Use of extracellular vesicle as marker for immune thrombocytopenia

Abstract

The use of an extracellular vesicle as a marker for immune thrombocytopenia. The extracellular vesicle (EV) is derived from platelets, and the extracellular vesicle is secreted by an anti-platelet membrane glycoprotein autoantibody by means of activating the AKT pathway. Provided is a detection kit for diagnosing immune thrombocytopenia, wherein the detection kit can be used for detecting extracellular vesicles.

Description

Use of extracellular vesicles as immune thrombocytopenia markers Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to use of an extracellular vesicle as an immune thrombocytopenia marker, wherein the extracellular vesicle is derived from platelets. Background Art

[0002] Immune thrombocytopenia (ITP) is a relatively rare autoimmune disease characterized by a low incidence of approximately 3 cases per 100,000 person-years. Notably, its prevalence peaks in men aged 75 years and older, reaching 9 cases per 100,000 persons. The etiological triggers of ITP remain largely elusive; however, an intriguing seasonal pattern has been documented. Disease incidence increases in winter, suggesting that viral infection may be a contributing factor. Despite these observations, 70% of adult ITP patients develop persistent, lasting longer than 3 months, or chronic, lasting more than 12 months from diagnosis. The incidence of these long-term cases exhibits remarkable stability across seasons, suggesting that factors other than seasonal pathogens may play a more significant role in the ongoing pathophysiology of the disease. This observation highlights the complexity of ITP, suggesting that acute ITP may be triggered by transient factors such as infection, while chronic ITP may involve more extensive immune dysregulation. This complexity requires further research to unravel the underlying mechanisms and triggers that lead to chronic disease. The pathogenesis of ITP involves a complex interplay of immune mechanisms, leading to increased platelet destruction and decreased platelet production. In patients with ITP, the immune system mistakenly produces autoantibodies against platelet membrane glycoproteins, such as GPIb / IX and GPIIb / IIIa. These bound autoantibodies lead to platelet phagocytosis by macrophages, which are primarily found in the spleen and, to a lesser extent, the liver. This phagocytic activity is the primary cause of the shortened platelet lifespan in patients with ITP. Furthermore, autoantibodies can bind to megakaryocytes (platelet precursors in the bone marrow), thereby impairing platelet production. This interference with megakaryocyte function and platelet release exacerbates thrombocytopenia. Single-cell transcriptome analysis of bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) revealed impaired megakaryopoiesis in ITP, particularly in immune progenitors. This finding suggests that megakaryocyte differentiation from CD9+ HSPCs is reduced in ITP, and subtype-specific gene expression changes in megakaryocyte progenitors provide new insights into the pathogenesis of ITP. Evidence suggests that T- and NK cell-mediated mechanisms also play a role in ITP. Autoreactive T cells may promote the destruction of platelets and megakaryocytes through cytotoxic activity and support B-cell-mediated autoantibody production. Advances in the understanding of their pathophysiology, particularly the role of autoantibodies, have led to the development of diagnostic methods designed to directly identify these autoantibodies.

[0003] Extracellular vesicles (EVs) are emerging as potential diagnostic biomarkers for various diseases, including ITP. EVs are membrane-bound vesicles released by cells into the extracellular space and can carry proteins, lipids, and nucleic acids that reflect the cellular state. EVs can reflect changes in the cellular environment, including changes in the immune response. In microbiology, several studies have demonstrated that EVs contribute to antibiotic resistance in bacteria. In ITP, the immune system targets platelets, and EVs released by platelets or immune cells may carry specific proteins or RNAs that indicate disease status. The presence and composition of EVs in the blood may be used to diagnose ITP. For example, if elevated levels of certain proteins or RNA molecules associated with ITP are found in EVs, they could serve as diagnostic markers. Because 125 EVs can be found in plasma, their analysis could provide a noninvasive method for diagnosing ITP, which is particularly beneficial for patients. Summary of the Invention

[0004] The present invention provides a use of an extracellular vesicle as a marker for diagnosing immune thrombocytopenia, wherein the extracellular vesicle is derived from platelets.

[0005] In some embodiments, the present invention provides a use of an extracellular vesicle as a marker for immune thrombocytopenia, wherein the extracellular vesicle (EV) is derived from platelets.

[0006] In the above-mentioned use of the present invention, the extracellular vesicles (EVs) are derived from platelets and are secreted by anti-platelet membrane glycoprotein autoantibodies through activation of the AKT pathway.

[0007] In another embodiment, the present invention provides a detection kit for diagnosing immune thrombocytopenia, wherein the detection kit can detect extracellular vesicles, which are secreted by anti-platelet membrane glycoprotein autoantibodies through activation of the AKT pathway.

[0008] In the above-mentioned detection kit of the present invention, the extracellular vesicles are used as a marker of immune thrombocytopenia.

[0009] In another embodiment, the present invention also provides a kit for diagnosing immune thrombocytopenia, which detects the content of anti-gpib / GPIX autoantibodies in extracellular vesicles.

[0010] In the above-mentioned kit of the present invention, the anti-GPIb / GPIX autoantibody serves as a biomarker for immune thrombocytopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows a schematic diagram of the platelet separation process (1A) and the appearance of isolated EVs (1B), EV size distribution, and counts (1C-1E) for patients in the healthy group, ITP group, and non-ITP group;

[0012] Figure 2 shows a comparison of autoantibodies in platelets and EVs of patients in the healthy group, ITP group, and non-ITP group (2A-2C);

[0013] Figure 3 is a comparison of anti-gpib autoantibodies inducing platelet apoptosis and EV secretion through AKT activation;

[0014] Figure 4 is a comparison of the anti-GPIb-induced ITP mouse model and the effects of the antibody on platelets and EVs;

[0015] Figure 5 is a comparison of the effects of SZ2 anti-gpib antibody treatment on MEG-01 and UT-7 cells on the stimulation of megakaryocyte apoptosis and EV production;

[0016] FIG6 shows platelet accumulation in the spleen and liver of mice 1 hour after intraperitoneal injection of 2.5 μg / g GW4869 and anti-gpib. DETAILED DESCRIPTION

[0017] The following examples are typical and are used to further illustrate and understand the essence of the present invention, but are not intended to limit the scope of the present invention in any way.

[0018] Sources of reagents and materials used in the following examples:

[0019] Materials and reagents:

[0020] Platelet receptor surface expression was assessed by flow cytometry using the following monoclonal antibodies:

[0021] Anti-cd41-fitc (GPIIb, Cat:984506, Biolegend, San Diego, USA), anti-cd42b-pe (GPIb, Cat: 303906, Biolegend), anti-cd62b-apc-cy7 (P-selectin, Cat: 304944, Biolegen d), anti-cd61-percp (GPIIIa, Cat: 336404, Biolegend), anti-cd107a (LAMP)-1-APC (Cat: 328620, Biolegend), anti-annexin V-APC(Cat:328620,Biolegend). 550474, BD Pharmingen, San Diego, USA), 10X Annexin V binding buffer (Cat: 556454, BD Pharmingen), anti-caspase-3-pe (Cat: 550821, BD Pharmingen), BD Cytofix / Cytoperm TMFixation / Transduction Kit (Cat: 554714, BD Pharmingen), GW4869 (Cat: hyy-19363, MedChemExpress,

[0022] Monmouth Junction, USA), AKT inhibitor (Cat: S7776, Selleck Chemicals,

[0023] Houston, USA), SZ2 anti-human GPIb (Cat: IM0409, Beckman Coulter, Brea, USA), R300 rat anti-mouse GPIb (Cat: R300, Emfret, w <s:1>werzburg, Germany).

[0024] EXOCET Exosome Quantification Kit (Cat: EXOCET96A, System Biosciences, Palo Alto, USA), Mouse TPO ELISA Kit (Cat: E-EL-M0640, Elabscience, Wuhan, China), Rabbit anti-human CD41 (Cat: PA5-79527, Thermo Scientific Fisher, Waltham, USA), Anti-CD42b (Cat: PA5-103118, Thermo Scientific Fisher), Anti-TSG101 (Cat: 72312S, CellSignaling Technology CST, Danvers, USA), anti-Alix (Cat: 92880, CST), anti-CD9 (Cat: 98327, CST), anti-CD63 (Cat: 52090, CST), anti-CD81 (Cat: 2433, CST), anti-F4 / 80 (Cat: 30325, CST), anti-rat IgG (H+L)-Alexa Fluor 488 (Cat: 4416, CST), anti-Akt (Cat: 9272, CST), and Phospho-Akt (Ser473) antibodies (Cat: 9271, CST).

[0025] Example 1 Preparation of platelet-derived exosomes

[0026] 1) Platelet separation

[0027] Whole blood was collected in EDTA-A2 anticoagulant vacuum tubes (4 ml of blood). To separate platelet-containing plasma from red blood cells and white blood cells, 4 ml of whole blood was centrifuged at 200 x g for 5 min in a 5 ml centrifuge tube. The separated plasma was then added to another tube and centrifuged again at 2000 x g for 2 min to obtain platelets (PLT). The platelets were washed with CGS buffer (0.123 M NaCl, 0.033 M 401 d-glucose, 0.013 M trisodium citrate); the washed platelets were adjusted to pH 6.5 and resuspended in modified Tyrode's buffer (2.5 mM HEPES, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, and 5.5 mM d-glucose), adjusted to pH 7.4, to a final concentration of 3 × 10 8 cells / mL.

[0028] 2) Plasma exosome extraction

[0029] Platelet-depleted plasma EVs (exosomes) or supernatants incubated with platelets were centrifuged at 10,000 x g for 30 min at 4°C. Pellets containing cell debris were discarded, and the supernatant containing EVs was ultracentrifuged at 100,000 x g for 70 min at 4°C (SW 40Ti swinging bucket rotor, Beckman Coulter) to obtain target EV microspheres, which were resuspended in PBS, aliquoted, and frozen at 80°C.

[0030] Example 2 Characterization of platelet-derived exosomes

[0031] Protein content was determined using the Bradford method using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Shanghai, China) with bovine γ-globulin as the standard, according to the manufacturer's protocol.

[0032] EV morphology was examined using a transmission electron microscope (Hitachi H-7500) following a previously described protocol. Isolated EVs were combined with 4% paraformaldehyde and adsorbed onto carbon-coated copper electromagnetic grids for 20 minutes. Samples were rinsed with PBS, fixed with 1% glutaraldehyde for 2 minutes, washed with deionized water, and stained with 1.5% uranyl acetate for 4 minutes. Images were then captured and analyzed using a transmission electron microscope (TEM). Samples were mounted on an NP100 membrane with a 44.5 mm filter and a voltage of 0.64 V. After dilution with 0.9% NaCl, the samples were pipetted into a pipette using consistent settings (camera: 161 frames / s, injection volume: 1 mL). The detection threshold was set to capture the maximum number of particles, ensuring that 10 to 100 red crosses were counted, with less than 10% being irrelevant to different particles. Recording and analysis were performed using NTA software (version 2.3, NanoSight).

[0033] Example 3 Effect of platelet-derived exosomes on immune thrombocytopenia

[0034] Patients and subjects

[0035] The Institutional Review Board approved this study and informed consent was obtained from patients from whom biological samples were extracted. The study was approved under the MEC Ethics Registration Number: NFEC-2023-394 of the Nanfang Hospital of Southern Medical University. All subjects were patients hospitalized in the Nanfang Hospital of Southern Medical University (Guangzhou, China) between September 2021 and September 2023. All patients agreed to participate in this study and signed a written informed consent. Platelet and blood cell counts were performed using a Sysmex XN9000 hematology analyzer (Sysmex Corporation). The patients were divided into three groups: i) Healthy control group (HC): 37 healthy adults from the Department of Laboratory Medicine, including 15 males and 22 females, with an average age of 357 (38.51±23.45) years and an average platelet count of 217.41±89.21×10 9 / L. None of the blood donors had a history of circulatory system diseases, immune system diseases, or malignant tumors, and none of the blood donors had a history of blood transfusion. All blood donors had no signs of infection one week before hospitalization and no medication one week before blood collection. The children's complete blood cell count was normal, and blood samples were collected; ii) ITP: All patients met the diagnostic criteria for ITP, including 34 males and 45 females. The average age was 39.15±20.84 years, the average onset time (from onset to hospitalization) was 1-6 days, and the average platelet count was 45.60±40.07×10 9 / L. This disease is not accompanied by obvious infectious symptoms and does not require other anti-infective drugs. ITP was confirmed by morphological analysis of bone marrow cells collected before administration; iii) Non-ITP with thrombocytopenia: 60 male and 75 female patients were included. The average age was 36.75±23.43 years, and the average platelet count was 40.58±36.32×10 9 There was no statistically significant difference in age and gender between the treatment group and the control group (p<0.05).

[0036] Cell culture

[0037] The human MEG-01 megakaryoblastic cell line was purchased from ATCC (Manassas, VA, USA); UT-7 cells (ACC 137) were purchased from DSMZ. MEG-01 cells were maintained in RPMI Medium 1640 (Gibco, Thermo Scientific) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Scientific) and 1% penicillin and streptomycin (Solarbio). UT-7 cells were maintained in 80–90% α-MEM (containing ribo- and deoxyribonucleosides) supplemented with 20% FBS and 5 ng / ml GM-CSF. Cells were tested for mycoplasma monthly. All cells were cultured in a 37°C, 5% CO2 incubator.

[0038] Cell proliferation assay

[0039] MEG-01 cells and UT-7 cells were seeded in 96-well flat-bottom plates for 1-3 days. The proliferation of MEG-01 and UT-7 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). Briefly, 1×10 4 Cells were seeded in 96-well plates and then exposed to different concentrations of VLZ (2.5, 5, and 10 μM) for 5 days at 37°C and 5% CO2. Each treated cell was incubated with CCK-8 solution at 37°C for 2 hours. The absorbance (OD) was measured at 450 nm.

[0040] Cell apoptosis assay

[0041] Apoptosis detection was performed using the Annexin V PI kit. Briefly, MEG-01 cells and UT-7 cells were seeded into 24-well plates and treated for 1-3 days. After the cells were removed from the plates, they were harvested and washed twice with PBS. The cells were resuspended in 500 μL Binding Buffer II / 1X Binding Buffer. 5 μL Annexin V-FITC and 5 μL propidium iodide (PI 50 μg / mL, optional) were then added. The cells were incubated in the dark at room temperature for 5 minutes. Cells were acquired on a BD Fortassa flow cytometer and analyzed using Flowjo V10 software (BD Bioscience).

[0042] Platelet separation

[0043] The separation process diagram is shown in Figure 1A.

[0044] Whole blood from all subjects in this study was collected in EDTA-A2 anticoagulant vacuum tubes (4 ml of blood). To separate platelet-containing plasma from red blood cells and white blood cells, 4 ml of whole blood was centrifuged at 200 x g for 5 min in a 5 ml centrifuge tube. The separated plasma was then added to another tube and centrifuged again at 2000 x g for 2 min to harvest PLTs. Platelets were washed with CGS buffer (0.123 M NaCl, 0.033 M 401d-glucose, 0.013 M trisodium citrate; pH 6.5) and resuspended in modified Tyrode buffer (2.5 mM HEPES, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3 and 5.5 mM d-glucose; pH 7.4) to a final concentration of 3 × 10 8 cells / mL.

[0045] Plasma exosome extraction

[0046] Platelet-depleted plasma EVs or supernatants incubated with platelets were centrifuged at 10,000 × g for 30 min at 4°C. Microspheres containing cell debris were discarded, and the supernatant containing EVs was ultracentrifuged at 100,000 × g for 70 min at 4°C (SW 40Ti swinging bucket rotor, Beckman Coulter) to obtain EV microspheres. These microspheres were resuspended in PBS, aliquoted, and stored frozen at 80°C. Protein content was determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Shanghai, China) using bovine γ-globulin as a standard, according to the manufacturer's protocol.

[0047] Exosome characteristics

[0048] EV morphology was examined using a transmission electron microscope (Hitachi H-7500) following a previously described protocol. Isolated EVs were combined with 4% paraformaldehyde and adsorbed onto carbon-coated copper electromagnetic grids for 20 minutes. Samples were rinsed with PBS, fixed with 1% glutaraldehyde for 2 minutes, washed with deionized water, and stained with 1.5% uranyl acetate for 4 minutes. Images were then captured and analyzed using a transmission electron microscope (TEM). Samples were prepared using an NP100 membrane with a 44.5 mm filter and a voltage of 0.64 V. After dilution with 0.9% NaCl, the samples were pipetted using a pipette using consistent settings (camera: 161 frames / s, injection volume: 1 mL). The detection threshold was set to capture the maximum number of particles, ensuring that 10 to 100 red crosses were counted, with less than 10% being irrelevant to distinct particles. The results were recorded and analyzed using NTA software (version 2.3, NanoSight). The results are shown in Figures 1B (EV appearance), 1C and 1D (particle size distribution), and 1E (EV particle count).

[0049] ITP-related autoantibody detection based on flow cytometry.

[0050] Platelet autoantibodies in ITP patients were detected using the FCIA 427 assay, detecting anti-gpib / GPIX, GPIIb / GPIIa, and GMP140 antibodies in platelets or platelets (PLTs) or platelet EVs (EVs). The fluorescence intensity of all five antibody-coated microbeads in ITP patient samples was higher than that in healthy controls, indicating the presence of platelet EV autoantibodies in ITP patients (Figures 2A-C). The MFI values ​​of the five antibody-coated microbeads in ITP patients were significantly higher than those in non-ITP patients or healthy controls. There was no statistically significant difference in MFI values ​​between non-ITP patients and healthy controls.

[0051] The above experimental results indicate that platelet EV autoantibodies exist in ITP patients, providing a theoretical basis for the treatment of immune thrombocytopenia with platelet EVs.

[0052] Example 4 Detection of ITP Autoantibodies on Platelets and Platelet-Derived EVs Using a Flow Cytometry-Based Method

[0053] ITP-related autoantibody detection based on flow cytometry.

[0054] Platelet autoantibodies in ITP patients were detected using the FCIA 427 assay, detecting anti-GPib / GPIX, GPIIb / GPIIa, and GMP140 antibodies in PLTs or EVs. The fluorescence intensity of all five antibody-coated microbeads in ITP patient samples was higher than that in normal controls, indicating the presence of platelet autoantibodies in ITP patients (Figure 2A). The MFI values ​​of these five antibody-coated microbeads in ITP patients were significantly higher than those in non-ITP patients or healthy controls. There was no statistically significant difference in MFI values ​​between non-ITP patients and healthy controls. Cells were acquired on a BD Fortassa flow cytometer and analyzed using Flowjo V10 software (BD Bioscience).

[0055] Statistical methods

[0056] Except for RNAseq analysis, all statistical tests used in this study were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The specific statistical test used for each comparison is specifically annotated in the respective figure legends.

[0057] In this study, we used flow cytometry to detect 130 ITP autoantibodies (anti-GPIX, anti-GPIB, anti-GPIIB, anti-GPIIA, and anti-GMP140) on platelets and platelet-derived EVs. We found that the positive rate of anti-GPIB / GPIX autoantibodies in EVs was higher than in platelets, suggesting that these antibodies could serve as biomarkers and provide a rational approach for ITP intervention. Furthermore, we investigated whether inhibiting EV secretion of platelets could exacerbate ITP.

[0058] Experimental results:

[0059] Anti-GPIb autoantibodies induce platelet apoptosis and EV secretion through AKT activation

[0060] To investigate the mechanism underlying elevated EV production in ITP patients, ITP conditions were mimicked by exposing platelets to anti-gpib autoantibodies. Incubation with either the SZ2 anti-human GPIb antibody or the exosome inhibitor GW4869 did not significantly alter CD42b expression. However, the SZ2 anti-gpib antibody reduced CD42b (GPIb) protein levels, similar to those observed in ITP patients. GW4869 prevented the SZ2 antibody-induced decrease in GPIb, suggesting a role for EVs in GPIb protein dissemination. Furthermore, the anti-gpib antibody upregulated the activation markers CD107a and CD62p and induced platelet apoptosis by upregulating caspase-3 (Figures 3A and 3B). The anti-gpib antibody further stimulated EV secretion to levels comparable to those observed in ITP patients (Figure 3C). To investigate the signaling pathways underlying anti-gpib-induced EV secretion, healthy donor platelets were incubated with various antibodies with or without GW4869. Anti-gpib antibodies reduced CD42b expression in healthy donor platelets188 (Figure 3D). Consistent with previous findings, anti-gpib antibodies activated AKT phosphorylation in platelets and induced apoptosis through the AKT pathway (Figures 3E-3F). In addition, nanoflow cytometry detected GPIb+EVs in healthy control platelets incubated with SZ2 anti-GPIb antibodies, while AKTi1 / 2 inhibitors reduced the generation of GPIb+EVs (Figures 3G-3H).

[0061] Anti-GPIB stimulates megakaryocyte apoptosis and EV production

[0062] To investigate the effects of anti-GPIb on megakaryocytes, we treated MEG-01 and UT-7 cells with the SZ2 anti-GPIb antibody for 24 hours (Figures 4A and 4B). Cell viability decreased after anti-GPIb treatment, and this decrease was further observed after co-treatment with anti-GPIb and GW4869. Anti-GPIb also induced the expression of caspase-3 (Figures 4C and 4D) and AKT mRNA (Figures 4E and 4F) in both cell lines. This induction correlated with decreased cell viability, as the combination of anti-GPIb and GW4869201 further increased caspase-3 and AKT mRNA expression. Anti-GPIb also stimulated the expression of the apoptosis marker Annexin V in both MEG-01 and UT-7 cells (Figures 4G–4I), and GW4869 further exacerbated cell death. Notably, serum from an ITP patient with anti-GPIb autoantibodies induced apoptosis in MEG-01 cells (Figure 4J). Quantification of EVs by ELISA showed that anti-GPIb antibodies significantly increased EV production in both cell lines, while GW4869 inhibited EV secretion (Figures 4K-4L). In megakaryocytes, AKT activation induced apoptosis and EV secretion, similar to its effects in platelets. Treatment of MEG-01 cells with the SZ2 anti-GPIb antibody and the AKT inhibitors AKTi1 / 2 reduced apoptosis (Figure 4M). Furthermore, knockout of the AKT gene in MEG-01 cells reduced anti-GPIb-induced apoptosis (Figure 4N). Anti-GPIb also induced the phosphorylation and expression of protein kinase cleavage Caspase-3 (Figures 4o-4q) and inhibited megakaryocyte maturation (Figures 4r-4s).

[0063] Inhibition of EV production aggravates ITP pathology

[0064] To investigate the impact of EV production on ITP progression, an anti-GPIb-induced ITP mouse model was established by intravenously administering the R300 anti-mouse GPIb antibody to C57BL / 6 mice (Figure 5A). Administration of the R300 antibody resulted in a decrease in platelet count 24 hours after injection, which recovered by day 7, likely due to antibody clearance (Figure 5B). Notably, co-administration of R300 with the EV inhibitor GW4869 resulted in a sustained decrease in platelet count from day 1 to day 7. Both liver TPO mRNA and serum TPO protein levels were significantly increased in the R300+GW4869 group (Figures 5C and 5D). Platelet-derived EVs were significantly increased compared with the IgG control group (Figure 5E). Co-administration of R300 and GW4869 also increased mortality (Figure 5F).

[0065] Inhibition of EV release promotes macrophage-mediated platelet consumption.

[0066] To elucidate the role of EVs in ITP, mice were intraperitoneally injected with 2.5 μg / g GW4869 8 hours before anti-GPIb injection. One hour after anti-GPIb administration, cryosections of spleen and liver tissue were immunofluorescently stained with anti-rat IgG and the F4 / 80 macrophage marker. R300 colocalized with F4 / 80+ macrophages, and EV inhibition enhanced the colocalization of R300 and F4 / 80+ macrophages. The results showed that platelets accumulated in areas of the spleen and liver enriched for F4 / 80+ macrophages (Figures 6A and 6B), indicating that macrophages captured platelets via Fc receptors. This result suggests that EVs may mitigate macrophage-mediated platelet consumption.

Claims

1. A method of using an extracellular vesicle as a marker for immune thrombocytopenia, wherein the extracellular vesicle is derived from platelets.

2. The use according to claim 1, wherein the extracellular vesicles are derived from platelets and are secreted by anti-platelet membrane glycoprotein autoantibodies through activation of the AKT pathway.

3. A detection kit for diagnosing immune thrombocytopenia, wherein the detection kit can detect extracellular vesicles, which are secreted by anti-platelet membrane glycoprotein autoantibodies through activation of the AKT pathway.

4. The detection kit according to claim 3, wherein the extracellular vesicles are used as a marker of immune thrombocytopenia.

5. A kit for diagnosing immune thrombocytopenia, wherein the kit detects the content of anti-gpib / GPIX autoantibodies in extracellular vesicles. The kit according to claim 5 , wherein the anti-GPIb / GPIX autoantibody is used as a biomarker for immune thrombocytopenia.

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