Method for evaluating long-term high onset risk of cerebrovascular, cardiovascular, and renal events using soluble CLEC-2

Abstract

The present invention provides a technique capable of accurately and simply predicting the long-term onset risk of cerebrovascular, cardiovascular, and renal events. By use of said technique, the present invention also provides a technique for selecting a treatment method for increasing preventative benefits for a subject patient.　Said technique involves evaluating the long-term high onset risk of cerebrovascular, cardiovascular, and renal events by using the concentration of soluble CLEC-2 in the subject's blood.

Description

Long-term high-risk assessment of cerebrovascular and renal events using soluble CLEC-2

[0001] The present invention relates to a method for long-term high risk assessment of cerebrovascular and renal events using soluble CLEC-2 (sCLEC-2).

[0002] Cerebrovascular and renal events refer to the development of cerebrovascular diseases such as stroke, ischemic heart disease (myocardial infarction, etc.), and arteriosclerosis obliterans, which may lead to the introduction of dialysis. Cerebrovascular diseases are a very important group of diseases that cause as many deaths as cancer in Japan and are the leading cause of death worldwide. It is extremely important to correctly assess the risk of developing these diseases and implement appropriate prevention and treatment measures.

[0003] Known risk factors for cerebrovascular and renal events include diabetes, chronic kidney disease (CKD), hypertension, dyslipidemia, obesity, and smoking, and managing each of these factors individually is important for risk reduction. Furthermore, the combined presence of these risk factors increases the risk. These risk factors have markers for assessment, and corresponding preventative measures and treatments are being implemented. For example, for dyslipidemia, there are risk markers such as LDL-cholesterol, and if these are abnormal, drugs such as statins are available to improve dyslipidemia. For diabetes, there are risk markers such as blood glucose levels and hemoglobin A1C, and if these are abnormal, measures such as diabetes medication, diet, and exercise are implemented. Furthermore, treatments and interventions tailored to each risk are being implemented to prevent cerebrovascular and renal events, such as antihypertensives for hypertension and smoking cessation for smokers. However, even with these individual treatments and prevention measures, patients with diabetes, hypertension, chronic kidney disease, etc. remain at risk for cerebrovascular and renal events, and risk assessment and interventions for cerebrovascular and renal events are important.

[0004] It is well known that the mechanism of onset of major cerebrovascular events such as cerebral infarction, myocardial infarction, and arteriosclerosis obliterans is the blockage of blood vessels by thrombi, and antithrombotic therapy has been established as a preventative measure. Antiplatelet drugs are used in cases where platelets are considered to be the main cause of thrombi, such as arterial thrombosis, and anticoagulants are used in cases where the coagulation system is considered to be the main cause of thrombi, such as venous thrombosis.

[0005] In this specification, the risk group and high-risk group for cerebrovascular and renal events are defined as follows. The risk group is defined as a group that is considered to be at higher risk of developing cerebrovascular and renal events than healthy individuals, but for whom antithrombotic therapy is not generally administered. This risk group includes patients with diabetes, chronic kidney disease, hypertension, dyslipidemia, obesity, smoking habits, etc. The high-risk group is a group that is at even higher risk of developing cerebrovascular and renal events 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, arteriosclerosis obliterans, angina pectoris, atrial fibrillation, or who have undergone artificial valve replacement surgery, etc.

[0006] While antithrombotic therapy is effective in preventing thrombosis by inhibiting thrombus formation, its side effect can be the induction of bleeding disorders due to the suppression of thrombus formation. Therefore, it is important to consider the balance between the preventive benefits and the risk of side effects when using antithrombotic therapy. Therefore, antiplatelet drugs are used for the treatment or secondary prevention of high-risk patients, such as those with high-risk diseases, such as angina pectoris, who are particularly susceptible to myocardial infarction, or those with a history of cerebral infarction, myocardial infarction, or arteriosclerosis obliterans. They are not generally used for primary prevention (prevention in people without a history of cerebrovascular disease) in patients at risk, such as those with diabetes, hypertension, or chronic kidney disease (CKD). For similar reasons, anticoagulant therapy is currently only used to prevent thrombosis in high-risk patients, such as those with atrial fibrillation or after prosthetic valve replacement. This is also stated in the Japanese guidelines for the treatment of CKD (Non-Patent Document 1), which states, "It is unclear whether the administration of aspirin is effective in preventing cardiovascular events in patients with CKD stages G3b to G5. On the other hand, it cannot be denied that the administration of aspirin may increase the risk of bleeding complications." As such, more accurate risk assessment is required for the administration of aspirin (antiplatelet drugs).

[0007] Because antiplatelet drugs are known to be effective against arteriosclerotic thrombosis, it can be assumed that a good risk prediction marker would be one that monitors changes in platelets. To this end, markers that monitor platelet activation and markers that measure platelet aggregation have been proposed. However, markers that measure platelet activity are difficult to measure and are not widely used clinically, and there are no markers that can predict cerebrovascular or renal events. Platelet aggregation was also expected to be a risk prediction marker, but its predictive ability was low and it has not been put to practical use. The technology described in Patent Document 1, which measures the rate of platelet count decline, has also not yet been put to practical use.

[0008] Patent Document 2 shows that the concentration of soluble CLEC-2 (sCLEC-2), a platelet activation marker, indicates platelet activation, and its measured value increases in thrombosis. Furthermore, if the platelet marker value changes, it is believed that thrombus formation has already occurred, and sCLEC-2 concentration is believed to indicate imminent danger, i.e., an increase in short-term risk (Patent Document 3). Furthermore, the technology described in Patent Document 2 has not yet been put to practical use. In other words, a platelet-based thrombosis risk marker technology for short-term cerebrovascular and renal events has not yet been established.

[0009] Activation of the coagulation system is also an important cause of thrombosis. Because thrombus formation progresses through the interaction between platelets and the coagulation system, it is said that platelets are the main players in the arterial system and the coagulation system (fibrin formation) is the main player in the venous system. However, any thrombus contains both platelets and fibrin. Therefore, coagulation system markers are also considered to be risk markers for the development of cerebrovascular and renal events. A representative example is D-Dimer. While D-Dimer can be a marker for the development of cerebrovascular and renal events such as cardiogenic cerebral infarction and aortic dissection, it is not generally used as a risk marker for cerebrovascular and renal events. For example, Non-Patent Document 2 assesses the risk of cardiovascular events using D-Dimer, and the tertiles of D-Dimer for patients with stable coronary artery disease (a high-risk disease for cardiovascular events) are (<0.32, 0.32-0.61, >0.62 μg / mL). Patients in this third quintile (>0.62 μg / mL) have a higher risk of developing cardiovascular events than patients in the first quintile (<0.32 μg / mL). The upper limit for healthy individuals for the D-Dimer measurement reagent used in this study was 1.0 μg / mL, so the risk assessment threshold was within the range of D-Dimer values ​​in normal individuals, and many healthy individuals would be included in the group assessed as high-risk, making it unsuitable as a risk assessment marker. Thus, D-Dimer is not considered an accurate marker for the risk of developing cerebrovascular or renal events.

[0010] As described above, there is currently no established technology that can evaluate the risk of developing cerebrovascular and renal events from the perspective of thrombosis, much less any suggestion of a thrombosis risk marker that can evaluate the long-term risk of developing cerebrovascular and renal events from the perspective of thrombosis.

[0011] Japanese Patent Publication No. 2022-73652 Japanese Patent No. 6078845 International Publication No. 2022 / 255348 International Publication No. 2021 / 172493

[0012] Clinical practice guidelines for preventing progression of kidney damage and smooth transition to renal replacement therapy for patients with CKD stage G3b-5. 2015 Japanese Society of Nephrology H. Naruse et al. Circulation Journal 2017 81: 1506-1513

[0013] The present invention aims to provide a technology that can accurately and simply predict the long-term risk of developing cerebrovascular and renal events, for which a large number of potential patients are expected but for which no useful methods have been found. In particular, the present invention aims to provide a technology that can predict patients with a high long-term risk of developing cerebrovascular and renal events from among patients with diseases considered to be at risk for cerebrovascular and renal events, such as diabetes, hypertension, and chronic kidney disease. Furthermore, the present invention aims to provide a technology that, based on these, can select a treatment method that will increase the preventive benefit for target patients.

[0014] The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they found that, among risk groups for cerebrovascular and renal events, groups with high blood sCLEC-2 concentrations have a particularly high long-term risk of developing cerebrovascular and renal events. In addition to this finding, they discovered that a patient group with a particularly high long-term risk of developing cerebrovascular and renal events can be extracted by using a combination of coagulation system markers D-Dimer and sCLEC-2, or a combination of sCLEC-2, D-Dimer, and platelet count, particularly an index value of (blood sCELC-2 concentration) × (blood D-Dimer concentration) or (blood D-Dimer concentration) × (blood sCLEC-2) / (platelet count), and thus completed the present invention.

[0015] Because many cardiovascular and renal events are primarily caused by vascular blockage due to thrombi, it is known that biomarkers related to fibrin (a product of the coagulation system), which is the main component of thrombi, and platelets often exhibit abnormal values ​​at the onset of cardiovascular and renal events. This is natural, as a clot is often present at the time of onset. These markers include D-Dimer, FDP, soluble fibrin, and thrombin-antithrombin complex in the fibrin system (coagulation system), and sCLEC-2, β-thromboglobulin, platelet factor 4, soluble p-selectin, and platelet aggregation in the platelet system. However, to be useful as long-term predictive markers for cardiovascular and renal events, these markers must exhibit abnormal values ​​outside the normal range well before the onset of cardiovascular and renal events. However, there is no evidence that these existing markers can serve as long-term predictive markers for cardiovascular and renal events, and they are not, of course, used. The surprising finding that the long-term high risk of developing cerebrovascular or renal events can be assessed by sCLEC-2 concentration, a combination of sCLEC-2 concentration and platelet count, a combination of sCLEC-2 concentration and D-Dimer concentration, and a combination of sCLEC-2 concentration, D-Dimer concentration, and platelet count is unpredictable based on conventional knowledge. Furthermore, it also makes it possible to easily recommend a treatment that will increase the preventive benefit for the target patient, making the present invention extremely effective and significant.

[0016] In a state where a thrombus is present or is forming, the above-mentioned markers exhibit abnormal values. However, the present inventors have found that when a new index based on sCLEC-2 concentration alone, a combination of sCLEC-2 concentration and platelet count, a combination of sCLEC-2 concentration and D-Dimer concentration, or a combination of sCLEC-2 concentration, D-Dimer concentration, and platelet count exhibits abnormal values, the incidence of cerebrovascular / renal events up to 24 months after measurement is 3 to 8 times higher than in cases where abnormal values ​​are not observed. Because thrombus formation is generally considered to occur immediately before the onset of cerebrovascular events, no thrombus-related marker has been found to be a marker that can predict the onset of cerebrovascular / renal events 24 months before the onset. In fact, existing platelet activation markers, platelet aggregation, etc., are not known to function alone as risk markers for cerebrovascular / renal events.

[0017] For example, it is generally believed that abnormalities in platelet-related markers such as sCLEC-2 or platelet counts indicate thrombus formation and therefore increase short-term risk (Patent Document 3). On the other hand, as described in the Examples below, we discovered that the cumulative incidence of cerebrovascular and cardiovascular events increases at a nearly constant rate over 24 months after measurement in the high sCLEC-2 group. If high sCLEC-2 levels represent thrombus formation causing vascular blockage at the time of blood collection or within a relatively short period thereafter, we would predict a relatively short-term increase in the frequency of cerebrovascular and cardiovascular events. However, the Kaplan-Meier curve did not reveal a short-term increase in incidence, and the cumulative incidence continued to increase almost linearly over 24 months. Therefore, we discovered that sCLEC-2 and related indicators in the present invention reflect a subject's long-term risk.

[0018] One example of a threshold (cutoff value) for determining long-term high risk of developing cerebrovascular or renal events based on sCLEC-2 concentration was 250 pg / mL. Because blood sCLEC-2 concentrations in healthy individuals are 200 pg / mL or less, very few healthy individuals would be determined to be at high risk at this threshold. It was also found that setting an appropriate sCLEC-2 cutoff value of 200 pg / mL or higher would significantly reduce false positives in which healthy individuals or other low-risk individuals are determined to be at high risk.

[0019] Furthermore, as described in the Examples below, in order to determine the long-term high risk of developing cerebrovascular and renal events, it is preferable to evaluate the platelet system, or the platelet system and the coagulation system simultaneously, as a thrombosis risk marker. Although sCLEC-2 concentration can evaluate the state of platelets and can be used alone as a long-term risk marker for developing cerebrovascular and renal events, it has been found that combining sCLEC-2 concentration with D-Dimer concentration can provide an even more useful marker for the long-term risk of developing cerebrovascular and renal events. Furthermore, it has been found that platelet activity can be accurately determined by combining sCLEC-2 concentration with platelet count (Patent Document 4), and furthermore, by combining sCLEC-2 concentration with D-Dimer concentration and / or platelet count, an index value of (blood sCLEC-2 concentration) × (blood D-Dimer concentration), (blood sCLEC-2 concentration) / (platelet count), or (blood D-Dimer concentration) × (blood sCLEC-2) / (platelet count) can be a long-term high-risk marker for the development of cerebrovascular, cardiovascular, and renal events.

[0020] In summary, the present inventors have found that blood sCLEC-2 concentration alone, or a combination of blood sCLEC-2 concentration and blood D-Dimer concentration, a combination of blood sCLEC-2 concentration and platelet count, or a combination of blood sCLEC-2 concentration, blood D-Dimer concentration and platelet count, and in particular, an index value of (blood sCLEC-2 concentration) × (blood D-Dimer concentration), (blood sCLEC-2) / (platelet count), or (blood D-Dimer concentration) × (blood sCLEC-2) / (platelet count), can identify a group at long-term high risk of developing cerebrovascular or renal events. The present invention is based on these findings.

[0021] That is, the present invention provides the following: [1] A method for assessing a subject's long-term high risk of developing cerebrovascular or renal events based on the concentration of soluble CLEC-2 in their blood. [2] A method for assessing a subject's long-term high risk of developing cerebrovascular or renal events based on the concentration of soluble CLEC-2 in their blood, the method of [1] comprising the steps of: (1) providing a blood sample from the subject; (2) determining the concentration of soluble CLEC-2 in the sample; and (3) correlating the concentration of soluble CLEC-2 with the long-term possibility of developing cerebrovascular or renal events in the subject. [3] The method of [1] or [2], which is a method for assessing the long-term risk of developing cerebrovascular or renal events in patients suspected of or diagnosed with a disease associated with a risk of cerebrovascular or renal events. [4] Any of the methods [1] to [3], wherein the disease associated with a risk of cerebrovascular and renal events is selected from chronic kidney disease, diabetes, hypertension, dyslipidemia, chronic maintenance dialysis, and arteriosclerotic disease. [5] Any of the methods [1] to [4], wherein the long-term high risk of developing cerebrovascular and renal events is assessed based on the D-Dimer concentration in the subject's blood in addition to the soluble CLEC-2 concentration in the subject's blood. [6] The method [5], wherein the long-term high risk of developing cerebrovascular and renal events is assessed based on the value obtained by multiplying the soluble CLEC-2 concentration in the subject's blood by the D-Dimer concentration. [7] Any of the methods [1] to [4], wherein the long-term high risk of developing cerebrovascular and renal events is assessed based on the soluble CLEC-2 concentration in the subject's blood in addition to the platelet count. [8] The method of [7], which evaluates the long-term high risk of developing cerebrovascular / renal events based on the value obtained by dividing the soluble CLEC-2 concentration in the blood of a subject by the platelet count. [9] The method of [7], which evaluates the long-term high risk of developing cerebrovascular / renal events based on the D-Dimer concentration in the blood of a subject in addition to the soluble CLEC-2 concentration and platelet count in the blood of a subject.

[10] The method of [9], which evaluates the long-term high risk of developing cerebrovascular / renal events based on the value represented by the following formula (1):

[11] A method for evaluating the long-term high risk of developing cerebrovascular and / or renal events by any of the methods [1] to

[10] , and selecting an antithrombotic drug.

[0022] The present invention also provides: a method for assessing a long-term high risk of developing cerebrovascular / renal events based on the concentration of soluble CLEC-2 in the blood of a subject (i.e., for example, the concentration of soluble CLEC-2 alone, or a combination of the concentration of soluble CLEC-2 with the D-Dimer concentration and / or the platelet count); a method for assessing a long-term high risk of developing cerebrovascular / renal events based on the concentration of soluble CLEC-2 in the blood of a subject, comprising: (1) providing a blood sample from the subject; (2) determining the concentration of soluble CLEC-2 in the sample; and (3) correlating the concentration of soluble CLEC-2 with the long-term possibility of developing cerebrovascular / renal events in the subject; - a method for assisting in the long-term high risk assessment of cerebrovascular / renal events by measuring the soluble CLEC-2 concentration in the blood of a subject (i.e., for example, the soluble CLEC-2 concentration alone or a combination of the soluble CLEC-2 concentration with the D-Dimer concentration and / or the platelet count); - a method for measuring (or determining) the soluble CLEC-2 concentration in the blood of a subject (i.e., for example, the soluble CLEC-2 concentration alone or a combination of the soluble CLEC-2 concentration with the D-Dimer concentration and / or the platelet count) in order to assess the long-term high risk of cerebrovascular / renal events; - an in vitro method for assessing the long-term high risk of cerebrovascular / renal events, characterized by measuring (or determining) the soluble CLEC-2 concentration in the blood of a subject (i.e., for example, the soluble CLEC-2 concentration alone or a combination of the soluble CLEC-2 concentration with the D-Dimer concentration and / or the platelet count); - Use of an antibody capable of measuring (or determining) soluble CLEC-2 concentration in the manufacture of a kit for assessing a high risk of developing cerebrovascular / renal events in the long term; - Use of an antibody capable of measuring (or determining) soluble CLEC-2 concentration and an antibody capable of measuring (or determining) D-Dimer concentration in the manufacture of a kit for assessing a high risk of developing cerebrovascular / renal events in the long term; and - A method for measuring (or determining) the soluble CLEC-2 concentration in the blood of a subject (i.e., for example, the soluble CLEC-2 concentration alone, or a combination of the soluble CLEC-2 concentration with the D-Dimer concentration and / or the platelet count) in order to provide information necessary for the long-term assessment of a high risk of developing cerebrovascular / renal events;The present invention also includes a method for evaluating a long-term high risk of developing cerebrovascular / renal events using any of these methods and selecting an antithrombotic drug.

[0023] The long-term risk of developing cerebrovascular and renal events can be easily and accurately evaluated using sCLEC-2 concentration alone or a combination of sCLEC-2 concentration with D-Dimer concentration and / or platelet count. Here, the combination of sCLEC-2 concentration with D-Dimer concentration and / or platelet count means a combination of sCLEC-2 concentration with either D-Dimer concentration or platelet count, as well as a combination of D-Dimer concentration, platelet count, and sCLEC-2 concentration, and more specifically, a combination of blood D-Dimer concentration and blood sCLEC-2 concentration, a combination of platelet count and blood sCLEC-2 concentration, and a combination of blood D-Dimer concentration, platelet count, and blood sCLEC-2 concentration.

[0024] Graph showing the tertiles of blood sCLEC-2 concentrations in 453 CKD patients and the frequency of cerebrovascular and renal events. Kaplan-Meier curves for tertiles of blood sCLEC-2 concentrations in 453 CKD patients. Graph showing the frequency of cerebrovascular and renal events for combinations of blood sCLEC-2 concentrations and blood D-Dimer concentrations in 453 CKD patients. Kaplan-Meier curves for combinations of blood sCLEC-2 concentrations and blood D-Dimer concentrations in 453 CKD patients. Graph showing the frequency of cerebrovascular and renal events for 453 CKD patients and the tertiles of (blood sCLEC-2 concentration) x (blood D-Dimer concentration). 1 shows Kaplan-Meier curves for 453 CKD patients based on tertiles of (blood sCLEC-2 concentration) x (blood D-Dimer concentration). 2 shows graphs of the incidence of cerebrovascular and renal events for 453 CKD patients based on tertiles of (blood D-Dimer concentration) x (blood sCLEC-2 concentration) / (platelet count). 3 shows Kaplan-Meier curves for 453 CKD patients based on tertiles of (blood D-Dimer concentration) x (blood sCLEC-2 concentration) / (platelet count).

[0025] Hereinafter, an embodiment of the present invention will be described in detail using the measurement and analysis of sCLEC-2 and the like in the blood of patients with chronic kidney disease (CKD) as an example of an embodiment of a method for evaluating a high risk of long-term onset of cerebrovascular and renal events, but the manner of use is not limited to this.

[0026] The method of the first aspect of the present invention includes a step of measuring the sCLEC-2 concentration in a blood sample collected from a subject (hereinafter referred to as a subject) for whom the risk of cerebrovascular and / or renal events is to be assessed. When assessing the long-term risk of developing cerebrovascular and / or renal events based solely on the sCLEC-2 concentration, for example, an appropriate cutoff value of 200 pg / mL or higher can be set, and a value equal to or greater than this cutoff value can be assessed as indicating a high long-term risk of developing cerebrovascular and / or renal events. A value below this cutoff value can be assessed as indicating a low long-term risk of developing cerebrovascular and / or renal events.

[0027] The method of the second aspect of the present invention includes measuring the sCLEC-2 concentration and the D-Dimer concentration in a blood sample collected from a subject. When evaluating the long-term risk of developing cerebrovascular / renal events using a combination of sCLEC-2 concentration and D-Dimer concentration, for example, appropriate cutoff values ​​are set for each, and if either or both exceed the cutoff value, the long-term risk of developing cerebrovascular / renal events can be evaluated as high. In this case, the cutoff value for sCLEC-2 concentration can be the value used when evaluating sCLEC-2 concentration alone. Furthermore, the cutoff value for D-Dimer concentration is preferably 1.0 μg / mL or higher, which is the upper limit for healthy individuals, and more preferably 1.45 μg / mL or higher.

[0028] In addition, as another evaluation method of the second aspect, when evaluation is performed using the value obtained by multiplying the sCLEC-2 concentration by the D-Dimer concentration, i.e., "(sCLEC-2 concentration) x (D-Dimer concentration)," an appropriate cutoff value can be set for the index value, and if the index value is equal to or higher, the long-term risk of developing cerebrovascular / cardiovascular and renal events can be evaluated as high, and if the index value is lower than that, the long-term risk of developing cerebrovascular / cardiovascular and renal events can be evaluated as low.

[0029] The method of the third aspect of the present invention includes the step of measuring the sCLEC-2 concentration and platelet count in a blood sample collected from a subject. In addition to the sCLEC-2 concentration and platelet count, the method of the third aspect of the present invention can also measure the D-Dimer concentration in the blood sample from the subject.

[0030] In the method of the third aspect of the present invention, when the long-term risk of developing cerebrovascular or renal events is evaluated using a combination of sCLEC-2 concentration and platelet count, for example, the evaluation method and cutoff value of the method of the first aspect (e.g., sCLEC-2 concentration alone) can be used, and the platelet count can also be added for evaluation, or an evaluation method and cutoff value that combines sCLEC-2 concentration and platelet count can be used for evaluation. Specific examples of the calculation formula for the evaluation method that combines sCLEC-2 concentration and platelet count include the use of an index value of "(sCLEC-2 concentration) / (platelet count)." In another method of the third aspect of the present invention, when the long-term risk of developing cerebrovascular or renal events is evaluated using a combination of sCLEC-2 concentration, D-Dimer concentration, and platelet count, for example, the evaluation method (e.g., a combination of sCLEC-2 concentration and D-Dimer concentration) and cutoff value in the method of the second aspect can be used, and the platelet count can be added to the evaluation, or the D-Dimer concentration can be added to the evaluation method and cutoff value that combines sCLEC-2 concentration and platelet count. Specific examples of the calculation formula for the evaluation method that combines sCLEC-2 concentration, D-Dimer concentration, and platelet count include the use of an index value of "(D-Dimer concentration) x (sCLEC-2 concentration) / (platelet count)".

[0031] Because sCLEC-2 is produced from platelets, there is a weak correlation between sCLEC-2 concentration and platelet count. For this reason, it has been reported that the index value "(sCLEC-2 concentration) / (platelet count)," which represents the sCLEC-2 value per platelet, better represents the degree of platelet activation than the sCLEC-2 concentration alone (Patent Document 4), and this can be referenced as appropriate. For example, an appropriate cutoff value can be set for the value "(sCLEC-2 concentration) / (platelet count)," which is the sCLEC-2 concentration divided by the platelet count, and a value exceeding this cutoff value can be evaluated as indicating a high long-term risk of developing cerebrovascular or renal events.

[0032] Furthermore, appropriate cutoff values ​​are set for each of the "(sCLEC-2 concentration) / (platelet count)" value and the D-Dimer concentration, and if either or both exceed the cutoff value, the long-term risk of developing cerebrovascular or renal events can be assessed as high. The cutoff value for "(sCLEC-2 concentration) / (platelet count)" in this case can be appropriately set based on the sCLEC-2 concentration and platelet count and used. The cutoff value for the D-Dimer concentration is preferably 1.0 μg / mL or higher, which is the upper limit for healthy individuals, and more preferably 1.45 μg / mL or higher.

[0033] In the above-mentioned specific examples, the method of using the platelet count was explained using the value obtained by dividing the sCLEC-2 concentration by the platelet count. However, the calculation formula explained in this specification, for example, the following formula (1): The order of calculations in formula (1) is arbitrary as long as the value represented by the formula (1) follows rules of calculation (rules for the order of calculations, rules for arithmetic operations) well known to those skilled in the art. In other words, as long as the calculation result is the same, there are no restrictions on the order of the items used in the calculation, and there are no restrictions on the combination of values ​​used to set the cutoff value.

[0034] For example, when each item described in formula (1) is used, the long-term high risk of developing cerebrovascular and renal events can be evaluated using the value obtained by multiplying the D-Dimer concentration by the value obtained by dividing the soluble CLEC-2 concentration in the subject's blood by the platelet count, or (2) multiplying the D-Dimer concentration in the subject's blood by the platelet count by the value obtained by multiplying the D-Dimer concentration in the subject's blood, or (3) multiplying the soluble CLEC-2 concentration in the subject's blood by the D-Dimer concentration (or (3') multiplying the D-Dimer concentration in the subject's blood by the soluble CLEC-2 concentration) by the platelet count.

[0035] As another evaluation method of the third aspect, when evaluation is performed using the value "(D-Dimer concentration) × (sCLEC-2 concentration) / platelet count" obtained by multiplying the sCLEC-2 concentration by the D-Dimer concentration and dividing the result by the platelet count, an appropriate cutoff value can be set for this value, and if the value is higher than this, it can be evaluated that the long-term risk of developing cerebrovascular / cardiovascular and renal events is high, and if the value is lower than this, it can be evaluated that the long-term risk of developing cerebrovascular / cardiovascular and renal events is low.

[0036] As used herein, cerebrovascular / renal events refer to the occurrence of symptoms or therapeutic intervention in cerebrovascular diseases, and include, for example, 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 reconstruction surgery, introduction of dialysis, arteriosclerosis obliterans, critical limb ischemia, aortic dissection, and the like.

[0037] In this specification, the specimen collected from the subject is preferably a blood sample collected from a human, plasma or whole blood to which an anticoagulant has been added, serum, or the like, but may also be collected from other mammals that may develop cerebrovascular or renal events, such as rats, mice, monkeys, dogs, etc. The subject is preferably a patient suspected of having a disease that is considered to be at high risk of developing cerebrovascular or renal events, or a patient diagnosed with such a disease. Examples of diseases at risk of cerebrovascular or renal events include patients with chronic kidney disease, diabetes, dyslipidemia, hypertension, chronic maintenance dialysis, patients with arteriosclerotic diseases, obesity, smokers, etc. Furthermore, patients with a history of the above-mentioned cerebrovascular or renal events may also be preferred subjects.

[0038] A known collection method can be used as a method for collecting a sample from a subject, depending on the measurement item. For example, in the case of measuring sCLEC-2 concentration, the sample can be collected using a blood collection tube for collecting plasma. A blood collection tube containing citrate, which has a low residual platelet content, is preferred, but those containing heparin or EDTA are also acceptable. In the case of measuring D-Dimer concentration, a blood collection tube containing citrate and EDTA is preferred. For measuring platelet count, a blood collection tube containing EDTA is preferred. The same blood collection tube may be used, or different blood collection tubes may be used for each measurement.

[0039] The method for measuring sCLEC-2 concentration is not particularly limited, but an immunological method using an antibody that recognizes sCLEC-2 is preferred. Immunological protein detection methods can be any known and commonly used method, such as immunoassays using labeled antibodies, such as enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent immunoassay, radioimmunoassay, immunochromatography, and Western blotting, or latex agglutination and immunoturbidimetry. However, a method with sufficient sensitivity for measuring sCLEC-2 concentrations in patients and healthy individuals should be used, and a highly sensitive method is desirable for this purpose, with chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent immunoassay, and the like being particularly preferred.

[0040] The method for measuring the D-Dimer concentration is not particularly limited, and may be a known immunoassay such as an enzyme immunoassay, a chemiluminescent enzyme immunoassay, a chemiluminescent immunoassay, a fluorescent immunoassay, a bioluminescent immunoassay, an electrochemiluminescent immunoassay, a latex agglutination method, or an immunochromatography method. The method for measuring the platelet count is not particularly limited, and any conventional known method may be appropriately employed.

[0041] As used herein, "CLEC-2" refers to a platelet-activating receptor belonging to the C-type lectin family. It is normally present on the platelet membrane but is released into the blood upon platelet activation. The term "soluble CLEC-2 (sCLEC-2)" as used herein refers to CLEC-2 or a CLEC-2-derived molecule released from platelets and detected in the blood. sCLEC-2 is believed to include proteins with molecular weights of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The proteins with molecular weights of approximately 40 kDa and approximately 32 kDa are presumed to be present on the platelet membrane surface and released in the form of microparticles produced upon platelet activation. These proteins are believed to have glycosylation attached. On the other hand, the protein with molecular weight of approximately 25 kDa is believed to be cleaved by proteases upon platelet activation and released from platelets. In the present invention, the amount of sCLEC-2 is measured. Regarding sCLEC-2, the protein with a molecular weight of about 40 kDa, the protein with a molecular weight of about 32 kDa, and the protein with a molecular weight of about 25 kDa may be detected together, or only the protein with a molecular weight of about 25 kDa may be detected.

[0042] The sCLEC-2 concentration measured in a subject's sample is usually expressed in pg / mL, but may be expressed in any unit, such as ng / L or ng / mL. The D-Dimer concentration is expressed in μg / mL or ng / mL, but may be expressed in any unit, such as ng / L. The D-Dimer concentration may be expressed in D-Dimer weight units or fibrinogen equivalent units (FEU). The platelet count is expressed in thousands / μL, 10,000 / μL, or the like, but may be expressed in any unit. The index value (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, but is essentially the same regardless of the unit combination and can be appropriately determined by referring to known methods. Also, similar index values ​​combining sCLEC-2 concentration, D-Dimer concentration, or platelet count can be used.

[0043] The cutoff value is set statistically and appropriately by comparing the measurement data of a group that experienced cerebrovascular or renal events after a certain period of time in a large population with those that did not. The cutoff value can be set appropriately by referring to known methods. For example, the measured sCLEC-2 concentration or D-Dimer concentration values, or index values ​​such as (sCLEC-2 concentration) x (D-Dimer concentration) or (D-Dimer concentration) x (sCLEC-2 concentration) / (platelet count), can be appropriately stratified using the 2nd, 3rd, or 4th quartile method, etc., to determine a particularly high-risk group, and the cutoff value can be determined from that threshold. The threshold may also be determined by setting a high-risk group without adhering to quantiles. Furthermore, an analysis can be performed to create an ROC curve (Receiver Operating Characteristic Curve), and the cutoff value can be determined from the sensitivity and specificity of risk prediction.

[0044] In the case of sCLEC-2 concentration alone, (sCLEC-2 concentration) × (D-Dimer concentration), or (D-Dimer concentration) × (sCLEC-2 concentration) / (platelet count), a high risk can be determined if the concentration is higher than the respective cutoff value, and a low risk can be determined if the concentration is lower than the respective cutoff value. In the case of a combination of sCLEC-2 concentration and D-Dimer concentration, a cutoff value can be set for each, and a high risk can be determined if both sCLEC-2 concentration and D-Dimer concentration are higher than the respective cutoff value, and a low risk can be determined if both are lower. Alternatively, a high risk can be determined if either concentration is higher than the respective cutoff value, and a low risk can be determined if either concentration is lower than the respective cutoff value. Furthermore, when stratifying into three or more categories such as high risk, intermediate risk, and low risk, two or more cutoff values ​​can be set.

[0045] The index value is likely to be calculated using measurements from a clinical testing device that measures sCLEC2 concentration or D-Dimer concentration and measurements from a hemocytometer that measures platelet count. In routine clinical practice, this calculation is preferably performed automatically on a hospital testing system, hospital system, or electronic medical record system connected to two or more measuring devices. However, a system that connects data from two or more measuring devices may also be constructed, or a machine that can simultaneously measure sCLEC2 concentration and D-Dimer concentration, or a machine that can simultaneously measure sCLEC2 concentration, D-Dimer concentration, and platelet count, may also be constructed. Alternatively, the index value may be calculated manually using two or more sets of data. Furthermore, the method for assessing the long-term risk of developing cerebrovascular and renal events according to an embodiment of the present invention is intended to be used as an aid to physicians in making diagnoses.

[0046] As used herein, the long-term risk of developing a cerebrovascular or renal event refers to the likelihood of developing a cerebrovascular or renal event several months or more, for example, six months or more, from the time of sample collection. For longer-term evaluation, it refers to the likelihood of developing a cerebrovascular or renal event 12 months, 18 months, or even 24 months from the time of sample collection.

[0047] According to an embodiment of the present invention, if a subject is assessed as being at high long-term risk for cerebrovascular or renal events, it is presumed that the subject has a thrombosis tendency, and antithrombotic therapy can be recommended to the subject. Antithrombotic therapy can be selected from antiplatelet drugs (e.g., aspirin, clopidogrel) and anticoagulants (e.g., warfarin, DOAC) based on the measured values ​​or index values ​​of sCLEC-2, D-Dimer, and / or platelets, and background information of the patient. Because antithrombotic therapy has side effects such as bleeding, it is highly desirable to be able to select patients for whom antithrombotic therapy can be recommended from among the subjects in order to increase the preventive benefit to the patient.

[0048] The present invention includes a system that can be used to carry out the method of the present invention described above, an electronic medical record, a clinical testing device, or an in-hospital testing system equipped with the system, and a kit that can be used to carry out the method of the present invention.

[0049] The systems of the present invention include, for example, a system for measuring (or determining) the aforementioned index value, a system for assessing a long-term high risk of developing cerebrovascular or renal events, a system for assisting in the assessment of a long-term high risk of developing cerebrovascular or renal events, a system for assessing (or detecting) a long-term high risk of developing cerebrovascular or renal events, and a system for measuring (or determining) the aforementioned index value for assessing a long-term high risk of developing cerebrovascular or renal events. Specific index values ​​include combinations of soluble CLEC-2 concentration and D-Dimer concentration and / or platelet count, more specifically, (sCLEC-2 concentration) × (D-Dimer concentration), (sCLEC-2 concentration) / (platelet count), (D-Dimer concentration) × (sCLEC-2 concentration) / (platelet count), etc.

[0050] Any of the systems of the present invention may include: (1) a storage means capable of storing the soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count in a blood sample derived from a subject; (2) a calculation means capable of calculating an index value combining the soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count; (3) a comparison means capable of comparing the index value obtained in (2) with a cutoff value (e.g., the index value of a comparison group); and (4) a display means capable of displaying the results obtained by the comparison.

[0051] Alternatively, any of the systems of the present invention may comprise a computer including a processor and a memory under the control of the processor, and the memory may have recorded therein programs for causing the computer to execute the following steps: (1) a storage step for storing in the memory the soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count in a blood sample derived from a subject; (2) a calculation step for calculating an index value combining the soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count; (3) a comparison step for comparing the index value obtained in the calculation step with a cutoff value (e.g., the index value of a comparison group); and (4) a display step for displaying the results obtained in the comparison step.

[0052] The electronic medical chart, clinical testing device, hospital testing system, etc. of the present invention can be equipped with the system of the present invention. The method of the present invention can be carried out using the system of the present invention, or the electronic medical chart, clinical testing device, hospital testing system, etc. of the present invention (hereinafter referred to as the system, etc.). The explanations given above regarding the method of the present invention can be applied as they are to the index values ​​that can be measured (or determined) using the system, etc. of the present invention, the "conditions, diseases, etc." that can be evaluated using the system, etc. of the present invention and their "evaluation, assessment, judgment criteria, etc.", and the methods for measuring the "soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count."

[0053] The kit of the present invention may include, for example, a soluble CLEC-2 measurement reagent, a D-Dimer measurement reagent, a platelet count measurement reagent, etc., and may include at least a soluble CLEC-2 measurement reagent. Furthermore, the kit may include a package insert describing the correlation between the soluble CLEC-2 concentration, the D-Dimer concentration, the platelet count, or an index value combining the soluble CLEC-2 concentration, the D-Dimer concentration, and / or the platelet count and a high long-term risk of developing cerebrovascular or renal events. The package insert may also include a method for calculating the index value.

[0054] Furthermore, the package insert included in the kit of the present invention is not particularly limited as long as it at least refers to the relationship between the soluble CLEC-2 concentration, or an index value formed by combining the soluble CLEC-2 concentration with the D-Dimer concentration and / or the platelet count, and the long-term high risk of developing cerebrovascular and / or renal events. In addition to the above-mentioned references, the package insert may include, for example, an explanation of the procedure for performing immunological measurements using the kit of the present invention, an explanation of the procedure for evaluating the long-term high risk of developing cerebrovascular and / or renal events based on the obtained measurement values, and precautions regarding the storage and handling of the kit itself.

[0055] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0056] Example 1 Measurement of sCLEC-2, D-Dimer, and Platelet Count in Patients with Chronic Kidney Disease, and Other Data Collection Example 1-1 Measurement of sCLEC-2 (Preparation of Reagent for Measuring sCLEC-2) Specimen diluent: A specimen diluent was prepared by combining 0.1 mol / L HEPES buffer (pH 7.5) containing a preservative with 2% sodium octanoate and 0.5% n-octyl-β-D-glucoside (OG).

[0057] The antibodies contained in the reagent were those described in the Examples of Japanese Patent No. 6078845 and were prepared as follows: First antibody solution: A mouse monoclonal antibody (11D5) that recognizes sCLEC-2 was bound to magnetic latex particles (JSR Corporation) and dispersed in 0.01 mol / L MES buffer (pH 6.0) containing a preservative. Second antibody solution: Another mouse monoclonal antibody (11E6) that recognizes sCLEC-2 was labeled with alkaline phosphatase (ALP) using the maleimide method and dispersed in 0.01 mol / L MES buffer (pH 6.5) containing a preservative. Luminescent substrate solution: 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo[3.3.1.13,7]decane}-4-yl)-1-phenylphosphate disodium (CDP-Star (registered trademark): Applied Biosystems) was used. B / F washing solution: A buffer containing 0.1 mol / L citric acid (pH 6.5), 0.15 mol / L NaCl, and 0.1% Triton X-100 was used.

[0058] (Measurement using assay reagents) sCLEC-2 was measured using a fully automated clinical testing system, STACIA (registered trademark, manufactured by LSI Medience Corporation). The prepared specimen dilution solution, first antibody solution (magnetic latex reagent), and second antibody solution (enzyme-labeled antibody reagent) were each filled into STACIA-specific bottles and placed in the instrument. Measurements were performed according to the operating method of the instrument. Specifically, 40 μL of specimen dilution solution was added to 10 μL of a specimen sample collected as citrated plasma, and the mixture was heated at 37°C for several minutes. 25 μL of first antibody solution (magnetic latex reagent) was then added and heated at 37°C for several minutes. Next, B / F separation was performed, 50 μL of second antibody solution (enzyme-labeled antibody reagent) was added, and the mixture was heated at 37°C for several minutes. B / F separation was then performed again, followed by the addition of 100 μL of luminescent substrate solution. After several minutes of reaction at 37°C, the signal intensity (counts) was measured.

[0059] Example 1-2: Measurement of D-Dimer D-Dimer was measured by the above STAICA using LPIA-Genesis D-Dimer reagent (manufactured by LSI Medience Corporation).

[0060] Example 1-3: Measurement of platelet count The platelet count was measured using an automated blood cell analyzer (Sysmex).

[0061] Example 1-4: Measurement of other items B-type natriuretic peptide (BNP) was measured using reagents manufactured by LSI Medience Corporation, and hemoglobin levels, blood glucose levels, and creatinine (for calculating eGFR) were measured using reagents in accordance with standard methods.

[0062] Example 1-5: Samples and Study Protocol The subjects of the measurement were 453 outpatients with chronic kidney disease (CKD) (age 64±15 years, estimated glomerular filtration rate (eGFR) 49±25 mL / min / 1.73 m ２ ) CKD stage 5 (eGFR: 15 mL / min / 1.73 m ２ Those under 85 years of age and those aged 85 or older were excluded. Blood samples were taken at the time of outpatient visits, and sCLEC-2, D-Dimer, platelet count, B-type natriuretic peptide (BNP), hemoglobin, blood glucose, and creatinine (for calculating eGFR) were measured. In addition, a medical history of myocardial infarction, stroke, heart failure, hypertension, diabetes, and dyslipidemia was investigated. The endpoint was cerebrovascular or renal events (new onset of cerebrovascular disease or initiation of artificial dialysis) within the 24-month observation period.

[0063] Example 2: Long-term risk assessment of cerebrovascular and renal events in CKD patients As a result of the study according to Example 1, 52 cases (11.5%) of cerebrovascular and renal events were observed during the 24-month observation period. sCLEC-2, D-Dimer, BNP, and blood glucose levels in the event-occurring group were significantly higher than those in the non-event-occurring group. On the other hand, eGFR and hemoglobin levels were significantly lower in the event-occurring group. Cox proportional hazards multivariate analysis revealed that sCLEC-2, D-Dimer, blood glucose levels, and a history of stroke were independent determinants of the occurrence of cerebrovascular and renal events.

[0064] Example 3: Long-term assessment of high risk of cerebrovascular and renal events in CKD patients based on sCLEC-2 concentration alone When CKD patients were divided into three tertiles based on sCLEC-2 levels, the event incidence rates for each tertile were as shown in Table 1.

[0065]

[0066] This is shown graphically in Figure 1. It was shown that when the sCLEC-2 level was 250 pg / mL or higher, the long-term risk over 24 months was approximately three times higher than when the level was 250 pg / mL or lower.

[0067] The Kaplan-Meier curve for this CDK case group is shown in Figure 2. According to this Kaplan-Meier curve, in cases where the sCLEC-2 value was 250 pg / mL or higher, the incidence of cerebrovascular and renal events increased almost linearly over 24 months (the proportion of subjects who did not develop the event decreased linearly), demonstrating that the sCELC-2 value can be used to evaluate a long-term high risk of developing cerebrovascular and renal events.

[0068] Example 4: Evaluation of long-term high risk of cerebrovascular and renal 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 levels of less than 250 pg / mL and ≥ 250 pg / mL, and further divided into tertiles of D-Dimer levels (< 0.6 μg / mL, 0.6-1.45 μg / mL, ≥ 1.45 μg / mL). The incidence of cerebrovascular and renal events in each group is shown in Figure 3. The Kaplan-Meier curve for this CKD case group is also shown in Figure 4. The group with sCLEC-2 levels of ≥ 250 pg / mL and D-Dimer levels of ≥ 1.45 μg / mL had an approximately 27% probability of developing cerebrovascular and renal events within 24 months, indicating a very high long-term risk. The upper limit for normal subjects using this D-Dimer reagent is 1.0 μg / mL.

[0069] Example 5: Assessment of Long-Term High Risk of Cerebrovascular and Renal Events in CKD Patients Using (sCLEC-2 Concentration) × (D-Dimer Concentration) The (sCLEC-2 concentration) × (D-Dimer concentration) ratio for each patient was calculated, and the incidence of cerebrovascular and renal events in the tertiles of this value (less than 180, 180-450, 450 or more) is shown in Figure 5. The Kaplan-Meier curve for this CKD case group is shown in Figure 6. The group with a (sCLEC-2 concentration) × (D-Dimer concentration) of 450 or more had a 19.7% probability of developing a cerebrovascular or renal event within 24 months, indicating a very high long-term risk, approximately five times higher than the group with a (sCLEC-2 concentration) × (D-Dimer concentration) ratio of 180 or less.

[0070] Example 6: Long-term high risk assessment of cerebrovascular and renal events in CKD patients using (D-Dimer concentration) × (sCLEC-2 concentration) / (platelet count) The ratio (D-Dimer concentration) × (sCLEC-2 concentration) / (platelet count) was calculated for each patient, and the incidence of cerebrovascular and renal events in the tertiles of this value (less than 0.84, 0.84-2.25, 2.25 or more) is shown in Figure 7. The Kaplan-Meier curves of cerebrovascular and renal events in these CKD patients are shown in Figure 8. The group with a (D-Dimer concentration) × (sCLEC-2 concentration) / (platelet count) ratio of 2.25 or more had a very high risk of developing a cerebrovascular or renal event within 24 months of 20.1%, which was approximately eight times higher than the group with a (D-Dimer concentration) × (sCLEC-2 concentration) / (platelet count) ratio of 0.84 or less.

[0071] As described above, the blood sCLEC-2 measurement value of the present invention, or the combination of blood sCLEC-2 measurement value and blood D-Dimer measurement value, or the combination of blood sCLEC-2 measurement value and platelet count, or the combination of blood sCLEC-2 measurement value, blood D-Dimer measurement value and platelet count, serves as an indicator for determining a long-term high risk of developing cerebrovascular / renal events, thereby enabling appropriate antithrombotic therapy to be recommended to increase the preventive benefit to patients. Measurement of blood sCLEC-2 is useful as a method for evaluating a long-term high risk of developing cerebrovascular / renal events.

Claims

1. A method for assessing a subject's long-term high risk of developing cerebrovascular and renal events based on the concentration of soluble CLEC-2 in their blood.

2. A method for assessing a subject's long-term high risk of developing cerebrovascular or renal events based on the concentration of soluble CLEC-2 in the subject's blood, comprising the steps of: (1) providing a blood sample from the subject; (2) determining the concentration of soluble CLEC-2 in the sample; and (3) correlating the concentration of soluble CLEC-2 with the possibility of the subject developing a long-term cerebrovascular or renal event.

3. The method according to claim 1 or 2, which is a method for evaluating the long-term risk of onset of a patient suspected of having a disease at risk of cerebrovascular / renal events or a patient diagnosed with such a disease.

4. The method according to any one of claims 1 to 3, wherein the disease associated with a risk of cerebrovascular / renal events is selected from chronic kidney disease, diabetes, hypertension, dyslipidemia, chronic maintenance dialysis, and arteriosclerotic disease.

5. The method according to any one of claims 1 to 4, wherein the long-term high risk of developing cerebrovascular and / or renal events is assessed based on the D-Dimer concentration in the subject's blood in addition to the soluble CLEC-2 concentration in the subject's blood.

6. The method according to claim 5, wherein the long-term high risk of developing cerebrovascular and / or renal events is evaluated based on the value obtained by multiplying the soluble CLEC-2 concentration in the subject's blood by the D-Dimer concentration.

7. The method according to any one of claims 1 to 4, wherein the long-term high risk of developing cerebrovascular / renal events is assessed based on the subject's platelet count in addition to the soluble CLEC-2 concentration in the subject's blood.

8. The method according to claim 7, wherein the long-term high risk of developing cerebrovascular / renal events is evaluated based on the concentration of soluble CLEC-2 in the subject's blood divided by the platelet count.

9. The method according to claim 7, wherein the long-term high risk of developing cerebrovascular / renal events is assessed based on the D-Dimer concentration in the subject's blood in addition to the soluble CLEC-2 concentration and platelet count in the subject's blood.

10. The method according to claim 9, wherein the long-term high risk of developing cerebrovascular or renal events is evaluated based on the value represented by the following formula (1):

11. A method for evaluating the long-term high risk of developing cerebrovascular / renal events and selecting an antithrombotic drug using the method according to any one of claims 1 to 10.

12. A system for assessing the long-term high risk of developing cerebrovascular and renal events, comprising: (1) a memory means capable of storing soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count in a blood sample derived from a subject; (2) a calculation means capable of calculating an index value combining the soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count; (3) a comparison means capable of comparing the index value obtained in (2) with a cutoff value; and (4) a display means capable of displaying the results obtained by the comparison.

13. A system for assessing a long-term high risk of developing cerebrovascular and renal events, comprising a computer including a processor and a memory under the control of the processor, wherein the memory has recorded therein programs for causing the computer to execute the following steps: (1) a storage step for storing in the memory the soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count in a blood sample derived from a subject; (2) a calculation step for calculating an index value combining the soluble CLEC-2 concentration, D-Dimer concentration, and / or platelet count; (3) a comparison step for comparing the index value obtained in the calculation step with a cutoff value; and (4) a display step for displaying the results obtained in the comparison step.

14. An electronic medical record, a clinical testing device, or an in-hospital testing system comprising the system according to claim 12 or 13.

15. A kit for evaluating the long-term high risk of developing cerebrovascular and / or renal events, comprising: (1) a soluble CLEC-2 measurement reagent; and (2) a package insert describing the correlation between the soluble CLEC-2 concentration or an index value consisting of a combination of soluble CLEC-2 concentration, D-Dimer concentration and / or platelet count, and the long-term high risk of developing cerebrovascular and / or renal events.

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