Method for predicting the formation or risk of thrombus in a medical device that performs blood circulation by pumping

Abstract

Provided is a means capable of predicting the formation of blood clots or the risk thereof in a medical device which performs blood circulation by a pump, by using a simple and minimally invasive technique. In this method for predicting the formation of blood clots or the risk thereof in a medical device which performs blood circulation by a pump, it is predicted that blood clots are formed or the risk thereof is present in the medical device when the concentration or expression amount of ADAM28 in a body fluid sample collected from a subject attached to the medical device is increasing from a baseline.

Description

[Technical field]

[0001] The present invention relates to a method for predicting the formation or risk of thrombus in a medical device that performs blood circulation by a pump. [Background technology]

[0002] When it is difficult to maintain life in patients with respiratory failure even with multidisciplinary treatment, extracorporeal membrane oxygenation (ECMO) therapy or percutaneous cardiopulmonary support (PCPS) is performed. ECMO / PCPS is a therapy that uses an artificial lung and a pump to replace the heart and lungs, and the circuit (ECMO / PCPS system) is composed of parts such as an artificial lung, a blood pump, a blood supply and disconnection blood vessel, a connecting tube, and a connector (Innovative Medicines, Medical Devices, and Regenerative Medicine Products Practical Promotion Project "Evaluation Method of Next-Generation Assisted Circulatory System" Working Group. Evaluation Guidelines for Mid- to Long-Term Respiratory / Circulatory Support (ECMO / PCPS) Systems (Draft)). The ECMO / PCPS systems currently in use basically use the same parts as those used in heart-lung machines for open-heart surgery, and most of these have been approved for use within 6 hours, which is the period of normal open-heart surgery. In normal open-heart surgery, the heart-lung machine only requires a few hours of treatment under strong anticoagulation therapy with systemic heparin administration, but long-term heparin administration increases the risk of bleeding. Unlike normal open-heart surgery, ECMO / PCPS often requires auxiliary circulation for several days to several weeks, during which high levels of anticoagulation and bleeding control are required. During ECMO, in addition to heparin treatment, platelets and coagulation factors are consumed, which increases the risk of bleeding, so preventing bleeding is extremely important. However, reducing the dose of anticoagulants will cause blood clots to form, so controlling bleeding / clotting is extremely important. Activated partial thromboplastin time (APTT) and activated clotting time (ACT) tests are performed to manage bleeding and blood clot formation.ELSO states that whole blood ACT is more reliable because blood cells affect heparin activity (Extracorporeal Life Support Organization (ELSO) Guidelines for Adult Respiratory Failure August, 2017), but on the other hand, it is said that APTT has a better correlation with heparin activity than ACT (Ujiie Yoshito, Respiratory ECMO Manual, 2014, Katsuseido Publishing). According to Moynihan et al., ACT was 224.5 seconds (95% confidence interval 169-304 seconds) in patients with bleeding complications and 229.4 seconds (180-317 seconds) in patients without bleeding, with no significant difference (p=0.177), but was 224.0 seconds (178-302 seconds) in patients with thrombotic complications and 235.3 seconds (178-302 seconds) in patients without thrombotic complications, with a significant difference (p =0.003), whereas the APTT was 81.8 seconds (37-200 seconds) in patients with bleeding complications and 127.6 seconds (50-200 seconds) in patients without bleeding, a significant difference (p<0.001), and 96.4 seconds (40-200 seconds) in patients with thrombotic complications and 137.3 seconds (54-200 seconds) in patients without thrombotic complications, a significant difference (p<0.001) (Moynihan K, Johnson K, Straney L, et al. Coagulation monitoring correlation with heparin dose in pediatric extracorporeal life support. Perfusion. 2017;32(8):675-685.).Conversely, in a clinical study that attempted to prevent bleeding and thrombosis using ACT and APTT in ECMO, the incidence of bleeding complications was 69% when managed with ACT and 80% with APTT (p=0.145), and the incidence of thrombotic complications was 41% when managed with ACT and 39% with APTT (p=0.85), showing no difference between the two test methods and indicating that these complications occurred frequently (Fitousis K, Klasek R, Mason PE, Masud F. Evaluation of a pharmacy managed heparin protocol for extracorporeal membrane oxygenation patients. Perfusion. 2017;32(3):238-244.).

[0003] As such, appropriate methods for preventing bleeding or anticoagulation therapy during ECMO have not yet been established (Esper SA, Levy JH, Waters JH, et al. Extracorporeal membrane oxygenation in the adult: a review of anticoagulation monitoring and transfusion. Anesth Analg. 2014;118:731-743.). For this reason, the biggest complication in ECMO / PCPS management is bleeding (Extracorporeal Life Support Organization: ELSO, https: / / www.elso.org / Resources / RisksandComplications.aspx). Brogan et al. have reported that bleeding occurs in 40-60% of patients as a complication of ECMO, and blood clots form in approximately 20% of patients (Brogan TV, Thiagarajan RR, Rycus PT, et al. Extracorporeal membrane oxygenation in adults with severe respiratory failure: a multi-center database. Intensive Care Med. 2009;35(12):2105-2114.). Sklar et al. also reported that bleeding occurred in 16% of patients and thrombosis occurred in 53% of patients with VV ECMO (Sklar MC, Sy E, Lequier L, et al. Anticoagulation Practices during Venovenous Extracorporeal Membrane Oxygenation for Respiratory Failure. A Systematic Review. Ann Am Thorac Soc. 2016;13(12):2242-2250.).

[0004] By the way, von Willebrand factor (vWF) acts when platelets adhere to the endothelial tissue of blood vessels and plays a central role in primary hemostasis (Sakazume, et al., Acquired von Willebrand syndrome caused by high shear stress, Artificial Organs, 2016;45(3):225-228.). That is, vWF binds to collagen in the subendothelial tissue, changes its three-dimensional structure under shear stress, interacts with the platelet membrane protein GPIb complex, induces platelet rolling on the vascular wall, and further binds to the activated platelet membrane protein GPIIb / IIIa to promote platelet aggregation (Horiuchi, Acquired von Willebrand syndrome associated with intractable cardiovascular disease). vWF also forms a complex with coagulation factor VIII to stabilize factor VIII. vWF is produced by endothelial cells and bone marrow megakaryocytes, and forms oligomers by disulfide bonds between molecules. vWF produced by endothelial cells forms a large polymeric multimer (unusually large-VWF multimer: UL-VWFM) with a molecular weight of over 15 million daltons. In the blood, however, its molecular structure changes due to shear stress, and it is degraded by ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13), and exists as a multimer consisting of 20 to 80 subunits. High molecular weight vWF multimers are important for primary hemostasis (Ruggeri ZM, Zimmerman TS. Variant von Willebrand's disease: characterization of two subtypes by analysis of multimeric composition of factor VIII / von Willebrand factor in plasma and platelets. J Clin Invest. 1980;65:1318-1325.), and in an environment where high blood shear stress is generated, a decrease in high molecular weight vWF multimers can lead to acquired von Willebrand syndrome and a tendency to hemorrhage.

[0005] ECMO often causes bleeding from the puncture site, and acquired von Willebrand syndrome developed in all cases within 24 hours of connection (Kalbhenn J, Schmidt R, Nakamura L, et al. Early diagnosis of acquired von Willebrand Syndrome (AVWS) is elementary for clinical practice in patients treated with ECMO therapy. J Atheroscler Thromb. 2015;22(3):265-271.). In addition, 10-33% of patients with implantable ventricular assist devices develop bleeding complications, and vWF is thought to be importantly involved (Draper KV, Huang RJ, Gerson LB. GI bleeding in patients with continuous-flow left ventricular assist devices: a systematic review and meta-analysis. Gastrointest Endosc. 2014;80:435-446.) (Sakatsume K, Akiyama M, Saito K, et al. Intractable Bleeding Tendency Due to Acquired Von Willebrand Syndrome After Jarvik 2000 Implant. J Artifficial Organs. 2016;9:289-292.). It has been reported that vWF, which plays an important role in primary hemostasis, is affected by the high mechanical shear stress. Therefore, in order to prevent bleeding during ECMO / PCPS or ventricular assist devices, the development of a diagnostic method using vWF is being considered (Hisanori Horiuchi. The AVec Study). On the other hand, UL-VWFM produced by endothelial cells etc. forms platelet thrombi, so to prevent this, ADAMTS13 degrades UL-VWFM to control thrombus formation (Masanori Matsumoto, Yoshihiro Fujimura. Von Willebrand factor cleaving enzyme (ADAMTS13). Journal of the Japanese Society of Internal Medicine. 2009;98(7):1586-1592.).If thrombus formation continues to progress, it will lead to blood vessel blockage and the surrounding tissue will become ischemic. To prevent this, when the blood vessel lumen narrows due to the growth of the platelet thrombus, high shear stress is generated, which results in a change in the structure of vWF and its degradation by ADAMTS13, preventing blood vessel blockage.

[0006] Recently, ADAM28 has been reported as a new enzyme that degrades vWF, but its relationship to thrombus formation control is unclear. ADAMTS13 degrades only vWF that has undergone structural changes, whereas ADAM28 degrades vWF that has not undergone structural changes (Mochizuki S, Soejima K, Shimoda M, et al. Effect of ADAM28 on carcinoma cell metastasis by cleavage of von Willebrand factor. J Natl Cancer Inst. 2012;104(12):906-922.). However, there have been no reports that ADAM28 is involved in thrombus formation. Summary of the Invention

[0007] Therefore, an object of the present invention is to provide a means for predicting the formation of thrombus or the risk of thrombus in medical devices that circulate blood using a pump (e.g., extracorporeal membrane oxygenator, ventricular assist device, etc.) using a simple and minimally invasive technique.

[0008] The present inventors have conducted extensive research to solve the above problems. As a result, they have surprisingly clarified for the first time the relationship between ADAM28 and thrombus formation in a medical device that circulates blood using a pump. Based on this finding, they have completed the present invention.

[0009] That is, according to one form of the present invention, there is provided a method for predicting the formation of a thrombus or the risk of it in a medical device that circulates blood using a pump, which predicts that a thrombus has formed or there is a risk of it in the medical device if the concentration or expression level of ADAM28 in a body fluid sample collected from a subject wearing the medical device is elevated above a reference value.

[0010] According to another aspect of the present invention, there is provided a method for providing data for predicting the formation of a thrombus or the risk of such a thrombus in a medical device that circulates blood using a pump, the method including predicting that a thrombus has formed or there is a risk of a thrombus in the medical device if the concentration or expression level of ADAM28 in a body fluid sample collected from a subject wearing the medical device is elevated above a reference value. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a photograph showing a thrombus attached to a centrifugal pump. [Diagram 2] FIG. 2 is a graph showing changes in concentrations of von Willebrand factor (vWF), ADAMTS13 and ADAM28. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] One aspect of the present invention relates to a method for predicting the formation or risk of a thrombus in a medical device that circulates blood using a pump, which predicts that a thrombus has formed or is at risk of forming in the medical device when the concentration or expression level of ADAM28 in a body fluid sample collected from a subject wearing the medical device is elevated above a reference value.

[0013] Another aspect of the present invention relates to a method for providing data for predicting the formation of a thrombus or the risk thereof in a medical device that circulates blood using a pump, the method comprising predicting that a thrombus has formed or there is a risk of a thrombus forming in the medical device when the concentration or expression level of ADAM28 in a body fluid sample collected from a subject wearing the medical device is elevated above a reference value.

[0014] According to the present invention, it is possible to predict the formation of thrombus or the risk of thrombus in a medical device that circulates blood using a pump, using a simple and minimally invasive technique.

[0015] Specifically, according to the method of one embodiment of the present invention, the formation of a thrombus or the risk thereof in a medical device that circulates blood by a pump can be easily predicted. Moreover, according to the method of one embodiment of the present invention, the formation of a thrombus can be detected before the blood level of vWF changes. Therefore, the formation of a thrombus can be predicted early. In addition, since it is only necessary to measure the concentration or expression level of ADAM28 in a body fluid sample, such a test can be easily performed. When a blood sample is used as the body fluid sample, a diagnosis can be made with a part of the blood collected in a normal blood test, and there is almost no burden on the subject. Therefore, according to the method of one embodiment of the present invention, the formation of a thrombus or the risk thereof in a medical device that circulates blood by a pump can be predicted easily and minimally invasively without using an expensive modality, and data for the prediction can be provided, and blood circulation by a pump can be safely performed.

[0016] An embodiment according to one aspect of the present invention will be described below, but the present invention is not limited to the following embodiment.

[0017] In this specification, the range "X to Y" means "X or more and Y or less." Furthermore, unless otherwise specified, the operations and measurements of physical properties are performed under conditions of room temperature (20 to 25°C) and relative humidity of 40 to 50% RH.

[0018] ADAM proteins (ADAMs: disintegrins and metalloproteinases) are multifunctional proteins involved in ectodomain shedding of transmembrane proteins, cell adhesion and invasion. (Edwards DR, Handsley MM, Pennington CJ. The ADAM Metalloproteinases. Mol Aspects Med. 2008;29(5):258-289.) (Murphy G. Regulation of the proteolytic disintegrin metalloproteinases, the 'Sheddases'. Semin Cell Dev Biol. 2009;20(2):138-145.) The human genome contains 25 ADAMs, including 4 pseudogenes, of which 21 are composed of 13 proteolytic ADAMs and 8 non-proteolytic ADAMs that exhibit proteolytic activity (Edwards DR, Handsley MM, Pennington CJ. The ADAM Metalloproteinases. Mol Aspects Med. 2008;29(5):258-289.) (Shiomi T, Lemaitre V, D'Armiento J, Okada Y. Matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non-neoplastic diseases. Pathol Int. 2010;60(7):477-496.).Proteolytic ADAMs share the metalloproteinase domain of matrix metalloproteinases (MMPs), and typical proteolytic ADAMs contain propeptide, metalloproteinase, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane and intracellular domains. Many proteolytic ADAMs, including ADAM8, ADAM9, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM28, are known to be overexpressed in human cancers and are associated with cancer growth and progression. However, it has not been suggested that ADAM28 could be a useful indicator for predicting the formation or risk of thrombus in medical devices that perform blood circulation by pumps.

[0019] ADAM28 is a known protein, and its amino acid sequence and cDNA sequence are also known. There are two types of ADAM28: membrane type (ADAM28m) and secretory type (ADAM28s). Representative amino acid sequences and cDNA sequences of human membrane type ADAM28 (and its mature form), as well as representative amino acid sequences and cDNA sequences of human secretory type ADAM28 (and its mature form) are disclosed in WO 2016 / 143702. Note that "human membrane type ADAM28" means that the amino acid sequence or nucleotide sequence of membrane type ADAM28 has an amino acid sequence or nucleotide sequence identical or substantially identical to the amino acid sequence or nucleotide sequence of membrane type ADAM28 naturally expressed in humans. "Substantially identical" means that the amino acid sequence or nucleotide sequence of interest has 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more) identity with the amino acid sequence or nucleotide sequence of membrane-type ADAM28 naturally expressed in humans, and has the function of human membrane-type ADAM28. The same interpretation applies to organisms other than humans, and proteins, genes, and fragments thereof other than membrane-type ADAM28.

[0020] In the method of the present invention, the concentration or expression level of ADAM28 in a body fluid sample collected from a subject is measured for use as a marker. In a preferred embodiment, in addition to ADAM28, the concentration or expression level of vWF and / or ADAMTS13 is measured for use as a marker. This can further improve the accuracy of predicting angiogenesis or the risk thereof.

[0021] vWF is a plasma protein that plays an important role in blood coagulation. vWF is mainly synthesized in the vascular endothelium and released into the blood in the form of a high molecular weight polymer. Wild-type human vWF is a polypeptide consisting of a total of 2813 amino acids including its signal peptide and pro-region. The amino acid sequence of wild-type human vWF and the amino acid sequence of the wild-type human mature vWF subunit are disclosed in WO 2004 / 035778.

[0022] ADAMTS13 is a known protein, and its amino acid sequence and cDNA sequence are also known. ADAMTS13 is a zinc-based metalloproteinase belonging to the ADAMTS family, and is thought to specifically cleave the bond between Tyr1605 and Met1606 of vWF, thereby reducing the multimer size of vWF and controlling the function of vWF. Detailed structure and sequence information on human ADAMTS13 are disclosed in Zheng X et al., J Biol Chem. 2001; 276 (44): 41059-63.

[0023] In this specification, examples of "medical devices that circulate blood using a pump" include artificial lungs (for example, extracorporeal membrane oxygenators), auxiliary artificial hearts, and the like.

[0024] As used herein, a "subject" is a subject in need of treatment with a medical device that circulates blood through a pump, preferably a human subject.

[0025] In the present specification, the term "body fluid sample" refers to any body fluid derived from a living organism (organism) that can measure the concentration or expression level of ADAM28, and, as necessary, the concentration or expression level of vWF and / or ADAMTS13. The body fluid sample is preferably a blood sample. The blood sample may be whole blood, plasma, or serum. The blood sample may be blood (whole blood) collected from a subject as is, or may be a sample to which an additive such as potassium EDTA salt, heparin, or sodium citrate has been added for the purpose of preventing coagulation. There is no particular limitation on the timing of collecting the body fluid sample from the subject. When the body fluid sample is a blood sample, the blood sample may be arterial blood or venous blood.

[0026] The body fluid sample used in the method of the present invention is preferably taken from the subject immediately after measurement, but may be stored. For example, the method of storing blood samples is not particularly limited as long as the values ​​of the above markers in the sample do not change, and low-temperature conditions such as 0 to 10°C without freezing are preferred, and frozen storage at -80°C or in liquid nitrogen is preferred for long-term storage.

[0027] In the method according to the present invention, the levels of the above markers can be measured using the conventionally known techniques described below.

[0028] As used herein, the "expression level" of a protein includes both the expression of RNA (transcription product) complementary to the gene encoding the protein, and the expression of the protein itself (translation product). Thus, as used herein, the expression level of a protein refers to the expression level or expression intensity of the transcription product or translation product. The expression level can usually be analyzed based on the production level of the transcription product, or the production level, activity, etc. of the translation product. In addition, the "concentration" of a protein refers to the amount (expression level) of the target protein present per unit volume of a body fluid sample (e.g., a blood sample).

[0029] The expression level of a protein may be measured by measuring the transcription product of a gene encoding the protein, i.e., mRNA, or by measuring the translation product of the gene, i.e., the protein itself. Preferably, the expression level is measured by measuring the translation product of the gene. The transcription product of a gene also includes cDNA obtained by reverse transcription from mRNA.

[0030] The expression level of the translation product can be measured by quantifying the translated protein or measuring the activity of the protein. Proteins can be quantified by, for example, electrophoresis, Western blotting, or chromatographic methods using affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, immunochromatography, or mass spectrometry, but can be easily and accurately measured by using an antibody specific to the protein.

[0031] The antibody can be prepared by a known method. Alternatively, the antibody can be provided by, for example, MyBioSource, Invitrogen, Santa Cruz Biotechnology, Sigma-Aldrich, etc., and can be appropriately obtained and used. The antibody for detection may be a polyclonal antibody or a monoclonal antibody.

[0032] Detection of proteins using antibodies can be performed by, but is not limited to, immunochromatography, Western blotting, EIA, ELISA, RIA, flow cytometry, and the like. Antibodies can be labeled with fluorescent labels, radioactive labels, enzymes, biotin, and the like, and secondary antibodies labeled in this way can also be used for detection. Incidentally, in clinical practice, the presence of myocardial injury is currently evaluated by detecting cardiac troponin T from blood samples by immunochromatography (Trop T Sensitive, Roche Diagnostics). Therefore, in the method according to the present invention, similarly, by measuring markers such as ADAM28 by immunochromatography, it is expected to be possible to construct a measurement method useful for predicting the formation or risk of thrombus in medical devices that circulate blood by pumps, which can be measured with minimal invasiveness and ease in clinical practice, can be universalized at low cost, has high accuracy and reproducibility, and allows for standardization of measurement methods. As a result, it is expected that appropriate treatment can be performed, and hemolysis and thrombus formation can be controlled. In addition, when the above measurement is performed by the immunochromatography method, it is preferable to use an immunochromatographic test paper. The immunochromatographic test paper preferably has a sample pad area for receiving a blood sample, a conjugate pad area containing a labeled antibody capable of binding to an indicator substance such as ADAM28, a membrane body for developing a sample, and a test line on the membrane body on which an antibody capable of binding to the indicator substance is immobilized and capable of binding to the indicator substance bound to the labeled antibody in the conjugate pad area. Furthermore, an immunochromatographic test paper having a control line that allows the amount of the label bound to the test line to be determined is more preferable. It is more preferable that the label is one that exhibits a stronger color depending on the concentration.

[0033] The expression level of the transcription product may be measured by, for example, measuring the level of gene expression in a sample using a nucleotide containing all or a part of the base sequence of mRNA as a probe or primer, and can be measured by, for example, a method using a microarray (microchip), a Northern blot method, a quantitative PCR method, etc. Quantitative PCR methods include agarose gel electrophoresis, polyacrylamide gel electrophoresis, a fluorescent probe method, a RT-PCR method, a real-time PCR method, an ATAC-PCR method, a Taqman PCR method, a SYVER (registered trademark) green method, a Body Map method, a Serial analysis of gene expression (SAGE) method, a Micro-analysis of Gene Expression (MAGE) method, etc., and these can be used appropriately. Evaluation may also be performed using a next-generation sequencer. Using these methods, the amount of mRNA can be measured by using a nucleotide probe or primer that hybridizes to the mRNA. The base length of the probe or primer used for measurement is preferably 10 to 50 mer, more preferably 15 to 25 mer.

[0034] When the expression of multiple genes, particularly the expression of multiple types of genes, is to be measured simultaneously, it is preferable to use a DNA microarray. A DNA microarray can be prepared by immobilizing nucleotides consisting of the base sequence of a gene or nucleotides containing a partial sequence thereof on a suitable substrate. Examples of the immobilization substrate include glass plates, quartz plates, and silicon wafers. Examples of the size of the substrate include 3.5 mm x 5.5 mm, 18 mm x 18 mm, and 22 mm x 75 mm, which can be set in various ways depending on the number of probe spots on the substrate and the size of the spots. As a method for immobilizing a polynucleotide or a fragment thereof, the charge of the nucleotide can be utilized to electrostatically bind the polynucleotide to a solid-phase carrier surface-treated with polycations such as polylysine, polyethyleneimine, and polyalkylamine, or a nucleotide having a functional group such as an amino group, an aldehyde group, a thiol group, or biotin can be covalently bound to a solid-phase surface having a functional group such as an amino group, an aldehyde group, or an epoxy group introduced therein. The immobilization can be performed using an array machine. At least one gene or a fragment thereof is immobilized on a substrate to prepare a DNA microarray, which is then contacted with mRNA or cDNA derived from a subject that has been labeled with a fluorescent substance and hybridized, and the type and amount of mRNA can be determined by measuring the fluorescence intensity on the DNA microarray.

[0035] In the method according to the present invention, the concentration or expression level of ADAM28 measured by the above-mentioned method is compared with a reference value to predict the formation or risk of thrombus in a medical device that circulates blood by a pump. That is, the present specification discloses a method for predicting the formation or risk of thrombus in a medical device that circulates blood by a pump. The present specification also discloses a method for providing data for predicting the formation or risk of thrombus in a medical device that circulates blood by a pump. In this case, as described above, when the concentration or expression level of ADAM28 is increased above the reference value, it is predicted that there is the formation or risk of thrombus in the medical device that circulates blood by a pump.

[0036] In addition, when the concentration or expression level of vWF and / or ADAMTS13 is used in combination with ADAM28 as a further marker, the accuracy of prediction based on ADAM28 can be improved by comparing the value of the marker measured by the above-mentioned method with a reference value.

[0037] In the present specification, the "reference value" is a numerical value that can be a standard for determining an increase or decrease in the expression level or concentration in the method according to the present invention. An example of the reference value is, for example, a measured value of the concentration or expression level of ADAM28 in a body fluid sample collected from a person in whom thrombus formation has not been confirmed (e.g., a healthy subject, a subject before blood circulation by a pump, a subject in a state in which thrombus formation is not expected except immediately after surgery, etc.). The reference value is preferably a measured value of the concentration or expression level of ADAM28 in a body fluid sample collected from a healthy subject or a subject before blood circulation by a pump. The same applies when the concentration or expression level of vWF and / or ADAMTS13 is used in combination as a marker in addition to ADAM28.

[0038] Additionally, a value of a marker previously measured in the same subject may be used as the reference value.

[0039] Marker values ​​may vary depending on the measurement method, so reference values ​​must be established for each measurement method.

[0040] According to yet another embodiment of the present invention, a detection reagent for use in the method according to one embodiment of the present invention described above is provided. Also, a kit for use in the method according to one embodiment of the present invention described above, which includes the detection reagent, is provided. The detection reagent enables detection of the concentration or expression level of ADAM28, and optionally vWF and / or ADAMTS13, in the body fluid sample described above. Examples of such detection reagents include antibodies for specific binding to a protein (e.g., ADAM28) in the body fluid sample, or DNA, RNA, or oligonucleotides for hybridization with the mRNA of a protein gene (e.g., ADAM28 gene) in the body fluid sample. Such DNA or RNA may be a probe capable of detecting hybridization by a fluorescent label or the like. Alternatively, the DNA or RNA may be a primer that can be used for amplifying mRNA. In addition to the above-mentioned detection reagent, the kit may contain other reagents necessary for detection, such as a buffer, various nucleotides, and other reagents necessary for hybridization or antibody binding. EXAMPLES

[0041] The effects of the present invention will be described using the following examples and comparative examples, although the technical scope of the present invention is not limited to the following examples.

[0042] [Creation of animal models] In the experiment, a sheep (female, 5 years old) was subjected to the test after a 7-day quarantine acclimation period.

[0043] A cannulation was made in the right jugular vein for blood inflow, and the other end of the cannulation was connected to an extracorporeal circulation device consisting of a centrifugal pump, and blood was removed via the cannulation in the right jugular vein. The blood circulation conditions using the centrifugal pump were 0.7 to 1.6 L / min, and anticoagulation was performed with heparin.

[0044] The experiments were carried out in accordance with the guidelines for animal experiments at Terumo Corporation.

[0045] [Blood collection] Blood was collected from the external jugular vein into EDTA blood collection tubes, centrifuged at 3000 rpm for 10 minutes, and plasma was collected and stored at -20°C for measurement of vWF, ADAMTS13, and ADAM28.

[0046] [Confirmation of thrombus formation] A thrombus attached to the centrifugal pump of the extracorporeal circulation device was visually confirmed (Figure 1).

[0047] [Measurement of von Willebrand factor (vWF) concentration] The concentration of vWF in plasma was measured using a Sheep von Willebrand Factor (vWF) Elisa kit (Competitive ELISA) (MyBioSource) according to the attached manual. The absorbance measurement in the ELISA analysis was repeated three times using a precision-controlled microplate reader (MICROPLATE READER SH9000, CORONA), and the average value was used as the final data.

[0048] [Measurement of ADAMTS13 concentration] The plasma ADAMTS13 concentration was measured using a Sheep Von Willebrand Factor cleaving protease (vWF-cp) ELISA Kit (MyBioSource) according to the attached manual. The absorbance measurement in the ELISA analysis was repeated three times using a precision-controlled microplate reader (MICROPLATE READER SH9000, CORONA), and the average value was used as the final data.

[0049] [Measurement of ADAM28 concentration] The plasma ADAM28 concentration was measured using the Sheep A Disintegrin and Metalloprotease 28 (ADAM28) ELISA Kit (MyBioSource) according to the attached manual. The absorbance measurement in the ELISA analysis was repeated three times using a precision-controlled microplate reader (MICROPLATE READER SH9000, CORONA), and the average value was used as the final data.

[0050] The results are shown in Figure 2. In Figure 2, the day when thrombus formation was confirmed is shown as day 0.

[0051] As shown in Figure 2, the concentration of ADAM28 increased first, followed by those of vWF and ADAMTS13. Then, thrombus formation was confirmed. From the above, it can be seen that by using ADAM28 as an indicator, it is possible to predict thrombus formation earlier than when using vWF or ADAMTS13 as indicators.

[0052] This application is based on Japanese Patent Application No. 2020-044275 filed on March 13, 2020, the disclosure of which is incorporated by reference in its entirety.

Claims

1. 1. A method for predicting the formation or risk of a thrombus in a medical device that circulates blood by a pump, comprising: A method in which, if the concentration or expression level of ADAM28 in a body fluid sample collected from a subject wearing the medical device is elevated above a reference value, it is predicted that a thrombus has formed in the medical device or there is a risk of it forming.

2. 1. A method for providing data for predicting the formation or risk of a thrombus in a medical device that circulates blood by a pump, comprising: The method includes predicting that a thrombus has formed or is at risk of forming in the medical device if the concentration or expression level of ADAM28 in a body fluid sample taken from a subject wearing the medical device is elevated above a reference value.

3. The method according to claim 1 or 2, wherein the reference value is the concentration or expression level of ADAM28 in a body fluid sample taken from a healthy subject or a subject before blood circulation by the pump.

4. The method according to any one of claims 1 to 3, wherein the body fluid sample is a blood sample.

5. A detection reagent for use in the method according to any one of claims 1 to 4, which enables detection of the concentration or expression level of ADAM28 in the body fluid sample.

6. The detection reagent according to claim 5 , which is an antibody against ADAM28, or a DNA or RNA or an oligonucleotide for hybridization with the mRNA of the gene encoding ADAM28.

7. A kit for use in the method according to any one of claims 1 to 4, comprising the detection reagent according to claim 6.

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