Use of integrin beta subunits in the diagnosis of venous thromboembolism

Abstract

The present invention provides a method for diagnosing venous thromboembolism (VTE), comprising: detecting the level of an integrin 1 subunit, an integrin 2 subunit, and/or an integrin 3 subunit in a blood sample. Also provided is a reagent kit for diagnosing VTE, comprising a substance capable of specifically binding to the integrin 1 subunit, the integrin 2 subunit, and/or the integrin 3 subunit.

Claims

1. A method for determining in a venous thromboembolism (VTE) subject undergoing continuous anti-coagulant treatment whether the subject is still in a thrombophilia state and treating the subject by adjusting the integrin subunit level, comprising: obtaining a blood sample from the subject undergoing continuous anti-coagulant treatment; detecting a level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in the blood sample of the subject by contacting the blood sample with a substance that specifically binds to integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit; and comparing the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in the blood sample of the subject with corresponding levels of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a health population, wherein a higher expression of integrin 1 subunit, integrin 2 subunit and/or integrin 3 subunit in the blood sample of the subject than that of the health population indicates that the venous thromboembolism (VTE) subject undergoing continuous anti-coagulant treatment is still in the thrombophilia state and in need of treatment, and determining that the subject is still in the thrombophilia state and treating the subject still in the thrombophilia state by administering a substance that can adjust the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit.

2. The method of claim 1, wherein the blood sample is a peripheral venous blood sample.

3. The method of claim 1, wherein the subject is a human.

4. The method of claim 1, wherein detecting integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit comprises binding at least one subunit of integrin 1 subunit, integrin 2 subunit, integrin 3 subunit, or combinations thereof with the substance.

5. The method of claim 1, wherein the venous thromboembolism is selected from the group consisting of acute pulmonary embolism (APE), chronic thromboembolic pulmonary hypertension (CTEPH), deep venous thrombosis and acute deep venous thrombosis.

6. The method of claim 1, wherein the venous thromboembolism is acute pulmonary embolism (APE).

7. The method of claim 1, wherein the venous thromboembolism is acute deep venous thrombosis.

8. The method of claim 1, wherein the substance that specifically binds is selected from the group consisting of an antibody, a ligand, an antagonist, and combinations thereof.

9. The method of claim 8, wherein the substance is labeled.

10. The method of claim 9, wherein the substance is fluorescently labeled.

11. The method of claim 1, wherein the substance that can adjust the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit specifically binds to integrin 1 subunit, integrin 2 subunit, and/or integrin 3.

12. The method of claim 11, wherein the substance that specifically binds is selected from the group consisting of an antibody, a ligand, an antagonist, and combinations thereof.

13. The method of claim 11, wherein the substance is labeled.

14. A method of determining a thrombophilia state in a venous thromboembolism (VTE) subject and treating the thrombophilia state, comprising: obtaining a blood sample from the subject; detecting a level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in the blood sample of the subject by contacting the blood sample with a substance that specifically binds to integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit; comparing the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in the blood sample of the subject with corresponding levels of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a health population, wherein a higher level of integrin 1 subunit, integrin 2 subunit and/or integrin 3 subunit in the blood sample of the subject than that of the health population indicates that the venous thromboembolism (VTE) subject is in the thrombophilia state and in need of treatment, and determining that the subject is still in the thrombophilia state and treating the subject still in the thrombophilia state by administering a substance that can adjust the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit.

15. The method of claim 14, wherein the venous thromboembolism is selected from the group consisting of acute pulmonary embolism (APE), chronic thromboembolic pulmonary hypertension (CTEPH), deep venous thrombosis and acute deep venous thrombosis.

16. The method of claim 14, wherein the substance that specifically binds is selected from the group consisting of an antibody, a ligand, an antagonist, and combinations thereof.

17. The method of claim 14, wherein the substance is labeled.

18. A method of for diagnosing and treating venous thromboembolism (VTE), comprising: detecting the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a blood sample of a subject using a diagnostic agent comprising a substance that specifically binds to integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit; comparing the detection level of the integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in the blood sample of the subject with corresponding level measured in control blood samples of a health subject; diagnosing the subject with venous thromboembolism (VTE) when a higher level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in the blood sample of the subject is detected in comparison with the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in the blood sample of the health subject, wherein a higher level of integrin 1 subunit, integrin 2 subunit and/or integrin 3 subunit in the blood sample of the subject than that of the health population indicates that the subject has venous thromboembolism (VTE) and is in need of treatment, and diagnosing the subject as having venous thromboembolism (VTE) and treating the subject that has venous thromboembolism (VTE) by administering a substance that can adjust the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit by specifically binding to integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit.

19. The method of claim 18, wherein the substance that specifically binds is selected from the group consisting of an antibody, a ligand, an antagonist and combinations thereof.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: The results of pre-isolation of thrombus samples by shotgun method.

(2) FIG. 2: The sequence information of the enzyme-digested peptide fragments and debris of thrombus protein samples.

(3) FIG. 3: The tandem protein mass spectrometry (MS) and information analysis of thrombus samples from acute PE patients, and results of Gene Oncology analysis.

(4) FIG. 4: The tandem protein MS and information analysis of thrombus samples from acute PE patients, and results of interaction network analysis, indicating that integrins comprising 2, 3, and 1 subunits are core proteins of red thrombus.

(5) FIG. 5: The tandem protein MS and information analysis of thrombus samples from acute lower limbs VTE patients, and results of Gene Oncology analysis.

(6) FIG. 6: The tandem protein MS and information analysis of thrombus samples from acute lower limbs VTE patients, and results of interaction network analysis, indicating that the integrin comprising 3 subunit and ITGA2B (i.e. integrin chain IIb) are core proteins of red thrombus.

(7) FIG. 7: The distribution of integrin 1, 2, and 3 subunits in control group of health population, wherein FIG. 7a indicates that integrin 1 subunit shows a normal distribution, FIG. 7b indicates that integrin 2 subunit shows an abnormal distribution, and FIG. 7c indicates that integrin 3 subunit shows a normal distribution.

(8) FIG. 8: The expression of integrin 1 subunit in control group of health population. FIG. 8a shows the expression average value and baseline range of integrin 1 subunit in the control group, wherein the average value is 8.182.69(%), and the baseline range is 2.8-13.56(%). FIG. 8b shows the distribution of expression level of integrin 1 subunit in the control group.

(9) FIG. 9: The expression of integrin 2 subunit in control group of health population. FIG. 9a shows the expression average value and baseline range of integrin 2 subunit in the control group, wherein the expression median is 88.6(%), and the baseline range is 71.1-95.8(%). FIG. 9b shows the distribution of expression level of integrin 2 subunit in the control group.

(10) FIG. 10: The expression of integrin 3 subunit in control group of health population. FIG. 10a shows the expression average value and baseline range of integrin 3 subunit in the control group, wherein the average value is 9.652.53(%), and the baseline range is 4.59-14.71(%). FIG. 10b shows the distribution of expression level of integrin 3 subunit in the control group.

DETAILED DESCRIPTION OF THE INVENTION

(11) Unless otherwise indicated, all the technical and scientific terms will have the common meanings known in the art. All the patents, patent applications, publications, sequences, and other disclosed material are incorporated herein as reference, unless otherwise indicated.

(12) In the present invention, it was discovered that, integrins containing certain subunits play important roles in VTE thrombogenesis, and are core proteins in venous thrombus protein network. Wherein said integrins include integrins of 1-3 subfamilies. Based on such a discovery, the present invention provides a novel method for diagnosing or treating venous thromboembolism, comprising detecting or adjusting the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a blood sample of a subject. Furthermore, the present invention also relates to a substance that can specifically bind to, detect, or adjust integrin subunit, and use of said substance in the preparation of a kit for diagnosing VTE.

(13) The term thrombus refers to a blood clot formed in blood vessel, which obstructs or blocks blood flow. When blood vessel is injured, platelets and some proteins (such as fibrin, fibrinogen, serum proteins, and cytoskeletal proteins, etc.) in blood will aggregate and form a blood clot to repair the injured site. If the clot falls off, it may become a thrombus.

(14) The term venous thromboembolism is a general term referring to thrombus formed in veins, comprising pulmonary arterial thromboembolism (PE) and deep venous thrombosis (DVT), wherein PE comprises acute pulmonary embolism (APE), chronic thromboembolic pulmonary hypertension (CTEPH). Deep venous thrombosis comprises acute and chronic deep venous thrombosis.

(15) The term integrin refers to a class of cell surface receptor proteins. Such class of proteins are heterodimers, which contain two different chains, called subunit and subunit respectively. Integrins containing the same subunit constitute integrin subfamilies, e.g. 1 subfamily containing integrin 1 subunit (also called CD29), 2 subfamily containing integrin 2 subunit (also called CD18), and 3 subfamily containing integrin 3 subunit (also called CD61). Said a subunit and subunit encompass variants formed by differential splicing, e.g. 4 variants of ITGB1.

(16) As used herein, differential splicing refers to a process in which different mRNA splicing isoforms are generated through different splicing manners (i.e. combining exons through different splicing sites) from the same mRNA precursor. The protein products obtained through differential splicing are isoforms each other, which can present different functional and structural properties, or lead to different phenotypes due to different expression levels in the same cell.

(17) The term subject refers to a mammal subject, preferably human.

(18) In one aspect, the present invention provides a novel method for diagnosing venous thromboembolism.

(19) In one embodiment, the method of the present invention comprises: detecting the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a blood sample of a subject, and comparing the detection results of said level with corresponding level measured in control blood sample of a health subject, wherein the increase of said level is an indication for diagnosing VTE.

(20) Quantitative detection for a protein in blood sample is conventional technique in the art. A person skilled in the art can select such technique based on specific requirements. For example, relevant antibody-based methods such as flow cytometry and ELISA, etc. can be used.

(21) The term blood sample refers to blood tissue sample taken from the circulatory system of a subject, preferably blood sample taken from peripheral veins. The blood sample of the present invention can contain haemocytes and extracellular fluid (i.e. blood plasma).

(22) Increased level as used herein can be determined using the following process, i.e. determining the range of normal level in a health population comprising a certain number of health subjects, then if the level of a subject to be tested is higher than said range of normal level, the level of the subject to be tested is considered as being increased.

(23) The range of normal level in health population can be determined using statistical methods. For example, Kolmogorov-Smirnov test can be used for a normality test, with P>0.05 as normal distribution. As for normal distribution data, the average value measured in health population combined with the standard deviation thereof (xS) is used to determine the normal level range. As for abnormal distribution data, the median combined with the P2.5-P97.5 in statistics is used to denote the normal level range.

(24) In the present invention, increased level, for example, means that the measured level increases by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared with the reference level in a blood sample of a health subject.

(25) Health subject refers to subject population who are normal in physical examinations. Reference level refers to the levels of integrin-related proteins measured in health subject population (statistical average is then obtained and combined with standard deviation to be used as reference level). The health subject population for sampling as control can comprise e.g. 50 subjects, 100 subjects, 150 subjects, 200 subjects, 250 subjects, 300 subjects, 500 subjects, or even more.

(26) The steps of the method of the present invention comprise: taking blood sample such as peripheral venous blood sample from a subject to be tested; then quantitatively detecting the amount of various integrin subunits in said blood sample by using substance(s) (e.g. an antibody) that specifically binds to various integrin subunits, wherein the method for quantitative detection can be, for example, conventional techniques like flow cytometry, and ELISA, etc.; taking peripheral blood samples from several (e.g. 200 or more) health subjects for detecting the amounts of various integrin subunits therein by using the same method as mentioned above, and calculating the statistical average value and combining it with standard deviation as reference level. The amounts of various integrin subunits in the subject to be tested are compared with corresponding reference levels, wherein the increased levels of various integrin subunits is an indication for diagnosing VTE.

(27) In one embodiment, the method of the present invention comprises detecting the levels of at least two subunits of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a blood sample of a subject. In one embodiment, the method of the present invention comprises detecting the levels of at least two subunits of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a blood sample of a subject. In another embodiment, the method of the present invention comprises detecting the levels of integrin 1 subunit, integrin 2 subunit, and integrin 3 subunit in a blood sample of a subject.

(28) Said venous thromboembolism in the present invention can be acute pulmonary embolism (APE), chronic thromboembolic pulmonary hypertension (CTEPH), or deep venous thrombosis (DVT), preferably acute pulmonary embolism or deep venous thrombosis.

(29) In one embodiment, the method of the present invention employs flow cytometry to detect the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit.

(30) Flow cytometry is a conventional technique in the art, and the detailed operations thereof can be seen in relevant publications, textbooks, or instructions of manufactures, e.g. (Loken M R (1990). Immunofluorescence Techniques in Flow Cytometry and Sorting (2nd ed.). Wiley. pp. 341-53).

(31) In another aspect, the present invention provides a substance for specifically detecting the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit. In one embodiment, the present invention provides a substance that specifically binds to integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit, wherein said substance is an antibody, a ligand, and/or an antagonist. In one embodiment, the antibody, ligand, and/or antagonist of the present invention is labeled. In one embodiment, the antibody, ligand, and/or antagonist of the present invention is fluorescently labeled. In one embodiment, the antibody, ligand, and/or antagonist of the present invention is labeled by phycoerythrin (PE).

(32) Ligands for integrin 1 subunit include, for example, laminin (LM), collagen protein (COL), fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1), etc. Ligands for integrin 2 subunit include, for example, fibrinogen (FB), intercellular adhesion molecule-1, intercellular adhesion molecule-2, intercellular adhesion molecule-3 (ICAM-1, ICAM-2, ICAM-3), factor X, ic3b, etc. Ligands for integrin 3 subunit include, for example, FB, FN, vitronectin (VN), von Willebrand factor (vWF), thrombospondin (TSP), etc.

(33) In another aspect, the present invention relates to use of a substance, which specifically binds to integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit, in the preparation of a diagnostic agent for diagnosing venous thromboembolism by detecting the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a blood sample of a subject.

(34) In one embodiment, the present invention relates to use of a substance, which specifically binds to at least two subunits of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit respectively, in the preparation of a diagnostic agent for diagnosing venous thromboembolism.

(35) In one embodiment, the present invention relates to use of at least two substances, which specifically bind to at least two subunits of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit respectively, in the preparation of a composition for diagnosing venous thromboembolism.

(36) In one embodiment, the substances that specifically bind to at least two subunits of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit refer to at least two different substances, wherein one of the substances specifically binds to one of 1 subunit, integrin 2 subunit, or integrin 3 subunit, and the other one specifically binds to another of integrin 1 subunit, integrin 2 subunit, or integrin 3 subunit.

(37) In one embodiment, the present invention relates to use of substances, which specifically binds to integrin 1 subunit, integrin 2 subunit, and integrin 3 subunit respectively, in the preparation of a composition for diagnosing venous thromboembolism.

(38) In one embodiment, the substances that specifically bind to 1 subunit, integrin 2 subunit, and integrin 3 subunit respectively refer to at least three different substances, wherein the first substance specifically binds to one of integrin 1 subunit, integrin 2 subunit, or integrin 3 subunit, the second substance specifically binds to the second of 1 subunit, integrin 2 subunit, or integrin 3 subunit, and the third substance specifically binds to the third of 1 subunit, integrin 2 subunit, or integrin 3 subunit.

(39) In one embodiment, the substance, that specifically binds to integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit, and thus can be used to specifically detect the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit in a blood sample, is an antibody, a ligand, and/or an antagonist, preferably a labeled antibody, ligand, and/or antagonist. In one embodiment, the antibody, ligand, and/or antagonist of the invention is fluorescently labeled, e.g. labeled by phycoerythrin (PE).

(40) In another aspect, the present invention provides a kit for diagnosing venous thromboembolism, which comprises a substance that specifically detects the level of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit, wherein the substance can specifically bind to integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit.

(41) In one embodiment, the kit of the present invention comprises a substance that specifically binds to at least two subunits of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit.

(42) In one embodiment, the kit of the present invention comprises the substances that respectively specifically bind to at least two subunits of integrin 1 subunit, integrin 2 subunit, or integrin 3 subunit.

(43) In one embodiment, the kit of the present invention comprises substances that specifically bind to integrin 1 subunit, integrin 2 subunit, and integrin 3 subunit, respectively.

(44) In another aspect, the present invention also relates to the ligand, antibody, antagonist and/or activity inhibitor of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit.

(45) The terms ligand, antibody, antagonist, activity inhibitor, etc. of the invention are commonly used terms in the art, and the meanings thereof are in accordance with the general understanding of a person skilled in the art, and the definitions thereof can also be referenced to commonly used textbooks and manuals.

(46) In the present invention, the substances that respectively specifically bind to at least two subunits of integrin 1 subunit, integrin 2 subunit, and/or integrin 3 subunit refer to at least two different substances, wherein one of the substances specifically binds to one of integrin 1 subunit, integrin 2 subunit, or integrin 3 subunit, while the other specifically binds to another one of integrin 1 subunit, integrin 2 subunit, or integrin 3 subunit. The substances that respectively specifically bind to integrin 1 subunit, integrin 2 subunit, and integrin 3 subunit refer to at least three different substances, wherein the first substance specifically binds to one of integrin 1 subunit, integrin 2 subunit, or integrin 3 subunit, the second substance specifically binds to the second of integrin 1 subunit, integrin 2 subunit, or integrin 3 subunit, while the third substance specifically binds to the third of integrin 1 subunit, integrin 2 subunit, or integrin 3 subunit.

(47) In another aspect, the present invention also relates to a device for performing diagnosis using the method of the present invention, e.g., a device that can performs clinical rapid diagnosis.

EXAMPLES

(48) The present invention will be described in details through the following Examples. These Examples are only used for illustrative purposes, and are not intended to limit the scope of the present invention. The present invention specifically contains the following Examples:

Example 1: Taking Acute VTE Thrombus Samples by Catheter

(49) By femoral venous puncture, a 7F right coronary catheter (Metronic, USA) was used to respectively take 3-5 red thrombus (5-15 mm in length, 10 g) from the pulmonary artery of a 39-year old male patient suffering from acute PE (APE) and from the acute lower limbs femoral vein of a 50-year old male (ADVT) patient, and the obtained fresh thrombus were used for the tandem protein mass spectrometry analysis in the following Example.

Example 2: Tandem Protein Mass Spectrometry and Data Analysis

(50) 1. Methodology:

(51) The flow charts for tandem protein mass spectrometry (MS/MS) detection and data analysis methods:

(52) Acute PE, DVT thrombusMS/MS (Model LTQ, Thermo Finnigan, USA)DatabaseProtein retrieveCorresponding genesGO (Gene Oncology) analysisGenes showing differencesProteins showing differencesKEGG Pathway analysis-Gene Network analysisIdentifying the core proteins in the network.

(53) 1.1 For thrombus protein shotgun experiment, the obtained peptide fragments data were subject to bioinformatics analysis, so as to precisely identify the proteins that the peptide fragment corresponding to, and said proteins were also analyzed for their functions and biological pathways.

(54) 1.2 GO Analysis:

(55) GO database comprises three types of function information: the biological processes that the genes involve in, the cellular location of the genes, and the molecular functions the genes exert. Differentially expressed proteins were used as the input for GO analysis. Genes showing differences were respectively mapped towards various nodes of GO database. The gene number at each node was calculated.

(56) 1.3 KEGG Pathway Analysis Method:

(57) Differentially expressed proteins were subject to KEGG pathway analysis.

(58) 1.4 Gene Network Analysis Method:

(59) Three different types of interaction relationships were integrated: (1) The relationships among genes in KEGG database (protein interactions, gene regulations, protein modifications, etc.); (2) The existing high throughput experiments; (3) The interactions between genes mentioned in the existing literatures.

(60) Through the KEGGSOAP software package under R, the signal pathway data in the KEGG database were analyzed, so as to identify the interactions between genes in the genome, including three types of relationships: ECrel Enzyme-enzyme relationship, indicating two enzymes that catalyze successive reaction steps PPrel Protein-protein interaction, such as binding and modification GErel Gene expression interaction, indicating the relationship between transcriptional factors and target gene products

(61) Data for protein-protein interaction can be obtained from MIPS data. The co-citation algorithm was adopted, specifically: PubMed Database publication abstracts were downloaded, each abstract was decomposed to sentences, and then the common gene name that exists in each sentence, i.e. the co-cited gene, was analyzed. For each pair of co-cited gene pair, the appearing frequency thereof was analyzed. The higher the appearing frequency of a gene pair, the higher the possibility that there exists interactions between said gene pair. The total number of PubMed Database publications was recorded as N, the respective independent appearing frequencies of the two genes of a gene pair in PubMed publication Database were recorded as m and n, respectively. The actual co-appearing number of the gene pair was k. Based on hypergeometric distribution, the possibility that more than k of co-citation can appear under completely random conditions was calculated:

(62) $p=1-\underset{i=0}{\stackrel{k-1}{.Math.}}p\left(i.Math.n,m,N\right)$$\mathrm{wherein}$$p\left(i.Math.n,m,N\right)=\frac{n.Math.\left(N-n\right).Math.m.Math.\left(N-m\right).Math.}{\left(n-i\right).Math.i.Math.\left(n-m\right).Math.\left(N-n-m+i\right).Math.N.Math.}$

(63) Finally, based on the data results, they were integrated into an interaction network among genes. The network was graphically displayed using medusa software.

(64) 2. Mass Spectrometry and Data Analysis:

(65) The APE, ADVT thrombus samples obtained in Example 1 were respectively analyzed by mass spectrometry, with the Mass spectrometer model LTQ (Thermo Finnigan USA).

(66) 2.1 LC-MS/MS

(67) Ettan MDLC system (GE Healthcare) was used for the desalting and isolation of trypsinized peptides mixture. In this system, the sample were desalted on RP trap column (Zorbax 300 SB C18, Agilent Technologies), and then isolated on RP column (150 m i.d., 100 mm in length, Column Technology Inc., Fremont, Calif.). Mobile phase A (0.1% formic acid in HPLC-grade water) and mobile phase B (0.1% formic acid in acetonitrile) were used. 20 g of the trypsinized peptides mixture was loaded onto the column, and the isolation was performed for 120 min by using 4-50% of B linear gradient at a flow rate of 2 L/min. Finnigan LTQ linear ion trap MS equipped with Electrospray interface (Thermo Electron) was connected to said LC equipment for detecting the diluted peptides. Data-dependent MS/MS spectrums were obtained. Each scanning cycle was composed by the following: one complete Profile mode MS scanning, followed by 5 Centroid mode MS/MS scanning, wherein the Dynamic Exclusion was set as following: repeat counting=2, repeat time=30 s, exclusion time=90 s. Each sample was analyzed in triplicate.

(68) 2.2 Data Analysis

(69) Bioworks Browser rev. 3.1 (Thermo Electron, San Jose, Calif.) was used to perform automatic retrieving for MS/MS spectrums against non-redundant International Protein Index (IPI) human protein database (version 3.26, 67687 items). BuildSummary was used to extract protein identification results from SEQUEST output file (Dai J, Shieh C H, Sheng Q H, Zhou H, Zeng R: Proteomic analysis with integrated multiple dimensional liquid chromatography/mass spectrometry based on elution of ion exchange column using pH steps. Anal Chem).

(70) The peptides were restricted as trypsinized peptide forms, and allowed to have up to two missing gaps. Carbamoyl methylation of Cysteine was considered as a fixed modification, while the oxidation of methionine residue was considered as a variable modification. The allowed mass tolerances were respectively as: precursor ion, 2.0 Da, while the fragment ion, 0.8 Da. Protein identification criteria was based on Delta CN (0.1) and cross-correlation score (Xcorr, one electric charge1.9, two electric charges2.2, three electric charges3.75).

(71) 3. Results and Analysis:

(72) The results were sent to Shanghai Sensichip Infotech Co. Ltd. (Shanghai, China) to perform the following bioinformatics analysis: (the preparation of the samples from the APE patient of Example 1, and the protein sample shotgun pre-isolation results of protein samples can be seen in FIG. 1, and the sequence information of the enzyme-digested peptide fragments and debris can be seen in FIG. 2) based on databaseprotein retrieveidentifying corresponding genesperforming Gene Oncology (GO) analysisidentifying genes showing differencesidentifying proteins showing differencesperforming signal pathway analysis of KEGG Pathwayobtaining protein interaction networkidentifying the core proteins in the network.

(73) Results:

(74) (1) GO analysis method: differentially expressed proteins were used as the input for GO analysis. Genes showing differences were respectively mapped towards various nodes of gene ontology database. The gene number at each node was calculated. The statistic results were as follows:

(75) TABLE-US-00001 TABLE 1 GO analysis results of APE thrombus samples (histogram was shown in FIG. 3) Protein Richness level Function category counts p-value Cell cycle and proliferation 20 0.002718086 Stress response 40 4.62E11 Transport 52 3.74E07 RNA Metabolism 14 0.999999301 DNA Metabolism 4 0.925590145 Protein Metabolism 17 0.394000101 Other metabolism processes 23 0.000119964 Development processes 6 0.183137052 Cell structure and biogenesis 82 9.92E13 Cell-cell signaling 17 0.686695527 Signal transduction 45 0.998147085 Cell adhesion 8 2.58E08 Death 9 0.353211866 Other biological processes 99 0.984977572 Unknown 32 0.873823656

(76) TABLE-US-00002 TABLE 2 GO analysis results of AVDT thrombus samples (histogram was shown in FIG. 5) Protein Richness level Function category counts p-value Cell cycle and proliferation 6 0.091311 Stress response 28 7.98E07 Transport 32 1.90E05 RNA Metabolism 4 0.999605 Protein Metabolism 2 0.06044 Other metabolism processes 18 0.08507 Development processes 2 0.239812 Cell structure and biogenesis 43 1.21E06 Cell-cell signaling 1 0.580049 Signal transduction 20 0.998072 Cell adhesion 5 0.000131 Death 9 0.46574 Other biological processes 27 0.794559

(77) (2) KEGG signal pathway analysis: GenMAPP v2.1 was used to map the genes towards the KEGG Pathway database, and the richness levels (p-value) of the genes in each pathway were counted. As for the APE thrombus samples, 35 relevant signal pathways were found. As for the AVDT thrombus samples, 94 relevant signal pathways were found.

(78) (3) Protein interaction network analysis: 3 different types of interaction relationships were combined together to obtain the protein interaction network, i.e. (a) the relationships of protein interactions, gene regulations, protein modifications, etc. in KEGG signal pathway analysis data; (b) protein-protein interactions proved by the existing high throughput experiments, such as yeast two-hybrid, etc.; (c) The interactions between genes and between proteins mentioned in existing publications.

(79) The bioinformatics analysis results for APE thrombus samples can be seen in FIGS. 3 and 4. The results indicated that 2, 3, and 1 subunit integrins were core proteins of red thrombus.

(80) The bioinformatics analysis results for AVDT thrombus samples can be seen in FIGS. 5 and 6. The results indicated that 3 subunit integrins and ITGA2B (i.e. integrin chain IIb) were core proteins of red thrombus.

Example 3: Tandem Protein Mass Spectrometry Analysis for Thrombus of Acute VTE Patients

(81) 1. Background Information of the Clinical Tested Subjects:

(82) (1) the patient group consisted of 24 cases of VTE, who were diagnosed and confirmed to have VTE by imaging (CTA, MRA, lung ventilation/perfusion scanning, angiography, or lower limbs venous vascular ultrasound).

(83) 12 patients were male, 12 patients were female, and the ages were from 25 to 82, with the average age 6216. 8 patients were acute VTE (within one month after onset); 9 cases were subacute VTE (within 1-3 months after onset); 7 case were chronic VTE (more than 3 months after onset), wherein 4 cases were readmitted patients whose CTEPH clinical symptoms were aggravated. 8 cases of acute phase VTE admitted patients were all subcutaneously injected with low molecular heparin 5000 unit/Bid for 7-10 days, and orally administered with 2.5 mG/Qd warfarin the first 3-4 days after stopping the low molecular heparin; 9 cases of admitted subacute phase VTE patients were subcutaneously injected with low molecular heparin 5000 unit/Qd for 7 days, and/or orally administered with 2.5 mG/Qd warfarin; 4 cases of readmitted CTEPH patients were treated with anticoagulant therapy as subacute phase, other 3 cases of chronic phase VTE patients were on outpatient follow-up, and orally administered with 2.5 mG/Qd warfarin for continuous treatment. During the detection process of the present investigation, none of the 24 cases VTE patients were found to have malignant tumor, autoimmune disease, or arterial thrombotic diseases.

(84) (2) 270 cases of healthy persons were used as control group, wherein 140 cases were male (age 20-75), 49 cases were in 20-39 years old group, 48 cases were in 40-59 years old group, and 43 cases were in over 60 years old group; 130 cases were female (age 21-72), 46 cases were in 20-39 years old group, 44 cases were in 40-59 years old group, and 40 cases were in over 60 years old group.

(85) The diagnosis for PE and DVT were all based on imaging (CT, MRA, ECT or angiography) proofs and clinical manifestations, wherein the criteria can be refer to (Torbicki A, Perrier A, Konstantinides S, et al. Guidelines on the diagnosis and management of acute pulmonary embolism: the Task Force for the Diagnosis and Management of Acute Pulmonary Embolism of the European Society of Cardiology (ESC)[J]. Eur Heart J, 2008, 29(18): 2276-2315; and Qaseem A, Snow V, Barry P, et al. Current diagnosis of venous thromboembolism in primary care: a clinical practice guideline from the American Academy of Family Physicians and the American College of Physicians [J]. Ann Intern Med, 2007, 146(6): 454-458).

(86) 2. Integrin Molecular Detection

(87) (1) 2 ml of peripheral venous blood was collected from the subjects (VTE patients, or control), 2 mg EDTA for anticoagulation and 20 l fluorescent antibodies were added into the test tubes; wherein the antibodies were respectively: antibodies against integrin 1 subunit (CD29) were PE-labeled, mouse-anti-human IgG2a, , clone No. HUTS-21, and the concentration is 50 mg/L; antibodies against integrin 2 subunit (CD18) were PE-labeled, mouse-anti-human IgG1, , clone No. 6.7, and the concentration is 50 mg/L; antibodies against integrin 3 subunit (CD61) were PE-labeled, mouse-anti-human IgG1, , clone No. VI-PL2, and the concentration is 50 mg/L; the fluorescent antibodies CD29-PE (1), CD18-PE (2), and CD61-PE (3) can be obtained from BD (Becton, Dickinson and Company, USA);

(88) (2) thoroughly mixing after adding into the samples and reacting in dark for 30 min at room temperature;

(89) (3) adding 500 l hemolysin into the test tubes; wherein the added hemolysin was used for dissolving erythrocytes, the model of which was OptiLyse C, and can be obtained from Beckman-Coulter, Inc., USA.

(90) (4) water bath at 37 C. for 30 min;

(91) (5) adding 100 l buffer (0.01M PBS, PH7.2) after washing, and detecting by flow cytometer, wherein the flow cytometer (EPICS XL-4) from BECKMAN-COULTER, Inc. was used;

(92) 3. Operation Procedure of Cell Immunization and Integrin Flow Cytometry:

(93) (1) In the experiment, 2 mg EDTA was added into the test tubes for anti-coagulation, and same types of control tubes were set at the same time, wherein 20 l of mouse IgG1-PC5, IgG1-FITC, IgG1-PE were respectively added according to the different fluorescent markers (20 l of mouse IgG2-PE was added for CD29 molecular detection, 50 mg/L). Subsequently, 20 l (50 mg/L) of corresponding fluorescent antibodies were respectively added. After thoroughly mixing, the mixture was kept in dark for 30 min at room temperature. Then, 500 l hemolysin (a product of BECKMAN-COULTER, Inc., USA, model OptiLyse C) was added, and the reaction was performed in water bath at 37 C. for 30 min. After being taken out and washed, 500 l of sheath reagent (obtained from Beckman-Coulter, Inc., USA, containing the active ingredients of sodium sulfate 9.84 g/L, sodium chloride 4.07 g/L, procaine hydrochloride 0.11 g/L) was added to each tube, followed by flow cytometry (a product of Beckman-Coulter, Inc., USA, model EPICS XL-4).

(94) (2) The BECKMAN-COULTER standard fluorescent microsphere was used to adjust the PMT voltage, fluorescence compensation, and sensitivity of the flow cytometry, so as to set up the detection protocol. 10000 cells were collected in each tube. The corresponding cell population in the scatter diagram of control tube of the same type was used to set the gate, and the percentage of positive cells (%) was determined according to the quadrant that the fluorescence corresponded to, and results were analyzed by the built-in SYSTEM-II software.

(95) 4. Statistical Analysis:

(96) SPSS18 statistical software was used for the statistical analysis of the control group. Kolmogorov-Smirnov test was used for normality test, with P>0.05 as normal distribution. The normal distribution data were represented as xS, variance analysis was used for the comparison among different age groups, and P<0.05 indicated a difference with statistical significance. Abnormal distribution data were represented by median and P2.5-P97.5, Wilcoxon rank-sum test was used for the comparison between different gender groups, and P<0.05 indicated a difference with statistical significance; Kruskal-Wallis H test was used for the comparison among different age groups, and P<0.05 indicated a difference with statistical significance.

(97) As for normal distribution data, the average value measured in health population was combined with its standard deviation (xS) to determine the range of normal level. As for abnormal distribution data, the median was combined with P2.5-P97.5 in statistics to indicate the range of normal level.

(98) The 1, 2 and 3 of VTE group were compared with those of the control group, and a value higher than the normal reference range was considered as increased expression.

(99) 5. Results:

(100) The results were shown as the percentage of cells containing a corresponding integrin subunit (positive cell) in the total cell counting, (i.e., 10000 cells were used as a unit, the percentage of positive cells). FIGS. 7-10 and the following table 3 are shown. In the 270 cases of control group, integrin 1, and 3 subunits showed a normal distribution, while integrin 2 subunit showed an abnormal distribution. There was no significant difference in the different genders, and with the increasing of age, 1 and 3 tend to decrease; the baseline region of integrin 1 subunit was 2.8-13.56%, of integrin 2 subunit was 71.1-95.8%; and of integrin 3 subunit was 4.59-14.71%.

(101) TABLE-US-00003 TABLE 3 Information and detection results of VTE patients Diagnostic 1 2 3 D-di CRP Fibrinogen No. Gender Age Diagnosis Phase method 2.8-13.56% 70.1-95.8% 4.59-14.71% <0.3 mg/L <2.87 mg/L 2-4.5 g/L 1 male 82 lower subacute phase ultrasound 11.37 97.5 14.5 1.06 132 4.613 limbs DVT 2 male 56 PE/lower subacute phase CTA/ 6.81 76.3 15.9 0.47 20.8 4.761 limbs ultrasound DVT 3 male 33 PE subacute phase CTA 11.4 75 18.9 0.22 51.8 4.553 4 female 58 lower chronic phase ultrasound 21.6 66.8 8.63 0.05 2.863 limbs DVT 5 female 66 CTEPH chronic phase CTA 12.8 96.4 12.9 0.05 0.3 3.25 6 female 54 lower subacute phase ultrasound 18.5 85 18.8 0.7 0.7 2.56 limbs DVT 7 female 56 lower chronic phase ultrasound 10.1 94.7 16 0.05 0.7 3.65 limbs DVT 8 male 68 CTEPH chronic phase CTA 7.27 98.2 12.3 0.14 24.7 3.825 9 female 25 PE subacute phase CTA 5.8 94.2 16.9 0.35 36 2.761 10 female 82 PE subacute phase1 CTA 17.3 99.5 5.6 0.05 0.2 2.495 11 male 65 lower subacute phase ultrasound 14.5 96.8 9.85 0.05 1.7 3.04 limbs DVT 12 female 61 PE acute phase CTA 4.5 96.4 17.3 2.495 13 male 71 PE acute phase PAG 14.3 95.6 13.1 1.48 16.4 3.761 14 female 81 PE chronic phase CTA 10.2 96.7 7.4 0.08 3.383 15 male 49 PE acute phase CTA 15.4 90.1 14.6 16 female 53 PE acute phase lung 13.1 97.2 6.4 1.25 0.6 3.576 ventilation/ perfusion 17 male 71 PE acute phase CTA 17.52 98.2 19 0.24 0.7 2.714 18 male 80 CTEPH chronic phase ventilation/ 23.5 99.4 18.7 0.75 32 3.264 perfusion scanning 19 female 75 PE acute phase ventilation/ 6.27 94.7 15.4 0.64 2.6 2.11 perfusion scanning 20 female 78 PE acute phase ventilation/ 6.92 96.9 13.4 0.96 38 4.481 perfusion scanning 21 male 64 lower subacute phase ultrasound 14.5 96.8 9.85 0.05 3.04 limbs DVT 22 female 80 CTEPH chronic phase CTA 17.3 99.5 5.6 0.09 2 2.607 23 male 48 PE acute phase CTA 15.49 90.1 14.6 0.89 4 2.00 24 male 32 PE subacute phase CTA 11.4 75 18.9 0.25 92 4.533 Total: 62 16 12.83 5.07 91.96 9.28 13.52 4.94 Note: 4 cases of CTEPH patients, with echocardiography systolic pressure between 65-74 mmHg; No. 4, 7, and 14 are follow-up patients.

(102) The integrin subunits of the 24 cases of the acute phase, subacute phase, and chronic phase VTE patients in the present investigation, though received different anti-coagulant protocol and different anti-coagulant time period, were all individually or jointly expressed in a level higher than the upper limit of the range in health population, wherein 15/24 cases (62.5%) of VTE patients had individually increased expression of integrin subunit, 11/24 cases (45.8%) had increased integrin 1 subunit, wherein acute phase 4/11 cases, subacute phase 4/11 cases, and chronic phase 3/11 cases; 13/24 cases (54.2%) had increased integrin 2 subunit, wherein acute phase 4/13 cases, subacute phase 4/13 cases, and chronic phase 5/13 cases; 10/24 cases (41.7%) had increased integrin 3 subunit, wherein acute phase 3/10 cases, subacute phase 6/10 cases, and chronic phase 1/10 cases. 9/24 cases had two or more jointly increased integrin subunits (37.5%), wherein 1, 2 and 3 jointly increased in 3/24 cases (12.5%), 1 and 2 jointly increased in 6/24 cases (25%), 1 and 3 jointly increased in 1/24 case (4.2%), and 2 and 3 jointly increased in 1/24 case (4.2%). The above cellular detection results indicated that, the higher expression of integrin subfamily in VTE patients can be used to diagnose VTE.

(103) The results in above Examples indicated that, the expressions of integrin 1, 2, and 3 subunits of blood cells in health population can be detected, wherein the expressions of 1 and 3 subunits showed a normal distribution, while the expression of 2 subunit showed an abnormal distribution. There was no statistical difference between genders, and with the increasing of age, the expressions of 1 and 3 subunits tend to decrease. For the 8 cases of acute phase, 9 cases of subacute phase, and 7 cases of chronic phase VTE patients of the present invention, integrin 1, 2, and 3 subunits individually or jointly increased in the expression, all higher than the upper limit of the reference range in control group, complying with the results of the thrombus tandem protein mass spectrometry analysis. The expression of integrin 1, 2, and/or 3 subunits in the VTE groups of the invention individually or jointly increased, indicating that the activation and signal transduction effects of integrin were increased; the higher expression of VTE patients' integrin 1, 2, and 3 subunits during the continuous anti-coagulant treatment indicated the stability, sensitivity, and specificity thereof, and also indicated that the patients were still in a thrombophilia state.