阅读说明：本技术 血小板膜蛋白在诊断急性冠脉综合征的应用 (Application of platelet membrane protein in diagnosis of acute coronary syndrome ) 是由 孟照辉 万雯 叶雨佳 李龙君 顾亚娟 于 2019-09-25 设计创作，主要内容包括：本发明公开了血小板膜蛋白在诊断急性冠脉综合征的应用,一种血小板膜蛋白为发明人首次发现在血小板膜上表达的蛋白-癌胚抗原细胞黏附分子5(carcinoembryonic antigenelated cell adhesion molecule 5,CEACAM5/CEA)。血小板膜表面CEA水平在诊断区分急性冠脉综合征患者和冠状动脉正常患者时,其受试者工作曲线(receiver operating characteristic curve,ROC)的曲线下面积(area under the curve,AUC)为0.86,具有临床诊断意义；联合肌钙蛋白cTnI诊断时,AUC达0.91。本发明提供的血小板膜蛋白可用于诊断急性冠脉综合征,且灵敏度高,特异性强。(The invention discloses an application of platelet membrane protein in diagnosing acute coronary syndrome, and the platelet membrane protein is protein-carcinoembryonic antigen cell adhesion molecule 5 (CEACAM 5/CEA) expressed on a platelet membrane for the first time discovered by an inventor. When a patient with acute coronary syndrome and a patient with normal coronary artery are diagnosed and distinguished by the CEA level on the surface of the platelet membrane, the area under the curve (AUC) of a Receiver Operating Curve (ROC) is 0.86, so that the CEA level has clinical diagnosis significance; when the combined troponin cTnI is diagnosed, the AUC reaches 0.91. The platelet membrane protein provided by the invention can be used for diagnosing acute coronary syndrome, and has high sensitivity and strong specificity.)

1. Use of platelet membrane protein for the diagnosis of acute coronary syndrome.

2. Use of platelet membrane protein as biochemical indicator in diagnosis of acute coronary syndrome is provided.

3. Use of platelet membrane proteins as biochemical markers in the diagnosis of acute coronary syndromes according to claim 2, characterized in that: use of platelet membrane proteins as biological markers for the diagnosis of acute coronary syndrome.

4. The platelet membrane protein is used as a reliable index for predicting risk stratification and adverse prognosis events of patients with early myocardial ischemia and acute coronary syndrome.

Technical Field

The invention relates to an application of platelet membrane protein in diagnosing acute coronary syndrome, belonging to the field of biochemistry.

Background

Acute Coronary Syndrome (ACS) is a type of Acute ischemic heart disease, the high morbidity and mortality of which have become the major health concerns in the world^[1]. Acute coronary syndrome is closely related to vulnerable plaque of coronary artery, and rupture of unstable plaque in coronary artery lumen can lead to platelet activation, thrombosis and even thrombotic occlusionAnd (6) a plug. It follows that platelets play an important role in the progression, instability and rupture of coronary plaque^[2-4]. The platelet surface membrane protein also participates in platelet activation, adhesion and aggregation, and can indirectly mediate the pathogenesis and clinical manifestation of acute coronary syndrome^[5,6]. Clinically, acute coronary syndrome is mainly diagnosed by the combination of typical chest pain symptoms, electrocardiogram change, or/and change of cardiac necrosis markers.

The cardiac necrosis marker, the most common cardiac troponin (cTn), is a fundamental tool in the assessment and diagnosis of acute coronary syndrome, and has become the gold standard for the diagnosis of myocardial infarction. Although troponins are highly specific and sensitive to myocardial injury, they fail to recognize specific mechanisms of myocardial injury, such as elevated troponin also occurs in a variety of disease states, e.g., acute pulmonary edema, pulmonary embolism, viral myocarditis, and the like. At the same time, troponin does not distinguish between stable and unstable angina pectoris^[7,8]. Therefore, the search for new biological markers with high sensitivity and strong specificity has great significance for the diagnosis of the acute coronary syndrome.

The research on novel biological markers for diagnosing the acute coronary syndrome is not stopped at home and abroad, and the research on the biological markers is not limited to the biochemical indexes of serum or plasma but also relates to the expression level of platelet membrane protein. Studies show that the platelet membrane surface collagen receptor glycoprotein VI (GPVI), P-selectin (P-selectin) and carcinoembryonic antigen cell adhesion molecule 1(CEACAM1/CD66a/Bgp) are expressed on the platelet membrane surface and play important roles in platelet-mediated physiological hemostasis and pathological thrombosis^[3,9,10]. In addition, the expression level of certain platelet membrane proteins in the acute coronary syndrome is obviously increased, and the platelet membrane proteins can be used as a new index for diagnosing the acute coronary syndrome^[3,6,9,11]。

Platelets play an important role in physiological hemostasis and pathological thrombosis, and platelet membrane proteins are key mediators in these processes^[12]. Carcinoembryonic antigen cell adhesion molecule 5(carcinoembryonic antigen associated cell adhesive)n-molecule 5, CEACAM5/CEA) and CEACAM1 are members of the carcinoembryonic antigen cell adhesion molecule (CEACAMs) family, which share some similarities in structure and ligand binding with homotropism or heterophilicity. CEA is widely expressed on various cell surfaces, including epithelial cells, endothelial cells, immune cells, etc., and plays an important role in cell adhesion, intercellular and intracellular signaling, and tumor progression^[13,14]. In addition, CEA as broad-spectrum tumor marker can reflect the existence of various tumors, and simultaneously, the high expression of CEA in the serum of patients with colorectal cancer, breast cancer, lung cancer and the like is closely related to tumor deterioration and subclinical metastasis^[14,15]. Platelets are also important players of tumor cell extravasation and tumor metastasis^[16,17]. Therefore, the development of the CEA as a new biological marker for predicting or diagnosing the acute coronary syndrome has very important value and clinical practical significance.

Reference to the literature

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[2]Gawaz M,Neumann FJ,Schomig A.Evaluation of platelet membraneglycoproteins in coronary artery disease:consequences for diagnosis andtherapy[J].Circulation.1999,99(1):E1-e11.

[3]Stellos K,Bigalke B,Stakos D,Henkelmann N,Gawaz M.Platelet-boundP-selectin expression in patients with coronary artery disease:impact onclinical presentation and myocardial necrosis,and effect of diabetes mellitusand anti-platelet medication[J].Journal of thrombosis and haemostasis:JTH.2010,8(1):205-7.

[4]Navas-Carrillo D,Marin F,Valdes M,Orenes-Pinero E.Decipheringacute coronary syndrome biomarkers:High-resolution proteomics in platelets,thrombi and microparticles[J].Critical reviews in clinical laboratorysciences.2017,54(1):49-58.

[5]Andrews RK,Gardiner EE,Shen Y,Berndt MC.Platelet interactions inthrombosis[J].IUBMB life.2004,56(1):13-8.

[6]Gremmel T,Frelinger AL,3rd,Michelson AD.Platelet Physiology[J].Seminars in thrombosis and hemostasis.2016,42(3):191-204.

[7]Katus H,Ziegler A,Ekinci O,Giannitsis E,Stough WG,Achenbach S,etal.Early diagnosis of acute coronary syndrome[J].European heart journal.2017,38(41):3049-55.

[8]del Val Martin D,Sanmartín Fernández M,Zamorano GómezJL.Biomarkers in acute coronary syndrome[J].IJC Metabolic&Endocrine.2015,8:20-3.

[9]Wong C,Liu Y,Yip J,Chand R,Wee JL,Oates L,et al.CEACAM1 negativelyregulates platelet-collagen interactions and thrombus growth in vitro and invivo[J].Blood.2009,113(8):1818-28.

[10]Bigalke B,Geisler T,Stellos K,Langer H,Daub K,Kremmer E,etal.Platelet collagen receptor glycoprotein VI as a possible novel indicatorfor the acute coronary syndrome[J].American heart journal.2008,156(1):193-200.

[11]Bigalke B,Stellos K,Weig HJ,Geisler T,Seizer P,Kremmer E,etal.Regulation of platelet glycoprotein VI(GPVI)surface expression and ofsoluble GPVI in patients with atrial fibrillation(AF)and acute coronarysyndrome(ACS)[J].Basic research in cardiology.2009,104(3):352-7.

[12]Ozaki Y,Suzuki-Inoue K,Inoue O.Platelet receptors activated viamulitmerization:glycoprotein VI,GPIb-IX-V,and CLEC-2[J].Journal of thrombosisand haemostasis:JTH.2013,11Suppl 1:330-9.

[13]Horst AK,Wagener C.CEA-Related CAMs[J].Handbook of experimentalpharmacology.2004,(165):283-341.

[14]Beauchemin N,Arabzadeh A.Carcinoembryonic antigen-related celladhesion molecules(CEACAMs)in cancer progression and metastasis[J].Cancermetastasis reviews.2013,32(3-4):643-71.

[15]Bramswig KH,Poettler M,Unseld M,Wrba F,Uhrin P,Zimmermann W,etal.Soluble carcinoembryonic antigen activates endothelial cells and tumorangiogenesis[J].Cancer research.2013,73(22):6584-96.

[16]Labelle M,Begum S,Hynes RO.Direct signaling between platelets andcancer cells induces an epithelial-mesenchymal-like transition and promotesmetastasis[J].Cancer cell.2011,20(5):576-90.

[17]Best MG,Sol N,Kooi I,Tannous J,Westerman BA,Rustenburg F,etal.RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer,Multiclass,and Molecular Pathway Cancer Diagnostics[J].Cancer cell.2015,28(5):666-76.

[18]Jneid H,Addison D,Bhatt DL,Fonarow GC,Gokak S,Grady KL,et al.2017AHA/ACC Clinical Performance and Quality Measures for Adults With ST-Elevation and Non-ST-Elevation Myocardial Infarction:A Report of the AmericanCollege of Cardiology/American Heart Association Task Force on PerformanceMeasures[J].Circulation Cardiovascular quality and outcomes.2017,10(10).

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Disclosure of Invention

The invention aims to provide application of platelet membrane protein in diagnosis of acute coronary syndrome, and aims to solve the problem that certain false positive exists when cardiac troponin is used as a marker for evaluating and diagnosing the acute coronary syndrome.

The invention provides an application of platelet membrane protein in diagnosing acute coronary syndrome.

The invention provides an application of platelet membrane protein as a biochemical index in diagnosing acute coronary syndrome.

The invention provides application of the compound as a reliable index in predicting risk stratification and adverse prognosis events of patients with early myocardial ischemia and acute coronary syndrome.

Further, the invention provides the application of the platelet membrane protein as a biological marker in diagnosing acute coronary syndrome.

The platelet membrane protein is derived from the surface of a platelet membrane and is CEA on the surface of the platelet membrane.

The platelet membrane protein is detected on washed platelets by two-color flow cytometry.

The platelet membrane protein is used for detecting the expression level of the platelet membrane protein on the platelets of patients with acute coronary syndrome and patients with normal coronary artery by two-color whole blood flow cytometry.

In the two-color flow cytometry experiment, the expression of CEA on the surface of the platelet membrane was measured in the resting state and after activation with thrombin (0.5U/ml, 1U/ml, 2U/ml) at different concentrations. Experiments prove that the CEA on the surface of the platelet membrane is expressed on the surface of the human platelet membrane, and the expression level of the CEA on the surface of the platelet membrane is increased after the thrombin with different concentrations is stimulated.

In the two-color whole blood flow cytometry experiment, the expression conditions of the CEA on the surface of the platelet membrane of patients with acute coronary syndrome and patients with normal coronary artery are measured. Experiments prove that the level of CEA on the surface of the platelet membrane of a patient with acute coronary syndrome is increased, and the serum albumin can be a novel, powerful and reliable biological marker for diagnosing the acute coronary syndrome.

The platelet membrane protein has high sensitivity and strong specificity, and has important significance for diagnosing acute coronary syndrome.

Drawings

FIG. 1 is a two-color flow cytometry assay for CEA expression on human platelets following resting or thrombin activation: FIG. 1A shows the expression of CEA on Resting (Resting) human platelets, expressed as Mean Fluorescence Intensity (MFI) + -Standard Error (SE), plotted on the abscissa as a grouping of washed platelets and on the ordinate as MFI (n-3;. P <0.01) of CEA on the surface of the platelet membrane; FIG. 1B is a representative flow cytometry histogram with FITC fluorescence intensity on the abscissa and platelet count on the ordinate (n-3;. P < 0.01); FIGS. 1C and 1D show the expression of CEA on the platelet membrane surface after thrombin stimulation at different concentrations, with the abscissa representing the treatment groups for washed platelets and the ordinate representing the MFI of CEA on the platelet membrane surface (n-3;. P < 0.01).

FIG. 2A is a graph showing the expression of platelet membrane surface CEA in patients with acute coronary syndrome and in patients with normal coronary arteries; FIG. 2B is a graph showing the expression of platelet membrane surface CEA in AMI, UA and coronary artery normal patients; fig. 2C shows the expression of platelet membrane surface CEA in STEMI, NSTEMI, UA and coronary normal patients, expressed as MFI ± SE, with the abscissa as a subgroup of patient diseases and the ordinate as MFI of platelet membrane surface CEA (. × P <0.01, n ═ 82).

Fig. 3A is a correlation of platelet membrane surface CEA levels with myocardial contraction markers (BNPs), MFI on the platelet membrane surface CEA on the abscissa and BNP concentration on the ordinate (n-82); fig. 3B-D are the correlation of platelet membrane surface CEA levels with myocardial injury markers (cTnI, CKMB, and MYO), respectively, plotted on the abscissa for MFI of platelet membrane surface CEA and plotted on the ordinate for the concentrations of cTnI, CKMB, and MYO, respectively (n-82).

FIG. 4A is the diagnostic value of platelet membrane surface CEA on acute coronary syndrome with false positive rate (1-specificity%) on the abscissa and true positive rate (sensitivity%) on the ordinate; FIG. 4B is a graph showing the comparison of the level of platelet membrane surface CEA, cTnI and their combined diagnostic efficacy in acute coronary syndrome. The abscissa is the false positive rate (1-specificity%), and the ordinate is the true positive rate (% sensitivity).

Detailed Description

The invention is further described below with reference to specific embodiments and the accompanying drawings. The examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

Subject inclusion and exclusion criteria in each example:

the study of the invention continuously included 82 patients who visited chest pain in the first hospital affiliated to Kunming medical university from 12 months to 3 months in 2018. All patients were diagnosed by coronary angiography according to the American Heart Association/American Heart Association (AHA/ACC) guidelines^[18]. Of the 82 patients, 62 were diagnosed with acute coronary syndrome, including ST-segment elevation myocardial infarction (ST-segment elevation myocardial infa)rction, STEMI) 21 patients, Non-ST elevation myocardial infarction (NSTEMI) 21 patients, Unstable Angina (UA) 20 patients; and 20 healthy subjects with normal coronary arteries as a control group. Patients younger than 18 years of age, with false positive markers of myocardial necrosis, patients who failed to give informed consent, and patients diagnosed with various types of tumors were excluded.

All procedures were performed according to the declaration of helsinki and were passed through the ethical committee of the first subsidiary hospital of the university of kunming medical, and all patients had signed informed consent prior to enrollment.

Acute coronary syndromes include STEMI, NSTEMI and UA; among them, STEMI and NSTEMI are also collectively called Acute Myocardial Infarction (AMI). The diagnostic criteria are as described above^[19]。