Method for evaluating high onset risk of cardiovascular / renal adverse events over long period of time using soluble CLEC-2

Abstract

Provided is a technique capable of accurately and simply predicting the risk of long-term onset of cardiovascular / renal adverse events. Further, by means of the above-described technique, a technique for selecting a treatment method for improving benefit in prevention of a subject patient is provided. According to the technology, the long-term high onset risk of cardiovascular and cerebrovascular / kidney adverse events is evaluated according to the concentration of soluble CLEC-2 in blood of a subject.

Description

Technical Field

[0001] This invention relates to a long-term high-incidence risk assessment method for cardiovascular / renal adverse events using soluble CLEC-2 (sCLEC-2). Background Technology

[0002] Cardiovascular / cerebrovascular / renal adverse events refer to the onset of cardiovascular and cerebrovascular diseases such as stroke, ischemic heart disease (myocardial infarction, etc.) caused by arteriosclerosis / thrombosis, or the need for interventional dialysis. Cardiovascular and cerebrovascular diseases are a leading cause of death in Japan, comparable to cancer, and are a very important category of diseases and the leading cause of death worldwide. Accurately assessing the risk of developing these diseases and implementing appropriate prevention and treatment are crucial.

[0003] As risk factors for cardiovascular / renal adverse events, diabetes, chronic kidney disease (CKD), hypertension, dyslipidemia, obesity, and smoking are known. Managing these factors individually is important for reducing risk. Furthermore, the risk increases further when these risk factors are present in combination. For each risk factor, there are biomarkers for evaluation, and corresponding prevention / treatment measures are implemented. For example, dyslipidemia has risk markers such as LDL cholesterol; if these are abnormal, medications such as statins are used to improve dyslipidemia. Regarding diabetes, there are risk markers such as blood glucose levels or hemoglobin A1C; if these are abnormal, diabetes medications or dietary and exercise strategies are implemented. Furthermore, for hypertension, as a preventative measure against cardiovascular / renal adverse events, treatments and management are implemented, such as using antihypertensive drugs and smoking cessation for smokers. However, even with such individualized treatment / prevention, patients with diabetes, hypertension, and CKD remain at risk for cardiovascular / renal adverse events, and risk assessment and management for these events remain crucial.

[0004] It is well known that the pathogenesis of major adverse cardiovascular and cerebrovascular events such as cerebral infarction, myocardial infarction, and occlusive arteriosclerosis is vascular occlusion caused by thrombosis, and antithrombotic therapy as a preventive measure has been established. In antithrombotic therapy, antiplatelet drugs are used in cases of arterial thrombosis where platelets are the main component of the thrombus, while anticoagulants are used in cases of venous thrombosis where the coagulation system is the main component of the thrombus.

[0005] In addition, the risk and high-risk groups for cardiovascular / renal adverse events are defined in this instruction manual as follows: The risk group is defined as those who, although considered to have a higher risk of cardiovascular / renal adverse events than healthy individuals, generally do not receive antithrombotic therapy. This risk group includes patients with diabetes, chronic kidney disease, hypertension, dyslipidemia, obesity, or a smoking habit. The high-risk group refers to those whose risk of cardiovascular / renal adverse events is higher than the risk group and for whom antithrombotic therapy is recommended. This high-risk group includes patients with a history of cerebral infarction, myocardial infarction, occlusive arteriosclerosis, angina pectoris, atrial fibrillation, or post-artificial valve replacement surgery.

[0006] Antithrombotic therapy has the effect of inhibiting thrombus formation and preventing thrombosis. However, as a side effect, inhibiting thrombus formation can sometimes trigger hemorrhagic diseases. Therefore, it is important to consider the balance between the preventive benefits and the risks of side effects when using antithrombotic therapy. Thus, antiplatelet drugs are used for the treatment or secondary prevention of high-risk diseases such as angina pectoris, which are particularly prone to myocardial infarction, or for high-risk patients with a history of cerebral infarction, myocardial infarction, or occlusive arteriosclerosis. They are generally not used for primary prevention (prevention in patients without a history of cardiovascular or cerebrovascular disease) in high-risk patients such as those with diabetes, hypertension, or chronic kidney disease (CKD). For the same reason, anticoagulant therapy is currently only used for thrombosis prevention in high-risk patients such as those with atrial fibrillation or after artificial valve replacement. Regarding this point, the Japanese version of the CKD treatment guidelines (non-patent literature 1) also states that "it is unclear whether the use of aspirin is effective in suppressing cardiovascular events in patients with CKD stages G3b-5. On the other hand, it cannot be ruled out that the risk of hemorrhagic complications may increase due to the use of aspirin." Therefore, a more accurate risk assessment is needed for the use of aspirin (an antiplatelet drug).

[0007] Antiplatelet drugs are known to be effective against atherosclerotic thrombosis, and therefore, observing changes in platelet counts can be considered a good risk predictor. To this end, measures have been proposed to observe markers of platelet activation and platelet aggregation. However, measuring markers of platelet activity is technically challenging, there are no widely used clinical markers, and no markers for predicting adverse cardiovascular / renal events are available. While platelet aggregation is expected to be used as a risk predictor, its predictive ability is low, making it impractical. The technique in Patent Document 1, which measures the rate of platelet count reduction, is also not practical.

[0008] Patent Document 2 illustrates that the concentration of soluble CLEC-2 (sCLEC-2), a marker of platelet activation, indicates platelet activation, and its measured value increases in thrombosis. Furthermore, a change in the value of platelet-based markers is considered an indication of thrombosis, and the sCLEC-2 concentration is considered to indicate imminent danger, i.e., a short-term increase in risk (Patent Document 3). However, the technology in Patent Document 2 is not practically applicable. Specifically, a risk marker technology for platelet-related thrombosis in short-term cardiovascular / renal adverse events has not yet been established.

[0009] Activation of the coagulation system is also an important cause of thrombosis. Thrombosis occurs through the interaction between platelets and the coagulation system. Therefore, although the arterial system is primarily platelet-based and the venous system is primarily coagulation-based (fibrin formation), all types of thrombi contain both platelets and fibrin. Thus, biomarkers of the coagulation system are considered potential risk markers for cardiovascular / renal adverse events. D-dimer is a representative biomarker. While D-dimer can be used as a marker for cardiovascular / renal adverse events such as cardioembolic stroke or aortic dissection, it is generally not used as a risk marker for these events. For example, although Non-Patent Literature 2 uses D-dimer for risk assessment of cardiovascular adverse events, the tertiary ranges for D-dimer in patients with stable coronary artery disease (a high-risk group for cardiovascular adverse events) are (<0.32 μg / mL, 0.32-0.61 μg / mL, >0.62 μg / mL). Patients in the third quartile (>0.62 μg / mL) had a higher risk of cardiovascular adverse events compared to patients in the first quartile (<0.32 μg / mL). The upper limit for D-dimer levels in healthy individuals used in this study was 1.0 μg / mL; therefore, the risk assessment threshold fell within the range of D-dimer values ​​typically found in healthy individuals, leading to many healthy individuals being classified as high-risk. Consequently, D-dimer is not considered a reliable biomarker for assessing the risk of cardiovascular / renal adverse events.

[0010] As mentioned above, there is currently no established technology to assess the risk of adverse cardiovascular / renal events from the perspective of thrombosis, nor are there any implications for thrombosis risk biomarkers that can assess the long-term risk of adverse cardiovascular / renal events from the perspective of thrombosis.

[0011] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2022-73652 Patent Document 2: Japanese Patent No. 6078845 Patent Document 3: International Publication No. 2022 / 255348 Patent Document 4: International Publication No. 2021 / 172493 Non-patent literature Non-patent document 1: Progress of renal impairment in patients with CKD G3b~5 Prevention and treatment of renal replacement therapy, transfer of kidney replacement therapy, diagnosis and treatment of kidney disease 2015 Japanese Nephrology Society Non-patent literature 2: H. Naruse et al. Circulation Journal 2017 81: 1506-1513 Summary of the Invention

[0012] (a) Technical problems to be solved This invention provides a technique for accurately and easily predicting the long-term risk of adverse cardiovascular / renal events, even in patients with a large potential patient population for whom no effective treatments have been found. Specifically, it provides a technique for further predicting patients with a high long-term risk of adverse cardiovascular / renal events, such as those with diabetes, hypertension, or chronic kidney disease, who are considered to be at risk for these events. Furthermore, this invention provides a technique for selecting treatment methods to improve the preventative benefits for target patients.

[0013] (II) Technical Solution The inventors of this application conducted in-depth research to solve the aforementioned technical problems. The results showed that, within the risk group for adverse cardiovascular / renal events, those with higher serum sCLEC-2 concentrations had a particularly high long-term risk of developing further adverse cardiovascular / renal events. In addition to this insight, it was discovered that by using the combination of D-dimer, a marker of the coagulation system, and sCLEC-2, and the combination of sCLEC-2, D-dimer, and platelet count, particularly the index value of (serum sCLEC-2 concentration) × (serum D-dimer concentration) or (serum D-dimer concentration) × (serum sCLEC-2) / (platelet count), it was possible to screen for patient groups with a particularly high long-term risk of adverse cardiovascular / renal events, thus completing this invention.

[0014] It is known that since the primary cause of most cardiovascular / renal adverse events is vascular occlusion due to thrombosis, biomarkers related to fibrin (a product of the coagulation system) or platelets, which are the main components of thrombi, often show abnormal values ​​at the onset of these events. This is understandable, as thrombi often have already formed by the time of onset. Among these biomarkers, D-dimer, FDP, soluble fibrin, and thrombin-antithrombin complex are known in the fibrin system (coagulation system), while sCLEC-2, β-thromboglobulin, platelet factor 4, soluble p-selectin, and platelet aggregation energy are known in the platelet system. However, for a biomarker to be useful as a long-term predictor of cardiovascular / renal adverse events, it needs to show abnormal values ​​outside the normal range at an early stage of the event. However, there is no insight into whether these existing biomarkers can serve as long-term predictors of cardiovascular / renal adverse events, and therefore they have not been used. The ability to evaluate the long-term high risk of adverse cardiovascular / renal events based on sCLEC-2 concentration, combinations of sCLEC-2 concentration and platelet count, combinations of sCLEC-2 concentration and D-dimer concentration, and combinations of sCLEC-2 concentration, D-dimer concentration, and platelet count is a surprising discovery that could not have been predicted with existing knowledge. Furthermore, the invention makes it easy to recommend treatments that help improve the preventive benefits for target patients, making its effectiveness and significance very high.

[0015] The aforementioned biomarkers show abnormal values ​​in the presence of thrombosis or in the process of thrombosis formation. However, the inventors of this application have discovered that when new indicators showing abnormal values ​​are derived from individual sCLEC-2 concentrations, combinations of sCLEC-2 concentrations and platelet counts, combinations of sCLEC-2 concentrations and D-dimer concentrations, or combinations of sCLEC-2 concentrations, D-dimer concentrations, and platelet counts, the incidence of adverse cardiovascular / renal events 24 months after measurement is 3 to 8 times higher than in the absence of abnormalities. Since thrombosis is generally considered to form shortly before the onset of disease, biomarkers previously unseen related to thrombosis can be predicted up to 24 months before the onset of disease. In fact, it is unknown whether existing platelet activation biomarkers or platelet aggregation energy, etc., can independently serve as risk biomarkers for adverse cardiovascular / renal events.

[0016] For example, it is generally believed that abnormalities in platelet-related markers such as sCLEC-2 or platelet counts indicate ongoing thrombosis, thus increasing short-term risk (Patent Document 3). On the other hand, as described in the examples described later, the cumulative incidence of adverse cardiovascular / renal events increased almost proportionally over 24 months following measurement in the high sCLEC-2 group. If a high sCLEC-2 value indicates thrombosis leading to vascular occlusion at or shortly after blood collection, a higher frequency of adverse cardiovascular / renal events can be predicted in a relatively short period. However, no increase in incidence was observed in the Kaplan-Meier curve; the cumulative incidence increased almost linearly over the 24-month period. Therefore, sCLEC-2 and related indicators in this invention reflect the long-term risk of the subjects.

[0017] In one case, the threshold (cutoff value) for determining a long-term high risk of cardiovascular / renal adverse events using sCLEC-2 concentration was 250 pg / mL. Since the sCLEC-2 concentration in the blood of healthy individuals is below 200 pg / mL, very few healthy individuals would be classified as high-risk at this threshold. It was also clarified that setting an appropriate sCLEC-2 cutoff value above 200 pg / mL would significantly reduce false positives in classifying healthy individuals and other low-risk individuals as high-risk.

[0018] Furthermore, as described in the embodiments below, in order to determine a long-term high risk of cardiovascular / renal adverse events, it is preferable to simultaneously evaluate the platelet system, or the platelet system and the coagulation system, as a risk marker for thrombosis. While sCLEC-2 concentration can assess platelet status and can serve as a long-term risk marker for cardiovascular / renal adverse events on its own, combining D-dimer concentration with sCLEC-2 concentration has shown it to be a more useful marker for the long-term risk of cardiovascular / renal adverse events. Furthermore, it was found that platelet activity can be accurately assessed by combining platelet count with sCLEC-2 concentration (Patent Document 4). In addition, by combining D-dimer concentration and / or platelet count with sCLEC-2 concentration, the index value of (sCLEC-2 concentration in blood) × (D-dimer concentration in blood), (sCLEC-2 concentration in blood) / (platelet count), or (D-dimer concentration in blood) × (sCLEC-2 in blood) / (platelet count) can serve as a long-term high-risk biomarker for the incidence of adverse cardiovascular / renal events.

[0019] In summary, the inventors of this application have discovered that the concentration of sCLEC-2 alone in the blood, or a combination of sCLEC-2 concentration and D-dimer concentration, a combination of sCLEC-2 concentration and platelet count, a combination of sCLEC-2 concentration and D-dimer concentration and platelet count, and particularly the index value of (sCLEC-2 concentration) × (D-dimer concentration), (sCLEC-2) / (platelet count), or (D-dimer concentration) × (sCLEC-2) / (platelet count) can screen for high-risk groups for long-term cardiovascular / renal adverse events. This invention is based on these findings.

[0020] That is, the present invention provides the following solution: [1] A method for assessing the long-term high risk of cardiovascular / renal adverse events based on the concentration of soluble CLEC-2 in the blood of subjects.

[0021] [2] The method according to [1] is a method for evaluating the long-term high incidence risk of cardiovascular / renal adverse events based on the concentration of soluble CLEC-2 in the blood of the subject, the method comprising: (1) The procedure of providing a blood sample from the subject; (2) The process of determining the concentration of soluble CLEC-2 in the sample; (3) A process of associating the concentration of the soluble CLEC-2 with the long-term incidence of adverse cardiovascular / renal events in the subject.

[0022] [3] The method according to [1] or [2] is a method for assessing the long-term risk of disease in patients who are suspected of having a disease at risk of adverse cardiovascular / renal events or who have been diagnosed with such a disease.

[0023] [4] According to any one of [1] to [3], wherein the disease with risk of cardiovascular / renal adverse events is selected from chronic kidney disease, diabetes, hypertension, dyslipidemia, chronic maintenance dialysis, and arteriosclerotic diseases.

[0024] [5] The long-term risk of adverse cardiovascular / renal events is evaluated based on the concentration of D-dimer in the blood of the subject, in addition to the concentration of soluble CLEC-2 in the subject's blood.

[0025] [6] The method of [5] is used to evaluate the long-term high risk of cardiovascular / renal adverse events based on the value obtained by multiplying the concentration of soluble CLEC-2 in the subject's blood by the concentration of D-dimer.

[0026] [7] The long-term risk of cardiovascular / renal adverse events is evaluated based on the platelet count of the subject, in addition to the concentration of soluble CLEC-2 in the subject's blood.

[0027] [8] The method of [7] was used to evaluate the long-term high risk of cardiovascular / renal adverse events based on the value obtained by dividing the concentration of soluble CLEC-2 in the subject's blood by the platelet count.

[0028] [9] According to the method of [7], in addition to the concentration of soluble CLEC-2 in the blood and the number of platelets, the long-term high risk of cardiovascular / renal adverse events was further evaluated based on the concentration of D-dimer in the blood of the subjects.

[0029]

[10] According to the method described in [9], the long-term high incidence risk of cardiovascular / renal adverse events is evaluated based on the value represented by the following formula (1). [Mathematical Expression 1]

[11] A method for evaluating the long-term high incidence risk of cardiovascular / renal adverse events and selecting antithrombotic drugs, wherein the method of any one of [1] to

[10] is used to evaluate the long-term high incidence risk of cardiovascular / renal adverse events and select antithrombotic drugs.

[0030] Furthermore, the present invention includes: - A method for evaluating the long-term high risk of cardiovascular / renal adverse events based on the concentration of soluble CLEC-2 in the subject's blood (i.e., soluble CLEC-2 concentration alone, or a combination of soluble CLEC-2 concentration with D-dimer concentration and / or platelet count).

[0031] - The method, which is a method for evaluating the long-term high incidence risk of cardiovascular / cerebrovascular / renal adverse events based on the concentration of soluble CLEC-2 in the blood of a subject, includes: (1) The procedure of providing a blood sample from the subject; (2) The process of determining the concentration of soluble CLEC-2 in the sample; (3) A process of associating the concentration of the soluble CLEC-2 with the long-term incidence of adverse cardiovascular / renal events in the subject.

[0032] - A method for assisting in the assessment of long-term high risk of cardiovascular / renal adverse events, which assesses the long-term high risk of cardiovascular / renal adverse events based on the concentration of soluble CLEC-2 in the subject's blood (i.e., soluble CLEC-2 concentration alone, or a combination of soluble CLEC-2 concentration with D-dimer concentration and / or platelet count).

[0033] - A method for assessing the long-term high risk of cardiovascular / renal adverse events by measuring (or determining) the concentration of soluble CLEC-2 in the blood of a subject (i.e., soluble CLEC-2 concentration alone, or a combination of soluble CLEC-2 concentration with D-dimer concentration and / or platelet count).

[0034] - An in vitro assessment method for long-term high risk of cardiovascular / renal adverse events, characterized by measuring (or determining) the concentration of soluble CLEC-2 in the blood of the subject (i.e., soluble CLEC-2 concentration alone, or a combination of soluble CLEC-2 concentration with D-dimer concentration and / or platelet count).

[0035] - The application of an antibody capable of measuring (or determining) soluble CLEC-2 concentration in an evaluation kit for assessing the long-term high risk of cardiovascular / renal adverse events; The application of an antibody capable of measuring (or determining) soluble CLEC-2 concentration and an antibody capable of measuring (or determining) D-dimer concentration in a kit for evaluating the long-term high risk of cardiovascular / renal adverse events; and - A method for determining (or identifying) the concentration of soluble CLEC-2 in the blood of a subject (i.e., soluble CLEC-2 concentration alone, or a combination of soluble CLEC-2 concentration with D-dimer concentration and / or platelet count) in order to provide information needed for assessing the long-term high incidence risk of cardiovascular / renal adverse events.

[0036] Furthermore, the present invention includes a method for evaluating the long-term high incidence risk of cardiovascular / renal adverse events according to any of the above methods, and for selecting antithrombotic drugs.

[0037] (III) Beneficial Effects Using sCLEC-2 concentration alone, or in combination with D-dimer concentration and / or platelet count, allows for a simple and accurate assessment of the long-term risk of adverse cardiovascular / renal events. The combination of sCLEC-2 concentration with D-dimer concentration and / or platelet count refers to any combination of D-dimer concentration or platelet count with sCLEC-2 concentration, or a combination of D-dimer concentration and platelet count with sCLEC-2 concentration. More specifically, it refers to combinations of serum D-dimer concentration with serum sCLEC-2 concentration, platelet count with serum sCLEC-2 concentration, and D-dimer concentration with platelet count with serum sCLEC-2 concentration. Attached Figure Description

[0038] Figure 1 This is a graph showing the tertiary range of serum sCLEC-2 concentration and the incidence of adverse cardiovascular / renal events in 453 CKD patients.

[0039] Figure 2 The Kaplan-Meier curves are based on the tertiary intervals of serum sCLEC-2 concentration in 453 CKD patients.

[0040] Figure 3 This is a chart showing the incidence of adverse cardiovascular / renal events based on the combination of serum sCLEC-2 concentration and serum D-dimer concentration in 453 CKD patients.

[0041] Figure 4 The Kaplan-Meier curve is based on the combination of serum sCLEC-2 concentration and serum D-dimer concentration in 453 CKD patients.

[0042] Figure 5 This is a chart showing the incidence of cardiovascular / renal adverse events in 453 CKD patients, based on the tertiary intervals of (serum sCLEC-2 concentration) × (serum D-dimer concentration).

[0043] Figure 6 The Kaplan-Meier curves are based on the tertiary intervals of (serum sCLEC-2 concentration) × (serum D-dimer concentration) of 453 CKD patients.

[0044] Figure 7 This is a chart showing the incidence of cardiovascular / renal adverse events in 453 CKD patients, based on the tertiary intervals of (serum D-dimer concentration) × (serum sCLEC-2 concentration) / (platelet count).

[0045] Figure 8The Kaplan-Meier curves are based on the tertiary intervals of (serum D-dimer concentration) × (serum sCLEC-2 concentration) / (platelet count) of 453 CKD patients. Detailed Implementation

[0046] The following is a detailed description of the implementation scheme of the present invention, using the measurement / analysis of sCLEC-2 and other substances in the blood of patients with chronic kidney disease (CKD) as an example, as an evaluation method for the long-term high risk of cardiovascular / cerebrovascular / renal adverse events. However, the implementation scheme of the method is not limited to this.

[0047] The first aspect of the present invention includes a step of determining the concentration of sCLEC-2 in a blood sample collected from a subject (hereinafter referred to as a subject) for assessing the risk of cardiovascular / renal adverse events. When assessing the long-term risk of cardiovascular / renal adverse events using sCLEC-2 concentration alone, a suitable threshold value is set, for example, above 200 pg / mL. If the concentration is above this threshold value, a high long-term risk of cardiovascular / renal adverse events can be assessed. If the concentration is below this threshold value, a low long-term risk of cardiovascular / renal adverse events can be assessed.

[0048] The second aspect of the present invention includes a step of measuring the concentrations of sCLEC-2 and D-dimer in a blood sample collected from a subject. When evaluating the long-term risk of cardiovascular / renal adverse events using a combination of sCLEC-2 and D-dimer concentrations, appropriate threshold values ​​are set for each, wherein a high long-term risk of cardiovascular / renal adverse events can be assessed when either or both exceed the threshold value. The threshold value for sCLEC-2 concentration can be the value used when evaluating with sCLEC-2 concentration alone. Furthermore, the threshold value for D-dimer concentration is preferably set to 1.0 μg / mL or higher, which is the upper limit for healthy individuals, and more preferably 1.45 μg / mL or higher.

[0049] In addition, as another evaluation method for the second scheme, when evaluating the value "(sCLEC-2 concentration)×(D-dimer concentration)" obtained by multiplying the sCLEC-2 concentration by the D-dimer concentration, an appropriate threshold value is set for this index value. If it is above the threshold value, it is evaluated as having a high long-term risk of cardiovascular / cerebrovascular / renal adverse events; if it is below the threshold value, it is evaluated as having a low long-term risk of cardiovascular / cerebrovascular / renal adverse events.

[0050] The third embodiment of the present invention includes a step of measuring the concentration of sCLEC-2 and the platelet count in a blood sample collected from a subject. Furthermore, in addition to the sCLEC-2 concentration and platelet count, the third embodiment of the present invention can further measure the concentration of D-dimer in the subject's blood sample.

[0051] In the third aspect of the present invention, when evaluating the long-term risk of adverse cardiovascular / renal events using a combination of sCLEC-2 concentration and platelet count, the evaluation method in the first aspect (e.g., based on sCLEC-2 concentration alone) and a threshold value can be used, with the platelet count further added for evaluation. Alternatively, an evaluation method combining sCLEC-2 concentration and platelet count, along with a threshold value, can be used for evaluation. Specifically, as a calculation formula for the evaluation method combining sCLEC-2 concentration and platelet count, an index value using "(sCLEC-2 concentration) / (platelet count)" can be listed.

[0052] In another method of the third aspect of the present invention, when evaluating the long-term risk of adverse cardiovascular / renal events using a combination of sCLEC-2 concentration, D-dimer concentration, and platelet count, for example, the evaluation method in the second aspect (e.g., based on a combination of sCLEC-2 concentration and D-dimer concentration) and a threshold value can be used, with the platelet count further added for evaluation; or, an evaluation method combining sCLEC-2 concentration and platelet count and a threshold value can be used, with the D-dimer concentration further added for evaluation. Specifically, as a calculation formula for the evaluation method combining sCLEC-2 concentration, D-dimer concentration, and platelet count, an index value of "(D-dimer concentration) × (sCLEC-2 concentration) / (platelet count)" can be listed.

[0053] Since sCLEC-2 is generated from platelets, sCLEC-2 concentration shows a weak correlation with platelet count. Therefore, the index value of "(sCLEC-2 concentration) / (platelet count)" (representing the sCLEC-2 value per unit of platelet) has been reported as more representative of the degree of platelet activation than sCLEC-2 concentration alone (Patent Document 4), which can be appropriately referenced.

[0054] For example, setting an appropriate threshold for the value "(sCLEC-2 concentration) / (platelet count)" obtained by dividing sCLEC-2 concentration by platelet count, when the threshold is exceeded, can be used to evaluate a high long-term risk of cardiovascular / cerebrovascular / renal adverse events.

[0055] Furthermore, appropriate threshold values ​​can be set for the "(sCLEC-2 concentration) / (platelet count)" value and D-dimer concentration. When either or both exceed the threshold value, a high long-term risk of cardiovascular / renal adverse events can be assessed. The threshold value for "(sCLEC-2 concentration) / (platelet count)" can be appropriately set and used based on the sCLEC-2 concentration and platelet count. Moreover, the threshold value for D-dimer concentration can preferably be set to 1.0 μg / mL or higher, which is the upper limit for healthy individuals, and more preferably to 1.45 μg / mL or higher.

[0056] Furthermore, in the specific examples above, the method of using platelet count by dividing the sCLEC-2 concentration by the platelet count was explained. However, the calculation formulas described in this specification, such as the value represented by the following formula (1), are arbitrary as long as they follow calculation rules (calculation order rules, arithmetic operation rules) well known to those skilled in the art. That is, as long as the calculation results are the same, there are no restrictions on the order of the items used in the calculation, and the combination of values ​​used to set the threshold is also unrestricted.

[0057] [Mathematical Expression 2] For example, when using the items described in formula (1), the long-term high risk of cardiovascular / renal adverse events can be evaluated by the following values: (1) Divide the concentration of soluble CLEC-2 in the subject's blood by the platelet count, and multiply the resulting value by the D-dimer concentration to obtain the value. value ; or (2) divide the concentration of D-dimer in the subject's blood by the platelet count, and multiply the resulting value by the concentration of soluble CLEC-2 to obtain the result. value; Or, (3) multiply the concentration of soluble CLEC-2 in the subject's blood by the concentration of D-dimer, and divide the resulting value (or (3') the value obtained by multiplying the concentration of D-dimer in the subject's blood by the concentration of soluble CLEC-2) by the platelet count to obtain the value.

[0058] As another evaluation method for the third approach, when evaluating the risk of cardiovascular / renal adverse events, the value obtained by multiplying the sCLEC-2 concentration by the D-dimer concentration and dividing the resulting value by the platelet count is "(D-dimer concentration) × (sCLEC-2 concentration) / platelet count". An appropriate threshold value is set for this value. If the value is above the threshold value, the risk of long-term cardiovascular / renal adverse events is evaluated as high. If the value is below the threshold value, the risk of long-term cardiovascular / renal adverse events is evaluated as low.

[0059] In this specification, cardiovascular / renal adverse events refer to the occurrence of symptoms or treatment interventions in cardiovascular and cerebrovascular diseases, such as cardiovascular and cerebrovascular death (fatal myocardial infarction, fatal stroke, sudden cardiac death), non-fatal myocardial infarction, unstable angina, ischemic stroke, transient ischemic attack, non-fatal stroke, percutaneous coronary intervention (PCI), AC bypass surgery, other cardiovascular reconstructive procedures, interventional dialysis, occlusive arteriosclerosis, severe limb ischemia, aortic dissection, etc.

[0060] In this specification, the specimens collected from the subject are preferably human blood samples, plasma or whole blood with added anticoagulants, serum, etc., or specimens collected from other mammals such as rats, mice, monkeys, and dogs that may cause adverse cardiovascular / renal events. Subjects are particularly preferred to be patients suspected of having or diagnosed with diseases that are considered to pose a high risk of adverse cardiovascular / renal events. Examples of diseases that pose a risk of adverse cardiovascular / renal events include chronic kidney disease, diabetes, dyslipidemia, hypertension, chronic maintenance dialysis, arteriosclerotic diseases, obesity, and smoking. Patients with a history of the aforementioned adverse cardiovascular / renal events are preferred candidates.

[0061] The method for collecting samples from subjects can be any known collection method appropriate to the assay. For example, when measuring sCLEC-2 concentration, blood collection tubes used for plasma collection can be used. Suitable tubes are those with low residual platelets and contain citric acid, or those containing heparin or EDTA. When measuring D-dimer concentration, blood collection tubes containing citric acid or EDTA are suitable. For platelet count measurement, blood collection tubes containing EDTA are appropriate. These can be the same type of tube or different types of tubes.

[0062] There are no particular limitations on the method for determining sCLEC-2 concentration, but immunological methods using antibodies that recognize sCLEC-2 are preferred. Any method can be used as an immunologically assay for protein detection, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CIA), electrochemiluminescent immunoassay (ECIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), immunochromatography, Western blotting, or other commonly used methods known to be conventional, such as latex agglutination or immunoturbidimetry. However, a method with sufficient sensitivity for determining sCLEC-2 concentration in patients or healthy individuals should be used. Therefore, a highly sensitive method is ideal, and chemiluminescent immunoassay, electrochemiluminescent immunoassay, or fluorescence immunoassay are particularly preferred.

[0063] The concentration of D-dimer can be determined using any well-known immunological assay, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent ELISA, chemiluminescent immunoassay, fluorescence immunoassay, bioluminescent immunoassay, electrochemiluminescent immunoassay, latex agglutination, and immunochromatography, without any particular limitation.

[0064] There are no particular restrictions on the method for determining platelet count; any existing, well-known methods may be used appropriately.

[0065] In this specification, "CLEC-2" refers to a platelet activation receptor belonging to the C-type agglutinin family. It is normally present in the platelet membrane but is released into the bloodstream upon platelet activation.

[0066] In this specification, 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 the blood.

[0067] sCLEC-2 includes proteins with a molecular weight of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa, as measured by SDS-PAGE under reducing conditions. Proteins with a molecular weight of approximately 40 kDa and approximately 32 kDa are present on the platelet membrane surface and are presumed to be released as particles generated during platelet activation. They are believed to contain added glycans. On the other hand, proteins with a molecular weight of approximately 25 kDa are believed to be cleaved by proteases and released from platelets during platelet activation. In this invention, the amount of such sCLEC-2 is measured. For sCLEC-2, proteins with a molecular weight of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa can be detected together, or only proteins with a molecular weight of approximately 25 kDa can be detected.

[0068] The concentration of sCLEC-2 measured from the subject's sample is usually expressed in pg / mL, but can also be expressed in any unit such as ng / L or ng / mL. D-dimer concentration is expressed in μg / mL or ng / mL, or can also be expressed in any unit such as ng / L. D-dimer concentration can also be expressed in weight units of D-dimer or fibrinogen equivalent units (FEU). Platelet count can be expressed in thousands / μL, tens of thousands / μL, or any other unit. The index value of (sCLEC-2 concentration) × (D-dimer concentration) or (D-dimer concentration) × (sCLEC-2 concentration) / (platelet count) can take various values ​​depending on the units used for each marker; any combination of units is essentially the same and can be appropriately set according to known methods. In addition, similar index values ​​combining sCLEC-2 concentration, D-dimer concentration, or platelet count can also be used.

[0069] For setting the cutoff value, the measurement data of the group that caused adverse cardiovascular / renal events after a certain period in most parent groups are compared with those of the group that did not cause adverse cardiovascular / renal events, and an appropriate cutoff value is set statistically. The cutoff value can be set appropriately using known methods. For example, based on the measured sCLEC-2 concentration or D-dimer concentration, or index values ​​such as (sCLEC-2 concentration) × (D-dimer concentration) or (D-dimer concentration) × (sCLEC-2 concentration) / (platelet count), appropriate stratification can be performed using quartile, ternary, or quartile methods to identify groups with particularly high risk, and the cutoff value can be determined based on their threshold. The threshold value is not limited to equiquartiles and can be set for groups with high risk. Alternatively, ROC curves (Receiver Operating Characteristic Curves) can be generated and analyzed to determine the cutoff value based on the sensitivity and specificity of risk prediction.

[0070] For individual sCLEC-2 concentrations, (sCLEC-2 concentration) × (D-dimer concentration), and (D-dimer concentration) × (sCLEC-2 concentration) / (platelet count), conditions above their respective thresholds can be classified as high-risk, and conditions below their respective thresholds as low-risk. When sCLEC-2 concentration and D-dimer concentration are combined, thresholds can be set for each. Conditions where both sCLEC-2 and D-dimer concentrations are above their respective thresholds are classified as high-risk, and conditions where both are below their respective thresholds are classified as low-risk. Alternatively, conditions where either concentration is above its respective threshold can be classified as high-risk, and conditions where either concentration is below its respective threshold can be classified as low-risk. Furthermore, when stratifying into three or more strata (high-risk, medium-risk, low-risk), two or more thresholds can be set.

[0071] It is believed that in most cases, the index value is calculated using measurements obtained from clinical testing equipment that measures sCLEC2 or D-dimer concentration and measurements obtained from a blood cell counter that measures platelet count. For this calculation, it is suitable for automated calculation in routine clinical practice using hospital testing systems, hospital systems, or electronic medical record systems connected to two or more measuring devices. However, it is also possible to build a system that connects data from two or more measuring devices, or a machine capable of simultaneously measuring sCLEC2 and D-dimer concentrations, or a machine capable of simultaneously measuring sCLEC2, D-dimer, and platelet counts. Alternatively, it can be calculated manually using two or more data points.

[0072] Furthermore, the method for evaluating the long-term risk of adverse cardiovascular / renal events according to the embodiments of the present invention can be used as an aid for physicians in diagnosis.

[0073] In this specification, the long-term risk of cardiovascular / renal adverse events refers to the likelihood of the event occurring several months or more after the sample collection, for example, more than 6 months later. For a longer-term assessment, it refers to the likelihood of the event occurring up to 12 months, then 18 months, and further up to 24 months later.

[0074] According to embodiments of the present invention, when a patient is assessed as having a high long-term risk of adverse cardiovascular / renal events, a thrombotic tendency can be presumed, and antithrombotic therapy can be recommended to the subject. As a choice of antithrombotic therapy, antiplatelet drugs (aspirin, clopidogrel, etc.) and anticoagulants (warfarin, DOAC, etc.) can be selected based on the measured values ​​or index values ​​of sCLEC-2, D-dimer, and / or platelets, and the patient's background information. Since antithrombotic therapy has side effects such as bleeding, it is highly preferable to select patients from the subjects for whom antithrombotic therapy can be recommended in order to maximize the patient's preventative benefits.

[0075] The present invention includes systems described herein that can be used to implement the methods of the present invention, electronic medical records having said systems, clinical examination devices, or hospital examination systems, reagent kits that can be used to implement the methods of the present invention, etc.

[0076] The systems of the present invention include, for example, the systems described above for measuring (or determining) index values, systems for evaluating the long-term high risk of cardiovascular / renal adverse events, systems for assisting in the evaluation of the long-term high risk of cardiovascular / renal adverse events, systems for evaluating (or detecting) the long-term high risk of cardiovascular / renal adverse events, and systems for measuring (or determining) the aforementioned index values ​​in order to evaluate the long-term high risk of cardiovascular / renal adverse events. Specific index values ​​include combinations of soluble CLEC-2 concentration, D-dimer concentration, and / or platelet count; more specifically, examples include (sCLEC-2 concentration) × (D-dimer concentration), (sCLEC-2 concentration) / (platelet count), (D-dimer concentration) × (sCLEC-2 concentration) / (platelet count), etc.

[0077] Any system of the present invention includes: (1) a storage mechanism capable of storing the concentration of soluble CLEC-2 and the concentration of D-dimer and / or the number of platelets in a blood sample from a subject; (2) a calculation mechanism capable of calculating an index value combining the concentration of soluble CLEC-2 and the concentration of D-dimer and / or the number of platelets; (3) a comparison mechanism capable of comparing the index value obtained in (2) with a critical value (e.g., the index value of a comparison group); and (4) a display mechanism capable of displaying the result obtained from the comparison.

[0078] Alternatively, any system of the present invention may include a computer comprising a processor and a memory under the control of the processor, the memory recording a program for causing the computer to perform the following steps: (1) a storage step that stores the concentration of soluble CLEC-2 and the concentration of D-dimer and / or the platelet count in a blood sample from a subject in the memory; (2) a calculation step that calculates an index value combining the concentration of soluble CLEC-2 and the concentration of D-dimer and / or the platelet count; (3) a comparison step that compares the index value obtained in the calculation step with a critical value (e.g., the index value of a comparison group); and (4) a display step that displays the result obtained in the comparison step.

[0079] The system of the present invention can be incorporated into electronic medical records, clinical examination devices, or hospital examination systems.

[0080] 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 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 can be directly applied to index values ​​that can be measured (or determined) using the system of the present invention, to "conditions, diseases, etc." that can be evaluated using the system of the present invention, and to their "evaluation / judgment / criteria, etc.", and to various measurement methods for "soluble CLEC-2 concentration, D-dimer concentration, and / or platelet count," etc.

[0081] The kit of the present invention may include, for example, a soluble CLEC-2 assay reagent, a D-dimer assay reagent, a platelet count assay reagent, etc., and may at least include a soluble CLEC-2 assay reagent. Additionally, it may include accompanying documentation describing the relationship between soluble CLEC-2 concentration, D-dimer concentration, platelet count, or an index value combining the soluble CLEC-2 concentration and D-dimer concentration and / or the platelet count, and the long-term high risk of cardiovascular / renal adverse events. The accompanying documentation may also include a pre-calculation method for the index value.

[0082] Furthermore, the accompanying documentation included in the kit of the present invention is not particularly limited as long as it at least mentions the relationship between the soluble CLEC-2 concentration, or the index value composed of the combination of the soluble CLEC-2 concentration and the D-dimer concentration and / or the platelet count, and the long-term high incidence risk of cardiovascular / renal adverse events. In addition to the above-mentioned content, it may also include, for example, instructions on the implementation steps of the immunological assay using the kit of the present invention, instructions on the steps of evaluating the long-term high incidence risk of cardiovascular / renal adverse events based on the obtained measurement values, and precautions for the storage / handling of the kit itself.

[0083] Example The present invention will be specifically described below through embodiments, but these embodiments do not limit the scope of the present invention.

[0084] Example 1: Measurement of sCLEC-2, D-dimer, platelet count, and other data collection in patients with chronic kidney disease Example 1-1: Determination of sCLEC-2 (Preparation of reagents for sCLEC-2 assay) • Specimen diluent: The specimen diluent was prepared by combining 2% sodium octanoate and 0.5% n-octyl-β-D-glucoside (OG) in 0.1 mol / L HEPES buffer (pH 7.5) containing preservatives.

[0085] The antibody contained in the reagent was prepared using the antibody described in the examples of Japanese Patent No. 6078845, as follows.

[0086] • First antibody solution: The mouse monoclonal antibody (11D5) recognizing sCLEC-2 is bound to magnetic latex particles (JSR) and dispersed in 0.01 mol / L MES buffer (pH 6.0) containing preservatives.

[0087] • Second antibody solution: Another mouse monoclonal antibody (11E6) recognizing sCLEC-2 was labeled with alkaline phosphatase (ALP) by maleimide method and dispersed in 0.01 mol / L MES buffer (pH 6.5) containing preservative.

[0088] • Luminescent matrix solution: 2-chloro-5-(4-methoxyspiro{1,2-dioxane-3,2'-(5'-chloro)-tricyclo[3.3.1.13,7]decane}-4-yl)-1-phenylphosphate disodium (CDP-Star (registered trademark): Applied Biosystems).

[0089] • B / F cleaning solution: Use a buffer solution containing 0.1 mol / L citric acid (pH 6.5), 0.15 mol / L NaCl, and 0.1% Triton X-100.

[0090] (Determination based on the reagents used) The sCLEC-2 assay uses the STACCIA (registered trademark, manufactured by LSI Medience Corporation) fully automated clinical testing system.

[0091] The prepared sample diluent, first antibody solution (magnetic latex reagent), and second antibody solution (enzyme-labeled antibody reagent) were respectively filled into STACCA-specific bottles and placed in the device. The following measurements were performed according to the operating procedure of the device.

[0092] Specifically, 40 μL of sample diluent was added to 10 μL of blood sample collected in the form of citrate plasma. After incubation at 37°C for several minutes, 25 μL of the first antibody solution (magnetic latex reagent) was added, and the mixture was incubated at 37°C for several minutes. Next, B / F separation was performed, and 50 μL of the second antibody solution (enzyme-labeled antibody reagent) was added. After incubation at 37°C for several minutes, B / F separation was performed again. Finally, 100 μL of luminescent matrix solution was added, and the mixture was reacted at 37°C for several minutes. The signal intensity (counting) was then measured.

[0093] Examples 1-2: Determination of D-dimer For the determination of D-dimer, the above-mentioned STAICA was used with the LPIA-Genesis D-dimer reagent (manufactured by LSI Medience Corporation).

[0094] Examples 1-3: Determination of platelet count Platelet count was determined using an automated blood cell analyzer (Sysmex).

[0095] Examples 1-4: Determination of other items B-type natriuretic peptide (BNP) is manufactured by LSI Medience Corporation. Hemoglobin levels, blood glucose levels, and creatinine (used to calculate eGFR) are measured using reagents according to standard methods.

[0096] Examples 1-5: Specimens and Research Protocols The subjects measured were 453 individuals (aged 64 ± 15 years, estimated glomerular filtration rate (eGFR) 49 ± 25 mL / min / 1.73 m). 2 Patients with chronic kidney disease (CKD) visiting the outpatient clinic were excluded. Additionally, patients with CKD stage 5 (eGFR: <15 mL / min / 1.73 mcg) were excluded. 2 Inclusion criteria included individuals aged 85 and older. Blood samples were collected during outpatient visits to measure sCLEC-2, D-dimer, platelet count, B-type natriuretic peptide (BNP), hemoglobin levels, blood glucose levels, and creatinine (for eGFR calculation). In addition, past medical history was investigated for myocardial infarction, stroke, heart failure, hypertension, diabetes, and dyslipidemia. The endpoint was defined as the occurrence of adverse cardiovascular / renal events (new regulations on onset of cardiovascular / cerebrovascular disease or interventional dialysis) within the 24-month observation period.

[0097] Example 2: Long-term risk assessment of adverse cardiovascular / renal events in CKD patients Following the results of the study in Example 1, 52 cases (11.5%) of adverse cardiovascular / renal events were identified during a 24-month observation period. Compared with the non-adverse event group, the adverse event group had significantly higher levels of sCLEC-2, D-dimer, BNP, and blood glucose. Conversely, the adverse event group had significantly lower levels of eGFR and hemoglobin. Cox proportional hazards multivariate analysis showed that sCLEC-2, D-dimer, blood glucose, and a history of stroke were independent determinants of the incidence of adverse cardiovascular / renal events.

[0098] Example 3: Long-term high incidence risk assessment of cardiovascular / renal adverse events in CKD patients at sCLEC-2 concentrations alone CKD patients were divided into three groups using the tertiary method of sCLEC-2 values. The incidence of adverse events in each tertiary is shown in Table 1.

[0099] [Table 1] Create a chart and display it Figure 1 Compared to sCLEC-2 values ​​below 250 pg / mL, sCLEC-2 values ​​above 250 pg / mL carry a long-term risk approximately three times higher over 24 months.

[0100] Furthermore, the Kaplan-Meier curves for this CDK syndrome group are shown in... Figure 2 According to the Kaplan-Meier curve, it can be observed that in cases with sCLEC-2 values ​​above 250 pg / mL, the incidence of adverse cardiovascular / renal events increases almost linearly over 24 months (while the proportion of subjects without events decreases linearly). This indicates that the long-term high risk of adverse cardiovascular / renal events can be evaluated based on sCLEC-2 values.

[0101] Example 4: Long-term high-incidence risk assessment of cardiovascular / renal adverse events in CKD patients using a combination of sCLEC-2 and D-dimer concentrations CKD patients were divided into two groups based on sCLEC-2 values ​​of less than 250 pg / mL and sCLEC-2 values ​​of 250 pg / mL and above. Further subgrouping was performed using the ternary intervals of D-dimer (less than 0.6 μg / mL, 0.6-1.45 μg / mL, and above 1.45 μg / mL). The incidence of adverse cardiovascular / renal events in each group was shown in the table. Figure 3 Furthermore, the Kaplan-Meier curves for this CKD group are shown in... Figure 4 The group with sCLEC-2 levels above 250 pg / mL and D-dimer levels above 1.45 μg / mL had an approximately 27% probability of experiencing adverse cardiovascular / renal events within 24 months, indicating a very high long-term risk. Furthermore, the upper limit for normal D-dimer levels is 1.0 μg / mL when using this reagent.

[0102] Example 5: Long-term high-incidence risk assessment of cardiovascular / renal adverse events in CKD patients at (sCLEC-2 concentration) × (D-dimer concentration) Calculate (sCLEC-2 concentration) × (D-dimer concentration) for each patient, and then display the incidence of adverse cardiovascular / renal events using the tertiary intervals of this value (less than 180, 180-450, and above 450). Figure 5Furthermore, the Kaplan-Meier curves for this CKD group are shown in... Figure 6 The group with (sCLEC-2 concentration) × (D-dimer concentration) above 450 had a 19.7% probability of experiencing adverse cardiovascular / renal events within 24 months, indicating a very high long-term risk, approximately 5 times higher than the group with (sCLEC-2 concentration) × (D-dimer concentration) below 180.

[0103] Example 6: Long-term high-incidence risk assessment of cardiovascular / renal adverse events in CKD patients at the ratio of (D-dimer concentration) × (sCLEC-2 concentration) / (platelet count) Calculate (D-dimer concentration) × (sCLEC-2 concentration) / (platelet count) for each patient, and use the tertiary intervals of this value (less than 0.84, 0.84-2.25, and above 2.25) to represent the incidence of adverse cardiovascular / renal events. Figure 7 Furthermore, Kaplan-Meier curves of adverse cardiovascular / renal events in this CKD patient are shown in... Figure 8 The group with a D-dimer concentration × (sCLEC-2 concentration) / (platelet count) of 2.25 or higher had a 20.1% probability of experiencing adverse cardiovascular / renal events within 24 months, which is a very high risk, approximately 8 times higher than the group with a D-dimer concentration × (sCLEC-2 concentration) / (platelet count) of 0.84 or lower.

[0104] Industrial applicability As described above, the serum sCLEC-2 measurement, or a combination of serum sCLEC-2 and serum D-dimer measurements, or a combination of serum sCLEC-2 and platelet count, or a combination of serum sCLEC-2, serum D-dimer, and platelet count, serves as an indicator capable of determining the long-term high risk of cardiovascular / cerebrovascular / renal adverse events. Therefore, to improve patient benefit in prevention, antithrombotic therapy can be appropriately recommended. Serum sCLEC-2 measurement is useful as a method for assessing the long-term high risk of cardiovascular / cerebrovascular / renal adverse events.

Claims

1. A method for evaluating the long-term high risk of cardiovascular / renal adverse events based on the concentration of soluble CLEC-2 in the blood of subjects.

2. The method according to claim 1, which is a method for evaluating the long-term high incidence risk of cardiovascular / renal adverse events based on the concentration of soluble CLEC-2 in the blood of a subject, the method comprising: (1) The procedure of providing a blood sample from the subject; (2) The process of determining the concentration of soluble CLEC-2 in the sample; (3) A process of associating the concentration of the soluble CLEC-2 with the long-term likelihood of cardiovascular / renal adverse events in the subject.

3. The method according to claim 1 or 2, wherein the subject is a method for long-term risk assessment of a disease suspected of having a risk of adverse cardiovascular / renal events or a patient diagnosed with such a disease.

4. The method according to any one of claims 1 to 3, wherein, Diseases at risk of cardiovascular / renal adverse events are selected from chronic kidney disease, diabetes, hypertension, dyslipidemia, chronic maintenance dialysis, and arteriosclerotic diseases.

5. The method according to any one of claims 1 to 4, wherein, In addition to the concentration of soluble CLEC-2 in the subjects' blood, the long-term risk of adverse cardiovascular / renal events was further evaluated based on the concentration of D-dimer in the subjects' blood.

6. The method according to claim 5, wherein, The long-term risk of adverse cardiovascular / renal events was evaluated by multiplying the concentration of soluble CLEC-2 in the subject's blood by the concentration of D-dimer.

7. The method according to any one of claims 1 to 4, wherein, In addition to the concentration of soluble CLEC-2 in the subjects' blood, the long-term risk of cardiovascular / renal adverse events was further evaluated based on the subjects' platelet count.

8. The method according to claim 7, wherein, The long-term risk of adverse cardiovascular / renal events is evaluated by dividing the concentration of soluble CLEC-2 in the subject's blood by the platelet count.

9. The method according to claim 7, wherein, In addition to the concentration of soluble CLEC-2 and platelet count in the subjects' blood, the long-term risk of adverse cardiovascular / renal events was further evaluated based on the concentration of D-dimer in the subjects' blood.

10. The method according to claim 9, wherein, The long-term high risk of cardiovascular / renal adverse events is evaluated based on the value expressed by the following formula (1): [Mathematical Expression 1] 。 11. A method for evaluating the long-term high risk of cardiovascular / cerebrovascular / renal adverse events and selecting antithrombotic drugs, wherein the method according to any one of claims 1 to 10 evaluates the long-term high risk of cardiovascular / cerebrovascular / renal adverse events and selects antithrombotic drugs.

12. A system for evaluating the long-term high risk of cardiovascular / cerebrovascular / renal adverse events, comprising: (1) A storage device capable of storing the concentration of soluble CLEC-2 and D-dimer and / or platelet count in a blood sample from a subject; (2) A calculation mechanism capable of calculating an index value that combines the concentration of soluble CLEC-2 with the concentration of D-dimer and / or the platelet count; (3) A comparison mechanism that can compare the index value obtained in (2) with the critical value; and (4) A display mechanism that can display the results obtained from the comparison.

13. A system for evaluating the long-term high risk of cardiovascular / cerebrovascular / renal adverse events, comprising a computer, the computer including a processor and a memory under the control of the processor, the memory storing a program for causing the computer to perform the following steps: (1) A storage process in which the concentration of soluble CLEC-2, the concentration of D-dimer, and / or the platelet count in a blood sample from the subject are stored in the memory. (2) The calculation process, which calculates the index value of the soluble CLEC-2 concentration, the D-dimer concentration and / or the platelet count; (3) A comparison step, which compares the index value obtained in the calculation step with the critical value; and (4) Display process, which displays the results obtained in the comparison process.

14. An electronic medical record, clinical examination device or hospital examination system comprising the system of claim 12 or 13.

15. A kit for evaluating the long-term high risk of cardiovascular / cerebrovascular / renal adverse events, comprising: (1) Soluble CLEC-2 assay reagent; and (2) The accompanying document describes the relationship between soluble CLEC-2 concentration, or an index consisting of a combination of soluble CLEC-2 concentration, D-dimer concentration, and / or platelet count, and the long-term high risk of cardiovascular / renal adverse events.

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