Methods of modulating immune thrombosis and biomarkers for diagnosing related conditions

Abstract

The present invention relates to novel biomarkers for NETosis-induced vascular occlusion comprising an immune thrombosis disorder. Accordingly, the present invention relates to compounds and methods for treating NETosis-induced vascular occlusion comprising an immune thrombosis disorder, as well as methods of diagnosing NETosis-induced vascular occlusion.

Description

[0001] This invention relates to novel biomarkers for NETosis-induced (or NETosis-related) vascular occlusion, including dysregulation of immune thrombosis. Therefore, this invention relates to compounds and methods for treating NETosis-induced vascular occlusion, including dysregulation of immune thrombosis, and methods for diagnosing NETosis-induced vascular occlusion.

[0002] Activation of the coagulation cascade represents a natural defense mechanism crucial for maintaining physiological hemostasis in response to infection or tissue damage. For example, during bacterial sepsis, the host's response to pathogen invasion involves the activation of multiple defense mechanisms. Among these, the involvement of innate immunity and activation of coagulation work together to combat infection. These close interactions between immunity and hemostasis are known as "immunothrombosis." This mechanism causes the formation of microthrombi within blood vessels, which allow pathogens to be captured, recognized, and destroyed, thus limiting their spread throughout the body. In some cases, these defense mechanisms can become dysregulated. Strong activation of vascular cellular components, including leukocytes, platelets, and endothelial cells, can lead to endothelial dysfunction, where endothelial cells become pro-adhesive and pro-thrombotic, while excessive coagulation activation results in inadequate anticoagulation and fibrinolytic regulation, ultimately leading to coagulopathy (Tschirhart DOI / 10.37051 / mir-00122). Therefore, under pathological conditions, excessive activation of the coagulation cascade may occur, leading to disseminated intravascular coagulation (DIC) and other thrombotic processes.

[0003] The inventors have dedicated years to studying the role of polymorphonuclear neutrophils (PMNs) (a subset of leukocytes) in disseminated intravascular coagulation (DIC) during septic shock. PMNs are early participants in the host's innate immune response to infection. They are activated at the site of infection and become capable of capturing pathogens and releasing their granular contents through phagocytosis. In this way, they help eliminate bacteria, but simultaneously create a hyperinflammatory state that can promote the development of tissue lesions. NETosis refers to the release of the contents of the neutrophils' nuclei in the form of structures called neutrophil extracellular traps (NETs). These NETs can both capture pathogens and circulate blood cells, thus contributing to the host's immune defense. The procoagulant surface of NETs allows them to activate the contact phase of coagulation. Therefore, NETosis of PMNs plays an important role in immune thrombotic dysregulation that can lead to DIC.

[0004] NET is associated with a variety of pathologies (Papayannopoulos V. doi:10.1038 / nri.2017.105), such as systemic autoimmune diseases and autoinflammatory diseases (Gustaf Wigerblad et al., https: / / doi.org / 10.1038 / s41577-022-00787-0), including Crohn's disease (Vincenzo Dinallo, DOI: 10.1093 / ecco- jcc / jjy215 Rheumatoid arthritis (Joanna Clarker et al., DOI: 10.1038 / s41584-020-0459-4), antiphospholipid syndrome, systemic lupus erythematosus (SLE) (Wirestam et al., DOI: 10.3389 / fimmu.2019.02734), thrombosis-related diseases (Zdanyte et al., doi: 10.3389 / fcvm.2023.1155512), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (Prasanna Misra et al., doi: https: / / doi.org / 10.1007 / s10067-021-05790-9), non-septic DIC (Hell L et al., DOI: 10.1016 / S0049-3848(16)30133-5 Viral diseases such as influenza, COVID-19 (Jiayu Li, DOI: 10.1186 / s12985-023-02116-w; Zhu et al., doi: 10.3389 / fimmu.2022.838011), and in cancer (Jingxuan Xia, DOI: 10.1002 / ijc.34750) and sickle cell disease vascular occlusion complications (Ravi Vats, doi: 10.1182 / blood.2021014552; Emilia A Barbu, DOI: 10.1016 / j.cyto.2019.154933).

[0005] The inventors have identified proteins that are highly differentially expressed between two cohorts of intensive care patients with and without DIC in septic shock. They demonstrated that the miniature chromosome maintenance complex (MCM complex), containing the proteins MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, is overexpressed in neutrophils of patients with septic shock-induced DIC.

[0006] However, these proteins have never been previously described as being involved in NETosis associated with DIC in septic shock. Therefore, the inventors developed a method based on bacterial stimulation of human neutrophils that induce NETosis. in vitro The model is similar to what is seen in septic shock-related disseminated intravascular coagulation (DIC): in fact, bacterial stimulation of human neutrophils triggers the emergence of NETs, ​​as seen in neutrophils of DIC patients. Using this model, it was demonstrated that significant overexpression of the MCM complex was observed under such stimulated conditions, and more importantly, inhibition of the MCM complex by an inhibitor called ciprofloxacin resulted in complete suppression of NETosis. Furthermore, further functional studies of stimulated and suppressed neutrophils showed that ciprofloxacin-treated neutrophils retained their ability to phagocytose bacteria, and that this treatment did not interfere with fibrinolysis, an important pathway in the anticoagulation system (see Experimental Section). Surprisingly, similar results were obtained by chemically stimulating NETosis in the PMN, suggesting that targeting NETosis could be a suitable therapeutic approach for modulating, and particularly partially suppressing, immune thrombosis and for treating immune thrombotic disorders other than sepsis.

[0007] These results confirm the major role of neutrophils in immune thrombosis dysregulation and provide a novel therapeutic target for NETosis-induced immune thrombosis dysregulation, such as DIC involved in septic shock.

[0008] Septic shock is the most severe form of host response to pathogenic microbial infection. Despite the development of effective anti-infective treatments and appropriate care, the mortality rate of septic shock remains high, at approximately 30% to 40%.

[0009] The pathophysiology of septic shock is characterized by an excessive and inappropriate systemic inflammatory response mediated by immune cells activated during pathogen invasion. Disseminated intraepithelial neoplasia (DIC) is a common complication of sepsis and septic shock, with one-third of patients developing DIC. DIC is associated with an increased mortality rate of up to 60%.

[0010] These results are particularly important given the current lack of consensus on the treatment of DIC associated with septic shock. Treatment primarily focuses on addressing the underlying pathology, namely the urgency of anti-infective therapy. However, various complementary approaches are currently being investigated to prevent thrombotic complications and restore the balance between coagulation and fibrinolysis (Helms, Iba et al., 2023).

[0011] Anticoagulation can reduce disseminated microthrombus formation by preventing excessive activation of coagulation and immunity. Against this backdrop, various studies have investigated combining the anticoagulant effects of unfractionated heparin (UFH) or low molecular weight heparin (LMWH) with the immunomodulatory and anti-inflammatory properties of heparin (Helms et al., 2023, https: / / link-springer-com.proxy.insermbiblio.inist.fr / article / 10.1007 / s00134-023-06980-6). However, these studies have yielded inconsistent results and have failed to conclude that this treatment approach is effective.

[0012] This is why some studies have turned to using thrombomodulin as a potential alternative for preventing thrombotic complications, but the efficacy of thrombomodulin in humans is a controversial topic (Vincent et al., 2019; Levi et al., 2020; François et al., 2021; Guo et Lin, 2023).

[0013] Another recognized goal in research on the treatment of septic DIC is to correct the imbalance between coagulation and fibrinolysis. Recent studies in patients with septic shock have shown that plasma protein supplementation or delivery via fresh plasma reduces the risk of bleeding and improves the efficacy of heparin, thereby improving survival and reducing the risk of progression to coagulopathy (El-Nawawy et al., 2021; Helms, Iba et al., 2023; Stahl et al., 2020; Weng et al., 2021).

[0014] Therefore, the treatment of DIC induced by septic shock remains a challenge due to the complexity of its pathophysiology.

[0015] In this invention, the inventors have identified the major role of the MCM complex in NETosis and demonstrated that using MCM inhibitors to modulate NETosis can restore the balance of immune thrombosis. Therefore, this invention relates to the use of MCM inhibitors for the prevention and / or treatment of immune thrombosis-related disorders.

[0016] Therefore, the present invention relates to an inhibitor of at least one protein in an MCM complex for use in regulating (preferably inhibiting or downregulating) immune thrombosis and for use in treating diseases associated with vascular occlusion and immune thrombosis dysregulation, the inhibitor acting as a NETosis regulator (preferably an inhibitor).

[0017] Regulation can refer to the enhancement or suppression of a phenomenon. In this invention, it preferably refers to partial or complete suppression. Complete suppression of a phenomenon causes the phenomenon to be completely interrupted, while partial suppression slows down the development of the phenomenon.

[0018] For example, in the case of suppressing NETosis, complete suppression means stopping the development of NET, while partial suppression means reducing the development of NET.

[0019] According to a specific embodiment, the present invention relates to an inhibitor of at least one protein in an MCM complex for use in the prevention and / or treatment of NETosis-induced vascular occlusion.

[0020] The MCM complex is a hexameric circular protein structure composed of six proteins (MSM2 to MCM7, SEQ ID NOs 1 to 6), whose amino acid sequences contain an ATPase domain and an N-terminal DNA-binding domain (Fletcher et al., 2003). These proteins play an indispensable role in the initiation and regulation of the S phase of DNA replication (Maine et al., 1984). The MCM complex also participates in chromatin elongation and remodeling, enabling DNA to unwind correctly through its helicase activity (Bochman & Schwacha, 2009). The MCM complex is highly conserved in eukaryotes and is essential for genome stability. Due to its indispensable function, the MCM complex has attracted considerable research interest, particularly in the field of cancer. MCM proteins have been identified as biomarkers in various types of cancer (Gonzalez et al., 2005) and are associated with rapid cell proliferation and poor survival in some cancers (Söling et al., 2005; Sun et al., 2022).

[0021] Inhibitors of the MCM complex are compounds that can prevent the expression of at least one protein in the MCM complex or reduce the biological activity of the MCM complex.

[0022] The inhibition of the MCM complex can be assessed by measuring the following: - Decreased expression of at least one protein in the MCM; The expression of a biomarker protein can be assessed using any method known in the art. The term "expression" refers to protein translation, mRNA transcription, or mass spectrometry analysis.

[0023] Suitable methods for detecting proteins include any suitable methods for detecting and / or measuring proteins from cells or cell extracts. Such methods include, but are not limited to, staining and / or sorting using flow cytometry, polymerase chain reaction, immunoblotting (e.g., Western blotting), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunohistochemistry and immunofluorescence, and mass spectrometry. Particularly preferred methods for detecting proteins include any single-cell assay, including staining and / or sorting using flow cytometry, immunohistochemistry, immunofluorescence, and mass spectrometry. Such methods are well known in the art. Furthermore, antibodies against cell surface or intracellular proteins described herein are known in the art and described in published literature, and methods for generating antibodies that can be developed against these proteins are also well known in the art.

[0024] Methods suitable for detecting mRNA include any appropriate method for detecting and / or measuring mRNA levels from cells or cell extracts. Such methods include, but are not limited to: polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), etc. In situ Hybridization, northern imprinting, sequence analysis, gene microarray analysis (gene chip analysis), RNA sequencing, and reporter gene detection. Such methods for detecting transcriptional levels are well known in the art, and many of these methods are described in detail (…). https: / / www.elsevier.com / books / rna-methodologies / farrell-jr / 978-0-12-804678-4） Mass spectrometry is being used in clinical laboratories to identify and quantify biomolecules in a variety of biological samples (Birhanu, Clin Proteomics, 2023).

[0025] - Decreased enzyme activity of the MCM complex was determined by helicase and ATP hydrolysis assays, ATP hydrolysis reactions coupled with NADH as a substrate, FRET assays, electrophoretic mobility shift assays (EMSA), and molecular beacon-based helicase assays (MBHA).

[0026] Non-limiting examples of inhibitors of the MCM complex include antibodies, antigen-binding proteins, peptides (optionally having non-natural or modified amino acids), polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, or heterocyclic compounds. Inhibitors of the MCM complex also include genetically engineered systems for reducing the expression of the MCM complex, such as the CRISPR / Cas system, zinc finger nuclease (ZFN) system, transcription activator-like effector nuclease (TALEN) system, siRNA, shRNA, antisense oligonucleotides, or miRNA… Compounds can also be obtained from a wide variety of sources, including synthetic and natural compound libraries. Synthetic compound libraries are commercially available from companies such as Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimac, New Hampshire), and Microsource (New Milford, Connecticut). Rare chemical libraries are available from Aldrich Chemical Company, Inc. (Milwaukee, Wisconsin). Natural compound libraries (including bacterial, fungal, plant, or animal extracts) are available from companies such as Pan Laboratories (Bosell, Washington). Furthermore, numerous methods are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides.

[0027] In a specific implementation, the inhibitor of the MCM complex may be selected from: - Quinolones, preferably fluoroquinolones -Statins, - Helicase inhibitors - Bromo-terminal domain and superterminal domain (BET) protein inhibitors, -Based on small molecules of furanonaphthoquinones -Polo kinase 1 inhibitor, -Polyphenolic compounds, 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitor, -Isoflavones, - Cantharidin and its derivatives -CDC7 kinase inhibitor, -Histone acetylation inhibitors, and - Post-translational modifications of proteins, such as proteasome inhibitors (Samuel Wolf Buchsbaum, DOI: 10.1038 / sj.onc.1210314). Preferably, the inhibitor of the MCM complex can be selected from: - Quinolones, preferably fluoroquinolones - Helicase inhibitors - Bromo-terminal domain and superterminal domain (BET) protein inhibitors, -Based on small molecules of furanonaphthoquinones -Polo kinase 1 inhibitor, 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitor, - Cantharidin and its derivatives -CDC7 kinase inhibitors, and - Post-translational modifications of proteins, such as proteasome inhibitors.

[0028] More precisely, the inhibitors can be selected from ciprofloxacin and its derivatives (such as Szosteck). et al. The following are listed in 2022: ciprofloxacin derived from menthol and thymol, trogostatin, biguanide, heliquomycin, β-carboline-3-carboxylic acid N-methylamide (CMA), widdrol, scutellarin, genistein, lovastatin, metformin, AS4583 and its derivatives RJ-LC-07-48, and proteasome inhibitors.

[0029] In a preferred embodiment, the inhibitor of the MCM complex is a fluoroquinolone of formula (I): (I) in: R1 represents a hydrogen atom or NH2; R2 represents a hydrogen atom or a halogen atom such as F, Cl, Br and I, preferably, the halogen atom is F; R3 represents a hydrogen atom or a C1-C4 alkyl group; R4 represents a C1-C6 alkyl group, preferably a C1-C4 alkyl group, and more preferably a cyclopropyl group or an ethyl group; R5 represents a hydrogen atom or a C1-C4 alkyl group, preferably a hydrogen atom.

[0030] According to the preferred embodiment, the fluoroquinolone of formula (I) is ciprofloxacin:

[0031] Or compound ChemBridge 5281925:

[0032] NETosis-induced vascular occlusion is a disorder that can occur in a variety of diseases. Depending on the specific implementation, diseases associated with NETosis-induced vascular occlusion, including immune thrombotic disorders, are selected from: septic DIC; non-septic DIC; autoimmune diseases such as antiphospholipid syndrome or systemic lupus erythematosus (SLE); inflammatory diseases such as Crohn's disease or rheumatoid arthritis; myocardial infarction, stroke, venous thromboembolism, thrombosis-related diseases, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; vascular occlusion in sickle cell disease and vascular occlusion complications of sickle cell disease; viral diseases such as influenza and COVID-19; and immune thrombosis associated with cancer and sickle cell disease.

[0033] In a preferred embodiment, the disease involves immune thrombosis disorder, and is DIC, more preferably septic DIC.

[0034] According to another embodiment, an inhibitor of at least one protein in the MCM complex is associated with a compound that directly targets the coagulation pathway (such as heparin, antithrombin, thrombotic regulatory proteins and / or selective inhibitors of neutrophil elastase (such as cevelex sodium)).

[0035] The present invention also relates to a method for treating NETosis-induced vascular occlusion in subjects with such need, the method comprising administering at least one inhibitor of at least one protein in an MCM complex.

[0036] In immune thrombosis, coagulation activation must be understood as an indispensable innate immune defense mechanism; hemostasis constitutes the first line of defense against nonspecific host disease, and regulated activation of hypothrombin production can benefit the host's defense against pathogens. This is why only patients with immune thrombosis disorders need treatment for NETosis-induced coagulopathy, highlighting the importance of proper patient stratification beforehand (Helms et al., 2020).

[0037] Therefore, the present invention relates to an inhibitor of at least one protein in an MCM complex for use in regulating immune thrombosis and for treating diseases associated with vascular occlusion and immune thrombosis dysregulation, the inhibitor acting as a NETosis modulator, wherein the inhibitor is administered to a patient suffering from NETosis-related vascular occlusion.

[0038] As defined in more detail below, such patients with NETosis-induced coagulopathy may exhibit the following: - Clinical signs of thrombosis and / or bleeding; and / or -ISTH score equal to or greater than 5 and / or JAAM score equal to or greater than 4; and / or - Elevated neutrophil DNA decompression as measured by neutrophil lateral fluorescence (NEUT-SFL) recorded by an automated hematology counter (Delabranche et al., (2017), Shock 47:313-317); and / or - Increased expression or enhanced biological activity of at least one protein in the MCM complex, see the methods below.

[0039] To date, there is no gold standard or specific laboratory test for diagnosing immune thrombotic disorders, particularly disseminated intravascular coagulation (DIC). Therefore, various diagnostic scores have been developed that consider classic hemostatic parameters. However, a diagnosis of DIC must first be made based on an underlying pathology consistent with its occurrence (such as sepsis). The diagnosis is then based on a combination of clinical and biological factors.

[0040] The clinical signs of DIC can be identified by the presence of the following symptom patterns: - Signs of thrombosis: characterized by diffuse microthrombus formation leading to purpuric skin lesions that can progress to local ischemia and necrosis, particularly in the extremities, and organ dysfunction associated with microthrombi that leads to visceral perfusion failure.

[0041] - Hemorrhagic signs: These may include petechiae, hemorrhagic bullae, patchy bleeding from mucous membranes or puncture sites, hematuria, or melena. This bleeding is due to the depletion of clotting factors and fibrinogen, as well as thrombocytopenia. It is even more common in DIC with high fibrinolytic components. In certain cases of septic DIC, it remains rare and occurs late.

[0042] Biological factors used to diagnose disseminated intravascular coagulation (DIC) in sepsis or septic shock (platelet count or rate of decline, prothrombin, fibrinogen, and D-dimer levels) are typically used to calculate ISTH and Japanese Society of Critical Care Medicine (JAAM) scores. Based on these scores, a diagnosis of DIC can be made if the ISTH score is equal to or greater than 5 (Taylor et al., 2001), or if the JAAM-DIC score is equal to or greater than 4 (Iba et al., 2016).

[0043] However, these scores are rarely used in clinical practice, primarily due to their relative limitations. None of these scores can detect patients with asymptomatic DIC, yet these patients have similar severity and mortality rates to those diagnosed with DIC upon intensive care admission (Delabranche et al., 2016). The diagnostic performance of the scores varies depending on the initial pathology and the nature of the DIC (Helms et al., 2020). Furthermore, these scores cannot distinguish between thrombotic and hemorrhagic phenotypes in patients with DIC. The management of patients with DIC is not standardized but can depend on the stage of DIC progression (thrombotic, hemorrhagic, fibrinolytic), highlighting the importance of proper prior stratification of patients (Helms et al., 2020).

[0044] Therefore, it is necessary to elucidate new biomarkers that can guide clinicians in the clinical diagnosis and monitoring of immune thrombotic disorders.

[0045] Therefore, the present invention relates to a method for diagnosing NETosis-related vascular occlusion involving immune thrombotic dysregulation in a subject, the method comprising: - Determined in biological samples from the subject, preferably blood samples, more preferably neutrophils; -Measure the biological activity or expression level of at least one protein in the MCM complex; - Compare the said bioactivity or said expression level of at least one protein in the MCM complex with the baseline bioactivity or expression level established from at least one healthy donor or at least one patient without vascular occlusion; The biological activity or expression of at least one protein in the MCM complex above the baseline level in the subject identifies the subject as having or potentially developing NETosis-related vascular occlusion, including immune thrombosis dysregulation.

[0046] The term "subject" refers to any animal subject, and in particular any vertebrate mammal, including but not limited to primates, rodents, livestock, and domestic pets. Preferred mammals for the method of the present invention include humans.

[0047] NETosis-related vascular occlusion, including immune thrombosis dysregulation, can be selected from: septic DIC; non-septic DIC; autoimmune diseases such as antiphospholipid syndrome or systemic lupus erythematosus (SLE); inflammatory diseases such as Crohn's disease or rheumatoid arthritis; myocardial infarction, stroke, venous thromboembolism, thrombosis-related diseases, antineutrophil cytoplasmic antibody (ANCA)-related vasculitis; vascular occlusion in sickle cell disease and vascular occlusion complications of sickle cell disease; viral diseases such as influenza and COVID-19; and cancer-related immune thrombosis.

[0048] In certain embodiments, the method of the present invention is combined with the following: - Observe for signs of thrombosis, characterized by diffuse microthrombus formation leading to purpuric skin lesions that can progress to local ischemia and necrosis, particularly in the extremities, and organ dysfunction associated with microthrombi leading to visceral perfusion failure; and / or -Observe for hemorrhagic clinical signs, which may include petechiae, hemorrhagic bullae, patchy bleeding from mucosa or puncture sites, hematuria, or melena; and / or - Assess ISTH and / or Japanese Society of Critical Care Medicine (JAAM) scores; and / or - Elevated neutrophil DNA decompression (Delabranche X, Stiel L, Severac F) was measured using neutrophil lateral fluorescence (NEUT-SFL) recorded by an automated hematology counter. et al. (2017), “Evidence of Netosis in Septic Shock-Induced Disseminated Intravascular Coagulation” (Shock 47:313-317).

[0049] Advantageously, the method of the present invention allows for the differentiation between DIC patients with sepsis or septic shock and non-DIC patients.

[0050] Healthy donors are individuals who have not yet been diagnosed with any disease. Preferably, a baseline level is established using at least 10 healthy donor samples, based on at least one healthy donor sample.

[0051] Each of MCM2, MCM3, MCM4, MCM5, MCM6 and / or MCM7 can be used as a biomarker in the method of the present invention.

[0052] The expression of a biomarker protein can be assessed using any method known in the art. The term "expression" refers to protein translation, mRNA transcription, or mass spectrometry analysis.

[0053] Methods applicable to the detection of proteins and mRNA are described above.

[0054] This invention also relates to the bioactivity or expression level of at least one protein in the MCM complex as a biomarker for diagnosing NETosis-related vascular occlusion. in vitro or In vitro use.

[0055] MCM helicase activity can be measured using helicase and ATP hydrolysis assays, ATP hydrolysis reactions coupled with NADH as a substrate, FRET assays, electrophoretic mobility shift assays (EMSA), and molecular beacon-based helicase assays (MBHA) (https: / / www.bmglabtech.com / en / application-notes / molecular-beacon-based-helicase-assays / ).

[0056] The present invention includes a kit for diagnosing NETosis-related vascular occlusion, the kit comprising at least one reagent for determining the bioactivity or expression level of at least one protein in the MCM complex in neutrophils.

[0057] The reagent may be, for example, a probe that hybridizes with a nucleic acid molecule encoding a marker protein under strict hybridization conditions; RT-PCR primers for amplifying mRNA or fragments thereof encoding the marker protein; and / or an antibody, its antigen-binding fragment, or other antigen-binding peptide that selectively binds to the marker protein.

[0058] The reagents in the kits of this invention can be conjugated with detectable tags or detectable labels. Such tags can be any suitable tags that allow detection of the reagents, and include, but are not limited to, any composition or label that can be detected by spectroscopic analysis, photochemical analysis, biochemical analysis, immunochemical analysis, electrical analysis, optical analysis, mass spectrometry (including multiplex, or parallel, or selected reaction monitoring (MRM / PRM / SRM)) (Lange V et al., “Selected reaction monitoring for quantitative proteomics: a tutorial.” Molecular Systems Biology, 2008;4(1):222.), or chemical means. Labels available in this invention include: biotin for staining with labeled streptavidin conjugates, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, etc.), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. In the case of sample preparation kits for mass spectrometry analysis, the kit may contain labeled lysine or arginine or other amino acids, or immunoprecipitation (including MRM / SRM / PRM) for LC-MS / MS.

[0059] Furthermore, the reagents in the kit can be immobilized on a substrate. Such substrates can include any suitable substrate for immobilizing the detection reagents, such as the substrates used in any of the detection methods described previously. In short, substrates suitable for immobilizing detection methods include any solid support (such as any solid organic support, biopolymer support, or inorganic support) that can form bonds with the detection method without significantly affecting the activity and / or ability of the detection method to detect the desired target molecule.

[0060] Exemplary organic solid carriers include polymers such as polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers (e.g., polyacrylamide), stabilized whole cells, and stabilized coarse whole cell / membrane homogenates.

[0061] Exemplary biopolymer carriers include cellulose, polydextran (e.g., Sephadex®), agarose, collagen, and chitin.

[0062] Exemplary inorganic carriers include glass beads (porous and non-porous), stainless steel, metal oxides (e.g., porous ceramics such as ZrO2, TiO2, Al2O3, and NiO), and sand.

[0063] Other existing kits include dry, pre-formulated antibody combinations for detecting rare events, immune function analysis, immune system research, and certain clinical applications, such as the DURAClone combination from Beckman Coulter.

[0064] According to another objective, the present invention relates to a method for evaluating the efficacy of a treatment for NETosis-related vascular occlusion in animal models or patients, the method comprising: - Prior to treatment, determine the bioactivity or expression of at least one protein in the MCM complex in biological samples obtained from animal models or patients, preferably blood samples, and more preferably neutrophils; - Following treatment administration, determine the expression of at least one protein in the MCM complex in neutrophils obtained from the patient; and - Compare the expression levels.

[0065] A decrease in the biological activity or expression level of a biomarker protein indicates that the treatment is effective. Attached Figure Description

[0066] Figure 1 Unstimulated neutrophils (A), with Escherichia coli ( Escherichia coli Representative immunofluorescence images of neutrophils stimulated with PMA (B) or neutrophils stimulated with PMA (C): Neutrophils were labeled with anti-myeloperoxidase antibody (see inset MPO) and anti-MCM2 antibody (see inset MCM2), and counterstained with DNA-labeled Hoechst (see inset Hoechst), original magnification x63. NETs were identified as extended DNA fibers associated with myeloperoxidase.

[0067] Figure 2 Based on DNA quantity, unstimulated neutrophils (black), using PMA or E. coli ( Escherichia coli Neutrophils stimulated (light gray), with PMA or E. coli ( Escherichia coli Neutrophils (dark gray) stimulated with 250 µM ciprofloxacin were used for NETosis quantification. A standard curve with known concentrations of fibrillary DNA and human neutrophil elastase allowed for the quantification of NETs in the sample.

[0068] Figure 3Ciprofloxacin in in vitro Dose-dependent inhibition of NETosis. (A) White light immunofluorescence image of neutrophils labeled with Hoechst, initial magnification x20. (B) Quantification of the dose-dependent inhibition of NETosis by ciprofloxacin.

[0069] Figure 4 : Using Escherichia coli ( Escherichia coli Representative immunofluorescence images of neutrophils stimulated (A) or PMA (B) and treated with ciprofloxacin: Neutrophils were labeled with anti-myeloperoxidase antibody (see inset MPO) and anti-MCM2 antibody (see inset MCM2) and counterstained with DNA-labeled Hoechst at x63 magnification.

[0070] Figure 5 Measurement of fluorescence intensity of FITC-labeled Escherichia coli on unstimulated neutrophils (A), neutrophils stimulated with PMA (B), and neutrophils stimulated with PMA and treated with 250 µM ciprofloxacin (C): Control tubes (black) were incubated on ice, while phagocytosis test tubes (gray) were incubated at 37 °C for 30 minutes.

[0071] Figure 6 Plasmin formation at different uPA doses (0–2–3–4, 5 nM) in unstimulated neutrophils (black), neutrophils stimulated with Escherichia coli or PMA (light gray), and neutrophils stimulated with PMA or Escherichia coli and treated with ciprofloxacin (dark gray). Plasmin formation from 0.5 nM plasminogen was monitored by measuring the release of a plasmin-selective chromogenic substrate from 0.75 mM pNA over 2 hours at 405 nM. Vi = initial reaction rate.

[0072] Example Materials and Methods patient This study included twenty patients with septic shock admitted to the Internal Medicine Intensive Care Unit of Strasbourg's Nouvel Hôpital Civil, nine of whom developed disseminated intravascular coagulation (DIC). Septic shock was diagnosed according to the latest international consensus definition (Singer et al., 2016), and DIC was further defined as a JAAM-DIC 2016 score of at least 4 within the first 24 hours following the onset of septic shock (Iba et al., 2016).

[0073] Blood sampling Ten milliliters of blood were drawn from BD Vacutainer Plus blood collection tubes (Becton Dickinson, Le Pondeclare, France) containing the anticoagulant EDTA (ethylenediaminetetraacetic acid) at admission, within 12 hours of diagnosis of septic shock, on day 3, and on day 7. Five milliliters of whole blood were also collected in a citrate-containing tube for routine hemostasis testing used to diagnose DIC.

[0074] Neutrophil isolation At each time point, neutrophils were isolated by negative immunomagnetic selection using two EDTA tubes with a commercial EasySep® kit (#19666, Stemcell Technologies, Canada) (Peters et al., 2005). Magnetic beads bound to antibodies specifically targeting surface proteins of all circulating hematopoietic cells except neutrophils were added to whole blood. The tube containing whole blood and beads was then placed on a magnet to separate labeled and unlabeled cells. Once the negative fraction was recovered, the collected neutrophils were washed twice with phosphate-buffered saline (PBS, Euromedex, France) to remove all plasma proteins. The cell suspension was examined using an automated fluorescence cytometer (Sysmex™ XN20® analyzer, Sysmex Corporation, Kobe, Japan) to count neutrophils and check their purity. The mean purity was 96.36% ± 0.363. 1,10 6 The neutrophils were aliquoted into dedicated Ependorf tubes in the form of dried pellets and stored at -80°C.

[0075] Proteomics analysis Proteins from isolated neutrophils were extracted into Laemmli buffer, and 7.5 µg of protein was concentrated into the stacking band of a 1D SDS-PAGE gel. Cysteine ​​reduction was then performed to break disulfide bonds, alkylation was performed to prevent disulfide bond reformation, and enzymatic digestion with trypsin solution was conducted to obtain peptides for mass spectrometry analysis. Analysis was performed in data-dependent acquisition mode on a nanoACQUITY UltraPerformance LC system (Waters Corporation, Milford, USA) coupled to a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, MASS). Label-free quantification was performed using MaxQuant software (v. 1.6.0.16, Max Planck Institute for Biochemistry, Martinsrid, Germany (Cox et al., 2014)). The STRING v.12 software (Szklarczyk et al., 2023) was used to perform gene ontology (GO) term enrichment analysis on differentially abundant proteins and protein networks.

[0076] Protein blot Proteins were extracted from patient neutrophils using RIPA lysis buffer (#10017003, Thermo Fisher Scientific, Waltham, USA) supplemented with protease inhibitors and phosphatase inhibitors (#04693116001 and #4906845001, Roche, Switzerland). Proteins (25 g) were separated by 12% SDS-PAGE gel (#161075, Biorad, USA) at 110 V for 2 hours, then transferred by electrophoresis to a nitrocellulose membrane (#IB23002, Thermo Fisher Scientific, USA). Non-specific binding sites on the membrane were blocked for 1 hour at room temperature with 5% bovine serum albumin (BSA, Euromédex, France) diluted in Tris saline buffer (TBS, Euromédex, France). Proteins of interest were labeled with specific primary antibodies and incubated overnight at 4°C. These antibodies were diluted in a 1 / 1000 blocking solution: rabbit monoclonal antibody PTX3 (ab125007), mouse monoclonal antibody PAD4 (ab128086), rabbit monoclonal antibody MMP9 (ab38898), and mouse monoclonal antibody NGAL (ab23477) (all from Abcam). The membrane was washed three times with TBS-T (TBS containing 0.1% Tween-20) and incubated for 1 h at room temperature with oxidase-labeled secondary antibodies (anti-rabbit and anti-mouse (#7074S and #7076S, Cell Signaling Technology, USA)). Immunochromatography was performed using chemiluminescent solutions. Chemiluminescence signals were recorded using an iBright CL100 and analyzed using ImageStudioLive software (V.5.2.5, Li-CORBiosciences, USA). Quantitative normalization relative to a GAPDH reference protein was performed for each protein of interest using the corresponding specific antibody (mouse monoclonal anti-GAPDH, ab8245).

[0077] In vitro induction and inhibition of NETosis In order to in vitro NETosis was induced by isolating neutrophils from healthy volunteers as previously described. After separation from other whole blood cells, the neutrophils were suspended in Hanks' Balanced Salt Solution buffer (HBSS, #P04-32505, Dominic Dutscher, France) at a concentration allowing distribution of 400,000 cells / well. Immunofluorescence assays were performed in 24-well plates with glass coverslips at the bottom, or plasminogenesis assays were performed in 96-well plates.

[0078] To induce NETosis, neutrophils were placed in Escherichia coli strains specifically selected for their resistance to fluoroquinolones. Escherichia coli The bacteria were incubated for 4 h at 37 °C and 5% CO2 in the presence of strain (MOI 100). These bacteria used protein kinase C (PKC) activation pathway to induce NET formation (Kenny et al., 2017), as did phorbol 12-myristate 13-acetate (PMA) (PMA was used as a positive control). Neutrophils were stimulated with 50 nM PMA (#P1585, Sigma-Aldrich, USA) under the same conditions as the bacteria.

[0079] In addition, under certain conditions, neutrophils are incubated with bacteria or PMA and simultaneously treated with ciprofloxacin (#17850, Merck, Germany).

[0080] Immunofluorescence Following stimulation, neutrophils were fixed onto slides for 30 minutes using 4% paraformaldehyde solution (PFA, #J19943-K2, Thermo Fisher Scientific, USA), followed by three washes with PBS. Once permeabilized with 0.2% Tween 20 solution for 15 minutes, the PNN antigen sites were blocked with 10% BSA solution for 3 hours. Cells were incubated with specific primary antibodies: rabbit anti-neutrophil elastase antibody (#481001, 1 / 1000, Merck, Germany) or anti-myeloperoxidase antibody (#MA-180878, 1 / 400, Thermo Fisher Scientific, USA), and rabbit anti-MCM2 antibody (#3619S, 1 / 500, Ccytok, USA), overnight at 4°C. After three washes, the cells were incubated in the dark for 1 hour at room temperature with appropriate secondary antibodies: Alexa Fluor 488 goat anti-mouse antibody and Alexa Fluor 647 goat anti-rabbit antibody (#A11001 and #A21245, respectively, Thermo Fisher Scientific, USA). After a further three washes, slides were counterstained with Hoechst 33342 solution for DNA labeling (NucBlue™ Live ReadyProbes, #R37605, Thermo Fisher Scientific, USA) and then mounted under coverslips with Prolong Gold mounting medium (#P10144). Immunofluorescence images were acquired using a Zeiss Axio Imager M2 Apotome microscope (PIC-STRA, CRBS, Strasbourg, France) and analyzed using ImageJ software.

[0081] In vitro measurement of NETosis Following stimulation with PMA or PMA and ciprofloxacin, NET in neutrophil suspensions was measured using a homemade capture ELISA developed by Dr. Angles-Cano. Briefly, 1 µg / mL of biotinylated anti-human neutrophil elastase (hNE) ELISA monoclonal antibody (Enzo Life Sciences, Lyon, France) was added to each well of a streptavidin-coated 96-well plate (Roche, Paris, France). After three washes, samples (50 µL / well) were dispensed into each well. After incubation at 37°C for 1 h, the plate was washed (3 times) and 50 µL of horseradish peroxidase-labeled anti-DNA monoclonal antibody (Cell DeathDetection ELISAPLUS kit) was added to each well, followed by another 1 h incubation at room temperature on a shaking device. After three washes, peroxidase substrate was added. Absorbance was measured at A405 nm for 1 hour using a microplate counter (VERSAmax microplate reader, Molecular Devices). The amount of NET was calculated based on a standard range of NET equivalents formed by fibrillary DNA coupled with neutrophil elastase.

[0082] Measurement of neutrophil phagocytic activity Neutrophils were stimulated with 50 nM PMA in 5 mL tubes and treated with ciprofloxacin. After 4 hours of stimulation, the tubes were centrifuged at 800 g for 15 minutes, and the neutrophils and NETs were resuspended in plasma from heparinized blood samples from healthy volunteers. FITC-labeled, conditioned E. coli (…) Escherichia coli Bacteria (Reagent B, PHAGOTEST™, Glycotope Biotechnology, Germany) were added to all tubes. Control tubes were incubated on ice, while test tubes were incubated at 37°C for 30 minutes. After adding blocking solution and performing three washes, flow cytometry analysis was performed to measure the relationship between the number of bacteria phagocytosed by each neutrophil and the mean FITC fluorescence intensity (MFI).

[0083] In vitro plasminogen activator Neutrophils were divided into 4 x 10 5 The concentration was inoculated into 96-well plates, and then E. coli ( Escherichia coliStimulation with either β-hydroxyprolyl arachidonic acid (PMA) or β-aminopropyl arachidonic acid (PPA) followed by ciprofloxacin treatment, as previously described. Four hours after stimulation, the plate was centrifuged at 800g for 15 minutes to allow removal of the supernatant and allow NETs to adhere to the bottom of the plate. NETosis was confirmed by visualizing the NETs with Hoechst staining and detecting neutrophil elastase activity. Next, Therasolv® urokinase was incubated on the NETs at four concentrations (0, 2, 3, 4.5 nM) at 37°C and 5% CO2 for 1 hour to allow binding to urokinase-type plasminogen activator receptor (uPAR). Following the centrifugation step to remove excess uPA, a solution containing plasminogen (0.5 µM) was added to trigger plasminogenesis. The kinetics of plasminogen to plasmin conversion were monitored for 2 hours by measuring the release of methyl(malonyl)hydroxy-prolyl-arginine-p-nitrobenzamide 169-135 (pNA) at 405 nm using a microplate reader. A specially designed computer program is used to calculate the initial velocity (Vi) of the reaction.

[0084] Statistical data Data expressed as mean ± standard deviation were analyzed using GraphPad Prism8® software (GraphPad Software, Inv., California, USA). “n” represents the number of patients or the number of trials. Means were compared using the Mann-Whitney test or the Kruskall-Wallis test. For plasminogen retardation, statistical variance between groups was determined by applying a two-way ANOVA test. A p-value < 0.05 was considered statistically significant. All measurements were performed independently at least three times.

[0085] result Patient characteristics This study included twenty patients, all of whom met the criteria for septic shock upon admission to the ICU. Nine patients were identified as having disseminated intravascular coagulation (DIC) based on a positive ISTH score (≥5).

[0086] Patients with septic shock and DIC have specific protein characteristics.

[0087] Proteomics analysis revealed key differences among the three groups at admission. In fact, the meta-analysis identified and quantified 1813 proteins in at least 70% of the samples, allowing us to categorize the samples into three groups, roughly corresponding to the three groups in our study: septic shock without DIC, septic shock with DIC, and controls. This unsupervised analysis confirmed that neutrophils exhibited different protein signatures depending on the diagnosis. On day 1, 224 proteins showed differential expression in patients with DIC compared to those without. Of these proteins, 115 were more abundant in neutrophils from septic shock patients with DIC, and 110 were less abundant.

[0088] Inhibition of MCM complex activity blocked NETosis in in vitro models of bacterial and non-bacterial stimulation.

[0089] Patients with disseminated intravascular coagulation (DIC) associated with septic shock are characterized by more severe NETosis than patients without DIC, and higher expression of MCM complex proteins was observed in neutrophils from patients with DIC. The MCM complex plays an indispensable role in DNA replication in eukaryotic cells. However, neutrophils are hematopoietic cells in the late stage of differentiation and do not divide. Therefore, we hypothesize that the expression of the MCM complex in neutrophils from DIC patients reflects the induction of NETosis in these patients.

[0090] Using E. coli ( Escherichia coli Four hours after stimulation, healthy neutrophils released NETs, ​​which were visualized using fluorescence microscopy. NETs were identified by their DNA chromatin structure associated with antimicrobial myeloperoxidase granules. Figure 1 B); These structures are similar to those observed after PMA stimulation ( Figure 1 C). MCM2 protein is present in the cytoplasm of unstimulated neutrophils ( Figure 1 A). Once infected with E. coli ( Escherichia coli ) Stimulation leads to the formation of NET ( Figure 1 B), the MCM2 protein remains uniformly expressed in the cytoplasm of activated neutrophils and is present on DNA filaments. Upon PMA stimulation, MCM2 localizes near the plasma membrane of neutrophils and on NETs (…). Figure 1 C).

[0091] To confirm our hypothesis regarding the role of the MCM complex in NETosis, the effect of one known inhibitor of the MCM2-7 complex (ciprofloxacin) on MCM complex inhibition was evaluated. Compared to unstimulated neutrophils, NETs were significantly increased in neutrophils stimulated by *Escherichia coli* and PMA (p<0.05). Figure 2 Treatment with ciprofloxacin significantly reduced NET formation (p<0.05). Figure 2 Therefore, after 4 hours of stimulation with PMA, the results showed that NETose was inhibited by ciprofloxacin, and this response appeared to be dose-dependent. Figure 3 ).

[0092] In addition, it is made from Escherichia coli (E. coli) Escherichia coli NETosis induced by fluoroquinolone-resistant strains (including ciprofloxacin) was also inhibited by 250 µM ciprofloxacin. Figure 4 A). These results are similar to those observed using PMA ( Figure 4 B). These neutrophils exhibit multilobed nuclei, with MPO present in the cytoplasm. The distribution of MCM2 protein in the cytoplasm is also uniform.

[0093] Our results indicate that inhibition of the MCM complex with ciprofloxacin can block NETosis induced by bacterial or non-bacterial stimuli. This inhibition opens a promising avenue for the partial suppression of NETosis and, consequently, immune thrombosis.

[0094] Inhibition of the MCM complex preserves neutrophil function.

[0095] Neutrophil function is impaired during disseminated intravascular coagulation (DIC). We investigated phagocytosis and fibrinolysis in neutrophils stimulated with and without MCM complex inhibitors.

[0096] After the MCM complex was inhibited, the phagocytic capacity of the stimulated neutrophils remained intact. Neutrophils play an indispensable role in the phagocytosis of pathogenic microorganisms. To determine whether this function is preserved in activated neutrophils whose MCM complex is inhibited by ciprofloxacin, we assessed their phagocytic capacity. Healthy neutrophils stimulated with PMA and treated with ciprofloxacin for 4 h were exposed to *E. coli* bacteria conjugated with the fluorescent dye FITC. After incubation at 37°C for 30 min, the FITC fluorescence intensity was measured by flow cytometry, reflecting the number of bacteria phagocytosed by neutrophils. To assess the phagocytic capacity of neutrophils, control tubes were used, subjected to the same experimental conditions but incubated on ice. Compared to the control tubes (incubated on ice with bacteria), unstimulated neutrophils in the test tubes (incubated at 37°C with bacteria) showed a significantly increased phagocytic activity towards FITC-conjugated bacteria. Figure 5 A, p<0.05. No change in neutrophil fluorescence intensity was observed after stimulation with the NETose inducer PMA, confirming that neutrophils lost their phagocytic capacity after this stimulation. Figure 5 B). However, in the presence of ciprofloxacin, phagocytic activity of PMA-activated neutrophils was observed as a control. Figure 5 C, p<0.01).

[0097] Therefore, these results indicate that ciprofloxacin inhibition of the MCM complex restores the phagocytic capacity of neutrophils after PMA stimulation.

[0098] The ability of neutrophils to produce plasmin is preserved after inhibition by the MCM complex.

[0099] Patients with septic shock and disseminated intravascular coagulation (DIC) exhibit a reduced ability to produce plasmin. This reduction may be associated with the NETosis phenomenon, as the DNA filaments of NETs contain elastase, which cleaves plasminogen into fragments, thereby impairing its ability to produce plasmin (Cruz et al., 2019; Delabranche et al., 2017). This mechanism leads to the hypofibrinolysis and microthrombi observed in DIC.

[0100] It can be done in vitro To assess plasmin production, this study involved depositing plasminogen ester and the chromogenic substrate pNA onto neutrophils previously incubated with escalating doses of urokinase. When plasminogen ester was exposed to uPA, it was converted to plasmin, which then catalyzed the conversion of p-nitrobenamide to p-nitroaniline, emitting a yellow light detectable by a spectrophotometer. This assay was used to monitor plasmin production for two hours.

[0101] Unstimulated neutrophils produce plasmin in proportion to their urokinase concentration. In contrast, when using Escherichia coli (… Escherichia coli When neutrophils are stimulated by PMA, the production of plasmin is lower. Figure 6 A, Escherichia coli ( E. coli Compared with the unstimulated group, p<0.01; Figure 6 B, PMA compared to the unstimulated group, p<0.0001). Interestingly, the presence of ciprofloxacin restored plasmin production ( Figure 6 A, Escherichia coli ( E. coli ) + Ciprofloxacin and Escherichia coli ( E. coli Compared to ), p < 0.0001; Figure 6 B, PMA + ciprofloxacin compared to PMA, p<0.001).

[0102] Therefore, these results suggest that the inhibition of the MCM complex by ciprofloxacin preserves the role of neutrophils in plasmin production, which may help improve hypofibrinolysis and actively regulate immune thrombosis.

[0103] Therefore, it has been demonstrated that inhibiting the MCM complex preserves phagocytic function and does not interfere with plasmin production in neutrophils stimulated by NETosis inducers. Thus, neutrophils participate in eliminating microbes and disrupting microthrombi by degrading fibrin clots.

[0104] sequence list SEQ. ID. N°1–Human MCM2 MAESSESFTMASSPAQRRRGNDPLTSSPGRSSRRTDALTSSPGRDLPPFEDESEGLLGTEGPLEEEEDGEELIGDGMERDYRAIPELDAYEAEGLALDDEDVEELTASQREAAERAMRQRDREAGRGLGRMRRGLLYDSDEEDEERPARKRRQVERATEDGEEDEEMIESIENLEDLKGHSVREWVSMAGPRLEIHHRFKNFLRTHVDSHGHNVFKERISDMCKENRESLVVNYEDLAAREHVLAYFLPEAPAELLQIFDEAALEVVLAMYPKYDRITNHIHVRISHLPLVEELRSLRQLHLNQLIRTSGVVTSCTGVLPQLSMVKYNCNKCNFVLGPFCQSQNQEVKPGSCPECQSAGPFEVNMEETIYQNYQRIRIQESPGKVAAGRLPRSKDAILLADLVDSCKPGDEIELTGIYHNNYDGSLNTANGFPVFATVILANHVAKKDNKVAVGELTDEDVKMITSLSKDQQIGEKIFASIAPSIYGHEDIKRGLALALFGGEPKNPGGKHKVRGDINVLLCGDPGTAKSQFLKYIEKVSSRAIFTTGQGASAVGLTAYVQRHPVSREWTLEAGALVLADRGVCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTSLQARCTVIAAANPIGGRYDPSLTFSENVDLTEPIISRFDILCVVRDTVDPVQDEMLARFVVGSHVRHHPSNKEEEGLANGSAAEPAMPNTYGVEPLPQEVLKKYIIYAKERVHPKLNQMDQDKVAKMYSDLRKESMATGSIPITVRHIESMIRMAEAHARIHLRDYVIEDDVNMAIRVMLESFIDTQKFSVMRSMRKTFARYLSFRRDNNELLLFILKQLVAEQVTYQRNRFGAQQDTIEVPEKDLVDKARQINIHNLSAFYDSELFRMNKFSHDLKRKMILQQF SEQ. ID. No. 2 – Human MCM3 MAGTVVLDDVELREAQRDYLDFLDDEEDQGIYQSKVRELISDNQYRLIVNVNDLRRKNEKRANRLLNNAFEELVAFQRALKDFVASIDATYAKQYEEFYVGLEGSFGSKHVSPRTLTSCFLSCVVCVEGIVTKCSLVRPKVVRSVHYCPATKKTIERRYSDLTTLVAFPSSSVYPTKDEENNPLETEYGLSVYKDHQTITIQEMPEKAPAGQLPRSVDVILDDDLVDKAKPGDRVQVVGTYRCLPGKKGGYTSGTFRTVLIACNVKQMSKDAQPSFSAEDIAKIKKFSKTRSKDIFDQLAKSLAPSIHGHDYVKKAILCLLLGGVERDLENGSHIRGDINILLIGDPSVAKSQLLRYVLCTAPRAIPTTGRGSSGVGLTAAVTTDQETGERRLEAGAMVLADRGVVCIDEFDKMSDMDRTAIHEVMEQGRVTIAKAGIHARLNARCSVLAAANPVYGRYDQYKTPMENIGLQDSLLSRFDLLFIMLDQMDPEQDREISDHVLRMHRYRAPGEQDGDAMPLGSAVDILATDDPNFSQEDQQDTQIYEKHDNLLHGTKKKKEKMVSAAFMKKYIHVAKIIKPVLTQESATYIAEEYSRLRSQDSMSSDTARTSPVTARTLETLIRLATAHAKARMSKTVDLQDAEEAVELVQYAYFKKVLEKEKKRKKRSEDESETEDEEEKSQEDQEQKRKRRKTRQPDAKDGDSYDPYDFSDTEEEMPQVHTPKTADSQETKESQKVELSESRLKAFKVALLDVFREAHAQSIGMNRLTESINRDSEEPFSSVEIQAALSKMQDDNQVMVSEGIIFLI SEQ. ID. No. 3 – Human MCM4 MSSPASTPSRRGSRRGRATPAQTPRSEDARSSPSQRRRGEDSTSTGELQPMPTSPGVDLQSPAAQDVLFSSPPQMHSSAIPLDFDVSSPLTYGTPSSRVEGTPRSGVRGTPVRQRPDLGSAQKGLQVDLQSDGAAAEDIVASEQSLGQKLVIWGTDVNVAACKENFQRFLQRFIDPLAKEEENVGIDITEPLYMQRLGEINVIGEPFLNVNCEHIKSFDKNLYRQLISYPQEVIPTFDMAVNEIFFDRYPDSILEHQIQVRPFNALKTKNMRNLNPEDIDQLITISGMVIRTSQLIPEMQEAFFQCQVCAHTTRVEMDRGRIAEPSVCGRCHTTHSMALIHNRSLFSDKQMIKLQESPEDMPAGQTPHTVILFAHNDLVDKVQPGDRVNVTGIYRAVPIRVNPRVSNVKSVYKTHIDVIHYRKTDAKRLHGLDEEAEQKLFSEKRVELLKELSRKPDIYERLASALAPSIYEHEDIKKGILLQLFGGTRKDFSHTGRGKFRAEINILLCGDPGTSKSQLLQYVYNLVPRGQYTSGKGSSAVGLTAYVMKDPETRQLVLQTGALVLSDNGICCIDEFDKMNESTRSVLHEVMEQQTLSIAKAGIICQLNARTSVLAAANPIESQWNPKKTTIENIQLPHTLLSRFDLIFLLLDPQDEAYDRRLAHHLVALYYQSEEQAEEELLDMAVLKDYIAYAHSTIMPRLSEEASQALIEAYVDMRKIGSSRGMVSAYPRQLESLIRLAEAHAKVRLSNKVEAIDVEEAKRLHREALKQSATDPRTGIVDISILTTGMSATSRKRKEELAEALKKLILSKGKTPALKYQQLFEDIRGQSDIAITKDMFEEALRALADDDFLTVTGKTVRLL SEQ. ID. No. 4 – Human MCM5 MSGFDDPGIFYSDSFGGDAQADEGQARKSQLQRRFKEFLRQYRVGTDRTGFTFKYRDELKRHYNLGEYWIEVEMEDLASFDEDLADYLYKQPAEHLQLLEEAAKEVADEVTRPRPSGEEVLQDIQVMLKSDASPSSIRSLKSDMMSHLVKIPGIIIAASAVRAKATRISIQCRSCRNTLTNIAMRPGLEGYALPRKCNTDQAGRPKCPLDPYFIMPDKCKCVDFQTLKLQELPDAVPHGEMPRHMQLYCDRYLCDKVVPGNRVTIMGIYSIKKFGLTTSRGRDRVGVGIRSSYIRVLGIQVDTDGSGRSFAGAVSPQEEEEFRRLAALPNVYEVISKSIAPSIFGGTDMKKAIACLLFGGSRKRLPDGLTRRGDINLLMLGDPGTAKSQLLKFVEKCSPIGVYTSGKGSSAAGLTASVMRDPSSRNFIMEGGAMVLADGGVVCIDEFDKMREDDRVAIHEAMEQQTISIAKAGITTTLNSRCSVLAAANSVFGRWDETKGEDNIDFMPTILSRFDMIFIVKDEHNEERDVMLAKHVITLHVSALTQTQAVEGEIDLAKLKKFIAYCRVKCGPRLSAEAAEKLKNRYIIMRSGARQHERDSDRRSSIPITVRQLEAIVRIAEALSKMKLQPFATEADVEEALRLFQVSTLDAALSGTLSGVEGFTSQEDQEMLSRIEKQLKRRFAIGSQVSEHSIIKDFTKQKYPEHAIHKVLQLMLRRGEIQHRMQRKVLYRLK SEQ. ID. NO. 5 – Human MCM6 MDLAAAAEPGAGSQHLEVRDEVAEKCQKLFLDFLEEFQSSDGEIKYLQLAEELIRPERNTLVVSFVDLEQFNQQLSTTIQEEFYRVYPYLCRALKTFVKDRKEIPLAKDFYVAFQDLPTRHKIRELTSSRIGLLTRISGQVVRTHPVHPELVSGTFLCLDCQTVIRDVEQQFKYTQPNICRNPVCANRRRFLLDTNKSRFVDFQKVRIQETQAELPRGSIPRSLEVILRAEAVESAQAGDKCDFTGTLIVVPDVSKLSTPGARAETNSRVSGVDGYETEGIRGLRALGVRDLSYRLVFLACCVAPTNPRFGGKELRDEEQTAESIKNQMTVKEWEKVFEMSQDKNLYHNLCTSLFPTIHGNDEVKRGVLLMLFGGVPKTTGEGTSLRGDINVCIVGDPSTAKSQFLKHVEEFSPRAVYTSGKASSAAGLTAAVVRDEESHEFVIEAGALMLADNGVCCIDEFDKMDVRDQVAIHEAMEQQTISITKAGVKATLNARTSILAAANPISGHYDRSKSLKQNINLSAPIMSRFDLFFILVDECNEVTDYAIARRIVDLHSRIEESIDRVYSLDDIRRYLLFARQFKPKISKESEDFIVEQYKHLRQRDGSGVTKSSWRITVRQLESMIRLSEAMARMHCCDEVQPKHVKEAFRLLNKSIIRVETPDVNLDQEEEIQMEVDEGAGGINGHADSPAPVNGINGYNEDINQESAPKASLRLGFSEYCRISNLIVLHLRKVEEEEDESALKRSELVNWYLKEIESEIDSEEELINKKRIIEKVIHRLTHYDHVLIELTQAGLKGSTEGSESYEEDPYLVVNPNYLLED SEQ. ID. No. 6 – Human MCM7 MALKDYALEKEKVKKFLQEFYQDDELGKKQFKYGNQLVRLAHREQVALYVDLDDVAEDDPELVDSICENARRYAKLFADAVQELLPQYKEREVVNKDVLDVYIEHRLMMEQRSRDPGMVRSPQNQYPAELMRRFELYFQGPSSNKPRVIREVRADSVGKLVTVRGIVTRVSEVKPKMVVATYTCDQCGAETYQPIQSPTFMPLIMCPSQECQTNRSGGRLYLQTRGSRFIKFQEMKMQEHSDQVPVGNIPRSITVLVEGENTRIAQPGDHVSVTGIFLPILRTGFRQVVQGLLSETYLEAHRIVKMNKSEDDESGAGELTREELRQIAEEDFYEKLAASIAPEIYGHEDVKKALLLLLV GGVDQSPRGMKIRGNINICLMGDPGVAKSQLLSYIDRLAPRSQYTTGRGSSGVGLTAAVLRDSVSGELLEGGALVLADQGVCCIDEFDKMAEADRTAIHEVMEQQTISIAKAGILTTLNARCSILAAANPAYGRYNPRRSLEQNIQLPAALLSRFDLLWLIQDRPDRDNDLRLAQHITY VHQHSRQPPSQFEPLDMKLMRRYIAMCREKQPMVPESLADYITAAYVEMRREAWASKDATYTSARTLLAILRLSTALARLRMVDVVEKEDVNEAIRLMEMSKDSLLGDKGQTARTQRPADVIFATVRELVSGGRSVRFSEAEQRCVSRGFTPAQFQAALDEYELNVWQVNASRTRITFV

Claims

1. An inhibitor of at least one protein in an MCM complex for use in the prevention and / or treatment of NETosis-induced vascular occlusion.

2. An inhibitor of at least one protein in the MCM complex for the stated purpose according to claim 1, wherein the inhibitor is selected from: - Quinolones, preferably fluoroquinolones - Helicase inhibitors - Bromo-terminal domain and superterminal domain (BET) protein inhibitors, -Based on small molecules of furanonaphthoquinones -Polo kinase 1 inhibitor, -Polyphenolic compounds, 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitor, - Cantharidin or its derivatives -CDC7 kinase inhibitors, and - Proteasome inhibitors.

3. An inhibitor of at least one protein in the MCM complex for the said use according to claim 1 or claim 2, wherein said inhibitor is a fluoroquinolone of formula (I): （I） in: R1 represents a hydrogen atom or NH2; R2 represents a hydrogen atom or a halogen atom such as F, Cl, Br and I, preferably, the halogen atom is F; R3 represents a hydrogen atom or a C1-C4 alkyl group; R4 represents a C1-C6 alkyl group, preferably a C1-C4 alkyl group, and more preferably a cyclopropyl group or an ethyl group; R5 represents a hydrogen atom or a C1-C4 alkyl group; preferably, a hydrogen atom.

4. An inhibitor of at least one protein in the MCM complex for the purpose according to any one of claims 1 to 3, wherein the NETosis-induced vascular occlusion occurs in a disease associated with immune thrombosis dysregulation, and said disease is selected from: septic DIC; non-septic DIC; autoimmune diseases, preferably antiphospholipid syndrome or systemic lupus erythematosus (SLE); inflammatory diseases, preferably Crohn's disease or rheumatoid arthritis; myocardial infarction, stroke, venous thromboembolism, thrombosis-related diseases, vascular occlusion in sickle cell disease, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; viral diseases, preferably influenza and COVID-19; and cancer.

5. An inhibitor of at least one protein in the MCM complex for the purpose according to any one of claims 1 to 4, wherein the disease associated with immune thrombosis dysregulation is DIC.

6. An inhibitor of at least one protein in the MCM complex for the purpose according to any one of claims 1 to 5, wherein the inhibitor is associated with a compound that directly targets the coagulation pathway, such as heparin, antithrombin, or thrombomodulin.

7. A method for diagnosing NETosis-induced vascular occlusion in a subject, the method comprising: - Determine the bioactivity or expression level of at least one protein in the MCM complex from a biological sample, preferably a blood sample, more preferably neutrophils, from a subject; - Compare the bioactivity or expression level of the at least one protein in the MCM complex with the baseline bioactivity or expression level established from at least one healthy donor or at least one patient without vascular occlusion; The biological activity or expression of at least one protein in the MCM complex above the baseline level in the subject identifies the subject as having or potentially developing NETosis-induced vascular occlusion.

8. The method according to claim 7, characterized in that, The method is combined with the following: - Observe for signs of thrombosis, characterized by diffuse microthrombus formation leading to purpuric skin lesions that can progress to ischemia and necrosis, particularly in the extremities, and organ dysfunction associated with microthrombi leading to visceral perfusion failure; and / or -Observe for hemorrhagic clinical signs, which may include petechiae, hemorrhagic bullae, patchy bleeding from mucosa or puncture sites, hematuria, or melena; and / or - Assess ISTH and / or Japanese Society of Critical Care Medicine (JAAM) scores; and / or Elevated neutrophil DNA decompression was measured by neutrophil lateral fluorescence (NEUT-SFL) recorded by an automated blood cell counter.

9. A kit for diagnosing DIC, the kit comprising at least one reagent for determining the expression level of at least one protein in the MCM complex in neutrophils.

10. A method for evaluating the efficacy of a treatment for NETosis-induced vascular occlusion in animal models or patients, the method comprising: - Prior to administering the treatment, determine the bioactivity or expression of at least one protein in the MCM complex in a biological sample, preferably a blood sample, obtained from the animal model or the patient; - Following administration of the treatment, determine the expression of at least one protein in the MCM complex in the biological sample obtained from the patient; and - Compare the expression levels.

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