WO2005059170A2

WO2005059170A2 - Method for the determination of fibrinolytic activity in blood

Info

Publication number: WO2005059170A2
Application number: PCT/GB2004/005249
Authority: WO
Inventors: Viveca Nerme; Karin Wahlander; Petter Bjorquist
Original assignee: Astrazeneca Ab; Astrazeneca Uk Limited
Priority date: 2003-12-17
Filing date: 2004-12-14
Publication date: 2005-06-30
Also published as: SE0303417D0; WO2005059170A3

Abstract

The present invention relates to an assay method for measuring the effect of an agent on fibrinolysis comprising the steps of: (a) providing a test sample comprising: (i) a fibrinolysis inhibitor and/or a zymogen of a fibrinolysis inhibitor; and (ii) an agent; (b) activating the coagulation system; and (c) measuring the amount of fibrin degradation products that are formed; wherein the difference between (i) the amount of fibrin degradation products formed in the presence of the agent and (ii) the amount of fibrin degradation products formed in the absence of the agent is indicative of the effect of said agent on fibrinolysis.

Description

SCREEN

FIELD OF INVENTION

The present invention relates to the field of biology and the coagulation and fibrinolysis of blood.

In particular, the present invention relates ter alia to a method for measuring the effect of an agent - such as a CPU inhibitor - on fibrinolysis.

BACKGROUND TO THE INVENTION

Coagulation and fibrinolysis are physiological pathways, which are involved in maintaining normal blood haemostasis in mammals.

The balance between the coagulation and the fibrinolytic pathways is essential for protection against excessive blood loss and excessive fibrin build-up in the vascular system, which would prevent normal blood flow.

The coagulation pathway comprises a series of enzymatic activations, which result in the formation of a fibrin clot.

Fibrinolysis is the result of a series of enzymatic reactions resulting in the degradation of fibrin by plasmin. The activation of plasminogen is the central process in fibrinolysis. The cleavage of plasminogen to produce plasmin is accomplished by the plasminogen activators, tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Initial plasmin degradation of fibrin generates carboxy-terminal lysine residues that serve as high affinity binding sites for plasminogen and t-PA. Since plasminogen bound to fibrin is much more readily activated to plasmin than free plasminogen this mechanism provides a positive feedback regulation of fibrinolysis.

One of the endogenous inhibitors to fibrinolysis is carboxypeptidase U (CPU). CPU is also known as plasma carboxypeptidase B, active thrombin activatable fibrinolysis inhibitor

(TAFIa), carboxypeptidase R or inducible carboxypeptidase activity.

CPU is formed during coagulation and fibrinolysis from its precursor proCPU by the action of proteolytic enzymes, such as thrombin, thrombin-thrombomodulin complex or plasmin. CPU cleaves basic amino acids at the carboxy-terminal of fibrin fragments. The loss of carboxy- terminal lysines and thereby of lysine binding sites for plasminogen and t-PA then serves to downregulate fibrinolysis. By inhibiting the loss of lysine binding sites for plasminogen and thus increase the rate of plasmin formation, effective inhibitors of carboxypeptidase U are expected to facilitate fibrinolysis. One assay to measure fibrinolytic activity has been reported in Fibrinolysis & Proteolysis (1999) 13, Suppl. 1, 1-10. This assay is reported to measure the global fibrinolytic capacity of a sample. A standardised fibrin clot is used containing silica to induce contact activation of the fibrinolytic system, in the presence of a small amount of t-PA. Lysis is quantified by measuring the amount of D-dimer that is formed. However, this method cannot be used to assay for inhibitors of CPU. Furthermore, this assay does not reflect individual variations in the coagulation cascade in different test samples.

There is a need in the art to study the effect of agents that stimulate Fibrinolysis, and one that takes into account interpatient variability of Fibrinolysis. Such agents may be used to further study the coagulation and fibrinolytic pathways and also to treat disorders associated with these pathways. SUMMARY ASPECTS OF THE PRESENT INVENTION

As described herein, the present invention relates inter alia to a simple and fast method for measuring the effect of an agent on fibrinolysis e.g., the fibrinolytic capacity, of a test sample. The method of the present invention relies in part on the use of an entity - such as calcium ions - for the activation of coagulation in a test sample and then measuring the amount of fibrin degradation products that are formed.

In a first aspect, the present invention relates to a method for measuring the effect of an agent on fibrinolysis comprising the steps of: (a) providing a test sample comprising: (i) a fibrinolysis inhibitor and/or a zymogen of a fibrinolysis inhibitor; and (ii) an agent; (b) activating the coagulation system; and (c) measuring the amount of fibrin degradation products that are formed; wherein a difference between (i) the amount of fibrin degradation products formed in the presence of the agent and (ii) the amount of fibrin degradation products formed in the absence of the agent is indicative of the effect of said agent on fibrinolysis.

Since each individual test sample contain a unique composition of coagulation components, each individual test sample will respond to an activation of coagulation, for example by the addition of calcium ions, in a unique way. Furthermore, since each individual test sample contains a unique composition of fibrinolytic components, each individual coagulated test sample will respond with a unique fibrinolytic response. The present invention provides a method that takes this into account. Each test subject serves as its own control when an individual test sample is assayed before and after the addition of an agent that modulates fibrinolysis. Advantageously, an optimal dose of said agent can be found for each test subject. Thus, the method of the present invention may be used for measuring the effect of an agent on fibrinolysis.

In a second aspect, the present invention relates to a method for measuring the effect of an activity on fibrinolysis in a subject comprising the steps of: (a) providing a test sample comprising a fibrinolysis inhibitor and/or a zymogen of a fibrinolysis inhibitor from the subject; (b) activating the coagulation system in the test sample; and (c) measuring amount of fibrin degradation products that are formed; wherein a difference between (i) the amount of fibrin degradation products formed before the activity and (ii) the amount of fibrin degradation products formed after the activity is indicative of the effect of the activity on fibrinolysis.

In a third aspect, the present invention relates to an assay method for identifying an agent that modulates clot lysis and/or fibrinolysis comprising the steps of: (a) providing a test sample; (b) contacting said test sample with an agent; (c) activating the coagulation system in the test sample; and (d) measuring the amount of fibrin degradation products that are formed by measuring the amount of D-dimer or fragment D of fibrin; wherein a difference between (i) the amount of fibrin degradation products formed in the presence of the agent and (ii) the amount of fibrin degradation products formed in the absence of the agent is indicative of an agent that modulates clot lysis and/or fibrinolysis. In one embodiment, the clot lysis reaction activated by step (a) is stopped before the amount of fibrin degradation products are measured.

In a fourth aspect, the present invention relates to an assay method comprising the steps of: (a) performing the assay method according to the third aspect of the present invention; (b) identifying one or more agents capable of modulating fibrinolysis; and (c) preparing a quantity of those one or more identified agents. In a fifth aspect, the present invention relates to a method comprising the steps of: (a) performing the assay method according to the third aspect of the present invention; (b) identifying one or more agents capable of modulating fibrinolysis; and (c) preparing a pharmaceutical composition comprising those one or more identified agents. In a sixth aspect, the present invention relates to an agent identified by the assay method according to the fourth aspect of the present invention.

In a seventh aspect, the present invention relates to a pharmaceutical composition prepared by the method of the fifth aspect of the present invention. In an eighth aspect, the present invention relates to a method of preventing and/or treating a disease comprising administering an agent according to the sixth aspect of the present invention and/or a pharmaceutical composition according to the seventh aspect of the present invention wherein said agent or pharmaceutical composition is capable of modulating fibrinolysis.

In a ninth aspect, the present invention relates to an agent according to the sixth aspect of the present invention and/or a pharmaceutical composition according to the seventh aspect of the present invention for use in modulating fibrinolysis for the prevention and/or treatment of a disease. In a tenth aspect, the present invention relates to the use of an agent according to the sixth aspect of the present invention and/or a pharmaceutical composition according to the seventh aspect of the present invention in the manufacture of a medicament for the prevention and/or treatment of a disease.

In an eleventh aspect, the present invention relates to a kit for performing the assay method according to the first aspect of the present invention, or the assay method according to the second aspect of the present invention comprising: a first vessel which comprises a component that stops clot lysis; a second vessel containing a plasminogen activator; and a third vessel containing an entity that activates the coagulation system.

Other aspects of the present invention are presented in the accompanying claims and in the following description and discussion. These aspects are presented under separate section headings. However, it is to be understood that the teachings under each section heading are not necessarily limited to that particular section heading.

PREFERRED EMBODIMENTS

Preferably, the coagulation system is activated (e.g. clot formation reaction started) using calcium ions and/or thrombin, or tissue factor. The person skilled in the art would be aware of the various ways of activating the coagulation system.

Preferably, the coagulation system is activated using calcium chloride and/or thrombin, or tissue factor.

Preferably, the fibrinolysis inhibitor is selected from the group consisting of: carboxypeptidase U (CPU), plasminogen activator inhibitor- 1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2) or alpha-2-antiplasmin.

Preferably, the test sample is/or comprises blood or a constituent thereof.

Preferably, the test sample is/or comprises plasma. Preferably, the amount of fibrin degradation products that are formed are measured by measuring the amount of D-dimer or fragment D of fibrin. The terms "D-dimer" and "fragment D of fibrin" are used interchangeably herein.

Preferably, the amount of D-dimer or fragment D of fibrin formed is measured using a D- dimer test selected from Biopool Auto-Dimer™ test, the TintElize® D-dimer test, the Accuclot™ D-dimer test, the Auto D-Dimer™ test, the Auto-Dimer™ test, the Minutex® D- dimer test, the NovoCard® D-dimer test, the MiniQuant™ D-dimer test or the Asserachrom® D-DI test.

Preferably, the activity is exercise, feeding, eating, mental stress or surgery. Preferably, the component that stops clot lysis is a plasmin inhibitor. More preferably, the component that stops clot lysis is aprotinin, D-Val-Phe-Lys chloromethyl ketone and/or alpha-2-antiplasmin.

Preferably, the kit contains an anti-heparin agent. ADVANTAGES

The present invention has a number of advantages. These advantages will be apparent in the following description.

By way of example, the present invention is advantageous since it provides a commercially useful method. By way of example, the present invention is advantageous since it provides a simple and fast method for measuring the effect of an agent on fibrinolysis.

By way of example, the present invention is advantageous since it provides a simple and fast method for measuring the effect of an activity on fibrinolysis.

By way of example, the present invention is advantageous since it provides a simple and fast assay method for identifying one or more agents that modulate clot lysis and/or fibrinolysis. Since each individual test sample contain a unique composition of coagulation components, each individual test sample will respond to an activation of coagulation, for example by the addition of calcium ions, in a unique way. Furthermore, since each individual test sample contains a unique composition of fibrinolytic components, each individual coagulated test sample will respond with a unique fibrinolytic response. The present invention provides a method that takes this into account. Each test subject serves as its own control when an individual test sample is assayed before and after the addition of an agent that modulates fibrinolysis. Advantageously, an optimal dose of said agent can be found for each test subject. Thus, the method of the present invention may be used for measuring the effect of an agent on fibrinolysis.

By way of further example, the present invention is advantageous since it provides a simple and fast method for measuring the fibrinolytic capacity in samples containing heparin by adding an anti-heparin agent - such as polybrene®.

DESCRIPTION OF THE FIGURES

Figure 1 is a bar graph illustrating the results of clot lysis in pooled human plasma using three different agents.

Figure 2 is a bar graph illustrating the results of clot lysis in pooled human plasma using three different agents, shown as a percentage of the control.

DETAILED DESCRIPTION OF THE INVENTION

"Fibrinolysis" refers, as it is referred to in the art, as the process of the dissolution of fibrin in blood clots resulting from the proteolytic action of plasmin or other enzymes.

As used herein, the term "fibrinolysis inhibitor" includes any endogenous inhibitor of fibrinolysis.

CPU is also known as active thrombin-activatable fibrinolysis inhibitor (TAFIa), plasma carboxypeptidase B, or carboxypeptidase R. Thus, for the avoidance of doubt, any reference to "CPU" also encompasses (TAFIa), plasma carboxypeptidase B and carboxypeptidase R.

CPU has been reviewed in J. Thromb. Haemost. (2003) 1, 1566-74 and Current Drug Targets

— Cardiovascular & Haematological Disorders (2001) 1, 59-74.

Fibrinolysis is the result of a series of enzymatic reactions resulting in the degradation of fibrin by plasmin. The activation of plasminogen is the central process in fibrinolysis. The cleavage of plasminogen to produce plasmin is accomplished by the plasminogen activators, tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA).

Initial plasmin degradation of fibrin generates carboxy-terminal lysine residues that serve as high affinity binding sites for plasminogen and t-PA. Since plasminogen bound to fibrin is much more readily activated to plasmin than free plasminogen this mechanism provides a positive feedback regulation of fibrinolysis.

CPU is formed during coagulation and fibrinolysis from its precursor proCPU by the action of proteolytic enzymes, such as thrombin, thrombin-thrombomodulin complex or plasmin. CPU cleaves basic amino acids at the carboxy-terminal of fibrin fragments. The loss of carboxy- terminal lysines and thereby of lysine binding sites for plasminogen then serves to downregulate fibrinolysis.

ProCPU contains 401 amino acids corresponding to a predicated peptide molecular mass of 45,999 Da (J. Biol. Chem (1991) 266, 21833-21838) and it is suggested that this protein is glycosylated because it runs on an SDS-PAGE gel with a molecular mass of about 60,000 Da. The gene encoding CPU has been given the name CPB2. CPB2 has been cloned and characterised (Biochemistry (1999) 38, 6547-6558) and is available in databases under Accession numbers AF080222 and AF080223. Thus, in a preferred embodiment, the present invention relates to a method for measuring the effect of a CPU inhibitor on clot lysis and/or fibrinolysis comprising the steps of: (a) providing a test sample comprising CPU and/or a zymogen of CPU which comprises or is contacted with the agent; (b) activating the coagulation system; and (c) measuring the amount of fibrin degradation products that are formed; wherein a difference between (i) the amount of fibrin degradation products formed in the presence of the CPU inhibitor and (ii) the amount of fibrin degradation products formed in the absence of the CPU inhibitor is indicative of the effect of said agent on fibrinolysis.

Suitably, the present invention may be therefore used to assay a test sample - such as human plasma - taken from a subject that has received or is receiving a CPU inhibitor. The effect that a given dose of the CPU inhibitor has on the lysis of a clot formed in the assay may then be determined. Thus, according to one aspect, the invention relates to a bioassay method for measuring the effect of a clot lysis agent in a patient by administering the agent to a patient and after a suitable period of time extracting blood from the patient, optionally extracting the plasma from the blood, activating the coagulation stystem and measuring the amount of fibrin degradation products. Such a method allows measurement of the efficacy of the agent within an individual patient.

In another preferred embodiment, the present invention relates to a method for measuring the effect of an activity on fibrinolysis in a subject comprising the steps of: (a) providing a test sample comprising CPU and/or a zymogen of CPU from the subject; (b) activating the coagulation system in the test sample; and (c) measuring amount of fibrin degradation products that are formed; wherein a difference between (i) the amount of fibrin degradation products formed before the activity and (ii) the amount of fibrin degradation products formed after the activity is indicative of the effect of the activity on fibrinolysis. Suitably, the present invention may be therefore used to assay a test sample - such as human plasma - taken from a subject that has undergone or is undergoing a particular activity. The effect of the activity on the lysis of a clot formed in the assay may then be determined. In another preferred embodiment, the present invention relates to an assay method for 5 identifying a CPU inhibitor that stimulates clot lysis and/or fibrinolysis comprising the steps of: (a) providing a test sample comprising CPU and/or a zymogen of CPU; (b) contacting said test sample with an agent; (c) activating the coagulation system in the test sample; and (d) measuring the amount of fibrin degradation products that are formed by measuring the amount of D-dimer or fragment D of fibrin; wherein a difference between (i) the amount of

10 fibrin degradation products formed in the presence of the agent and (ii) the amount of fibrin degradation products formed in the absence of the agent is indicative of a CPU inhibitor that stimulates clot lysis and/or fibrinolysis. Plasminogen activator inhibitor-1 (PAI-1) is a major regulatory component of the plasminogen-plasmin system and is the principal physiologic inhibitor of both tissue-type

15 plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). It is a single chain glycoprotein with a molecular weight of 50 kDa (Van Mourik J A et al., J Biol Chem (1984) 259: 14914-14921) and is the most efficient inhibitor known of the single- and two- chain forms of tPA and of uPA (Table 1) (Lawrence D et al., Eur J Biochem (1989) 186:523- 533). PAI-1 also inhibits plasmin and trypsin (Hekman C M et al., Biochemistry (1988)

20 27:2911-2918) and also inhibits thrombin and activated protein C, though with much lower efficiency. PAI-1 cDNA encodes a protein of 402 amino acids that includes a typical secretion signal sequence (Ny et al., supra; Ginsburg et al., 1986, supra). Mature human PAI-1 isolated from cell culture is composed of two variants of 381 and 379 amino acids in approximately equal

25 proportions. PAI-1 is a glycoprotein with three potential N-linked glycosylation sites containing between 15 and 20% carbohydrate (Van Mourik J A et al., supra). Alpha-2-antiplasmin is believed to be the primary inhibitor of plasmin in the blood (Aoki et al., J Clin. Invest. 60: 361, 1977; and Collen and Wiman, Blood 51: 563-569, 1978). The reaction between alpha-2-antiplasmin and plasmin occurs in two steps, with the first being a

30 rapid, reversible interaction and the second being a slower intra-molecular rearrangement that results in the formation of a covalent bond between alpha-2-antiplasmin and plasmin. At clotting sites, alpha-2-antiplasmin becomes covalently attached to fibrin via activity of the same enzyme, factor XIII, that crosslinks fibrin. The term "test sample" as used herein, has its natural meaning.

The sample may be or may be derived from a mammal.

Preferably, the test sample is or is derived from an animal or a human. Most preferably, the test sample is or is derived from a human. The sample may be or may be derived from biological material.

The test sample may be or may be derived from blood or a component thereof - for example, plasma.

In a preferred embodiment, the test sample is fresh plasma.

If the test sample is in the form of blood then it may be prepared using the following method. Blood is collected on citrate and plasma decanted after centrifugation. It is then aliquoted into vials, deep-frozen and thawed just before analysis. Alternatively, fresh plasma can be used.

As described herein, the test sample may comprise one or more agents. Thus, for example, the test sample may be plasma prepared from blood taken from a subject that has received an agent that modulates - such as stimulates - fibrinolysis. In this embodiment, the assay serves as a biomarker of the effects of an agent on blood clot inhibition in a patient (subject). This may be useful in a clinical trial setting or to monitor a patient under therapy.

Alternatively, if the test sample does not already contain such an agent, then one or more agents may be contacted with the test sample.

The test sample comprises an endogenous fibrinolysis inhibitor. In accordance with the present invention, the coagulation system is activated in the test sample and a fibrin clot is formed. The fibrin clot is then dissolved by the fibrinolytic system, such that the D-dimer or fragment D of fibrin measurement reflects the fibrinolytic capacity of the test sample.

Suitably, the activation of the coagulation system may also lead to the activation of an inactive fibrinolysis inhibitor - such as pro-CPU - to form an active enzyme. Since an active fibrinolysis inhibitor is formed, it is possible to study, for example, the effects of inhibitors of fibrinolysis inhibitors - such as CPU - in a test sample.

In a preferred embodiment the coagulation system is activated using calcium ions and/or thrombin, or tissue factor. More preferably, the coagulation system is activated using calcium ions - such as calcium chloride - and/or thrombin. Most preferably, the coagulation system is activated using calcium chloride.

Typically, an agent will be added at the same time or before activating coagulation. The degree (e.g., the amount) of clot lysis may be measured using various methods that are known in the art.

Preferably, the degree of clot lysis is measured by measuring the amount of fibrin degradation products that are formed in the test sample. In this embodiment of the invention, fibrinolysis is therefore measured by measuring the amount of fibrin degradation products that are formed in the test sample.

More preferably, the amount of fibrin degradation products that is formed in the test sample is determined by measuring the D-dimer or fragment D derived from the degradation of cross- linked fibrin by plasmin. In this embodiment of the invention, fibrinolysis is therefore measured by measuring the amount of D-dimer or fragment D derived from the degradation of cross-linked fibrin by plasmin in the test sample.

The amount of D-dimer formed at a given time reflects the fibrinolytic capacity of the test sample.

More preferably, the amount of D-dimer or fragment D of fibrin formed is measured using the Biopool Auto-Dimer™ test, the TintElize® D-dimer test, the Accuclot™ D-dimer test, the

Auto D-Dimer™ test, the Auto-Dimer™ test, the Minutex® D-dimer test, the NovoCard® D- dimer test, the MiniQuant™ D-dimer test or the Asserachrom® D-DI test.

Most preferably, the amount of D-dimer or fragment D of fibrin formed is measured using the

Biopool Auto-Dimer™ test or the Asserachrom® D-DI test. METHOD

In accordance with the present invention, the methods and assay methods of the invention may be performed as follows.

To a vial containing a plasminogen activator, Start reagent and human plasma is added.

Alternatively, the plasminogen activator may be contained in the Start reagent. Preferably, the Start reagent comprises or contains a solution of calcium ions - such as CaCl2.

The Start reagent may also contain a plasminogen activator.

Optionally, the Start reagent may contain polybrene® and/or Tween-80.

As used herein, the term "plasminogen activator" has its conventional meaning as used in the art i.e., any serine protease that converts plasminogen into plasmin. Examples of suitable plasminogen activators include, but are not limited to, t-PA, u-PA, streptokinase and bat plasminogen activator.

In a preferred embodiment, the plasminogen activator is tissue plasminogen activator - such as t-PA - preferably, single chain tPA (sct-PA). Preferably, the plasminogen activator is a recombinant protein - such as recombinant sct-PA Typically, the Start reagent will be an isotonic solution with a pH of about 7.4.Preferably, the assay concentration of CaCl2 is 1-100 mmol/L. More preferably, the assay concentration of CaCl2 is 5-50 mmol/L. Most preferably, the assay concentration of CaCl2 is about 15 mmol/L.

Preferably, the assay concentration of sct-PA is 0.1-3 nM. More preferably, the assay concentration of sct-PA is 0.5-1 nM. Most preferably, the assay concentration of sct-PA is about 0.7 nM. Optionally, the Start reagent may contain a polyoxyethylenesorbitan, such as Tween-80. Preferably, the assay concentration of Tween-80 is about 0.001-0.01%. Most preferably, the assay concentration of Tween-80 is about 0.005%.

Optionally, the Start reagent may contain an anti-heparin agent - such as polybrene®. Preferably, the assay concentration of polybrene® is 0.0001-0.1 mg/mL. More preferably, the assay concentration of polybrene® is 0.001-0.01 mg/mL. Most preferably, the assay concentration of polybrene® is about 0.005 mg/mL.

The mixture is then incubated for about 5 minutes-24 hours at about 37 °C and incubation is stopped by adding Stop reagent. Preferably, the mixture is incubated for 15-120 min at 37 °C and incubation is stopped by adding Stop reagent. More preferably, the mixture is incubated for 30-50 minutes at about 37 °C and incubation is stopped by adding Stop reagent. Optionally, the temperature can be 4-50°C.

Typically, the Stop reagent will be an isotonic solution with a pH of about 7.4.

Suitably, the Stop reagent comprises a component that stops clot lysis - such as aprotinin, D-

Val-Phe-Lys chloromethyl ketone and/or alpha-2-antiplasmin.

Preferably, the assay concentration of aprotinin is 10-3000 KIU/mL. More preferably, the assay concentration of aprotinin is 50-1000 KIU/mL. Most preferably, the assay concentration of aprotinin is about 200 KIU/mL.

The vial is then centrifuged at, for example, 2000 x g for 10 min at 4 °C, and the supernatant is withdrawn. Alternatively, a sample is withdrawn without performing the centrifugation step. D-dimer and/or fragment D may be measured in the supernatant using a D-dimer test - such as the Biopool Auto-Dimer™ test, the TintElize® D-dimer test, the Accuclot™ D-dimer test, the Auto D-Dimer™ test, the Auto-Dimer™ test, the Minutex® D-dimer test, the NovoCard® D- dimer test, the MiniQuant™ D-dimer test or the Asserachrom® D-DI test. Most preferably, the amount of D-dimer or fragment D of fibrin formed is measured using the Biopool Auto- Dimer™ test or the Asserachrom® D-DI test.

If an agent - such as a CPU inhibitor - is used in the method, then the agent may be added at the start of the assay with the Start reagent. Alternatively, the agent may already be contained in the test sample.

Typically, each agent concentration will be tested in duplicate.

Suitably, the present invention may be used to assay human plasma taken from a subject that has received or is receiving an agent that stimulates fibrinolysis - such as a CPU inhibitor.

The effect that a given dose of the agent has on the lysis of a clot formed in the assay may then be determined.

The assay method that is described herein may also be used for measuring the effect of an activity on fibrinolysis.

The activity may include any activity performed by or upon a mammal - such as a human or an animal - that may affect clot lysis and/or fibrinolysis. Such activities may include, but are not limited to exercise - such as walking, running, feeding, eating, mental stress or a medical procedure - such as surgery.

The term "modulating" may refer to preventing, suppressing, inhibiting, alleviating, restoring, elevating, stimulating or otherwise affecting clot lysis and/or fibrinolysis.

For some aspects of the present invention, the term "modulating" may refer to restoring, elevating or stimulating clot lysis and/or fibrinolysis. By way of example, the assay method described herein may be used to measure the effect of compounds that stimulate fibrinolysis.

The prevention and/or suppression and/or inhibition and/or alleviation and/or restoration and/or stimulation of clot lysis and/or fibrinolysis may be useful in the treatment of disorders associated with imbalances in the coagulation and/or fibrinolytic pathways. The elevation and/or restoration and/or stimulation of clot lysis and/or fibrinolysis may elevate and/or restore and/or stimulate the dissolution and removal of fibrin clots, for example. As a result, the susceptibility to diseases - such as stroke, deep vein thrombosis, and myocardial infarction - may be minimised or prevented.

The prevention and/or suppression and/or inhibition of clot lysis and/or fibrinolysis may be useful in the treatment of disorders associated with excessive fibrinolysis and decreased coagulation, which may result in haemorrhage.

In a preferred embodiment of the present invention, the term modulating means stimulating.

Accordingly, in this embodiment, the present invention relates to an assay method for identifying an agent - such as a CPU inhibitor - that stimulates clot lysis and/or fibrinolysis comprising the steps of: (a) providing a test sample; (b) contacting said test sample with an agent; (c) activating the coagulation system in the test sample; and (d) measuring the amount of fibrin degradation products that are formed by measuring the amount of D-dimer or fragment D of fibrin; wherein a difference between (i) the amount of fibrin degradation products formed in the presence of the agent and (ii) the amount of fibrin degradation products formed in the absence of the agent is indicative of an agent that stimulates fibrinolysis. Typically, the disease as described herein is a disorder associated with coagulation or fibrinolysis.

Advantageously, the method of the present invention may be useful for the study of the effect of agents used in those conditions where inhibition of fibrinolysis inhibitors - such as CPU - is beneficial - such as in the treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Conditions associated with hypercoagulability and thrombo-embolic diseases include, but are not limited to, protein C resistance and inherited or acquired deficiencies in anti-thrombin III, protein C, protein S and heparin cofactor II.

Other conditions associated with hypercoagulability and thrombo-embolic disease include, but are not limited to, circulatory and septic shock, circulating anti-phospholipid antibodies, hyperhomocysteinemia, heparin induced thrombocytopenia and defects in fibrinolysis.

Other disease states may include the therapeutic and/or prophylactic treatment of venous thrombosis and pulmonary embolism, arterial thrombosis (for example in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis) and systemic embolism usually from the atrium during atrial fibrillation or from the left ventricle after transmural myocardial infarction.

Further indications include the therapeutic and/or prophylactic treatment of disseminated intravascular coagulation caused by bacteria, multiple trauma, intoxication or any other mechanism, fibrinolytic treatment when blood is in contact with foreign surfaces in the body, such as vascular grafts, vascular stents, vascular catheters, mechanical and biological prosthetic valves or any other medical device, and fibrinolytic treatment when blood is in contact with medical devices outside the body, such as during cardiovascular surgery using a heart-lung machine or in haemodialysis. Advantageously, the method of the present invention may also be useful for the identification of agents that modulate fibrinolysis. As used herein, the term "agent" may be a single entity or it may be a combination of entities.

The agent may be an organic compound or other chemical. The agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial. The agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof. The agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule. The agent may even be an antibody. The agent may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.

By way of example, the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi- synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised agent, a peptide cleaved from a whole protein, or a peptides synthesised synthetically (such as, by way of example, either using a peptide synthesiser or by recombinant techniques or combinations thereof), a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof. The agent may be in the form of a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof. For a review on suitable salts see Berge et al, J. Pharm. Sci., 1977, 66, 1-19.

The agent of the present invention may be capable of displaying therapeutic properties. For some aspects, preferably, the agent is an inhibitor of a fibrinolysis inhibitor - such as an inhibitor of CPU. Several such inhibitors of CPU are known in the art as reviewed in Current Drug Targets - Cardiovascular & Haematological Disorders (2001) 1, 59-74. By way of example, analogues of lysine or arginine act as effective competitive inhibitors of active CPU, including the lysine analogue ε-aminocaproic acid, 2-mercaptomethyl-3- guanidinoethylthiopropanoic acid and 2- guanidinoethylmercaptosuccinic acid (J. Biol. Chem. (1998) 273, 2127-2135; Biochem. Biphys. Acta (1990), 1034, 86-92; and Biocheniistij (1995) 34, 5811-5816). Others inhibitors include, but are not limited to, potato tuber carboxypeptidase inhibitor (J Gin. Invest. (1995) 96, 2534-2538); leech carboxypeptidase inhibitor (J. Biol. Chem. (1998) 273, 32927-32933). CPU inhibitors are also disclosed in WO 00/66550, WO 00/66557, WO 03/013526 and WO 03/027128 and a pharmaceutical formulation containing a CPU inhibitor and a thrombin inhibitor is disclosed in WO 00/66152.

Inhibitors of plasma carboxypeptidase B are disclosed in, for example, WO 01/19836. Inhibitors of TAFIa are disclosed in, for example, WO 02/14285, WO 03/061652 and WO 03/061653.

The agent may be administered in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are well known to those skilled in the art, and for example include those mentioned by Berge et al, in J. Pharm. Sci., 66, 1-19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p-toluenesulphonate salts. It will be appreciated by those skilled in the art that an agent may be derived from a prodrug. Examples of prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form an agent of the present invention which are pharmacologically active. It will be further appreciated that certain moieties known as "pro-moieties", for example as described in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985 (the disclosured of which is hereby incorporated by reference), may be placed on appropriate functionalities of agents. Such prodrugs are also included within the scope of the invention. An agent or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesize an agent in whole or in part. For example, if they are peptides, then peptides may be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).

Synthesis of peptide agents may be performed using various solid-phase techniques (Roberge JY et al. (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising an agent or any part thereof may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant agent. In an alternative embodiment of the invention, the coding sequence of a peptide agent (or variants, homologues, derivatives, fragments or mimetics thereof) may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al. (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al. (1980) Nuc Acids Res Symp Ser 225- 232). An agent may be prepared by recombinant DNA techniques.

Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratoiy Manual, Second Edition, Books 1- 3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, H Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

The method of the present invention may be a screen, whereby a number of agents are tested for modulating clot lysis and/or fibrinolysis.

The assay methods of the present invention may be suitable for both small and large-scale screening of agents as well as in quantitative assays. Agents identified by the method of the present invention may be used as therapeutic agents - i.e., in therapy applications.

As with the term "treatment", the term "therapy" includes curative effects, alleviation effects, and prophylactic effects. The therapy may be on mammals such as humans or animals. The therapy may be for treating disorders associated with coagulation or fibrinolysis.

In vivo models may be used to investigate and/or design therapies to modulate clot lysis and/or fibrinolysis.

The models could be used to investigate the effect of various tools/lead compounds on coagulation and/or fibrinolysis.

Animal test models may be used as, or in, the methods of the present invention.

The animal test model will be a non-human animal test model.

In a further aspect, the present invention relates to a kit for performing the methods and assay methods of the present invention.

Typically, the kit may comprise a first vessel which comprises a plasminogen activator; a second vessel containing an entity that activates the coagulation system; and a third vessel containing a component that stops clot lysis.

Preferably, the coagulation system is activated using calcium ions and/or thrombin, or tissue factor. More preferably, the coagulation system is activated using calcium chloride and/or thrombin. Most preferably, the coagulation system is activated using calcium chloride.

Preferably, the amount of D-dimer or fragment D formed is measured using a D-dimer test, such as the Biopool Auto-Dimer™ test, the TintElize® D-dimer test, the Accuclot™ D-dimer test, the Auto D-Dimer™ test, the Auto-Dimer™ test, the Minutex® D-dimer test, the NovoCard® D-dimer test, the MiniQuant™ D-dimer test or the Asserachrom® D-DI test.

Biopool Auto-Dimer™ test or the Asserachrom® D-DI test.

Preferably, the component that stops clot lysis is a plasmin inhibitor - such as aprotinin, D-

Val-Phe-Lys chloromethyl ketone and/or alpha-2-antiplasmin. Optionally, the kit may contain an anti-heparin agent - such as polybrene®.

Optionally, the kit may comprise a vessel containing a known stimulator of fibrinolysis as a control. The known stimulator of fibrinolysis may include, but is not limited to, 2- mercaptomethyl-3-guanidinoethylthiopropanoic acid, 2- guanidinoethylmercaptosuccinic acid, potato tuber carboxypeptidase inhibitor, leech carboxypeptidase inhibitor or any stimulators of fibrinolysis described in WO 00/66550, WO 00/66557 or WO 03/013526.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES Example 1

Materials - The reagents and vials that are used in Example 2 and 3 of the present invention can be obtained from the following sources: sct-PA (Biopool, Umea, Sweden); Aprotinin (Pentapharm); Tween-80 (Sigma Molecular

Biology); CaCl2 (Merck); Hepes (Merck); NaCl (Fresenius Kabi Norge AS).

Vials (1.5 mL, Treff Lab, Degersheim, Switzerland)

The following components are used in the assay method of the present invention: Start reagent

30 mmol/L CaCb, 1.44 nM sct-PA in 20 mM Hepes, 0.01% Tween-80, pH 7.4

Stop reagent

600 KIU/mL aprotinin, NaCl (9 mg/mL)

Plasma Human plasma is prepared as follows. Blood is collected on 0.129 M citrate and plasma decanted within 2 hours following a centrifugation at 2,500 x g. It is then aliquoted into vials, deep frozen at -80 °C and thawed just before analysis.

D-dimer kit

Biopool Auto-Dimer™, Biopool, Umea, Sweden. The assay concentration for each component is as follows: sct-PA, 0.72 nmol/L; CaCl₂, 15 mmol/L; Hepes, 10 mmol/L; Tween-80, 0.005 %; Aprotinin,

200 KIU/mL; Plasma 50 %.

Example 2

Methods To each vial 150 μL of Start reagent and 150 μL human plasma are added.

The mixture is incubated for 40 min at 37 °C and incubation is stopped by adding 150 μL

Stop reagent.

The tube is centrifuged at 2000 X g for 10 min at 4 °C and the supernatant is withdrawn.

The supernatant is then removed from the tube and the Biopool Auto-Dimer™ kit used to measure the amount of fibrin degradation products.

Example 3

Effect of three agents that stimulate lysis

The assay method is performed as described in Examples 1 and 2, with the exception that

3 μL of agent solution is added to the start reagent. Three different agents are tested at three different concentrations of 0.1, 0.5 and 5 μmol/L.

Control reactions containing no agent are also performed.

The results obtained using the three different agents are shown in Table 1 and in Figures 1 and

2. These results show that increasing the concentration of each of the three agents in the assay increases the amount of D-dimer that is generated, indicating that these agents stimulate lysis.

Table 1

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in biology or related fields, are intended to be within the scope of the following claims.

Claims

1. A method for measuring the effect of an agent on fibrinolysis comprising the steps of:

(a) providing a test sample comprising: (i) a fibrinolysis inhibitor and/or a zymogen of a fibrinolysis inhibitor; and (ii) an agent;

(b) activating the coagulation system; and

(c) measuring the amount of fibrin degradation products that are formed; wherein a difference between (i) the amount of fibrin degradation products formed in the presence of the agent and (ii) the amount of fibrin degradation products formed in the absence of the agent is indicative of the effect of said agent on fibrinolysis.

2. A method according to claim 1 wherein the coagulation system is activated using calcium ions and/or thrombin, or tissue factor.

3. A method according to any of the preceding claims, wherein the coagulation system is activated using calcium chloride.

4. A method according to any of the preceding claims, wherein the fibrinolysis inhibitor is selected from the group consisting of: carboxypeptidase U (CPU), plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2) or alpha-2-antiplasmin.

5. A method according to any of the preceding claims, wherein the test sample comprises blood or a constituent thereof.

6. A method according to any of the preceding claims, wherein the test sample comprises plasma.

7. A method according to any one of the preceding claims wherein the amount of fibrin degradation products that are formed are measured by measuring the amount of D-dimer or fragment D of fibrin.

8. A method for measuring the effect of an activity on fibrinolysis in a subject comprising the steps of: (a) providing a test sample comprising a fibrinolysis inhibitor and/or a zymogen of a fibrinolysis inhibitor from the subject;

(b) activating the coagulation system in the test sample; and

(c) measuring amount of fibrin degradation products that are formed; wherein a difference between (i) the amount of fibrin degradation products formed before the activity and (ii) the amount of fibrin degradation products formed after the activity is indicative of the effect of the activity on fibrinolysis.

9. A method according to claim 8 wherein the activity is exercise, feeding, eating, mental stress or surgery.

10. An assay method for identifying an agent that modulates clot lysis and/or fibrinolysis comprising the steps of:

(a) providing a test sample; (b) contacting said test sample with an agent;

(c) activating the coagulation system in the test sample; and

(d) measuring the amount of fibrin degradation products that are formed by measuring the amount of D-dimer or fragment D of fibrin; wherein a difference between (i) the amount of fibrin degradation products formed in the presence of the agent and (ii) the amount of fibrin degradation products formed in the absence of the agent is indicative of an agent that modulates clot lysis and/or fibrinolysis.

11. A kit for performing the method according to any one of claims 1-9, or the assay method according to claim 10 comprising: a first vessel which comprises a component that stops clot lysis; a second vessel containing a plasminogen activator; and a third vessel containing an entity that activates the coagulation system.

12. A kit according to claim 11 wherein the entity comprises or contains calcium ions, preferably, calcium chloride and/or thrombin.

13. A kit according to any of claims 11 or 12, wherein the component that stops clot lysis is a plasmin inhibitor, such as: aprotinin, D-Val-Phe-Lys chloromethyl ketone and/or alpha-2- antiplasmin.