect thrombin inhibitor. Prolonged clotting times increases measuring sensitivities and thus differences between samples containing different amounts of indirect thrombin inhibitors. Using several samples containing the indirect thrombin inhibitor in concentrations within the desired measuring range generally allow one to adapt the sensitivity of the assay and the detection times.

[0057] Prothrombin activators can be added to the blood at different concentrations (units/ml). Typical concentrations for prothrombin activators derived, for example, from snake venoms are about 0.001 U/ml, about 0.0015 U/ml, about 0.002 U/ml, about 0.0025 U/ml, 0.003 U/ml, about 0.0035 U/ml,

about 0.004 U/ml, about 0.0045 U/ml, about 0.005 U/ml, about 0.0055 U/ml, about 0.006 U/ml, about 0.0065 U/ml, 0.007 U/ml, about 0.0075 U/ml, about 0.008 U/ml, about 0.0085 U/ml, about 0.009 U/ml, about 0.0095 U/ml, about 0.01 U/ml, about 0.015 U/ml, about 0.02 U/ml, about 0.025 U/ml, 0.03 U/ml, about 0.035 U/ml, about 0.04 U/ml, about 0.045 U/ml, about 0.05 U/ml, about 0.055 U/ml, about 0.06 U/ml, about 0.065 U/ml, 0.07 U/ml, about 0.075 U/ml, about 0.08 U/ml, about 0.085 U/ml, about 0.09 U/ml, about 0.095 U/ml, about 0.1 U/ml, about 0.15 U/ml, 0.2 U/ml, about 0.25 U/ml, about 0.3 U/ml, about 0.35 U/ml, about 0.4 U/ml, about 0.45 U/ml, about 0.5 U/ml, about 0.55 U/ml, 0.6 U/ml, about 0.65 U/ml, about 0.7 U/ml and about 0.75 U/ml. In many embodiments of the present invention, the prothrombin activator is part of a reagent that may be combined with an appropriate sample for analysis. Such a reagent might be a fluid reagent, in particular a liquid reagent, more in particular an aqueous reagent (also referred to herein as "reagent prepared in aqueous solution"). As will be discussed in more detail below, the reagent may also be a dry reagent, which contains one or more prothrombin activators and, optionally, other substances, in, e.g., dried or lyophilized form. In certain embodiments the volume of reagent, for example, an aqueous reagent and the volume of sample containing at least one blood component are about the same, for example, but not limited to, both the reagent and the sample are having a volume of about 20μl, about 40μl, about 80μl, about 160μl, about 320μl, about 640μl, about 1ml, about 2ml, about 5ml, about 10ml or 20ml. If the sample is whole blood, the sample amounts lie often between about 100μl and about 1ml, if the sample is plasma, the sample amounts lie often between about 50μl and 150μl. Using similar amounts of reagent/sample allows, e.g., for the use of the same pipette for pipetting blood and reagent and, in certain embodiments, may facilitate the mixing of the two solutions (see Example 4). In other embodiments, the volume of reagent, such as an aqueous reagent, containing one or more prothrombin activators is somewhat or substantially smaller than the sample volume. This is, for example, advantageous when whole blood clotting kinetics are assessed via thromboelastography, thromboelastometry or other whole blood methods that are used to measure coagulation of a patient's blood during surgical procedures. As the person skilled in the art will appreciate, the desirable relative volumes of

reagent to sample will vary widely depending on the application but includes for example relative volumes of reagent to sample of about 1:15, about 1:10, about 1:5, about 1:2, preferably about 1:1 for the assessment of plasma (to minimize sample volume) and similar relative volumes for other sample types including whole blood, preferably about 1 :15 for the assessment of whole blood (in order to determine the appropriate whole blood kinetics without disturbing clot formation by high dilution). As the person skilled in the art will also appreciate, embodiments in which the relative volumes of reagent to sample are reversed are also within the scope of the present invention.

[0058] When used in combination, one prothrombin activator might be present at much lower concentrations than the other prothrombin activator. For example, noscarin might be present in a concentration of 0.005 to 0.002 U/ml and textarin might be present and a concentration of 0.25 to 0.30. However, as the person skilled in the art will appreciate, a wide variety of combinations of concentrations of prothrombin activators is possible, which will dependent on the type of prothrombin activator and the purpose for which it is to be used. In one embodiment of the present invention combinations of different prothrombin activators can be used to improve and/or optimize the kinetics for indirect thrombin inhibitor, in particular heparin, monitoring. For example, using various combinations of the two snake venoms textarin and noscarin can increase the sensitivity of the assay as well as allows one to adjust the time required for the testing. In preferred embodiments, at least about 10 seconds, preferably about 10 seconds elapse between the mixing of the sample and reagent until clotting is initiated in a sample containing no indirect thrombin inhibitor. This time period allows for the mixing of sample and reagent to be completed and optical or mechanical variables of the mixture to equilibrate before the clotting is detected. However, extended time periods prior to the detection of clotting generally leads, in mixtures comprising indirect thrombin inhibitors at higher concentrations, to long turn-around times (that are, times until results are available) and also limits the throughput of the analyzer (i.e. the amount of samples that can be tested during a certain time period, e.g., 1 hour). Thus, the time period prior to clotting is usually limited so that testing times to not exceed about 1000 seconds,

preferably not exceed 300 seconds and most preferably not exceed 200 seconds. In certain embodiments, calcium is added. In other embodiments, phospholipids are added to facilitate the formation of coagulation factor complexes and to promote the activity of prothrombin activators such as textarin. Other snake venoms, such as RW-V, that activate auxiliary factors of the prothrombin activators derived from snake venom may also be added. For example, RW-V activates factor V which enhances the activity of noscarin. Which, if any, of the above substances will be added will, at least in part, depend on the system used for analysis and/or whether, e.g., whole blood samples or plasma samples are used. For example, when whole blood samples are analyzed in, e.g., a ROTEM-system, as described, e.g., in United States Patent 5,777,215 and which will be discussed in more detail later on, the addition of phospholipids is not preferred. In fact, additional prospholipids are preferably avoided. If, however, a plasma sample is used, the addition of phospholipids may be advantageous since plasma itself is devoid of phospholipids that could make up surfaces for prothrombin activators. In certain embodiments, such additional substances are not part of the initial reagent containing the prothrombin activator, but are only mixed with this reagent at a later point in time. For example, in certain embodiments using dry reagent, any of these additional substances may be applied to, e.g., a test strip, so that the prothrombin activator containing reagent and, e.g., the phospholipids, are only mixed upon contact with a liquid sample. In certain embodiments, plasma proteins such as additional prothrombin and/or antithrombin and/or FV and/or fibrinogen may be added, preferably to the reagent containing the prothrombin activator, in order to standardize the amount of these proteins in methods and systems of the present invention and thus to render measurements of the amount/concentration of indirect thrombin inhibitor present in the sample more specific towards the amount of indirect thrombin inhibitor tested. In certain embodiments, prothrombin is preferably added to the reagent either at the time or shortly before the reagent is added to the sample. Alternatively, prothrombin may be provided as part of the reagent but in a form that is only susceptible to the action of the prothrombin activator upon addition to the sample. Standardization of the amount of these proteins entails that the measurement will be more accurate,

since the presence of plasma proteins in the reagent generally compensates for any deficiency or superabundance of plasma protein in the patient sample tested. For example, an amount of 0.1-5 U of prothrombin, FV and/or antithrombin may be added/ml of plasma sample tested. An amount 0.1-1 mg/ml fibrinogen may be added/ml of plasma sample tested. It is evident to the ones skilled in the art that any plasma protein which participates in a reaction forming a part of the methods and systems of the invention can be purified from human and/or non-human animal plasma and added to the sample and/or reagent.

[0059] As mentioned above, low and high concentrations of indirect prothrombin inhibitor can be determined and/or quantified via the methods and systems of the present invention. What constitutes a low or high concentration of an indirect prothrombin inhibitor depends on the nature of the inhibitor. However, about 0.8 U unfractionated heparin/ml; about 0.75 U/ml; about 0.7 U/ml; about 0.65 U/ml; about 0.6 U/ml; about 0.55 U/ml; about 0.5 U/ml; about 0.45 U/ml; about 0.4 U/ml; about 0.35 U/ml; about 0.3 U/ml; about 0.25 U/ml; about 0.2 U/ml and less are considered low doses of unfractionated heparin. Concentrations in excess of about 2.0 U unfractionated heparin/ml; about 2.5 U/ml; about 3.0 U/ml; about 3.5 U/ml; about 4.0 U/ml; 4.5 U/ml; about 5.0 U/ml; about 5.5 U/ml; about 6.0 U/ml; about 6.5 U/ml; about 7.0 U/ml; about 7.5 U/ml; about 8.0 U/ml; 8.5 U/ml; about 9.0 U/ml; about 9.5 U/ml; about 10.0 U/ml are considered moderate and high concentrations of unfractionated heparin. Thus, assays can be designed which can quantify low and/or high concentrations of heparin.

[0060] In certain embodiments of the invention, the method can be easily standardized by using highly purified enzymes for the activation of prothrombin.

Thus, in certain embodiments only very low concentrations such as about 0,005 U/ml of prothrombin activators are required, in particular when purified, highly active enzymes are used. A low amount of activator is easier to dry on the test strips or cartridges of point of care analysers and is more easily dissolved within the sample during the assay, therefore producing more accurate and reproducible results. Thus, the concentrations of prothrombin activator employed

will depend at least partially on the system used for analysis. In two preferred of such systems, namely the clinical coagulation analyzer Amelung KC4-MICRO and whole blood analyzers based on the ROTEM (Rotational Thromboelastogram) principal (United States Patent 5,777,215; R. J. LUDDINGTON; Review: Thrombelastography/thromboelastometry, Clinical & Laboratory Haematology 27(2), pp. 81 (April 2005); http://www.anaesthesiauk. com/article.aspx?articleid=100102 (as of December 21 , 2005); Calatzis et al., A new technique for fast and specific coagulation monitoring, European Surgical Research 28:S1 (89), 1996), typical amounts used are, e.g., 0.005 U/ml noscarin and 0.3U/ml texarin for KC4-MICRO and up to 40U/ml noscarin and 10U/ml texarin for the ROTEM-system.

[0061] As the person skilled in the art will appreciate, the method of the present invention can be performed using fluid, in particular, liquid or dry reagent including lyophilized reagent comprising the prothrombin activator(s) and, optionally, any other substances that might be added to, e.g., facilitate the processing of the prothrombin in a sample via the prothrombin activator of the present invention. Often the use dry reagent is advantageous since its components, such as the prothrombin activator(s), have a better stability in dry form. In certain embodiments in which dry reagent are used, the reagent can be provided ready to use for analysis, that is, without the need for any reagent preparation. Dry reagent may be applied to test strips or cartridges for near- patient testing. A sample, such as a blood sample, may be added to such a test strip or cartridge. The reagent is reconstituted by the blood sample and the reaction starts immediately or almost immediately. Such a set up allows for the detection and quantification of indirect thrombin inhibitors, in particular heparin, by persons without any laboratory skills and permits for short turnaround times for sample testing.

[0062] Thrombin activity and/or activation can be measured using different parameters such as, but not limited to, the amount of clotting, which is often assessed by the change of viscosity or elasticity of the sample. Other parameters

include the change of optical density or turbidity of the sample. Indirect parameters include thrombin induced changes of thrombin substrates such as a fluorigenic or amperogenic substrates or changes to the reading of an electrode that can detect thrombin activity and/or activation.

[0063] Material and Methods [0064] Experiment 1 :

[0065] Normal plasma was spiked with rising amounts of heparin. The prothrombin activator textarin derived from snake venom was used to activate the prothrombin in the sample. A concentration of 0.3 U/ml plasma of textarin was used for the experiment, A volume of 75 μl of plasma and 75 μl of textarin solution were employed. Clotting was detected by means of a KC4™ coagulometer produced by TRINITY BIOTECH. As seen in Fig. 3 increasing amounts of heparin resulted in an almost linear rise in the clotting time. Here up to 3 U of heparin/ml could be detected within 400 seconds.

[0066] Experiment 2:

[0067] The prothrombin activator noscarin was used, also at concentrations of 0.3 U/ml of blood. The action of noscarin is dependent on the availability of activated FV. Therefore the FV activator, RW-V ₁ was added in a concentration of 5 U/ml blood. Again, normal plasma was spiked with rising heparin concentrations and tested as described in Example 1. Fig. 4 shows that the quantification of more than 5 U heparin/ml within 500 seconds. However, Fig. 4 also shows that the sensitivity towards concentrations lower than 3 U/ml was relatively low, as indicated by the short clotting times.

[0068] Experiment 3:

[0069] The prothrombin activators textarin and noscarin were combined in varying concentrations. In particular, 0.005-0.002 U of noscarin/ml was combined with 0.25-0.3 U of textarin/ml. In addition phospholipids where added in a concentration of 50 micro-g/ml, together with the addition of the snake venom RW-V. RW-V is a specific activator of the platelet factor V, which enhances the activity of noscarin. Finally, an addition of 25 mM of CaCl2 optimized the activity

of the formed thrombin. The addition of phospholipids facilitates the formation of coagulation factor complexes and promotes the activity of textarin. Fig. 5 shows the results of the experiment in a plasma sample. Depending on the amount of noscarin or textarin used, the shape of the dose-response curve changed. A higher amount of noscarin, lead to a flatter dose response curve at high heparin concentrations and therefore enhanced the measuring range of the assay. Fig. 5 also shows that the dose response curves were in all instances steep at low heparin concentration. Thus, already relatively small amounts of heparin could be reliably detected. Thus, quantification at ranges around 0.2 U unfractionated heparin/ml was possible.

[0070] Experiment 4:

[0071] In this experiment two reagents were prepared in aqueous solution:

Reagent 1 : 0,025U/ml Noscarin in 25mM CaCI ₂ / 2% HEPES-buffer (pH7.4)

Reagent 2: 0,3U/ml Textarin + 50μg/ml PL + 10U/ml RW-V in 2% HEPES-buffer

(pH7.4)

[0072] The reagents were pipetted into a cuvette (50μl each) of a coagulometer

(e.g. KC4™ by TRINITY BIOTECH) and the measurement were started without any further incubation by adding 50μl sample (plasma).

[0073] A coefficient of variation of 3-5% was obtained in repeated measurements of samples spiked with rising amounts of heparin as depicted in

Fig. 6.

[0074] Similarly precise results were obtained when the testing was performed with fresh plasma from healthy donors or patients.

[0075] Experiment 5:

[0076] Two reagents are prepared in aqueous solution: Reagent 1 : 0,216U/ml Noscarin in 141mM CaCI ₂ / 2% HEPES-buffer (pH7.4) Reagent 2: 6,4U/ml Textarin + 283μg/ml PL + 57U/ml RW-V in 2% HEPES- buffer (pH7.4)

[0077] The reagents were pipetted into the test cell (20μl each) and the measurement was started without any further incubation by adding 300μl sample (plasma or whole blood).

[0078] Experiment 6:

[0079] A single reagent was prepared in aqueous solution:

Reagent: Noscarin 80 U/ml + Textarin 480 U/ml + RW-V 240 U/ml + CaCI ₂

0.06mmol/ml in HEPES 5OmM 0.5% BSA pH 7.4

[0080] The reagent was pipetted into the test cell (20μl) of a ROTEM-system and the measurement was started without any further incubation by adding 300μl whole blood (sample), resulting in the following concentrations of the reagent components in the sample mixture: noscarin 5.3U/ml sample mixture, textarin

32U/ml sample mixture, RW-V 16U/ml sample mixture, Calcium 0.06mmol/ml sample mixture. The reagent is stable at room temperature (Fig. 10). Different concentrations of unfractionated heparin (UFH) could be detected in an appropriate whole blood sample (Fig. 9).

[0081] It will be appreciated that the methods and systems of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.