Method for platelet activation assay based on soluble CLEC-2 and platelet count

Abstract

The present application develops a biomarker that better reflects platelet activation in vivo, and provides a method for assisting in grasping the state of platelet activation that can be used for the examination of thrombotic hemostatic diseases, etc. The method for assisting in grasping the state of platelet activation includes: (1) a process of measuring the concentration of soluble CLEC-2 and the number of platelets in a test sample from a test subject; (2) a process of calculating the value of the concentration of soluble CLEC-2 / number of platelets by dividing the concentration of soluble CLEC-2 by the number of platelets; and (3) a process of comparing the value of the concentration of soluble CLEC-2 / number of platelets with the value of the concentration of soluble CLEC-2 / number of platelets obtained using a test sample from a healthy subject, as needed.

Description

Technical Field

[0001] The present invention relates to a platelet activation determination method based on soluble CLEC-2 and platelet count and a method for detecting thrombotic hemostatic diseases. Background Art

[0002] Thrombosis formed in blood vessels is considered a major cause of life and death in a wide range of human diseases. Platelet-induced thrombus enlargement or increased platelet aggregation can lead to conditions such as severe myocardial infarction, chronic unstable angina, transient ischemic stroke, peripheral vascular disease, arterial thrombosis, occlusive arteriosclerosis, pulmonary thromboembolism, and in-stent restenosis. In developed countries, coronary artery occlusion or cerebral circulation disorders caused by thrombosis are recognized as the leading cause of death, making the treatment of thrombosis a pressing issue worldwide. Furthermore, during natural disasters, people are urged to prevent deep vein thrombosis and pulmonary embolism (a condition in which venous thrombosis travels with the bloodstream and blocks the pulmonary arteries) during disaster evacuation, and preventive measures are also receiving attention. Platelets contain granules and a large number of membrane glycoproteins on their surface. Platelet activation leads to the release of granules and structural changes in membrane glycoproteins, some of which are used as platelet activation markers.

[0003] Platelet factor 4 (PF4) and β-thromboglobulin (βTG) are contained in platelet α-granules and released upon platelet activation, thus they have long been used as platelet activation markers. However, since substances in the granules are released even by mild stimulation, such as during blood collection, there are many restrictions on blood collection and sample handling. For example, they require the use of specialized blood collection tubes containing a mixture of platelet activation inhibitors and thicker blood collection needles of 20 gauge or greater, as well as the cumbersome process of immediately cooling blood after collection without the use of a tourniquet. Furthermore, due to concerns about data reliability, these methods are rarely used in clinical practice.

[0004] Furthermore, in recent years, the use of membrane glycoproteins, which are cleaved by proteases as platelets activate, as markers of platelet activation has been increasing. Specifically, representative molecules include the von Willebrand factor (vWF) receptor GPIba and the collagen receptor GPVI. However, little is known about their clinical significance. Furthermore, since these molecules rely on the release of certain ligands, their ability to accurately reflect the activation state of platelets remains unclear.

[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 and 2). Furthermore, recent research has shown that CLEC-2 is released from the platelet membrane surface upon platelet activation, becoming soluble and being released (Non-Patent Document 1, Patent Document 3). Furthermore, although literature has been published on the measurement of soluble CLEC-2 in several disease states, its clinical application remains largely unknown (Non-Patent Document 2).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent No. 4961595

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 2007-070359

[0010] Patent Document 3: Japanese Patent No. 6078845

[0011] Non-patent literature

[0012] Non-patent literature 1: Platelets 2015; 26(8): 711-719

[0013] Non-Patent Literature 2: Thrombosis Research, 2019, Vol. 178, pp. 54-58 Summary of the Invention

[0014] Technical Problems to be Solved by the Invention

[0015] The technical problem of the present invention is to develop a biomarker that better reflects platelet activation in vivo and use it for the examination of thrombotic hemostatic diseases, etc.

[0016] Technical means to solve technical problems

[0017] The inventors of the present application conducted in-depth research to solve the above-mentioned technical problems. They found that while the reported concentration of soluble CLEC-2 (hereinafter sometimes referred to as sCLEC-2) in plasma can reflect the degree of platelet activation in an organism, accurate diagnosis may not be possible based solely on this concentration. sCLEC-2 is a molecule released from platelets into plasma upon platelet activation, and it was expected that measuring the concentration of sCLEC-2 in plasma would reflect the degree of platelet activation. However, studies using blood from various conditions revealed that while the concentration of sCLEC-2 in plasma can reflect platelet activation to a certain extent, this concentration is also positively correlated with the number of platelets in the blood. The number of platelets in the blood varies from person to person and sometimes increases or decreases depending on the condition. When platelets are activated but the platelet count is low, the plasma sCLEC-2 concentration decreases. Conversely, when platelet activation is weak but the platelet count increases, the plasma sCLEC-2 concentration increases. This finding suggests that the plasma sCLEC-2 concentration may not necessarily reflect the degree of platelet activation in an organism. Under these circumstances, the inventors of this application realized that dividing the plasma sCLEC-2 concentration by the platelet count could reveal the amount of sCLEC-2 released per platelet, and that using this as an indicator could better reflect platelet activation, leading to in-depth research. They found that the ratio of plasma sCLEC-2 concentration to platelet count is more reflective of thrombotic disease than the plasma sCLEC-2 concentration itself, and that this indicator can also be used to detect thrombotic hemostatic diseases. Based on these findings, the present invention was completed.

[0018] The present invention provides the following solutions:

[0019] [1] A method for assisting in understanding the activation state of platelets, comprising:

[0020] (1) a step of measuring the concentration of soluble CLEC-2 and the number of platelets in a test sample from a subject; and

[0021] (2) A step of determining the platelet activation state based on the soluble CLEC-2 concentration and the platelet count.

[0022] [2] A method for assisting in understanding the activation state of platelets, comprising:

[0023] (1) a step of measuring the concentration of soluble CLEC-2 and the number of platelets in a test sample from a subject; and

[0024] (2) A step of dividing the soluble CLEC-2 concentration by the platelet count to calculate the value of soluble CLEC-2 concentration / platelet count.

[0025] [3] The method according to [2], further comprising: comparing the value of soluble CLEC-2 concentration / platelet count with a test sample obtained from a healthy subject.

[0026] [4] The method according to [2] or [3], wherein the soluble CLEC-2 concentration / platelet value is a marker reflecting the condition of a thrombotic hemostatic disease.

[0027] [5] The method according to [4], wherein the thrombotic hemostatic disease is any one of disseminated intravascular coagulation (DIC), myocardial infarction, angina pectoris, cerebral infarction, arteriosclerosis obliterans, deep vein thrombosis, pulmonary thromboembolism, cardiogenic cerebral infarction, antiphospholipid syndrome, and sepsis.

[0028] [6] The method according to any one of [1] to [5], wherein the method is used as a method for monitoring the prognosis or treatment process of a patient with a thrombotic hemostatic disease.

[0029] [7] A system for understanding the activation state of platelets, comprising:

[0030] (1) a recording device capable of recording the concentration of soluble CLEC-2 and the number of platelets in a test sample from a subject;

[0031] (2) a calculation mechanism capable of calculating the value of soluble CLEC-2 concentration / platelet number by dividing the soluble CLEC-2 concentration by the platelet number;

[0032] (3) a comparison mechanism capable of comparing the obtained value of the soluble CLEC-2 concentration / platelet count of the test subject with the value of the soluble CLEC-2 concentration / platelet count of a healthy subject; and

[0033] (4) A display unit capable of displaying the result obtained by the comparison.

[0034] [8] A system for understanding the activation state of platelets, comprising a computer including a processor and a memory under the control of the processor, wherein the memory stores a program for causing the computer to execute the following steps:

[0035] (1) a recording step of recording the soluble CLEC-2 concentration and platelet count in a test sample from a subject in the memory;

[0036] (2) a calculation step of dividing the soluble CLEC-2 concentration by the platelet count to calculate a value of soluble CLEC-2 concentration / platelet count;

[0037] (3) a step of comparing the soluble CLEC-2 concentration / platelet count value obtained in the calculation step with the soluble CLEC-2 concentration / platelet count value of a healthy subject; and

[0038] (4) A display step of displaying the result obtained through the comparison step.

[0039] [9] An electronic medical record, clinical examination device or in-hospital examination system comprising the system described in [7] or [8].

[0040]

[10] A kit for determining the activation state of platelets, comprising:

[0041] (1) soluble CLEC-2 assay reagent and / or platelet assay reagent; and

[0042] (2) Instructions describing the correlation between the value of soluble CLEC-2 concentration / platelet number and the platelet activation state.

[0043] Effects of the Invention

[0044] According to the present invention, the amount of sCLEC-2 released per platelet can be determined, allowing accurate assessment of the platelet activation state in the blood. This allows for thorough verification of the correlation between sCLEC-2 concentration and platelet count and clinical efficacy, and is expected to be used as a marker for thrombotic hemostatic diseases, particularly arterial thrombotic hemostatic diseases such as myocardial infarction, cerebral infarction, arteriosclerosis obliterans, disseminated intravascular coagulation (DIC), and sepsis, for which there are few effective marker tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Graph showing the correlation between platelet count (PLT) and sCLEC-2 concentration (sCLEC-2) in sepsis patients without DIC (n-DIC), sepsis patients with DIC (DIC), and all sepsis patients (all sepsis).

[0046] Figure 2 Graphs showing sCLEC-2 concentration (sCLEC-2), platelet count (PLT), and sCLEC-2 concentration / platelet count (sCLEC-2 / PLT) in healthy individuals, patients with sepsis who did not develop DIC (without DIC), and patients with sepsis who developed DIC (with DIC).

[0047] Figure 3 The ROC curves are related to sCLEC-2 concentration (solid line) and sCLEC-2 concentration / platelet number (dashed line). DETAILED DESCRIPTION

[0048] The methods of the present invention include but are not limited to the following methods, namely, a method for determining (or measuring) the soluble CLEC-2 concentration / platelet count, a method for assisting in understanding the activation state of platelets, a method for diagnosing (or detecting) thrombotic hemostatic diseases, a method for assisting in diagnosing thrombotic hemostatic diseases, a method for determining (or measuring) the soluble CLEC-2 concentration / platelet count for the purpose of diagnosing thrombotic hemostatic diseases, etc., all of which comprise: (1) determining the soluble CLEC-2 concentration and platelet count in a test sample from a subject; (2) determining the platelet count based on the soluble CLEC-2 concentration and platelet count in the test sample; and (3) determining the platelet count based on the platelet count. The process of determining the platelet activation state based on the soluble CLEC-2 concentration and the platelet count, more specifically, includes: (1) measuring the soluble CLEC-2 concentration and platelet count in a test sample from a subject; and (2) calculating the value of soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count. If necessary, the process may further include comparing the value of soluble CLEC-2 concentration / platelet count with the value of soluble CLEC-2 concentration / platelet count obtained using a test sample from a healthy subject. The process of determining the platelet activation state based on the soluble CLEC-2 concentration and platelet count can be implemented, 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. Alternatively, the platelet activation state can be comprehensively determined using the soluble CLEC-2 concentration and platelet count. Hereinafter, the description will be mainly made by taking as an example a scheme using the value of soluble CLEC-2 concentration / platelet number, but the present invention is not limited to this scheme.

[0049] As used herein, the term "CLEC-2" refers to a platelet-activating receptor belonging to the C-type lectin family, which is normally present on the platelet membrane but released into the blood upon platelet activation. As used herein, the term "soluble CLEC-2 (sCLEC-2)" refers to CLEC-2 or molecules derived from CLEC-2 that are released from platelets and detected in blood (or, in a buffer when cultured in a buffer).

[0050] sCLEC-2 includes proteins with a molecular weight of about 40 kDa, about 32 kDa, and about 25 kDa when subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.

[0051] It is estimated that proteins with a molecular weight of approximately 40 kDa and proteins with a molecular weight of approximately 32 kDa are present on the platelet membrane surface and are released in the form of microparticles generated with platelet activation. These proteins are believed to have sugar chains attached to them.

[0052] On the other hand, it is believed that a protein with a molecular weight of approximately 25 kDa is cleaved by proteases as platelets are activated and released from the platelets.

[0053] In the present invention, the amount of soluble CLEC-2 is measured. Regarding sCLEC-2, 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 can be detected in combination, or only the protein with a molecular weight of about 25 kDa can be detected.

[0054] The sample used in the measurement is preferably human, but samples from animals other than humans may also be used to understand the disease condition of the experimental animal. The experimental animal is not particularly limited, and examples thereof include guinea pigs, rats, mice, and chinchillas.

[0055] The method of the present invention is also preferably used to examine thrombotic hemostatic disorders. "Hemostasis," as used herein, refers to the effective and appropriate prevention of bleeding or hemorrhage through the interaction of platelets and coagulation factors. "Thrombotic hemostatic disorders," as used herein, include, but are not limited to, conditions or diseases involving excessive bleeding or abnormal blood coagulation. Abnormal blood coagulation is associated with, but is not limited to, severe coronary insufficiency syndrome, myocardial infarction, unstable angina, refractory angina, coronary embolism after thrombolytic therapy, coronary embolism after coronary angioplasty, cerebrovascular disease caused by thrombosis, cerebral infarction, embolic stroke, thrombotic stroke, transient ischemic attack, venous thrombosis, deep vein thrombosis, pulmonary embolism, coagulopathy, disseminated intravascular coagulation syndrome, thrombotic microangiopathy, thrombotic thrombocytopenic purpura, occlusive thromboangiitis, thrombosis associated with heparin-induced thrombocytopenia, thrombotic complications caused by extracorporeal circulation, thrombotic complications caused by devices such as cardiac or other intravascular catheters, intra-aortic balloon pumps, arterial stents, or heart valves, and conditions requiring implantation of artificial organs, antiphospholipid antibody syndrome, and severe infections associated with abnormal thrombotic hemostasis such as sepsis.

[0056] For example, if the sCLEC-2 concentration / platelet count ratio is higher than that of healthy individuals or a group without a thrombotic hemostatic disease, it can be said that the likelihood of having a thrombotic hemostatic disease or the risk of developing a thrombotic hemostatic disease is higher. Based on this comparison, the correlation between the degree of platelet activation, as reflected by the sCLEC-2 concentration / platelet count ratio, and various diseases can be used as standardized data.

[0057] To give a specific example, the sCLEC-2 concentration and platelet count are measured in patients with myocardial infarction or cerebral infarction. If the ratio is high, it can be determined that platelet activation has occurred in the body, and countermeasures such as the administration of antiplatelet drugs, increased or additional administration of different types of antiplatelet drugs can be formulated.

[0058] In addition, it is believed that the following usage can also be implemented: sCLEC-2 concentration and platelet count can be measured in patients at high risk of myocardial infarction or cerebral infarction, such as diabetic patients, and antiplatelet drugs such as aspirin can be administered as primary prevention to patients with high sCLEC-2 concentration / platelet count ratios. For serious infections such as sepsis accompanied by thrombotic hemostatic abnormalities, it can also be used to diagnose them based on the degree of thrombotic hemostatic abnormalities. Furthermore, sCLEC-2 concentration and platelet count can be measured in patients taking antiplatelet drugs such as aspirin and clopidogrel. If the values ​​are high, it is also possible to consider increasing the antiplatelet drug dosage, switching to a different type of antiplatelet drug, or implementing additional medication.

[0059] Furthermore, there are reports that platelet microparticles can be a contributing factor to the progression of chronic rheumatoid arthritis. Since CLEC-2 is also expressed on microparticles, the sCLEC-2 concentration / platelet count may be elevated in patients with chronic rheumatoid arthritis, making this method potentially useful for the detection of chronic rheumatoid arthritis.

[0060] Furthermore, there are reports that CLEC-2 binds to a membrane protein called podoplanin, which is expressed in certain tumor cells, and promotes tumor metastasis. In patients with podoplanin-expressing tumors, the tumor in the blood may activate platelets, leading to increased sCLEC-2 concentrations and platelet counts. Therefore, sCLEC-2 concentrations and platelet counts may be useful as cancer metastasis markers.

[0061] 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 sometimes referred to as an "anti-sCLEC-2 antibody") is preferred. As immunological methods for detecting proteins, any method using a labeled antibody, such as enzyme immunoassay (ELISA), chemiluminescent immunoassay, fluorescent antibody method, radioimmunoassay, or immunochromatography, or a commonly known method such as Western blotting, latex agglutination, or immunoturbidimetry, can be used. However, from the perspective of operational simplicity and measurement accuracy, immunoassays using labeled antibodies are preferred. Rapid results are desirable for intraoperative diagnosis, and therefore, chemiluminescent immunoassays or immunochromatography are particularly preferred.

[0062] The platelet count is usually measured using an automatic blood cell counter (hemocytometer), but can also be counted using a hemocytometer and a microscope.

[0063] For example, a specimen is collected from a subject (especially a patient) using a blood collection tube for plasma collection. If the platelet count is also taken into account, a blood collection tube with the addition of EDTA is preferably used, but a blood collection tube with the addition of heparin or citric acid may also be used. A single blood collection tube can be used to obtain samples for measuring the sCLEC-2 concentration in plasma and for measuring the platelet count, but if blood is collected at the same time, respective blood collection tubes may also be used. The sCLEC-2 concentration in plasma can be measured using, for example, plasma centrifuged at 2000g for about 20 minutes, but the centrifugation conditions are not limited thereto, and a measurement system using whole blood may also be used. The following will be described using the determination of the sCLEC-2 concentration in plasma as an example, but is not limited thereto. The platelet count in the blood is measured using whole blood to which an anticoagulant such as EDTA has been added.

[0064] 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 1000 / mm 3 The sCLEC-2 concentration used here can be expressed as ng / mL, ng / L, or any other unit, and the platelet count can be expressed as 10,000 / mm 3 Although arbitrary units such as sCLEC-2 and platelet count can be used, they should be used consistently for comparison. Using various units can result in different values ​​for sCLEC-2 concentration / platelet count, but the concept is essentially the same.

[0065] The ratio is calculated using the values ​​obtained by a clinical testing device for measuring sCLEC-2 concentration and the values ​​obtained by a blood cell counter for measuring platelet count. From the perspective of routine diagnosis and treatment, this calculation is preferably performed automatically on a hospital's testing system, hospital system, or electronic medical record system that is connected to the two measuring devices. However, a system that connects the data from the two measuring devices can also be constructed, and a device that can simultaneously measure sCLEC-2 concentration and platelet count can also be constructed. Alternatively, manual calculation can be performed using these two sets of data.

[0066] The standard data obtained in the present invention is not particularly limited as long as it indicates the correlation between the plasma soluble CLEC-2 concentration / platelet count and the degree of platelet activation or various diseases. Examples thereof include threshold values ​​for determination, raw data used to calculate the threshold values ​​for determination, or statistically processed data. The standard data may be recorded in the instructions or attached separately as a data sheet. The attached instructions may be in the form of paper, electronic media such as CD-ROMs, or downloaded from a homepage.

[0067] sCLEC-2 is released into the blood upon platelet activation. Existing platelet activation markers, such as PF4 and βTG, suffer from the problem of nonspecific release due to granule stimulation caused by the physical pressure of blood collection. However, the release mechanism of sCLEC-2 relies on signal transduction that triggers platelet activation, making it a more accurate marker for platelet activation in vivo. Furthermore, since CLEC-2 expression in the human body is largely restricted to the platelet-megakaryocyte system, it is a specific marker with fewer false positives.

[0068] While soluble GPVI, released by a similar mechanism, is produced in large quantities in response to GPVI receptor-specific agonists, its production is very low in response to other known platelet-activating agonists. This means that since production varies depending on the type of stimulus, it is difficult to consider it as a marker. However, sCLEC-2 is produced at comparable levels not only in response to specific agonists but also to known agonists, making it a highly suitable molecule for use as a quantitative marker of platelet activation.

[0069] One of the advantages of sCLEC-2 is that it is not restricted by blood sampling procedures. That is, unlike PF4 and βTG, which are released by mild stimulation such as blood sampling, sCLEC-2 is released due to strong platelet activation, so no special blood sampling procedures are required. Furthermore, data showing that the concentration of sCLEC-2 in the plasma of patients with acute coronary syndrome is significantly higher than that of healthy subjects has been obtained. It can be considered that its utility as a monitoring object for predicting the onset and prognosis or determining the therapeutic effect is also high, and it is a marker that is significantly superior to existing activation markers. In addition, the concentration of sCLEC-2 in the plasma of patients with cerebral infarction also increases, and it has been observed that patients with high sCLEC-2 concentrations in their plasma have a poorer prognosis.

[0070] The present invention discovered that the plasma sCLEC-2 concentration is positively correlated with the platelet count in the blood. Specifically, a trend was observed where the plasma sCLEC-2 concentration increased in individuals with a high platelet count, while the plasma sCLEC-2 concentration decreased in individuals with a low platelet count. Consequently, the plasma sCLEC-2 concentration may be affected by the platelet count in the blood and may not necessarily reflect platelet activation.

[0071] Therefore, when the sCLEC-2 concentration in plasma is divided by the number of platelets in the blood to calculate the amount of sCLEC-2 released per platelet, and this is used as an indicator for diagnosing thrombotic diseases, the degree of platelet activation can be evaluated independently of the number of platelets in the blood. Specifically, for example, the sCLEC-2 concentration in plasma (A) is expressed in pg / mL, and 1000 platelets / mm 3 The platelet count (B) in the blood is expressed as A divided by B, and the value obtained is used as an indicator of platelet activation.

[0072] Disseminated intravascular coagulation (DIC) is a typical thrombotic hemostatic disease characterized by platelet activation. In patients with this disease, platelet count gradually decreases with platelet activation. A study of plasma sCLEC-2 concentrations and the ratio of sCLEC-2 concentration to platelet count was conducted using a DIC group (those with sepsis and DIC) and a non-DIC group (those with sepsis but without DIC). The results showed that the sCLEC-2 concentration / platelet count ratio showed a higher diagnostic efficiency for DIC. Specifically, the sCLEC-2 concentration / platelet count ratio showed significantly higher values ​​in the DIC group. In contrast, while the sCLEC-2 concentration in the plasma of the DIC group showed higher values ​​than that of the non-DIC group, no statistically significant difference was found. Furthermore, a significant correlation was observed between the sCLEC-2 concentration / platelet count ratio and the severity of the DIC condition in the DIC group. Therefore, the sCLEC-2 concentration / platelet count ratio can be considered a far more effective and useful diagnostic indicator than the sCLEC-2 concentration in plasma. Furthermore, compared to data from healthy individuals, the sCLEC-2 concentration / platelet count ratio in sepsis patients, regardless of whether they had DIC, was significantly higher. Therefore, it can also be considered a useful diagnostic indicator for sepsis, a severe infection associated with abnormal thrombotic hemostasis.

[0073] This indicator is more efficient and useful than sCLEC-2 concentration because it can determine platelet activity regardless of the number of platelets in the blood for thrombotic hemostatic diseases caused by platelet thrombosis, such as acute coronary syndrome (angina pectoris, myocardial infarction) and cerebral infarction.

[0074] The present invention also includes: a system that can be used to implement the method of the present invention described above; an electronic medical record, clinical examination device or hospital examination system equipped with the system; a kit that can be used to implement the method of the present invention, etc.

[0075] The systems of the present invention include, for example: a system for determining (or measuring) the soluble CLEC-2 concentration / platelet count, a system for understanding the platelet activation state, a system for assisting in the diagnosis of thrombotic hemostatic diseases, a system for diagnosing (or detecting) thrombotic hemostatic diseases, a system for determining (or measuring) the soluble CLEC-2 concentration / platelet count for the purpose of diagnosing thrombotic hemostatic diseases, and the like.

[0076] Any system of the present invention may include: (1) a recording unit capable of recording the soluble CLEC-2 concentration and platelet count in a test sample from a subject; (2) a calculation unit capable of calculating a value of soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count; (3) a comparison unit capable of comparing the obtained value of the soluble CLEC-2 concentration / platelet count of the subject with the value of the soluble CLEC-2 concentration / platelet count of a healthy subject; and (4) a display unit capable of displaying the result obtained by the comparison.

[0077] Alternatively, any system of the present invention includes a computer including a processor and a memory under the control of the processor, wherein the memory may store a program for causing the computer to execute the following steps: (1) a recording step of recording the soluble CLEC-2 concentration and the platelet count in a test sample from a subject in the memory; (2) a calculation step of calculating a value of soluble CLEC-2 concentration / platelet count by dividing the soluble CLEC-2 concentration by the platelet count; (3) a comparison step of comparing the value of soluble CLEC-2 concentration / platelet count obtained in the calculation step with the value of soluble CLEC-2 concentration / platelet count of a healthy subject; and (4) a display step of displaying the result obtained in the comparison step.

[0078] The system of the present invention can be installed in the electronic medical record, clinical examination device, or in-hospital examination system of the present invention.

[0079] The method of the present invention can be implemented using the system of the present invention, or the electronic medical record, clinical examination device, or in-hospital examination system of the present invention (hereinafter referred to as the system of the present invention, etc.). The above description of the method of the present invention is directly applicable to the "soluble CLEC-2 concentration / platelet count value" that can be measured (or measured) using the system of the present invention, etc., the "conditions, diseases, etc." and their "determination, judgment criteria, etc." that can be grasped using the system of the present invention, etc., and the various measurement methods for "soluble CLEC-2 concentration and platelet count."

[0080] The kits of the present invention include, for example, kits for determining (or measuring) the soluble CLEC-2 concentration / platelet count, kits for understanding the platelet activation state, kits for diagnosing (or detecting) thrombotic hemostatic diseases, and kits for determining (or measuring) the soluble CLEC-2 concentration / platelet count for diagnosing thrombotic hemostatic diseases.

[0081] The kit of the present invention may include (1) a soluble CLEC-2 measurement reagent and / or a platelet measurement reagent, and (2) instructions describing the correlation between the solubilized CLEC-2 concentration / platelet count value and the platelet activation state.

[0082] It is also important to describe in advance in the instructions the method for calculating the sCLEC-2 concentration / platelet count. This kit can be used to create a kit for evaluating platelet activation or diagnosing hemostatic disorders by using the sCLEC-2 concentration in plasma and the sCLEC-2 concentration / platelet count. Using this kit to calculate the sCLEC-2 concentration / platelet count allows for simple and rapid testing when needed for diseases such as abnormal platelet activation or thrombosis. The results are useful for distinguishing between other diseases and determining treatment strategies.

[0083] Furthermore, the instructions included in the kit of the present invention are not particularly limited as long as they at least mention the relationship between the plasma sCLEC-2 concentration / platelet count and the degree of platelet activation or various diseases. In addition to the aforementioned contents, the instructions may also include, for example, instructions regarding the steps for performing immunological assays using the kit of the present invention, instructions regarding the steps for detecting the plasma sCLEC-2 concentration / platelet count and the degree of platelet activation or various diseases based on the obtained measurement values, and precautions regarding the storage and handling of the kit itself.

[0084] Example

[0085] Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

[0086] Example 1: Determination of sCLEC-2 Concentration in Human Plasma

[0087] According to Example 6 of Japanese Patent No. 6078845, the concentration of sCLEC-2 in human plasma (pg / mL) was measured.

[0088] A sandwich ELISA system was constructed using mouse anti-human CLEC-2 monoclonal antibodies (1-11D5 and 3-11E6 antibodies) prepared in the examples of Japanese Patent No. 6078845. Specifically, the purified 1-11D5 antibody (F(ab)'2) was diluted to 10 μg / mL using 0.05 mol / L carbonate buffer (pH 9.5) and added to an ELISA plate (Maxisorp, NUNC) at 100 μL / well. After reacting overnight at 4°C, the plate was washed three times with borate-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 for blocking. Next, the human CLEC-2 (hCLEC-2) protein prepared in Example 3 of Japanese Patent No. 6078845 and used as a standard was diluted in 10% SuperBlock, 0.1% sodium octanoate, and 0.14 mol / L sodium chloride / PBS. When using plasma as a measurement sample, it was diluted fivefold or more and added at 100 μL / well. The cells were reacted at 37°C with shaking for 1.5 hours, followed by three washes in the same manner. The biotin-labeled 3-11E6 antibody (F(ab)'2-biotin) prepared in Example 5 of Japanese Patent No. 6078845 was diluted to 1.0 μg / mL in 10% SuperBlock, 0.1% sodium octanoate, and 0.14 mol / L sodium chloride / PBS, and 100 μL was added to each well. The cells were reacted at 37°C with shaking for 1 hour, followed by three washes in the same manner. Next, AMDEX horseradish peroxidase-conjugated streptavidin (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. The reaction was allowed to proceed at 37°C with shaking for 1 hour, followed by washing five times using the same method. 100 μL of tetramethylbenzidine (TMB) solution was added to each well. The reaction was allowed to proceed at room temperature for approximately 20 minutes, and then the reaction was stopped with 2N sulfuric acid solution. The absorbance at 450 nm (-620 nm) was measured using a well plate spectrophotometer (BIO-TEK INSTRUMENTS / EL312e).

[0089] Example 2: Determination of platelet count in human whole blood

[0090] The platelet count in human whole blood was measured using an automatic blood cell counter (XN-550) from Sysmex. Platelet counts were expressed as 1000 / mm3 .

[0091] Example 3: Calculation of sCLEC-2 concentration / platelet count

[0092] The measured values ​​of sCLEC-2 concentration measured in Example 1, the platelet count measured in Example 2, and the values ​​of sCLEC-2 concentration / platelet count calculated based on these values ​​are shown in Table 1. The specimens for measurement were 32 cases of sepsis (20 cases with disseminated intravascular coagulation (DIC) and 12 cases without DIC), and the diagnosis was made according to the diagnostic criteria for acute DIC of the Japanese Society of Emergency Medicine. In addition, each measured value is the measured value of the specimen collected on the first day of hospitalization. Furthermore, the sCLEC-2 concentration in the plasma and the platelet count in the blood of 37 healthy subjects were measured, and the sCLEC-2 concentration / platelet count was calculated. The mean ± standard deviation of the sCLEC-2 concentration, platelet count, and sCLEC-2 concentration / platelet count of healthy subjects were 87 ± 38.9 (pg / mL), 263 ± 63.4 (1000 / mm2), and 1000 / mm3, respectively. 3 ), 0.34±0.15.

[0093] [Table 1]

[0094] Non-DIC / DIC Plasma sCLEC-2 (pg / mL) <![CDATA[Platelet count (per 1000 / mm 3 )]]> sCLEC-2 / platelet count Non-DIC Case 1 145.9 410.0 0.4 Non-DIC Case 2 357.9 148 2.4 Non-DIC Case 3 204.8 249 0.8 Non-DIC Case 4 99.4 93 1.1 Non-DIC Case 5 165.5 178 0.9 Non-DIC Case 6 659.2 407 1.6 Non-DIC Case 7 485.8 389 1.2 Non-DIC Case 8 58.7 120.0 0.5 Non-DIC Case 9 271.7 193.0 1.4 Non-DIC Case 10 244.4 263 0.9 Non-DIC Case 11 74.4 90 0.8 Average of non-DIC group 251.6 230.9 1.10 Standard deviation of non-DIC group 185.8 123.2 0.57 DIC Case 1 336.4 217 1.6 DIC Case 2 290.7 120 2.4 DIC Case 3 216.2 140 1.5 DIC Case 4 372.5 168 2.2 DIC Case 5 349.0 96 3.6 DIC Case 6 232.4 197 1.2 DIC Case 7 129.3 96 1.3 DIC Case 8 264.1 134 2.0 DIC Case 9 186.1 22 8.5 DIC Case 10 665.1 471 1.4 DIC Case 11 321.3 90 3.6 DIC Case 12 161.9 318 0.5 DIC Case 13 156.7 84 1.9 DIC Case 14 264.1 122 2.2 DIC Case 15 323.4 113 2.9 DIC Case 16 194.5 67 2.9 DIC Case 17 393.9 180 2.2 DIC Case 18 411.2 155 2.7 DIC Case 19 576.4 73.0 7.9 DIC Case 20 315.1 24.2 13.0 DIC Case 21 86.9 73 1.2 DIC group average 297.5 141.0 3.2 DIC group standard deviation 140.2 101.4 3.0 P value non-DIC vs DIC 0.25 0.035 0.0008

[0095] The correlation between the sCLEC-2 concentration measured in Example 1 (sCLEC-2) and the platelet count (PLT) measured in Example 2 was investigated for all sepsis patients, sepsis patients with DIC (DIC), and sepsis patients without DIC (n-DIC). Statistically significant correlation was observed between sCLEC-2 concentration and platelet count ( Figure 1 The sCLEC-2 concentration / platelet count value, calculated from the sCLEC-2 concentration and platelet count, was calculated and compared between patients with sepsis who developed DIC and those who did not develop DIC. No significant difference was observed between the two groups in sCLEC-2 concentration (p = 0.25), but a significant difference was observed between the two groups in sCLEC-2 concentration / platelet count (p = 0.0008). ( Figure 2 This demonstrates that sCLEC-2 is affected not only by platelet activation but also by platelet count, and that the amount of sCLEC-2 relative to platelet count better reflects platelet activity. Furthermore, in sepsis, both sCLEC-2 concentrations and sCLEC-2 concentration / platelet ratios were higher than those in healthy subjects, regardless of whether they had DIC, demonstrating that sCLEC-2 concentrations and sCLEC-2 concentration / platelet ratios can also be used to diagnose sepsis.

[0096] Example 4: Evaluation as a biomarker based on ROC curve

[0097] A ROC (Receiver Operating Characteristic) curve was prepared using the measured values ​​and calculated values ​​obtained in Example 3, and the area under the curve (AUC) was calculated. Figure 3 The results for sCLEC-2 concentration (solid line) and sCLEC-2 concentration / platelet count (dashed line) are shown. The AUC of the ROC curve was 0.628 for sCLEC-2 concentration, 0.866 for sCLEC-2 concentration / platelet count, and 0.732 for platelet count. It is believed that the closer the AUC is to 1.0, the higher the diagnostic performance, and the sCLEC-2 concentration / platelet count ratio shows the best diagnostic performance.

[0098] Industrial Applicability

[0099] The present invention can be used to grasp the activation state of platelets and to examine thrombotic hemostatic diseases.

Claims

1. A method for assisting in understanding the activation state of platelets, the method not being intended for diagnosing a disease and comprising: (1) a step of measuring the concentration of soluble CLEC-2 and the number of platelets in a test sample from a subject; and (2) A step of dividing the soluble CLEC-2 concentration by the platelet count to calculate the value of soluble CLEC-2 concentration / platelet count.

2. The method according to claim 1, further comprising: A step of comparing the soluble CLEC-2 concentration / platelet count value with a test sample obtained from a healthy subject.

3. A system for understanding platelet activation status, comprising: (1) a recording device capable of recording the concentration of soluble CLEC-2 and the number of platelets in a test sample from a subject; (2) a calculation mechanism capable of calculating the value of soluble CLEC-2 concentration / platelet number by dividing the soluble CLEC-2 concentration by the platelet number; (3) a comparison mechanism capable of comparing the obtained value of the soluble CLEC-2 concentration / platelet count of the test subject with the value of the soluble CLEC-2 concentration / platelet count of a healthy subject; and (4) A display unit capable of displaying the result obtained by the comparison.

4. A system for determining the activation state of platelets, comprising a computer including a processor and a memory under the control of the processor, wherein the memory stores a program for causing the computer to execute the following steps: (1) a recording step of recording the soluble CLEC-2 concentration and platelet count in a test sample from a subject in the memory; (2) a calculation step of dividing the soluble CLEC-2 concentration by the platelet count to calculate a value of soluble CLEC-2 concentration / platelet count; (3) a step of comparing the soluble CLEC-2 concentration / platelet count value obtained in the calculation step with the soluble CLEC-2 concentration / platelet count value of a healthy subject; and (4) A display step of displaying the result obtained through the comparison step.

5. A clinical examination device or an in-hospital examination system comprising the system according to claim 3 or 4.

6. A kit for determining the activation state of platelets, comprising: (1) Soluble CLEC-2 assay reagent and / or platelet assay reagent; and (2) Instructions describing the correlation between the value of soluble CLEC-2 concentration / platelet number and the platelet activation state.

Images