Method for measuring platelet activation based on available CLEC-2 and platelet count

Abstract

We develop a biomarker that better reflects platelet activation in vivo and provide a method to assist in determining the platelet activation status, which can be used for testing thrombostatic diseases. The above method for assisting in determining the platelet activation status comprises (1) a process of measuring the soluble CLEC-2 concentration and the number of platelets in a test sample derived from a subject, (2) a step of calculating the soluble CLEC-2 concentration value by dividing the soluble CLEC-2 concentration by the number of platelets, and, if desired, further comprises (3) a step of comparing with the value of the soluble CLEC-2 concentration / number of platelets obtained using a test sample derived from a normal healthy subject.

Description

Technology Field

[0001] The present invention relates to a method for measuring platelet activation based on soluble CLEC-2 and platelet count, and a method for testing thrombotic hemostatic diseases. Background Technology

[0002] Blood clots formed within blood vessels are considered a critical causative factor in life-or-death situations among a wide range of human diseases. The enlargement of blood clots by platelets or the accumulation of platelet aggregation lead to conditions such as severe myocardial infarction, chronic unstable angina, transient ischemia, peripheral vascular disease, arterial thrombosis, obstructive arteriosclerosis, pulmonary thromboembolism, and stent restenosis. In developed countries, coronary artery occlusion and cerebral circulatory disorders caused by blood clots are recognized as leading causes of death, making the control of thrombosis a global urgent task. Furthermore, in the event of natural disasters, the prevention of deep vein thrombosis and pulmonary embolism (a disease in which a venous thrombus gets trapped in the bloodstream and occludes the pulmonary artery) during disaster evacuation is highlighted, and effective response to these conditions is considered crucial. In platelets, there are granules within the platelet and many membrane glycoproteins on the surface of the platelet, and along with platelet activation, the release of granules or structural changes in membrane glycoproteins occur, some of which are used as platelet activation markers.

[0003] Platelet factor IV (PF4) and β-thromboglobulin (βTG) are contained within the α-granules of platelets and are released upon platelet activation, so they have been used as platelet activation inhibitors for a long time. However, because substances within the granules are released even by mild stimulation such as blood collection, there are many restrictions on blood collection surgery and specimen processing, such as the cumbersome process of collecting blood without a tourniquet using a dedicated blood collection tube containing a platelet activation inhibitor cocktail and a thick lancet of 20 gauge (G) or larger, and then immediately cooling with ice. In addition, they are not widely used in clinical practice due to the reliability of the data.

[0004] In addition, recently, membrane glycoproteins cleaved by proteases accompanying platelet activation are gaining value as platelet activation markers. Specifically, von Willebrand factor (vWF) receptor GPIba and collagen receptor GPVI are cited as representative molecules, but knowledge regarding the clinical significance of any of them is lacking, and furthermore, because they are specific ligand-dependent release responses, it is unclear whether they accurately reflect the activated state of platelets.

[0005] CLEC-2 (C-type lectin-like receptor 2) is a platelet-activating receptor belonging to the C-type lectin family, identified by Inoue et al. in 2006 (Patent Documents 1, 2). Furthermore, recent research results have reported that CLEC-2 is released from the membrane surface accompanying platelet activation, becomes soluble, and is released (Non-patent Document 1, Patent Document 3). In addition, while literature measuring soluble CLEC-2 in several pathological conditions has been published, there are many unclear aspects regarding its clinical applications (Non-patent Document 2). Prior art literature

[0006] [Patent Document 1] Patent Publication No. 4961595 [Patent Document 2] Patent Publication No. 2007-070359 [Patent Document 3] Patent Publication No. 6078845

[0007] [Non-patent Literature 1] Platelets 2015; 26(8): 711-719 [Non-patent Literature 2] Thrombosis Research, 2019, Vol. 178, p. 54-58 The problem to be solved

[0008] The objective of the present invention is to develop a biomarker that better reflects platelet activation in vivo and to use it for testing thrombotic hemostatic diseases. means of solving the problem

[0009] The inventors have conducted a thorough examination to solve the above problem. As a result, they discovered that the previously reported concentration of soluble CLEC-2 in plasma (hereinafter referred to as sCLEC-2) reflects the degree of platelet activation in vivo, but it cannot necessarily provide an accurate diagnosis based on that alone. sCLEC-2 is a molecule released from platelets into the plasma when platelets are activated, and it was expected that measuring the concentration of sCLEC-2 in plasma would reflect the degree of platelet activation. However, when examining blood samples from various pathological conditions, it was found that while the concentration of sCLEC-2 in plasma reflects platelet activation to some extent, the concentration also exhibits a positive correlation with the number of platelets in the blood. The number of platelets in the blood varies from person to person and may increase or decrease depending on the pathological condition. It was discovered that the sCLEC-2 concentration in plasma decreases when the number of platelets is low even if platelets are activated, and conversely, increases when the number of platelets increases even if platelet activation is weak, so it may not necessarily reflect the degree of platelet activation in vivo. Therefore, by dividing the sCLEC-2 concentration in plasma by the number of platelets, the amount of sCLEC-2 released per platelet could be determined, and it was hypothesized that using this as an indicator might better reflect platelet activation, so an investigation was conducted. As a result, it was discovered that the ratio of sCLEC-2 concentration in plasma to the number of platelets reflects the pathological state of thrombotic disease more than the sCLEC-2 concentration in plasma itself, and it was found that this indicator can also be used for testing thrombostatic diseases. Based on the above findings, the present invention was completed.

[0010] The present invention provides the following:

[0011] [1] (1) A process for measuring the concentration of soluble CLEC-2 and the number of platelets in a test sample derived from the subject, and

[0012] (2) A method for assisting in determining platelet activation status, comprising a process for determining platelet activation status from the above-mentioned soluble CLEC-2 concentration and the above-mentioned platelet count.

[0013] [2] (1) A process for measuring the concentration of soluble CLEC-2 and the number of platelets in a test sample derived from the subject, and

[0014] (2) A method for assisting in determining platelet activation status, comprising a process of calculating the value of soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count.

[0015] [3] The method of [2] further comprises the step of comparing the value of soluble CLEC-2 concentration / platelet count obtained using a test sample derived from a normal healthy subject.

[0016] [4] The above available CLEC-2 concentration / platelet value is a marker reflecting the pathological condition of thrombostatic disease, [2] or [3] method.

[0017] [5] The above thrombostatic disease is any one of disseminated intravascular coagulation syndrome (DIC), myocardial infarction, angina pectoris, cerebral infarction, obstructive arteriosclerosis, deep vein thrombosis, pulmonary thromboembolism, cardiogenic cerebral infarction, antiphospholipid syndrome, or sepsis, [4] method.

[0018] [6] Any one of [1] to [5] methods for use as a method to monitor the prognosis or treatment progress of a patient with a thrombostatic disease.

[0019] [7] (1) A memory means capable of remembering the concentration of soluble CLEC-2 and the number of platelets in a test sample derived from the subject,

[0020] (2) A calculation means capable of calculating the value of soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count,

[0021] (3) a comparison means capable of comparing the value of the soluble CLEC-2 concentration / platelet count of the obtained subject with the value of the soluble CLEC-2 concentration / platelet count of a normal healthy subject, and

[0022] (4) A display means capable of displaying the result obtained by the above comparison,

[0023] A system for determining platelet activation status including

[0024] [8] A computer comprising a processor and a memory in control of the processor, wherein the memory includes the following process:

[0025] (1) A memory process for storing the concentration of soluble CLEC-2 and the number of platelets in a test sample derived from the subject in the memory,

[0026] (2) A calculation process for calculating the value of soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count,

[0027] (3) A comparison process for comparing the value of the soluble CLEC-2 concentration / platelet count obtained by the above calculation process with the value of the soluble CLEC-2 concentration / platelet count of a normal healthy subject, and

[0028] (4) A display process for displaying the results obtained from the above comparison process,

[0029] A system for determining the platelet activation state, having a program stored in the computer for executing the above.

[0030] [9] Electronic chart, clinical examination device, or in-hospital examination system equipped with the system of [7] or [8].

[0031]

[10] (1) Soluble CLEC-2 measuring reagent and / or platelet measuring reagent, and

[0032] (2) A kit for determining platelet activation status, comprising an attached document stating the relationship between the available CLEC-2 concentration / platelet count value and the platelet activation status. Effects of the invention

[0033] According to the present invention, the amount of sCLEC-2 released per platelet can be determined, and it becomes possible to accurately determine the activation status of platelets in the blood. By doing so, sufficient verification of the clinical utility of the sCLEC-2 concentration / platelet value becomes possible, and it is expected to be used as a marker reflecting the pathological condition of thrombostatic diseases. In particular, it is expected to be used as a marker in arterial thrombostatic diseases such as myocardial infarction, cerebral infarction, obstructive arteriosclerosis, disseminated intravascular coagulation syndrome (DIC), and sepsis, for which there are few effective tests available to use as markers. Brief explanation of the drawing

[0034] [Figure 1] This is a graph showing the correlation between platelet count (PLT) and sCLEC-2 concentration (sCLEC-2) in non-DIC cases in sepsis patients (n-DIC), DIC cases in sepsis patients (DIC), and pre-sepsis patients (pre-sepsis). [Figure 2] This is a graph showing sCLEC-2 concentration (sCLEC-2), platelet count (PLT), and sCLEC-2 concentration / platelet count (sCLEC-2 / PLT) in a normal healthy person, a non-DIC case (no DIC) in a septic patient, and a DIC case (with DIC) in a septic patient. [Fig. 3] ROC curves related to sCLEC-2 concentration (solid line) and sCLEC-2 concentration / platelet count (dotted line). Specific details for implementing the invention

[0035] The method of the present invention includes, but is not limited to, a method for measuring (or determining) soluble CLEC-2 concentration / platelet count, a method for assisting in identifying the platelet activation state, a method for diagnosing (or detecting) thrombostatic disease, a method for assisting in the diagnosis of thrombostatic disease, a method for measuring (or determining) soluble CLEC-2 concentration / platelet count for the diagnosis of thrombostatic disease, etc. Any method comprises (1) a process of measuring the soluble CLEC-2 concentration and platelet count in a test sample derived from a subject, and (2) a process of identifying the platelet activation state from the soluble CLEC-2 concentration and the platelet count. More specifically, the method comprises (1) a process of measuring the soluble CLEC-2 concentration and platelet count in a test sample derived from a subject, and (2) a value of soluble CLEC-2 concentration / platelet count obtained by dividing the soluble CLEC-2 concentration by the platelet count. The method includes a calculation process and, if desired, may further include a process of comparing the result with the value of soluble CLEC-2 concentration / platelet count obtained using a test sample derived from a normal healthy subject. The process of determining the platelet activation status from the soluble CLEC-2 concentration and platelet count may be carried out, for example, by calculating the value of soluble CLEC-2 concentration / platelet count and comparing it with the value of soluble CLEC-2 concentration / platelet count of a healthy subject, but the platelet activation status may also be determined integrally using the soluble CLEC-2 concentration and platelet count. The following describes examples using the value of soluble CLEC-2 concentration / platelet count, but the present invention is not limited to these examples.

[0036] In this specification, the term “CLEC-2” refers to a platelet-activating receptor belonging to the C-type lectin family, which is normally present on the membrane of a platelet but is released into the blood upon activation of the platelet. In this specification, the term “soluble CLEC-2 (sCLEC-2)” means CLEC-2 or a molecule derived from CLEC-2 that is released from such platelets and detected in the blood (in the buffer if incubated in a buffer).

[0037] sCLEC-2 includes proteins with a molecular weight of about 40 kDa, proteins with a molecular weight of about 32 kDa, proteins with a molecular weight of about 25 kDa, etc., in SDS-PAGE under reducing conditions.

[0038] Proteins with a molecular weight of approximately 40 kDa and proteins with a molecular weight of approximately 32 kDa are present on the surface of platelet membranes and are presumed to be released contained within microparticles produced in conjunction with platelet activation. It is thought that sugar chains are attached to these.

[0039] Meanwhile, it is thought that proteins with a molecular weight of about 25 kDa are cleaved by proteases upon platelet activation and released from platelets.

[0040] In the present invention, the amount of soluble CLEC-2 as described above is measured. sCLEC-2 may be detected by combining a protein with a molecular weight of about 40 kDa, a protein with a molecular weight of about 32 kDa, and a protein with a molecular weight of about 25 kDa, or only by detecting a protein with a molecular weight of about 25 kDa.

[0041] While it is preferable for the samples used for measurement to be of human origin, samples derived from animals other than humans may be used to assess the pathological condition of experimental animals. There are no specific restrictions on the experimental animals; examples include guinea pigs, rats, mice, and chinchillas.

[0042] The method of the present invention is also suitably used for the examination of thrombostatic diseases. As used herein, "hemostasis" means that platelets and coagulation factors jointly effectively and appropriately stop bleeding or hemorrhage. As used herein, "thrombostatic disease" includes, but is not limited to, conditions or diseases involving excessive bleeding or abnormal blood coagulation. Abnormal blood coagulation is associated with severe coronary insufficiency syndrome, myocardial infarction, unstable angina, refractory angina, thrombotic occlusion within the coronary artery after thrombolytic therapy, thrombotic occlusion within the coronary artery after coronary angioplasty, thrombotic vascular disease, cerebral infarction, embolic stroke, thrombotic stroke, transient cerebral ischemic attack, venous thrombosis, deep vein thrombosis, pulmonary embolism, coagulation disorders, disseminated intravascular coagulation syndrome, thrombotic microvascular disorder, thrombotic thrombocytopenic purpura, obstructive thromboangiitis, thrombosis associated with heparin-induced thrombocytopenia, thrombotic complications due to extracorporeal circulation, cardiac or other intravascular catheters, intra-aortic balloon pumps, and arteries It is associated with, but is not limited to, thrombotic complications caused by devices such as stents or heart valves, conditions requiring the installation of artificial organs, antiphospholipid antibody syndrome, and severe infections accompanied by thrombostatic abnormalities such as sepsis.

[0043] For example, compared to normal healthy individuals or groups with non-thrombotic hemostatic diseases, a high value of sCLEC-2 concentration / platelet count indicates a high likelihood of having a thrombotic hemostatic disease or a high risk of developing the disease. Based on this comparison, the correlation between the degree of platelet activation, expressed as the sCLEC-2 concentration / platelet count value, and various diseases can be used as standard data.

[0044] For example, in patients with myocardial infarction or cerebral infarction, the sCLEC-2 concentration and platelet count are measured, and if the ratio is high, it can be determined that platelet activation is occurring in vivo, and measures such as administering, increasing, or administering additional antiplatelet drugs of other types can be taken.

[0045] In addition, it is considered possible to use the method by measuring sCLEC-2 concentration and platelet count in high-risk patients for myocardial infarction or cerebral infarction, such as diabetic patients, and administering antiplatelet drugs such as aspirin as primary prevention to patients with high sCLEC-2 concentration / platelet count values. In severe infections accompanied by thrombostatic abnormalities, such as sepsis, it may also be used for diagnosis based on the degree of thrombostatic abnormality. Furthermore, in patients taking antiplatelet drugs such as aspirin or clopidogrel, it is possible to measure sCLEC-2 concentration and platelet count, and if the values ​​are high, consider increasing the dosage of the antiplatelet drug, changing to a different type of antiplatelet drug, or administering additional drugs.

[0046] In addition, there are reports that platelet microparticles are a factor that increases exacerbation in chronic rheumatoid arthritis, and since CLEC-2 is expressed on microparticles, there is a possibility that the sCLEC-2 concentration / platelet count may increase in patients with chronic rheumatoid arthritis, so it is also useful for testing chronic rheumatoid arthritis.

[0047] In addition, CLEC-2 has been reported to bind to a membrane protein called podoplanin, which is expressed in certain tumor cells, in vivo, thereby promoting metastasis. In patients with tumors expressing podoplanin, the tumor in the blood can activate platelets, which can increase the sCLEC-2 concentration / platelet count. Therefore, the sCLEC-2 concentration / platelet count has the potential to be used as a cancer metastasis marker.

[0048] The method for detecting the presence of sCLEC-2 is not particularly limited, but an immunological method using an antibody that recognizes sCLEC-2 (hereinafter referred to as an "anti-sCLEC-2 antibody") is preferred. As a method for immunologically detecting the protein, any known and commonly used method may be used, such as enzyme immunoassay (ELISA), chemiluminescent immunoassay, fluorescent antibody method, radioimmunoassay, immunochromatography, or other immunoassays using labeled antibodies, or Western blotting, latex agglutination, or immunoturbidimetry; however, among these, immunoassays using labeled antibodies are preferred due to their ease of operation and measurement precision. Since it is desirable to obtain results quickly for diagnosis during surgery, chemiluminescent immunoassay and immunochromatography are particularly preferred.

[0049] The platelet count is usually measured using an automatic hemocytometer (hemocytometer), but it is also possible to count using a hemocytometer and a microscope.

[0050] A specimen is collected from the subject (especially a patient) using, for example, a blood collection tube intended for plasma collection. While a blood collection tube containing EDTA is suitable for platelet count measurement, tubes containing heparin or citric acid are also acceptable. Samples for measuring sCLEC-2 concentration in plasma and platelet count may be obtained from a single blood collection tube, but if collected simultaneously, separate tubes may be used for each. Plasma sCLEC-2 concentration is measured using plasma centrifuged at 2000g for approximately 20 minutes, for example; however, centrifugation conditions are not limited to this, and a measuring system using whole blood may also be used. The following explanation uses the measurement of sCLEC-2 concentration in plasma as an example, but it is not limited to this. For blood platelet count, whole blood containing an anticoagulant such as EDTA is used.

[0051] The concentration of sCLEC-2 in plasma is expressed, for example, in pg / mL, and the number of platelets in blood is expressed, for example, in 1,000 / mm³, and the sCLEC-2 concentration / platelet count is calculated. The sCLEC-2 concentration used here may be expressed in any unit such as ng / mL or ng / L, and the number of platelets may be expressed in any unit such as 10,000 / mm³; however, unified units must be used for comparison. Although the sCLEC-2 concentration / platelet count may take various values ​​by using various units, they are essentially the same concept.

[0052] The ratio is calculated using the measurement from a clinical testing device that measures sCLEC-2 concentration and the measurement from a hemolytic analyzer that measures platelet count. While it is preferable for routine clinical practice to perform this calculation automatically on a system such as a hospital testing system, a hospital system, or an electronic chart connected to both measuring devices, it is also possible to establish a system that links the data from the two measuring devices, or to build a machine capable of simultaneously measuring sCLEC-2 concentration and platelet count. Alternatively, the calculation may be performed manually using both sets of data.

[0053] The standard data obtained in the present invention is not particularly limited as long as it indicates the correlation between the soluble CLEC-2 concentration / platelet count in plasma and the degree of platelet activation or various diseases, but examples include a threshold for determination, original data for calculating the threshold for determination, or statistical processing data. The standard data may be described in an attached document or attached separately as a data sheet. Furthermore, the form of the attached document includes paper, electronic media such as CD-ROMs, and downloads from websites, etc.

[0054] sCLEC-2 is released into the bloodstream in conjunction with platelet activation. While existing platelet activation markers (MICA), such as PF4 and βTG, have a problem in that their granules are stimulated by physical pressure during blood collection, leading to non-specific release, sCLEC-2 has a signal-transmission-dependent release mechanism that induces platelet activation and is thought to be a marker that more accurately reflects platelet activation in vivo. Furthermore, since CLEC-2 expression is almost exclusively limited to the platelet-megakaryotypal lineage in humans, it serves as a specific marker with low false positives.

[0055] Soluble GPVI, released through a similar mechanism, is strongly produced upon stimulation by a GPVI receptor-specific agonist, but its production is very weak upon stimulation by other known platelet-activating agonists. In other words, if the production amount varies depending on the type of stimulation, it becomes very difficult to interpret it as a marker. However, sCLEC-2 is produced at similar levels under stimulation by known agonists as well as specific agonists, making it an optimal molecule for quantitative markers of platelet activation.

[0056] One of the advantages of sCLEC-2 is that it is not subject to restrictions on blood collection procedures. That is, unlike PF4 and βTG, which are released by mild stimuli such as blood collection, sCLEC-2 is released through strong platelet activation, so it does not require special blood collection procedures. Furthermore, data has been obtained showing that the concentration of sCLEC-2 in the plasma of patients with acute coronary syndrome is significantly higher than that of normal healthy individuals. This gives it high value as a monitor for predicting onset and prognosis or evaluating treatment efficacy, and it is considered a marker that is dramatically superior compared to conventional activation markers. Additionally, sCLEC-2 concentrations are elevated in the plasma of patients with cerebral infarction, and it indicates that patients with high plasma sCLEC-2 concentrations have a poor prognosis.

[0057] In the present invention, it was discovered that the concentration of sCLEC-2 in the plasma has a positive correlation with the number of platelets in the blood. That is, in individuals with a high number of platelets, the concentration of sCLEC-2 in the plasma tends to be high, while in individuals with a low number of platelets, the concentration of sCLEC-2 in the plasma tends to be low. As a result, the concentration of sCLEC-2 in the plasma is affected by the number of platelets in the blood, so there are cases where platelet activation does not necessarily occur.

[0058] Therefore, by dividing the sCLEC-2 concentration in plasma by the number of platelets in blood to calculate the sCLEC-2 release amount per platelet and using this as an indicator to diagnose thrombotic disease, the degree of platelet activation can be evaluated without relying on the number of platelets in blood. Specifically, for example, the sCLEC-2 concentration in plasma is expressed as pg / mL (A), the number of platelets in blood is expressed as 1000 / mm² (B), and the number obtained by dividing A by B is used as an indicator of platelet activation.

[0059] Disseminated intravascular coagulation syndrome (DIC) is a typical thrombostatic disorder involving platelet activation. In patients with this disease, the number of platelets decreases along with platelet activation. This pathological condition was examined using both plasma sCLEC-2 concentration and sCLEC-2 concentration / platelet count in a DIC group complicated by sepsis (patients with sepsis having DIC) and a non-DIC group (patients without sepsis). The results showed that sCLEC-2 concentration / platelet count demonstrated high diagnostic efficiency for DIC. Specifically, while sCLEC-2 concentration / platelet count showed significantly higher values ​​in the DIC group, plasma sCLEC-2 concentration was higher in the DIC group compared to the non-DIC group, but no statistically significant difference was found. Furthermore, in the DIC group, sCLEC-2 concentration / platelet count also showed a significant correlation with the severity of the DIC condition. Therefore, it can be concluded that sCLEC-2 concentration / platelet count can serve as a much more efficient and useful diagnostic indicator compared to plasma sCLEC-2 concentration. Additionally, when compared to data from normal healthy individuals, sepsis Patients showed high sCLEC-2 concentration / platelet ratios regardless of the presence or absence of DIC. Therefore, it can be said that it is a useful diagnostic indicator for sepsis, a severe infection accompanied by thrombotic hemostasis abnormalities.

[0060] This indicator is a more efficient and useful indicator compared to sCLEC-2 concentration because it can determine platelet activity without being affected by the number of platelets in the blood, even in thrombostatic diseases caused by platelet thrombosis such as acute coronary syndrome (angina pectoris, myocardial infarction) and cerebral infarction.

[0061] The present invention includes a system that can be used to carry out the method of the present invention described above, an electronic chart equipped with said system, a clinical examination device or an in-hospital examination system, a kit that can be used to carry out the method of the present invention, etc.

[0062] The system of the present invention includes, for example, a system for measuring (or determining) the soluble CLEC-2 concentration / platelet count, a system for determining the platelet activation state, a system for assisting in the diagnosis of thrombostatic diseases, a system for diagnosing (or detecting) thrombostatic diseases, a system for measuring (or determining) the soluble CLEC-2 concentration / platelet count for the diagnosis of thrombostatic diseases, etc.

[0063] Any system of the present invention may include (1) a memory means capable of storing the soluble CLEC-2 concentration and the number of platelets in a test sample derived from a test subject, (2) a calculation means capable of calculating the value of the soluble CLEC-2 concentration / number of platelets by dividing the soluble CLEC-2 concentration by the number of platelets, (3) a comparison means capable of comparing the value of the soluble CLEC-2 concentration / number of platelets obtained from the test subject with the value of the soluble CLEC-2 concentration / number of platelets of a normal healthy subject, and (4) a display means capable of displaying the result obtained by the comparison.

[0064] Alternatively, any system of the present invention may include a computer comprising a processor and a memory under the control of said processor, and said memory may have a program stored in the memory for executing the following steps: (1) a memory process for storing the soluble CLEC-2 concentration and the number of platelets in a sample of a subject of examination in said memory; (2) a calculation process for calculating the value of the soluble CLEC-2 concentration / number of platelets by dividing the soluble CLEC-2 concentration by the number of platelets; (3) a comparison process for comparing the value of the soluble CLEC-2 concentration / number of platelets obtained by said calculation process with the value of the soluble CLEC-2 concentration / number of platelets of a normal healthy subject; and (4) a display process for displaying the result obtained by said comparison process.

[0065] The electronic chart, clinical examination device, or in-hospital examination system of the present invention may be equipped with the system of the present invention.

[0066] The method of the present invention may be carried out using the system of the present invention, or the electronic chart, clinical examination device, or in-hospital examination system of the present invention (hereinafter referred to as the system, etc. of the present invention). Regarding the "value of soluble CLEC-2 concentration / platelet count" that can be measured (or determined) using the system, etc. of the present invention, the "condition, disease, etc." that can be identified using the system, etc. of the present invention, the "judgment, judgment criteria, etc." thereof, and the measurement methods, etc. of "soluble CLEC-2 concentration and platelet count," the description above regarding the method of the present invention may be applied as is.

[0067] The kit of the present invention includes, for example, a kit for measuring (or determining) soluble CLEC-2 concentration / platelet count, a kit for determining platelet activation status, a kit for diagnosing (or detecting) thrombostatic disease, a kit for measuring (or determining) soluble CLEC-2 concentration / platelet count for diagnosing thrombostatic disease, etc.

[0068] The kit of the present invention may include (1) a soluble CLEC-2 measuring reagent and / or a platelet measuring reagent, and (2) an attached document describing the correlation between the soluble CLEC-2 concentration / platelet count value and the platelet activation status.

[0069] It is also important to include the method for calculating sCLEC-2 concentration / platelet count in the attached document above. By using this kit, both the sCLEC-2 concentration in plasma and the sCLEC-2 concentration / platelet count can be used to evaluate platelet activation or to diagnose hemostatic diseases. By calculating the sCLEC-2 concentration / platelet count using the above reagent kit, diseases such as platelet activation disorders or thrombosis can be detected simply and quickly when necessary, and the results can be helpful in differentiating them from other diseases or determining treatment guidelines.

[0070] In addition, the attached document included in the kit of the present invention is not particularly limited, provided that it mentions the relationship between the sCLEC-2 concentration / platelet count in plasma and the degree of platelet activation or various diseases. In addition to the above mention, it may include, for example, an explanation regarding the procedure for performing immunological measurements using the kit of the present invention, an explanation regarding the procedure for detecting the sCLEC-2 concentration / platelet count in plasma and the degree of platelet activation or various diseases based on the obtained measurements, and precautions regarding the preservation and handling of the kit itself.

[0071] [Example]

[0072] The present invention will be specifically described below by way of examples, but these do not limit the scope of the present invention.

[0073] Example 1: Measurement of sCLEC-2 concentration in human plasma

[0074] According to Example 6 of Patent No. 6078845, the sCLEC-2 concentration (pg / mL) in human plasma was measured.

[0075] A sandwich ELISA system was constructed using mouse anti-human CLEC-2 monoclonal antibodies (1-11D5, 3-11E6 antibodies) prepared in the example of Patent Publication No. 6078845. Specifically, 1-11D5 antibody (F(ab)'2), purified with 0.05 mol / L carbonic acid buffer (pH 9.5), was diluted to 10 μg / mL and added to an immunoplate (Imnoplate, Maxisorp, NUNC) at a rate of 100 μL / well. After reacting overnight at 4°C, the plates were washed three times with boric acid-buffered saline (BBS) containing 0.05% Tween-20, and 200 μL of phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) was added to each well and blocked. Next, as a 'sample' and 'standard sample,' the human CLEC-2 (hCLEC-2) protein prepared in Example 3 of Patent Publication No. 6078845 was diluted using 10% SuperBlock, 0.1% sodium octanoate, and 0.14 mol / L sodium chloride / PBS. In addition, when plasma was used as the measurement sample, it was diluted by more than 5 times, added at a rate of 100 μL / well, and shaken at 37 ℃ for 1.5 hours ( After reacting while ), the wells were washed three times in the same manner. The Biotin-labeled 3-11E6 antibody (F(ab)'2-Biotin) prepared in Example 5 of Patent Publication No. 6078845 was diluted to 1.0 μg / mL with 10% SuperBlock, 0.1% sodium octanoate, and 0.14 mol / L sodium chloride / PBS, and 100 μL was added to each well. The wells were reacted at 37°C for 1 hour while shaking, and then washed three times in the same manner. Next, AMDEX streptavidin-conjugated horseradish peroxidase (GE Healthcare) was diluted with 10% SuperBlock, 0.1% sodium octanoate, and 0.14 mol / L sodium chloride / PBS, and 100 μL was added to each well. After reacting with shaking at 37°C for 1 hour, the plates were washed 5 times in the same manner, and 100 μL of tetramethylbenzidine (TMB) solution was added to each well. After reacting at room temperature for about 20 minutes, the reaction was stopped with 2 N sulfuric acid solution. The absorbance at 450 nm (-620 nm) was measured using a plate spectrophotometer (BIO-TEK INSTRUMENTS / EL312e).

[0076] Example 2: Measurement of Platelet Count in Human Whole Blood

[0077] The number of platelets in human whole blood was measured using a Sysmex automated blood cell counter (XN-550). The platelet count was expressed as 1,000 cells / mm³.

[0078] 《Example 3: Calculation of sCLEC-2 Concentration / Platelet Count》

[0079] Table 1 shows the measured values ​​of sCLEC-2 concentration in Example 1, the platelet count measured in Example 2, and the sCLEC-2 concentration / platelet count calculated from these values. The measurement specimens were 32 septic cases (20 cases with disseminated intravascular coagulation syndrome (DIC) and 12 cases without DIC), diagnosed according to the acute phase DIC diagnostic criteria of the Japanese Society of Emergency Medicine. In addition, each measurement value was taken from specimens collected on the first day of hospitalization. Furthermore, the sCLEC-2 concentration in the plasma and the blood platelet count of 37 normal healthy individuals were measured, and the sCLEC-2 concentration / platelet count was calculated. The mean ± standard deviation of sCLEC-2 concentration, platelet count, and sCLEC-2 concentration / platelet count in normal healthy people were 87 ± 38.9 (pg / mL), 263 ± 63.4 (1000 plates / mm3), and 0.34 ± 0.15, respectively.

[0080]

[0081] For pre-septic patients, cases of DIC in septic patients (DIC), and cases of non-DIC in septic patients (n-DIC), the correlation between the measured sCLEC-2 concentration (sCLEC-2) in Example 1 and the platelet count (PLT) measured in Example 2 was investigated, and a statistically significant correlation was observed between the sCLEC-2 concentration and the platelet count (Fig. 1). When the values ​​of sCLEC-2 concentration / platelet count calculated from the sCLEC-2 concentration and platelet count were calculated and compared between cases of DIC in septic patients and cases of non-DIC in septic patients, no significant difference was observed between the two groups in sCLEC-2 concentration (p = 0.25), but a significant difference (p = 0.0008) was confirmed between the two groups in sCLEC-2 concentration / platelet count (Fig. 2). Through this, it was confirmed that sCLEC-2 was affected not only by platelet activation but also by the platelet count, and that a higher sCLEC-2 level per platelet count indicated greater platelet activation. Additionally, since sCLEC-2 concentration and sCLEC-2 concentration / platelet ratio showed higher values ​​than in healthy individuals in cases of sepsis, regardless of the presence or absence of DIC, these results were shown to be useful for the diagnosis of sepsis.

[0082] Example 4: Evaluation as a Biomarker Based on ROC Curve

[0083] Using the measured and calculated values ​​obtained in Example 3, a Receiver Operatorating Characteristic (ROC) curve was constructed, and the Area Under the Curve (AUC) was calculated. Figure 3 shows the results for sCLEC-2 concentration (solid line) and sCLEC-2 concentration / platelet count (dotted line). The AUC values ​​for the ROC curves were 0.628 for sCLEC-2 concentration, 0.866 for sCLEC-2 concentration / platelet count, and 0.732 for platelet count. It is considered that the closer the AUC is to 1.0, the higher the diagnostic capability, and sCLEC-2 concentration / platelet count showed the best diagnostic capability.

[0084] The present invention can be used to determine the platelet activation status and to test for thrombotic hemostatic diseases, etc.

Claims

Claim 1 (1) a process for measuring the concentration of soluble CLEC-2 and the number of platelets in a sample of a test subject, wherein the sample for measuring the concentration of soluble CLEC-2 is plasma or whole blood and the sample for measuring the number of platelets is whole blood; and 2) a process for determining the platelet activation state from the concentration of soluble CLEC-2 and the number of platelets, the method of assisting in determining the platelet activation state. Claim 2 (1) A process for measuring the soluble CLEC-2 concentration and platelet count in a sample of a subject of examination, wherein the sample for measuring the soluble CLEC-2 concentration is plasma or whole blood and the sample for measuring the platelet count is whole blood; and (2) A process for calculating the value of soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count, a method for assisting in determining the platelet activation state. Claim 3 The method of claim 2, further comprising the step of comparing with the value of soluble CLEC-2 concentration / platelet count obtained using a test sample derived from a healthy subject. Claim 4 A method according to claim 2, wherein the value of the soluble CLEC-2 concentration / platelet count is a marker reflecting the pathological state of a thrombostatic disease. Claim 5 A method according to claim 4, wherein the thrombostatic disease is any one of disseminated intravascular coagulation syndrome (DIC), myocardial infarction, angina pectoris, cerebral infarction, obstructive arteriosclerosis, deep vein thrombosis, pulmonary thromboembolism, cardiogenic cerebral infarction, antiphospholipid syndrome, and sepsis. Claim 6 A method according to claim 3, wherein the value of the soluble CLEC-2 concentration / platelet count is a marker reflecting the pathological state of a thrombostatic disease. Claim 7 A method according to claim 6, wherein the thrombostatic disease is any one of disseminated intravascular coagulation syndrome (DIC), myocardial infarction, angina pectoris, cerebral infarction, obstructive arteriosclerosis, deep vein thrombosis, pulmonary thromboembolism, cardiogenic cerebral infarction, antiphospholipid syndrome, and sepsis. Claim 8 A method according to any one of claims 1 to 7, for use as a method for monitoring the prognosis or treatment progress of a patient with a thrombostatic disease. Claim 9 (1) a memory means capable of storing the soluble CLEC-2 concentration and platelet count in a sample of a subject, wherein the sample for measuring the soluble CLEC-2 concentration is plasma or whole blood and the sample for measuring the platelet count is whole blood; (2) a calculation means capable of calculating the value of the soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count; (3) a comparison means capable of comparing the value of the soluble CLEC-2 concentration / platelet count of the obtained subject with the value of the soluble CLEC-2 concentration / platelet count of a normal healthy subject; and (4) a display means capable of displaying the result obtained by the comparison, a system for determining the platelet activation state. Claim 10 A system for determining platelet activation status, comprising a computer including a processor and a memory in control of the processor, wherein the memory has a program recorded therein for executing the following processes: (1) a memory process for storing the soluble CLEC-2 concentration and platelet count in a test sample derived from a test subject in the memory, wherein the sample for measuring the soluble CLEC-2 concentration is plasma or whole blood and the sample for measuring the platelet count is whole blood; (2) a calculation process for calculating the value of soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count; (3) a comparison process for comparing the value of soluble CLEC-2 concentration / platelet count obtained from the calculation process with the value of soluble CLEC-2 concentration / platelet count of a normal healthy subject; and (4) a display process for displaying the result obtained from the comparison process. Claim 11 A system according to claim 9 or claim 10, wherein the system is an electronic chart, a clinical examination device, or an in-hospital examination system. Claim 12 A kit for determining platelet activation status comprising (1) a soluble CLEC-2 measuring reagent, and (2) an attached document stating the correlation between the soluble CLEC-2 concentration / platelet count value and the platelet activation status.

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