KR101608134B1 - Function assay for protein Z anticoaogulant system - Google Patents

Abstract

The present invention relates to a novel biomarker for blood coagulation diagnosis based on the anticoagulation system of protein Z and its use, and more particularly to a biomarker for the diagnosis of coagulation factor Xa which is involved in the inhibition of blood coagulation factor Xa And the diagnostic use thereof and the use thereof. BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 > [2] < / RTI >

Description

Function assay for protein Z anticoaogulant system < RTI ID = 0.0 >

The present invention relates to a novel biomarker for blood coagulation ability and thrombocyte function diagnosis based on an anticoagulant system of protein Z and its use and more specifically to a blood coagulation factor Xa The present invention relates to a method for analyzing the blood coagulation function using the anticoagulant system and its diagnostic use and its use.

If the blood flow is abnormal due to thrombosis such as deep vein thrombosis, pulmonary embolism, prosthetic valve graft or atrial fibrillation, anticoagulant treatment will be given. These patients need to continuously control the amount of anticoagulant they take to prevent bleeding or embolism. At this time, blood coagulation test is essential in order to check whether the blood coagulation process is normally maintained in the body.

The blood coagulation test can be carried out for various purposes such as measurement of hemorrhagic susceptibility of an operated patient and monitoring of an anticoagulant therapy patient to prevent blood coagulation. A number of coagulation tests are currently in use, one of which is the prothrombin time (PT) test. The PT test relies on activation of the exogenous coagulation pathway by thromboplastin added to the blood sample subjected to the PT test. Activation of the exogenous coagulation pathway leads to the production of thrombin, a proteolytic enzyme that catalyzes the conversion of fibrinogen to fibrin.

Thromboplastin (TF) is a membrane bound glycoprotein that complexes with Factor VIIa and the Factor VIIa / TF complex initiates the blood coagulation process. Once formed, the Factor VIIa / TF complex activates a series of specific enzymes involved in the exogenous and endogenous pathways of the coagulation cascade, which ultimately leads to the formation of thrombin, fibrin, platelet activation, and finally coagulation.

A conventional PT test utilizes a series of enzymatic reactions described above under in vitro conditions and controlled conditions to diagnose dysfunction or deficiency of the patient's blood coagulation system. The time it takes for blood clot formation to occur is a blood coagulation test such as prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time.

PT is the time until coagulation after addition of a mixed solution of tissue thromboplastin and Ca ^{2 + to} the test plasma, and is the time required for the coagulation factor VII, factor X, factor V, prothrombin, fibrinogen And the coagulation activity of the cells. Furthermore, APTT is not a time until solidification after addition of a sufficient amount of phospholipids and an activating agent (kaolin, silicic anhydride, Era geusan (ellagic acid), and so on) with an appropriate amount of Ca ^{2 +} in the test plasma, in taking over the solidification The coagulation activity of factor XII, factor XI, prekallikrein, high molecular kininogen, factor IX, factor VIII, factor X, factor V, prothrombin, fibrinogen, .

Generally, the abnormality in these blood coagulation tests indicates that the coagulation time is prolonged. The abnormality of the blood coagulation system reflects a sign of bleeding tendency or blood clotting tendency (blood coagulation tendency) in vivo.

In the blood coagulation system in the human body, proteins that promote blood coagulation and proteins that inhibit blood coagulation are normally balanced with each other. When protein (protein) Z that inhibits blood coagulation decreases, coagulation of blood proceeds excessively, Arrhythmia thrombosis, pulmonary embolism, stroke).

The present inventors have focused on a functional PZ system in cirrhosis coagulation disorders and attempted to exploit the potential function of the potential use of protein Z in determining the thrombus formation tendency.

Therefore, the present inventors measured the decrease of PZ level, the increase of ZPI level, the ratio of lag time and the ratio of peak thrombin to the protein Z (PZ) blood coagulation inhibition system in liver cirrhosis patients, And the present invention has been completed.

Zhang D, Hao J, Yang N. Protein C and D-dimer are related to portal vein thrombosis in patients with liver cirrhosis. J Gastroenterol Hepatol. 2010 Jan; 25 (1): 116-21. PubMed PMID: 19686413. Epub 2009/08/19. eng. Tripodie, Chantarangkul V, Mannucci PM. Acquired coagulation disorders: revised using global coagulation / anticoagulation testing. Br J Haematol. 2009 Oct; 147 (1): 77-82. PubMed PMID: 19659548. Epub Aug 8, 2009. eng. 3. Vasse M. The protein Z / protein Z-dependent protease inhibitor complex. Systemic or local control of coagulation Hamostaseologie. 2011 Aug; 31 (3): 155-8, 60-4. PubMed PMID: 21691673. Epub 06/22/2011. eng.

It is an object of the present invention to provide a composition for a marker for in vitro diagnosis of blood coagulation ability containing protein Z and a method for in vitro diagnosis of blood coagulation ability using the same.

Another object of the present invention is to provide various uses for diagnosing the blood coagulation ability of the protein Z.

It is another object of the present invention to provide the use of protein Z as a diagnostic marker in the cirrhosis progression stage.

In order to solve the above problems, the present invention provides a novel use of protein Z (Protein Z, PZ) which inhibits the activity of blood coagulation factor Xa.

In one embodiment, the present invention provides a composition for diagnosing thrombotic thrombosis-related blood coagulation disorders, said protein Z comprising Protein Z (PZ), which provides information on the function of thrombus formation.

In particular, the protein Z (Protein Z, PZ) is characterized in that the expression is reduced in a blood coagulation disorder associated with thrombin thrombosis, wherein the diagnosis is made by distinguishing whether the function of thrombus formation in the subject is enhanced or suppressed .

In another embodiment,

And measuring the expression level of protein Z (Protein Z, PZ) from the blood sample separated from the patient, in order to provide information for diagnosis of abnormal blood coagulation disease. At this time, when the expression of protein Z (PZ) in the blood sample isolated from the patient appears to be lower than that of the normal control sample, it provides information that the function of blood coagulation and / or thrombus formation is enhanced.

The blood sample to be used is preferably whole blood or plasma, more preferably platelet-depleted plasma.

The blood coagulation disorder is a disease associated with thrombotic thrombosis, particularly in a group consisting of hemophilia, von Willebrand disease, liver disease, and disseminated intravascular coagulation (DIC) And may be one or more selected diseases. Most preferably liver disease including liver cirrhosis and the like.

The method of the present invention may further include the step of measuring the protein Z-dependent protease inhibitor (ZPI) level to further enhance the reliability.

At this time, when the level of protein Z-dependent protease inhibitor (ZPI) in the blood sample isolated from the patient is increased as compared with the normal control sample, information can be provided that the function of thrombus formation is enhanced.

Meanwhile, in another embodiment of the present invention, in order to provide information for diagnosis of abnormal blood coagulation disease,

Expressing protein Z (Protein Z, PZ) in a blood sample separated from the patient; And a step of measuring a change in thrombin production amount.

Here, the change of the thrombin production amount is a ratio of (a) a lag time ratio as a ratio to a thrombin generation time, and (b) a peak to a peak thrombin value, for a blood sample separated from a normal control sample and a patient, Thrombin ratio. ≪ / RTI > For further details, please refer to the following description.

When performing the above method, it is possible to provide information that the function of thrombus formation is enhanced when the lag time ratio is increased or the peak thrombin ratio is decreased.

The blood sample used or blood coagulation abnormalities are as described above.

As described above, the present invention provides a biomarker for diagnosing thrombotic thrombosis-related blood coagulation disorders, which provides new blood coagulation ability and thrombogenic ability based on the anticoagulant system of protein Z, and its various uses .

As described above, the present invention relates to a new method for measuring the function of blood coagulation, wherein the function of the blood coagulation inhibition system, protein Z, is measured in the blood of a patient to check whether the function of thrombus formation has been enhanced or inhibited And the results can be used as a finding reflecting the thrombotic tendency or bleeding tendency of the patient. By measuring the thrombus formation tendency in the patient's blood, the blood coagulation abnormality diagnosis related to the thrombotic thrombosis Which can be used for clinical purposes in the future.

1 is a schematic diagram (A) of a coagulation pathway involved in the protein C and protein Z anticoagulation systems; And (B) a functional analysis method for the PZ anticoagulation system of the present invention.
FIG. 2 is a graph showing the plasma concentration of PZ and ZIP according to the MELD score in cirrhosis patients and normal controls.
FIG. 3 is a graph showing the results of analysis based on lag time, peak thrombin and endogenous thrombin potential (ETP) parameters in the thrombin generation assay (TGA) according to the severity of cirrhosis. Graph.
FIG. 4 shows the result of analyzing the correlation with PZ or ZIP according to the ratio of the thrombin generation assay parameter obtained according to the presence or absence of PZ.
Figure 5 shows the results of analysis of the levels of lag time ratio-PZ, peak thrombin ratio-PZ and ETP ratio-PZ according to PZ and ZIP in cirrhosis patients.
Figure 6 shows the analysis by lag time, peak thrombin and endogenous thrombin potential (ETP) parameters in a thrombin generation assay (TGA) for a thrombin case. Graph.

The terms used in the present invention are defined as follows.

"Diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, blood coagulation ability and thrombotic tendency are to be confirmed. Particularly, it is useful for diagnosing blood clotting ability and thrombus disposition related to liver disease.

"A diagnostic marker or diagnosis marker is a substance that can diagnose blood coagulation ability and thrombosis tendency differently from a normal case and is a polypeptide or nucleic acid capable of diagnosing the coagulation abnormality (Such as mRNA), lipids, glycolipids, glycoproteins, sugar (monosaccharides, disaccharides, polysaccharides, etc.) and the like.

"Blood clotting" refers to the process of plasma coagulation in vertebrates centering on thrombin generation. During thrombin generation, a small amount of thrombin is incorporated into the fibrin chunk / network (at enlargement), and the liberated catalytic site of the protease can amplify the chunk growth (Liu, Nossel et al. Vali and Scheraga 1988). Thrombin interacts with a variety of substrates and activates various lump promoting factors, such as (a) the thrombin causes platelet activation by cleavage of the kinase receptor, (b) feedback of factors V, VIII and XI (C) activates transglutaminase, Factor XIII, and (d) converts fibrinogen to fibrin. Once the mass is formed and stabilized by strand-liver cross-linking under pathological conditions, the mass interferes with normal blood flow in the blood vessel leading to blockage of arteries and veins.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Protein" also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein

"Label" or "label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.

"Antibody" is used in its broadest sense and specifically includes intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (e. G. Bispecific antibodies) formed from at least two intact antibodies, and And antibody fragments exhibiting the desired biological activity.

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel function of a particular protein Z, and is based on the discovery of new functions of protein Z, in particular measuring blood coagulation ability according to the functional evaluation of the PZ anticoagulation system.

In particular, the present invention is a first method for measuring the function of a protein Z coagulation inhibiting system in the human body. More particularly, the present invention relates to a method for determining the thrombin formation level by measuring a protein Z level having a suppressive effect on the coagulation factor Xa, The present invention relates to a method for determining whether or not a function has been enhanced and determining a thrombotic tendency or a hemorrhagic tendency of a subject (patient) to provide information and diagnosis.

<Diseases to be diagnosed>

In the present invention, the disease to provide and diagnose the function of the thrombus formation is a blood coagulation disorder. In particular, the present invention relates to the diagnosis of abnormal blood coagulation disorders associated with thrombotic thrombosis.

The abnormality of the blood coagulation system reflects a sign of bleeding tendency or blood clotting tendency (blood coagulation tendency) in vivo. In the blood coagulation system in the human body, proteins that promote blood coagulation and proteins that inhibit blood coagulation are normally balanced with each other. Protein Z is a protein that inhibits blood coagulation. When the expression amount of protein Z decreases, blood coagulation is excessively The risk of thrombosis (deep vein thrombosis, pulmonary embolism, stroke, etc.) is increased.

Blood clotting disorders are largely congenital and acquired. It is mainly caused by deficiency of congenital blood clotting factor. Typical are hemophilia A and B. Other von Willebrand disease and diseases associated with deficiency of coagulation factors (I, II, V, VII, X, XI, XII, XIII) Acquired coagulation factor deficiency is associated with severe liver disease, disseminated intravascular coagulation (DIC), circulating anti-coagulant, vitamin K deficiency, and drugs. Particularly, in one embodiment of the present invention, liver disease, particularly liver cirrhosis disease, was targeted.

Cirrhosis is defined as histopathologically, clinically classified as symptomatic cirrhosis and noncancerous cirrhosis, and noncancerous cirrhosis is associated with ascites, variceal hemorrhage, hepatic encephalopathy and jaundice. CT, abdominal ultrasonography, and MRI are available for the diagnosis of liver cirrhosis. There are typical findings such as nodular liver surface, spleen enlargement, presence of intra-abdominal collateral vessel showing portal hypertension, or upper gastrointestinal endoscopy The diagnosis of liver cirrhosis can be made without a liver biopsy if esophageal or gastric varices are present and clinical signs of cirrhosis are present. However, imaging studies are not satisfactory in diagnosing cirrhotic cirrhosis, and there are no established criteria for diagnosing cirrhosis using imaging tests, even in the case of non-cirrhotic cirrhosis.

However, in addition to imaging studies, it is helpful to diagnose cirrhosis by assessing albumin, bilirubin, and prothrombin time and platelet counts, which are the criteria for classifying the Child-Pugh score among the blood tests commonly performed in clinical practice. Recently, there are many reports that predict liver cirrhosis by noninvasive methods such as blood test and liver elasticity measurement. However, there are no guidelines for blood markers and their use that are ideal for the diagnosis of cirrhosis.

The present invention is basically based on a new function of protein Z capable of diagnosing blood coagulation ability and thrombus formation tendency and is not only used as a marker for blood coagulation ability but also as a diagnostic marker for a cirrhosis progression stage closely related thereto .

The selection and application of significant diagnostic markers determines the reliability of diagnostic results. Significant diagnostic markers are those markers that are highly reliable with high validity and consistency in repeated measurements. A new blood coagulation ability in vitro diagnostic marker of the present invention protein Z exhibits the same result in repeated experiments with genes whose expression is directly or indirectly changed with thrombus formation according to the change of thrombin production amount, Are highly reliable markers that are very large when compared to the control group and have little chance of producing false results. Therefore, the results diagnosed on the basis of the results obtained using the expression characteristics of the significant diagnostic marker of the present invention can be reasonably reliable.

<Protein Z Applications>

Therefore, in one aspect, the present invention relates to a novel blood coagulation ability of protein Z and its use as a thrombogenic ability in vitro diagnostic marker.

Protein Z (Protein Z, PZ) is an anticoagulant that accelerates the inhibitory effect of protein Z-dependent protease inhibitor (ZPI) on coagulation factor Xa. Protein Z of the present invention is known, For example, a known human-derived sequence can be obtained from a known DB, but is not limited thereto and includes functional equivalents thereof. At least 90% identical, more preferably at least 95%, 96%, 97%, 98% or 99% identical to the protein Z sequence of the present invention; Or isolated polynucleotides comprising nucleoside complementary to such exemplary sequences.

The present invention can diagnose whether or not a thrombus formation tendency is formed by measuring the amount of change in thrombin production in plasma using the correlation between the expression of protein Z and the production of thrombin.

Therefore, from the same viewpoint, the present invention relates to a blood coagulation ability and a thrombus-forming ability for in vitro diagnostic using the expression characteristics of protein Z, and a blood coagulation ability and a thrombus formation in vitro diagnostic method using the same.

In one embodiment, a composition for diagnosing abnormal blood coagulation associated with thrombotic thrombosis, which contains protein Z (Protein Z, PZ), can be provided.

At this time, the protein Z (Protein Z, PZ) may be used as a criterion for determining the decrease in expression in a blood coagulation disorder related to thrombotic thrombosis, and the diagnosis may be made based on the function of the thrombus formation Lt; RTI ID = 0.0 &gt; inhibited, &lt; / RTI &gt;

<Diagnosis and analysis method>

The present invention also provides, from another point of view, a blood coagulation function comprising a step of measuring the expression level of protein Z (Protein Z, PZ) from a blood sample isolated from a patient, in order to provide information for diagnosing abnormal blood coagulation disorders And the like.

At this time, when the expression of protein Z (PZ) in the blood sample separated from the patient is decreased as compared with the normal control sample, it is informed that the function of thrombus formation is enhanced.

And, in order to increase the reliability of the judgment, it is preferable that the method further comprises the step of measuring the level of the protein Z-dependent protease inhibitor (ZPI, protein Z-dependent protease inhibitor).

At this time, when the level of protein Z-dependent protease inhibitor (ZPI) in the blood sample separated from the patient is increased as compared with that of the normal control sample, it can also be informed that the function of thrombus formation is enhanced.

In yet another aspect, the present invention provides a method for diagnosing blood coagulation disorders comprising the steps of: (i) expressing protein Z (Protein Z, PZ) in a blood sample separated from a patient; And (ii) measuring a change in thrombin production amount.

At this time, the expression of protein Z (Protein Z, PZ) can be carried out by a person skilled in the art by appropriately selecting a known arbitrary method. An expression vector may be used, and the protein Z may be added directly, and there is no great limitation.

The change in the thrombin production amount is determined by measuring the ratio of (a) a lag time ratio as a ratio to the thrombin generation time, and (b) a ratio of a peak to a peak thrombin value for a blood sample separated from a normal control sample and a patient, Thrombin ratio. &Lt; / RTI &gt; More specific details can be understood with reference to the following figure.

At this time, when the lag time ratio increases or the peak thrombin ratio decreases, information indicating that thrombus formation function is enhanced can be provided.

Hereinafter, a protocol for evaluating blood coagulation ability and blood clotting ability from a blood sample usable in the present invention is exemplified.

For the production of blood samples, tissues or cell samples from mammals (generally human patients) can be used. Samples may be obtained by a variety of procedures known in the art including, but not limited to surgical excision, aspiration, or biopsy. The sample can be fixed (i.e. preserved) by conventional means.

The blood sample used in the present invention may be whole blood or plasma. Preferably, blood is used rather than blood is used as it is, and it is more preferable to use platelet-removing plasma. A known method may be used for the platelet removal, and for example, centrifugation, platelet-removing filter treatment, or the like can be used. As a commercial product, Pooled Normal Plasma (manufactured by Precision BioLogic), Platelet Poor Plasma (Technoclone), and LAtrol (Trade Mark) Normal Control (American Diagnostica) are known.

First, the level of protein Z (PZ) and protein Z-dependent protease inhibitor (ZPI) is measured in plasma. Any of the known methods may be used, and enzyme immunoassay is used in one embodiment of the present invention.

In addition, if the PZ level decreases and the ZPI level increases, the thrombus formation tendency can be diagnosed and the development of cirrhosis can be diagnosed and related information can be provided. That is, as the liver damage becomes severe, the plasma PZ level decreases and the ZPI level tends to increase. As the plasma PZ level and / or the ZPI level are used, the degree of progression of the liver damage and the information Can be obtained.

In addition, it is preferable to confirm the change in the thrombin production amount by adding an extrinsic protein Z (Protein Z, PZ) to the blood sample of the control group and the test group.

In contrast, the lag time ratio and / or the peak thrombin ratio for the control and experimental blood samples are calculated using the following equation. In patients with reduced protein Z function, the amount of thrombin produced is significantly reduced when protein Z is added, and this degree of reduction reflects the tendency to thrombogenesis.

Particularly, in the presence of cirrhosis, the lag time ratio -PZ is higher than that of the control group, and the peak thrombin ratio -PZ is significantly lower than that of the control group.

Since the lag time ratio -PZ and the peak thrombin ratio -PZ have a very important relationship with the tendency of thrombotic thrombosis in particular, the results of this parameter analysis show that the relationship with the blood coagulation factors and information on the presence or absence of thrombosis .

That is, the assay method using the anticoagulant system of protein Z of the present invention is particularly useful for providing accurate information for diagnosing the propensity for ongoing anticoagulation.

It is possible to indirectly identify the causes of bleeding in patients with cirrhosis who had no bleeding symptoms rather than other coagulation indexes.

From a similar point of view, the present invention relates to a kit for in vitro diagnosis of blood clotting ability and thrombus formation for providing information necessary for diagnosing abnormal blood coagulation diseases associated with thrombin thrombosis using protein Z.

For this purpose, the kit may comprise, for example, a primer or probe that specifically binds to a gene sequence encoding protein Z; Or an antibody that specifically binds to protein Z. The kits of the present invention may be made from a number of separate packaging or compartments containing the required reagent components.

Preferably, the diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method comprising means for measuring changes in thrombin production.

<Combined analysis method>

Since the coagulation inhibitory action in the human body occurs simultaneously and coexistently with respect to various coagulation factors, the conventional technique and the present invention can be used in combination to provide a rich and accurate blood coagulation ability, thrombus formation It can provide information about the tendency and bleeding propensity.

That is, the method of analyzing blood coagulation function using protein Z of the present invention is based on the coagulation factor Xa to be inhibited, and in the blood coagulation function test method, the coagulation factors V and VIII were assayed using thrombomodulin Method, it is possible to provide information considering the influence on different blood coagulation factors together.

In particular, while conventional markers such as PT and aPTT have been widely used only in clinical trials to measure hemorrhagic tendencies, the functional evaluation of our advanced PZ system provides information on ongoing coagulation-inhibitory activity, The reliability and efficiency of information on blood coagulation ability and thrombogenic ability can be improved.

As described above, the present invention has the characteristics of expression in blood plasma of protein Z, which is involved in the inhibition of blood coagulation factor Xa, and the method for analysis and diagnosis of blood coagulation function using anticoagulation system, It is suggested that this method can be used as an in vitro diagnostic test method which is useful for clinical use as a new method of measuring the thrombus formation tendency in the blood of a patient by variously utilizing the use of the protein as a disease diagnosis marker related to thrombosis .

<Examples>

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Materials and methods

1. Experimental group

A total of 158 patients with liver cirrhosis were included in the study (Table 1).

Liver cirrhosis was diagnosed by pathological examination of liver biopsy and / or clinical, experimental and radiological examination. Exclusion criteria were obstruction of congenital hemostasis, use of anticoagulant within 7 days of blood collection, or body weight less than 30kg. The median age was 59 years (range 24-88 years).

The severity of liver disease was assessed according to the MELD (model for end-stage liver disease) score. The MELD score was used for cirrhosis severity assessment and was calculated as follows: MELD score - 10 [0.957 ln (creatinine, mg / dl) + 0.378 ln (bilirubin, mg / dl) + 1.12 ln (INR) .

The severity of liver disease was also scored according to the Child-Pugh classification: 81 cases (51.3%) were class A, 47 cases (29.8%) were class B, and 30 cases (19.0% Thrombosis cases including portal vein thrombosis, pulmonary embolism or deep vein thrombosis were investigated and a total of 50 patients (31.6%) were followed up for 3 months follow-up period Thrombosis. In addition, 59 healthy adults with normal physical examinations were included as controls. This experiment was approved by the Institutional Audit Committee of Seoul National University Hospital.

2. Blood samples and analysis of results

Peripheral venous blood samples were collected in commercial tubes containing 0.109 mol / L sodium citrate (Becton Dickinson, San Jose, Calif., USA). Plasma was isolated by centrifugation of whole blood for 15 minutes at 1550 g within 1.5 hours after blood collection. Plasma samples were stored at -80 ° C.

Plasma plasma was applied for enzyme immunoassay (Asserachrom Protein Z, Diagnostica Stago, Asnieres, France) for the determination of PZ. ZPI was measured using an enzyme-linked immunosorbant assay kit (ZPI ELISA kit, MyBioSource, San Diego, Calif., USA). Coagulation tests including PT, aPTT, and coagulation factor analysis were performed on an automated coagulation analyzer, ACL3000 (Instrumentation Laboratory SpA, Milan, Italy). Anticoagulants are PT-based clathing assays using HemosIL RecombiPlasTin reagents for factor (F) II, FV, FVII, and FX, and aPTT based assays using SynthASil reagents (FVIII, FXI, FXI, and FXII) And analyzed by a clotting analysis.

Antithrombin and protein C were measured by chromogenic assays (Stachrom ATIII and Stachrom Protein C, Diagnostica Stago). The number of platelets and white blood cells (WBC) was measured by a Sysmex XE-2100 analyzer (Sysmex, Kobe, Japan). Serum levels of total protein, albumin, aspartate transaminase, alanine transaminase (ALT), alkaline phosphatase (ALP) and bilirubin were measured using a Toshiba 200FR automatic analyzer (Toshiba Medical Systems, , Japan).

3. Thrombin generation assay

Thrombin generation in TF-triggered platelet-deficient plasma was measured by a scaled automated thrombogram method (Thrombinoscope BV, Maastricht, The Netherlands).

Briefly, 20 μL of reagent containing TF and a phospholipid or thrombin meter at a final concentration of 1 pM (PPP Reagent Low, Thrombinoscope BV) was dispersed in each well of a round bottom 96-well plate, and 80 μL of test Plasma was added. After addition of 20 μL of fluorogenic substrate to HEPES buffer with CaCl ₂ , the fluorescence signal was read on a Fluoroskan Ascent fluorometer (Thermo Labsystems OY, Helsinki, Finland) and the thrombin generation curve was calculated with software (Thrombinoscope BV). The thrombin generation curves were evaluated using parameters including the initiation, propagation and termination phases of thrombin generation, namely lag time, ETP and peak thrombin concentration (peak thrombin &lt; RTI ID = 0.0 &gt; )

In the same way, PZ (Haematologic Technologies, Essex Junction, VT, USA) or TM (Haematologic Technologies) was added to the second sample at a final concentration of 300 nM or 2.5 nM and the test was repeated.

The new parameter of ETP ratio-PZ was calculated by dividing the ETP value with PZ by the ETP value without PZ x 100. The lag time ratio -PZ and peak height ratio -PZ were calculated in the same way.

 The ETP ratio -TM was calculated by dividing the ETP value with the TM by the ETP value without TM x 100. The exogenous PZ adduct can significantly reduce peak thrombin and ETP values, and only with a low PZ level in plasma and a long lag time The increased lag time ratio -PZ and reduced peak thrombin ratio -PZ and ETP ratio -PZ will appear in cirrhotic plasma with low PZ levels. This represents a high sensitivity to exogenous PZ and ultimately a functional deficiency of the endogenous PZ anticoagulation system (FIG. 1B).

Similarly, the lag time ratio - TM and peak height ratio - TM were also calculated. Since the TM is an anticoagulant, the external addition of the TM trigger reduces the ETP value. Therefore, the reduced lag time ratio -TM and the increased peak thrombin ratio -TM and ETP ratio -TM reflect the high resistance to the anticoagulant action of TM, which is a hyper- coagulability.

4. Statistical analysis

Data were compared using the Kruskal-Wallis analysis and the Mann-Whitney U test for continuous variables and the chi-square test for categorical variables. Correlation is expressed as Pearsons coefficients.

PT and aPTT, multiple linear regression analysis were performed to evaluate the relative effects of coagulation and anticoagulation factors on thrombin generation.

In each model, the standardized regression coefficients (β) of the independent variables and the corrected R2 were calculated. All analyzes were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). A p value of less than 0.05 was considered significant.

Example  1: solidification and Anticoagulation  Plasma level of the factor

Clinical and experimental characteristics of patients with cirrhosis according to the MELD score are shown in Table 2.

PT and aPTT persisted significantly longer at higher MELD scores (> 20) compared to lower MELD scores (<9). All coagulation factors and anticoagulants, including antithrombin and protein C, decreased gradually with increasing MELD score.

PZ levels in patients with liver cirrhosis were significantly lower than those in healthy controls (Table 2). The PZ concentration gradually decreased as the MELD score increased (FIG. 2A). The concentration of PZ was calculated as FII (r = 0.463), FV (r = 0.307), FVII (r = 0.391), FIX = 0.334), antithrombin (r = 0.261), and protein C (r = 0.376), respectively. PZ levels were also significantly correlated with PT (r = -0.420) and aPTT (r = -0.308).

Median levels of ZPI in patients with liver cirrhosis were slightly higher when compared to controls (Table 2). Interestingly, ZPI levels were significantly increased at high MELD scores (> 20) (FIG. 2B). ZPI levels did not show any significant correlation with coagulation and anticoagulant factors as well as PT and aPTT (data not shown).

Example 2: Functional evaluation of PZ anticoagulant system using TGA

In 1 pM TF-induced TGA, the lag time median, peak thrombin and ETP values in cirrhotic patients were significantly different from the control (Table 3).

As the severity of cirrhosis grew, lag time increased and corresponding peak thrombin and ETP decreased (FIGS. 3A-3C).

The lag time ratio-PZ in patients with cirrhosis was significantly higher than in healthy controls and was related to the severity of liver cirrhosis (Table 3, Figure 3D). Peak thrombin ratio-PZ and ETP ratio-PZ in patients with liver cirrhosis were significantly lower than in healthy controls, and cirrhosis severity was also associated (Figures 3E-3F).

Example  3: TGA Using Protein  Functional evaluation of C anticoagulant system

The lag time rate-TM in patients with cirrhosis was significantly higher than in healthy controls (Table 3). However, this did not show a linear correlation with cirrhosis severity (Table 3, Fig. 3G). Peak thrombin ratio-TM and ETP ratio-TM were much higher in the cirrhotic group than in the control group (Table 2). Peak thrombin ratio-TM and ETP ratio-TM were significantly higher in cirrhotic patients with a MELD score of 10-19 (Fig. 3H-3I).

Example  4 : TGA  Parameters and solidification and Anticoagulation  Correlation with Factors

The lag time ratio-PZ and peak thrombin ratio-PZ were in intermediate correlation with plasma PZ levels (Figs. 4A-4B). However, the ETP ratio-PZ showed no significant correlation with the PZ level (FIG. 4C). Plasma ZPI levels did not show any correlation with lag time ratio -PZ, peak thrombin ratio -PZ and ETP ratio -PZ (Fig. 4D-4F).

To investigate the effect of PZI concentration on ratio-PZ, we divided the cirrhotic patients into three groups according to the respective PZ and PZI levels (FIG. 5). The low group was defined as having a PZ or ZPI level below 30% (PZ, 0.79 μL / mL; ZPI, 4.77 μL / mL) Or ZPI level (PZ, 1.45 μL / mL; ZPI, 11.71 μL / mL).

In patients with low PZ levels, patients with high or normal ZPI levels showed a high value of lag time ratio-PZ (Fig. 5A), indicating that in patients with low PZ levels, high levels of ZPI were found in PZ Suggesting that it contributes to the florid anticoagulation function of the system.

In patients with normal PZ levels, patients with high ZPI levels showed a higher lag time rate-PZ than patients with normal or low ZPI levels. Interestingly, in patients with high PZ levels, no significant difference in lag time ratio-PZ with respect to ZPI level was seen, indicating that PZI levels were influenced by the presence of sufficient endogenous PZ levels in patients with high PZ levels .

Similarly, in patients with low PZ levels, low peak thrombin ratio-PZ was found in patients with high or normal levels of ZPI compared to patients with low levels of PZ (FIG. 5B).

In patients with normal PZ levels, the peak thrombin ratio-PZ gradually decreased with increasing ZPI levels. However, there was no significant difference in peak thrombin ratio-PZ in terms of ZPI levels in patients with high PZ levels. In the case of ETP ratio-PZ, the ratio change was not significant depending on the PZ or ZPI level (FIG. 5C).

In the linear correlation analysis, the lag time ratio -PZ and the peak time ratio -PZ were significantly related to the levels of all coagulation and anticoagulant factors, while the ETP ratio -PZ did not (Table 4).

ETP ratio-TM was also correlated with FII, FV, FVII, FXI, antithrombin, and protein C, while rat-to-rate and peak-to-thrombin ratio-TM were not.

Example 5: Relationship between TGA parameters and thrombin

We compared TGA parameters between patients with and without thrombosis (Figure 6). There was no difference in lag time, peak thrombin, and ETP levels between patients with and without thrombosis (Figs. 6A-6C).

The lag time ratio -PZ was significantly higher in patients with cirrhosis with trombosis than in those without cirrhosis (median, 119.7 vs 114.4; P = 0.006, Fig. 6D). Correspondingly, the peak thrombin ratio-PZ was much lower in patients with thrombosis (median, 49.6 vs 62.9; P = 0.002, FIG. 6E). However, ETP ratio-PZ did not show significant differences among patients with or without thrombosis. (Fig. 6F).

The lag time ratio -TM, peak thrombin ratio -TM, and ETP ratio -TM did not show any significant difference in terms of thrombosis (FIG. 6G-6I). There was no significant difference in plasma PZ and ZPI levels between patients with and without thrombosis (median PZ, 1.05 [range, 0.09-2.33] vs 1.23 [0.27-3.28], P = 0.121; median ZPI, 8.85 [0.26-55.21] vs 6.84 [0.42-163.48], P = 0.056).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (15)

In order to provide information on the cause of thrombosis, a method of measuring the function of protein Z (Protein Z, PZ) in a blood sample isolated from a patient,
Wherein the function of the protein Z is measured by thrombin-forming ability. The method according to claim 1,
Wherein the function of the thrombus formation is enhanced when the function of the protein Z (Protein Z, PZ) appears to be lower than that of the normal control sample. The method according to claim 1,
The thrombin-forming ability can be determined by measuring (a) a lag time ratio as a ratio to the thrombin generation time, and (b) a peak thrombin ratio as a ratio to a peak thrombin value for a blood sample separated from a normal control sample and a patient peak thrombin ratio). &lt; / RTI &gt; The method of claim 3,
Wherein the function of the thrombus formation is enhanced when the lag time ratio is increased or the peak thrombin ratio is decreased. The method according to claim 1,
Wherein the blood sample is whole blood or plasma. A method for determining the occurrence of thrombosis in liver cirrhosis disease using the method for measuring the increase or decrease of the protein Z of claim 1. delete delete delete delete delete delete delete delete delete

Images