Methods of modulation of immunothrombosis and biological markers of diagnosis of associated conditions

Abstract

The present invention relates to new biological markers of NETosis-induced vascular occlusion, including immunothrombosis dysregulation. Accordingly, the present invention is directed to compounds and to methods for the treatment of NETosis-induced vascular occlusion including immunothrombosis dysregulation and to methods of diagnosis of NETosis-induced vascular occlusion.

Description

[0001] Methods of modulation of immunothrombosis and biological markers of diagnosis of associated conditions

[0002] The present invention relates to new biological markers of NETosis-induced (or NETosis-associated) vascular occlusion, including immunothrombosis dysregulation. Accordingly, the present invention is directed to compounds and to methods for the treatment of NETosis-induced vascular occlusion including immunothrombosis dysregulation and to methods of diagnosis of NETosis-induced vascular occlusion.

[0003] The activation of the coagulation cascade represents a natural mechanism of defense, critical to keep physiological hemostasis in response to an infection or tissue damage. For instance, during bacterial sepsis the host's response to pathogen invasion involves the activation of numerous defense mechanisms. Among them, the engagement of innate immunity and the activation of coagulation act together against the infection. These close interactions between immunity and hemostasis are referred to as "immunothrombosis". This mechanism results in the formation of microthrombi inside blood vessels, which allows for the capture, recognition, and destruction of pathogens in order to limit their spread within the body. In some cases, defense mechanisms can become dysregulated. The intense activation of vascular cellular components, including leukocytes, platelets, and endothelium, is then responsible for endothelial dysfunction, with an endothelium that becomes pro-adhesive and prothrombotic, along with excessive coagulation activation, leading to a deficiency in anticoagulant and fibrinolytic regulatory systems, ultimately resulting in coagulopathy (Tschirhart DOI / 10.37051 / mir-00122). So, in pathological situations, excessive activation of the coagulation cascade may occur, leading to disseminated intravascular coagulopathy (DIC) and other thrombotic processes.

[0004] The Inventors have been working for several years on the role of Polymorphonuclear Neutrophils (PMN), a subpopulation of leukocytes, in disseminated intravascular coagulation (DIC) during septic shock. PMNs are early players in the host's innate immune response to infection. They are activated at the site of infection and become capable of capturing the pathogen by phagocytosis and releasing the contents of their granules. In this way, they help to eliminate the bacteria, but at the same time create a hyper-inflammatory state that can contribute to the appearance of tissue lesions. NETosis refers to the release by neutrophils of the contents of their nuclei in the form of structures known as Neutrophil Extracelular Traps (NETs). These NETs are capable of capturing both pathogens and circulating blood cells, thereby contributing to the host's immune defense. NETs' pro-coagulant surface enables them to activate the contact phase of coagulation. NETosis of PMNs thus plays an important role in dysregulation of immunothrombosis that can lead to DIC.

[0005] NETs are associated with several pathologies (Papayannopoulos V. doi:10.1038 / nri.2017.105) such as systemic autoimmune 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-icc / iiy215), rheumatoid arthritis (Joanna

[0006] 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) and with thrombo-inclusive 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 / sl0067-021-05790-9), non-septic DIC (Hell L et al. DOI: viral diseases such as nfluenza, COVID- 19 (Jiayu Li, DOI:

[0007] 10.1186 / sl2985-023-02116-w, Zhu et al. doi:10.3389 / fimmu.2022.838011), as well as in cancer (Jingxuan Xia, DOI: 10.1002 / ijc.34750) and sickle-cell disease vaso-occlusive complications (Ravi Vats, doi: 10.1182 / blood.2021014552; Emilia A Barbu, DOI: 10.1016 / j.cyto.2019.154933).

[0008] The Inventors have identified proteins that are highly differentially expressed between two cohorts of intensive care patients in septic shock with or without DIC. They validated that the Mini Chromosome Maintenance complex (MCM complex), which contains proteins MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, is overexpressed in neutrophils of patients in septic shock-induced DIC.

[0009] However, the involvement of these proteins in NETosis associated with DIC in septic shock has never been previously described. For this reason, Inventors developed an in vitro model based on the bacterial stimulation of human neutrophils that induces NETosis, similarly as seen in septic shock-associated DIC: indeed bacterial stimulation of human neutrophils provoked the appearance of NETs as seen in neutrophils of DIC patients. Using this model, they demonstrated the significant overexpression of the MCM complex in this stimulated conditions and on the top of it that the inhibition of MCM complex by an inhibitor called ciprofloxacin has resulted in the total inhibition of NETosis. Moreover, through a further functional study of stimulated and inhibited neutrophils, they showed that ciprofloxacin-treated neutrophils retained their ability to phagocytose bacteria and this treatment did not interfere with fibrinolysis, an important pathway in the anticoagulant system (see experimental part). Surprisingly, similar results were obtained with chemical stimulation of NETosis in PMNs, showing that targeting NETosis can be a suitable therapeutic approach to modulate, in particular inhibit partially, immunothrombosis and treat immunothrombosis disorders other than septic.

[0010] This result confirms the major role of neutrophils in the dysregulation of immunothrombosis and provides a new therapeutic target in NETosis-induced immunothrombosis dysregulation, such as DIC involved in septic shock.

[0011] Septic shock is the most severe form of host response to infection by a pathogenic microorganism. Mortality in septic shock is high, around 30 to 40%, despite the development of effective anti-infective treatments and appropriate care.

[0012] The pathophysiology of septic shock is characterized by an exaggerated and inappropriate host systemic inflammatory response mediated by immune cells activated during the response to pathogen invasion. DIC is a frequent complication of sepsis and septic shock with one-third of patient developing DIC. DIC is associated with increased mortality of up to 60%.

[0013] These results are all the more important that, at present, there is no consensus on the treatment of DIC associated with septic shock. Management focuses primarily on the treatment of the underlying pathology, i.e. the urgency of anti-infective therapy. However, various complementary approaches are currently under investigation to prevent thrombotic complications and restore the balance between coagulation and fibrinolysis (Helms, Iba, et aL, 2023).

[0014] Anticoagulation could reduce disseminated micro-thrombosis by preventing excessive activation of coagulation and immunity. In this context, various studies have examined the use of unfractionated heparin (UFH) or low-molecular-weight heparins (LMWH) to combine the anticoagulant effects, which inactivate factor Xa and certain coagulation proteases, 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). The results of these studies do not converge and do not allow to conclude that this therapeutic approach is effective.

[0015] This is why some research has turned to the use of thrombomodulin as a potential alternative to prevent thrombotic complications, but efficacy of thrombomodulin in humans was a subject for debates (Vincent et al. 2019, Levi et al. 2020, Francois et al. 2021, Guo et Lin, 2023).

[0016] Another recognized objective in therapeutic research in the face of septic DIC is to correct the imbalance between coagulation players and fibrinolysis. Studies on patients in septic shock have recently shown that plasma protein supplementation or delivery via the administration of fresh plasma reduces the risk of bleeding and increases 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).

[0017] The treatment of septic-chock induced DIC thus remains a challenge due to the complexity of its pathophysiology.

[0018] In the present invention, Inventors have identified the major role of MCM complex in NETosis, and demonstrated that the use of MCM inhibitors for NETosis modulation leads to restoring a balanced immunothrombosis. The present invention thus relates to the use of MCM inhibitors for the prevention and / or treatment of immunothrombosis dysregulation associated disorders.

[0019] Accordingly, the present invention relates to inhibitor of at least one protein of the MCM complex, acting as a NETosis modulator, preferably inhibitor, for its use for modulation of immunothrombosis, preferably inhibition or downregulation, and for the treatment of diseases associated with vascular occlusion and immunothrombosis dysregulation.

[0020] Modulation can designate an increase or an inhibition of a phenomenon. In the present invention, it preferably designates an inhibition that can be partial or total. A total inhibition of a phenomenon results in the total interruption of said phenomenon, whereas a partial inhibition slows the development of said phenomenon. For example, in the case of inhibition of the NETosis, a total inhibition means the stop of the development of NETs and a partial inhibition the decrease of the development of NETS.

[0021] According to a particular embodiment, the present invention relates to inhibitor of at least one protein of the MCM complex for its use forthe prevention and / or treatment of NETosis-induced vascular occlusion.

[0022] The MCM complex is a hexameric, ring-shaped protein structure composed of six proteins (MCM2 to MCM7, of SEQ. ID. N°1 to 6, respectively) with an ATPase domain in their amino acid sequences, as well as an N-terminal DNA-binding domain (Fletcher et al., 2003). These proteins play an essential role in the initiation and regulation of the S phase of DNA replication (Maine et al., 1984). The MCM complex is also involved in chromatin elongation and remodeling, enabling the proper unwinding of DNA through its helicase activity (Bochman & Schwacha, 2009). Highly conserved in eukaryotes, the MCM complex is essential for genome stability. Thanks to its essential functions, the MCM complex has attracted considerable research interest, particularly in the field of cancer. MCM proteins have been identified as biomarkers in several types of cancer (Gonzalez et aL, 2005), being associated with rapid cell proliferation and poor survival in some cancers (Soling et al., 2005; Sun et aL, 2022).

[0023] An inhibitor of the MCM complex is a compound able to prevent at least one protein of MCM complex expression or to decrease the MCM complex biological activity.

[0024] The inhibition of the MCM complex may be assessed by measuring:

[0025] - the decrease of expression of at least one protein of MCM;

[0026] Expression of marker proteins may be assessed using any known methods in the art. The term expression refers to protein translation or mRNA transcription, or mass spectrometry.

[0027] Methods suitable for the detection of protein include any suitable method for detecting and / or measuring proteins from a cell or cell extract. Such methods include, but are not limited to, staining and / or sorting using flow cytometry, polymerase chain reaction, immunoblot (e.g., Western blot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunohistochemistry and immunofluorescence, mass spectrometry. Particularly preferred methods for detection of proteins include any single cell assay, including staining and / or sorting using flow cytometry, immunohistochemistry, immunofluorescence assays 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 are described in the public literature, and methods for production of antibodies that can be developed against these proteins are also well known in the art.

[0028] Methods suitable for detecting mRNA include any suitable method for detecting and / or measuring mRNA levels from a cell or cell extract. Such methods include, but are not limited to: polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), in situ hybridization, Northern blot, sequence analysis, gene microarray analysis (gene chip analysis), RNA sequencing and detection of a reporter gene. Such methods for detection of transcription levels are well known in the art, and many of such methods are described in deta i I (https: / / www.elsevier.com / books / rna-methodologies / farrell-jr / 978-0-12-804678- 4}. Mass spectrometry is being used implemented in clinical laboratories to identify and quantify biomolecules in a variety of biological specimens (Birhanu, Clin Proteomics, 2023).

[0029] - the decrease of enzymatic activity of the MCM complex by unwinding and ATP hydrolysis assays, ATP hydrolysis reaction coupled to reactions that use NADH as a substrate, FRET assays, electrophoretic mobility shift assay (EMSA), The molecular beaconbased helicase assay (MBHA).

[0030] Non limiting examples of inhibitor of the MCM complex include antibodies, antigen-binding proteins, polypeptides optionally with non-natural or modified amino acids, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, or heterocyclic compounds. Inhibitors of the MCM complex also include genetic engineering system for decreasing the expression of the MCM complex such as CRISPR / Cas system, a zinc-finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, a siRNA, a shRNA, an antisense oligonucleotide or a miRNA...

[0031] Compounds can also be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Synthetic compound libraries are commercially available from, for example, Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich Chemical Company, Inc. (Milwaukee, Wis.). Natural compound libraries comprising bacterial, fungal, plant or animal extracts are available from, for example, Pan Laboratories (Bothell, Wash.). In addition, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides.

[0032] In a particular embodiment, the inhibitor of the MCM complex may be selected among:

[0033] - quinolones, preferably fluoroquinolones,

[0034] - statins,

[0035] - helicase inhibitors,

[0036] - Bromodomain and Extraterminal (BET) protein inhibitors,

[0037] - small molecules based on furanonaphthoquinones,

[0038] - Polo-kinase 1 inhibitors,

[0039] - polyphenolic compounds,

[0040] - 3-hydroxy-3-methylglutatyl CoA (HMG-CoA) reductase inhibitors,

[0041] - iso-flavones,

[0042] - Cantharidin and derivatives thereof,

[0043] - CDC7 kinase inhibitors,

[0044] - histone acetylation inhibitors, and

[0045] - post-translational modifications of the protein such as proteasome inhibitors (Samuel Wolf Buchsbaum, DOI: 10.1038 / sj. one.1210314); preferably, among:

[0046] - quinolones, preferably fluoroquinolones,

[0047] - helicase inhibitors,

[0048] - Bromodomain and Extraterminal (BET) protein inhibitors,

[0049] - small molecules based on furanonaphthoquinones,

[0050] - Polo-kinase 1 inhibitors,

[0051] - 3-hydroxy-3-methylglutatyl CoA (HMG-CoA) reductase inhibitors,

[0052] - Cantharidin and derivatives thereof,

[0053] - CDC7 kinase inhibitors, and - post-translational modifications of the protein such as proteasome inhibitors.

[0054] Even more precisely, the inhibitor may be selected among ciprofloxacin and its derivatives (as menthol and thymol derived ciprofloxacin described by Szosteck et al., 2022), trichostatin, biguanide, heliquomycin, P-carboline-3-carboxylic acid N-methylamide (CMA), widdrol, breviscapine, genistein, lovastatin, metformin, AS4583 and its derivative RJ-LC-07-48, proteasome inhibitors.

[0055] In a preferred embodiment, the inhibitor of the MCM complex is a fluoroquinolone of formula (I): wherein:

[0056] Ri represents a hydrogen atom or NH2;

[0057] R2 represents a hydrogen or a halogen atom such as F, Cl, Br, and I, preferably, the halogen atom is F;

[0058] R3 represents a hydrogen atom or a C1-C4 alkyl radical;

[0059] R4 represents a Ci-Ce alkyl radical; preferably a C1-C4 alkyl radical and more preferably a cyclopropyl or an ethyl radical;

[0060] Rs represents a hydrogen atom or a C1-C4 alkyl radical; preferably a hydrogen atom.

[0061] According to a preferred embodiment, the fluoroquinolone of formula (I) is ciprofloxacin: or compound ChemBridge 5281925: NETosis-induced vascular occlusion is a disorder that can occurs in various diseases. According to a particular embodiment, the disease associated with NETosis- induced vascular occlusion, including immunothrombosis dysregulation is selected amongst 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, thrombo-inclusive diseases, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis; vaso-occlusion in sickle-cell disease and sickle-cell disease vaso-occlusive complications, viral diseases, such as influenza, COVID-19, and immunothrombosis associated to cancers, and sickle-cell disease.

[0062] In a preferred embodiment, said disease involves immunothrombosis dysregulation and is DIC, more preferably a septic DIC.

[0063] According to another embodiment, the inhibitor of at least one protein of the MCM complex is associated with a compound directly targeting coagulation pathways, such as heparin, antithrombin, thrombomodulin and / or selective inhibitor of neutrophil elastase, such as sivelestat.

[0064] The present invention also relates to a method of treating NETosis-induced vascular occlusions in a subject in need thereof comprising the administration of at least one inhibitor of at least one protein of the MCM complex.

[0065] In immunothombosis, coagulation activation must be understood as an essential innate immune defense mechanism; hemostasis constitutes a first line of non-specific host defense, and a regulated activation of low thrombin production may be beneficial for host defense against the pathogen. This is the reason why only patients with dysregulation of immunothrombosis are to be treated against NETosis-induced coagulopathies, highlighting the importance of appropriate prior stratification of patients (Helms et aL, 2020).

[0066] The present invention thus relates to inhibitor of at least one protein of the MCM complex, acting as a NETosis modulator, for its use for modulation of immunothrombosis, and for the treatment of diseases associated with vascular occlusion and immunothrombosis dysregulation, wherein said inhibitor is administered to a patient suffering from NETosis-associated vascular occlusion. As defined in more details below, such patient suffering from NETosis-induced coagulopathy may show:

[0067] - thrombotic and / or hemorrhagic clinical signs; and / or

[0068] - an ISTH score equal to or higher than 5 and / or a JAAM score is equal to or higher than 4; and / or

[0069] - an increased neutrophil DNA decompaction by neutrophil-side fluorescence light (NEUT-SFL) recorded by an automated blood cell cytometer (Delabranche et al. (2017) Shock 47: 313-317); and / or

[0070] - an increased expression or an increased biological activity of at least one protein of the MCM complex, see the method below.

[0071] To date, there is no gold-standard or specific laboratory test for diagnosing immunothrombosis dysregulation, in particular DIC. For this reason, several diagnostic scores have been developed, taking into account classical haemostasis parameters. But to make the diagnosis of DIC, there must first be an underlying pathology compatible with its occurrence, such as sepsis. The diagnosis is then based on a combination of clinical and biological factors.

[0072] Clinical signs of DIC is marked by the appearance of a symptomatology that may be :

[0073] - Thrombotic: characterized by disseminated microthrombosis resulting in purpuric skin lesions that can progress to ischemia and necrosis, particularly in the extremities, as well as organ dysfunction related to microthrombi, leading to visceral perfusion failure.

[0074] - Hemorrhagic: which may involve petechiae, hemorrhagic bullae, sheet bleeding from mucous membranes or puncture sites, hematuria or melena. This bleeding is secondary to the consumption of coagulation factors and fibrinogen, as well as thrombocytopenia. They are even more frequent in DIC with a hyperfibrinolytic component. In the particular case of septic DIC, they remain rare and of late onset.

[0075] Biological factors (blood platelet content or decrease rate, prothrombin, fibrinogen and D-dimers content) to diagnose DIC in sepsis or septic shock are usually used to calculate scores of the ISTH and the Japanese Society of Intensive Medicine (JAAM). According to these scores, a diagnosis of DIC can be made if the ISTH score is equal to or higher than 5 (Taylor et al., 2001), or if the JAAM-DIC score is equal to or higher than 4 (I ba et al., 2016).

[0076] However, these scores are rarely used in clinical practice, mainly because of their relative imperfections. None of these scores can detect patients with asymptomatic DIC, yet these patients have a similar severity and mortality rate to those with DIC diagnosed on intensive care admission (Delabranche et al., 2016). The diagnostic performance of the scores varies according to the initial pathology, as well as DIC (Helms et al., 2020). Furthermore, these scores do not distinguish between thrombotic or hemorrhagic phenotypes in patients with DIC. The therapeutic management of patients with DIC is not standardized, but may depend on the progressive stage of DIC (thrombotic, hemorrhagic, fibrinolytic), highlighting the importance of appropriate prior stratification of patients (Helms et aL, 2020).

[0077] It is therefore essential to shed light on new biological markers that could guide clinicians in the clinical diagnosis and monitoring of immunothrombosis dysregulation.

[0078] The present invention thus relates to a method of diagnosing NETosis-associated vascular occlusion, including immunothrombosis dysregulation in a subject, comprising :

[0079] - determining in biological sample, preferably a blood sample, more preferably neutrophils, from a subject;

[0080] - measuring the biological activity or the level of expression of at least one protein of the MCM complex;

[0081] - comparing said biological activity or level of expression of the at least one protein of the MCM complex to a baseline biological activity or level of expression established from at least one healthy donor or one patient without vascular occlusion; wherein a biological activity or an expression of the at least one protein of the MCM complex in said subject higher than said baseline level identifies the subject as having or likely to develop NETosis-associated vascular occlusion, including immonothrombosis dysregulation.

[0082] The term subject refers to any animal subject, and particularly, any vertebrate mammals, including, but not limited to, primates, rodents, livestock and domestic pets. Preferred mammals for the method of the present invention include humans. NETosis-associated vascular occlusion, including immunothrombosis dysregulation may be selected amongst septic DIC; non-septic DIG; 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, thrombo-inclusive diseases, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis; vaso-occlusion in sickle-cell disease and sickle-cell disease vaso-occlusive complications; viral diseases, such as influenza, COVID-19, and immunothrombosis associated to cancers.

[0083] In a particular embodiment, the method of the invention is combined with:

[0084] - observation of thrombotic signs characterized by disseminated microthrombosis resulting in purpuric skin lesions that can progress to ischemia and necrosis, particularly in the extremities, as well as organ dysfunction related to microthrombi, leading to visceral perfusion failure: and / or

[0085] - observation of hemorrhagic clinical signs which may involve petechiae, hemorrhagic bullae, sheet bleeding from mucous membranes or puncture sites, hematuria or melena; and / or

[0086] - assessment of the score of the ISTH and / or the Japanese Society of Intensive Medicine (JAAM) ; and / or

[0087] - measure of the increased neutrophil DNA decompaction by neutrophil-side fluorescence light (NEUT-SFL) recorded by an automated blood cell cytometer (Delabranche X, Stiel L, Severac F, et al (2017) Evidence of Netosis in Septic Shock-Induced Disseminated Intravascular Coagulation. Shock 47: 313-317).

[0088] Advantageously, the present method allows to differentiate DIC and non-DIC patient with sepsis or septic shock.

[0089] A healthy donor is an individual for whom no disease has been diagnosed. Preferably, the baseline level established from at least one healthy donor sample is established with at least 10 healthy donor samples.

[0090] Each of MCM2, MCM3, MCM4, MCM5, MCM6 and / or MCM7 can be used as a biological marker in the method of the invention. Expression of marker proteins may be assessed using any known methods in the art. The term expression refers to protein translation or mRNA transcription, or mass spectrometry.

[0091] Methods suitable for the detection of protein and mRNA are as described above.

[0092] The present invention also relates to the in vitro or ex vivo use of the biological activity orthe level of expression of at least one protein of the MCM complex as a biological marker for diagnosing NETosis-associated vascular occlusion.

[0093] MCM helicase activity can be measured using unwinding and ATP hydrolysis assays, ATP hydrolysis reaction coupled to reactions that use NADH as a substrate, FRET assays, electrophoretic mobility shift assay (EMSA), the molecular beacon-based helicase assay (MBHA) (https: / / www.bmglabtech.com / en / application-notes / molecular-beacon- based-helicase-assays / ).

[0094] Included in the present invention are kits for the diagnosis of NETosis-associated vascular occlusion comprising at least one reagent being used to determine the biological activity or the expression level of at least one protein of the MCM complex in neutrophils.

[0095] Said reagent may be e.g., a probe that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding the marker proteins; RT-PCR primers for amplification of mRNA encoding the marker proteins or a fragment thereof; and / or an antibody, antigen-binding fragment thereof or other antigen-binding peptide that selectively binds to the marker proteins.

[0096] The reagents of the kit of the present invention can be conjugated to a detectable tag or detectable label. Such a tag can be any suitable tag which allows for detection of the reagents and includes, but is not limited to, any composition or label detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, mass spectrometry including multiple or parallel or selected reaction monitoring (MRM / PRM / SRM) (Lange V, et al. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol. 2008;4(l):222.), or chemical means. Useful labels in the present invention include biotin for staining with labelled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. In case of sample preparation kits for mass spectrometry, kits may contain labelled lysine or arginine or other aminoacid, or immunoprecipitation for LC-MS / MS including MRM / SRM / PRM.

[0097] In addition, the reagents of the kit can be immobilized on a substrate. Such a substrate can include any suitable substrate for immobilization of a detection reagent such as would be used in any of the previously described methods of detection. Briefly, a substrate suitable for immobilization of a means for detecting includes any solid support, such as any solid organic, biopolymer or inorganic support that can form a bond with the means for detecting without significantly effecting the activity and / or ability of the detection means to detect the desired target molecule.

[0098] Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers (e.g., polyacrylamide), stabilized intact whole cells, and stabilized crude whole cell / membrane homogenates.

[0099] Exemplary biopolymer supports include cellulose, polydextrans (e.g., Sephadex®), agarose, collagen and chitin.

[0100] Exemplary inorganic supports include glass beads (porous and nonporous), stainless steel, metal oxides (e.g., porous ceramics such as ZrO2, TiO2, A1203, and NiO) and sand.

[0101] Other existing kits consist of dried, pre-formulated antibody panels for the detection of rare events, immune function analysis, research on the immune system and certain clinical applications, such as the DURACIone panel from Beckman Coulter.

[0102] According to another object, the present invention relates to a method to evaluate the efficacy of a treatment of NETosis-associated vascular occlusion in an animal model or a patient, comprising :

[0103] - determining the biological activity or the expression of at least one protein of the MCM complex in as biological sample, preferably a blood sample, and more preferably neutrophils, taken from the animal model or the patient before administering the treatment;

[0104] - determining the expression of at least one protein of the MCM complex in neutrophils taken from the patient after administering the treatment ; and - comparing said levels of expressions.

[0105] The decrease of the biological activity or of the expression level of the marker proteins indicates that the treatment is efficient.

[0106] FIGURES

[0107] Figure 1: Representative immunofluorescence images of unstimulated neutrophils (A), stimulated with Escherichia coli (B) or with PMA (C): Neutrophils were labelled with anti-myeloperoxidase antibody (see panels MPO) and anti-MCM2 antibody (see panels MCM2) and counterstained with DNA-labeling Hoechst (see panels Hoechst), original magnification x63. NETs were identified as elongated DNA fibers, associated with myeloperoxidase.

[0108] Figure 2: NETosis quantification based on DNA quantity on unstimulated neutrophils (black), neutrophils stimulated with PMA or Escherichia coli (light grey), neutrophils stimulated with PMA or Escherichia coli and treated with 250 pM ciprofloxacin (dark grey). A standard curve with known concentrations of fibrillar DNA and human neutrophil elastase allow the quantification of NETs in samples.

[0109] Figure 3: Dose-dependent inhibition of NETosis by ciprofloxacin in vitro. (A) Whitelight immunofluorescence images of neutrophils labelled with Hoechst, initial magnification x20. (B) Quantification of dose dependant inhibition of NETosis by ciprofloxacin.

[0110] Figure 4: Representative immunofluorescence images of neutrophils stimulated with Escherichia coli (A) or PMA (B) and treated with ciprofloxacin : neutrophils were labelled with anti-myeloperoxidase antibody (see panels MPO) and anti-MCM2 antibody (see panels MCM2) and counterstained with DNA-labelling Hoechst original magnification x63.

[0111] Figure 5: Measurement of fluorescence intensity of FITC-labeled Escherichia coli bacteria on unstimulated neutrophils (A), stimulated with PMA (B), stimulated with PMA and treated with 250 pM ciprofloxacin (C): Control tubes (black) were incubated on ice, while phagocytosis test tubes (grey) at 37°C for 30 minutes.

[0112] Figure 6: Plasmin formation at different doses of uPA (0 - 2 - 3 - 4,5 nM) in unstimulated neutrophils (black) or neutrophils stimulated with Escherichia Coli or PMA (light grey), stimulated with PMA or Escherichia Coli and treated with Ciprofloxacin (dark grey). Plasmin formation from 0,5 nM plasminogen was monitored by measuring the release of pNA from, the plasmin-selective chromogenic substrate at 0.75 mM for 2 hours at 405 nM. Vi= initial velocities of the reaction.

[0113] EXAMPLE

[0114] Material and methods

[0115] Patients

[0116] Twenty patients in septic shock admitted to the medical intensive care units of Strasbourg's Nouvel Hopital Civil were included in this study, 9 of whom presented with disseminated intravascular coagulation (DIC). Septic shock was diagnosed according to the definition of the latest international consensus conference (Singer et aL, 2016), and DIC if the JAAM-DIC 2016 score was at least 4 during the first 24 hours of septic shock (Iba et al., 2016).

[0117] Blood sampling

[0118] Ten milliliters of blood were drawn on admission, within 12 hours of the diagnosis of septic shock, on day 3 and on day 7 using BD Vacutainer Plus blood collection tubes (Becton Dickinson, Le pont de Claix, France) containing the anticoagulant EDTA (Ethylene Diamine Tetra Acetic Acid). Five milliliters of whole blood was also collected in citrate-containing tubes to perform the routine haemostasis tests used to diagnose DIC.

[0119] Isolation of neutrophils

[0120] At each time point, two EDTA tubes were used to isolate neutrophils by negative immunomagnetic selection using a commercial EasySep® kit (#19666, Stemcell Technologies, Canada) (Peters et aL, 2005). Magnetic beads, bound to antibodies specifically targeting the surface proteins of all circulating hematopoietic cells except neutrophils, were added to whole blood. Next, the tube containing whole blood and beads was placed in a magnet to separate labeled from unlabeled cells. Once the negative fraction had been recovered, the collected neutrophils were washed twice with phosphate- buffered saline (PBS, Euromedex, France) to remove all plasma proteins. The cell suspension was checked by automated fluorocytometry (SysmexTM XN20® analyzer, Sysmex Corporation, Kobe, Japan) to count the neutrophils and check their purity. Average purity was 96.36% ± 0.363. l,106neutrophils were aliquoted as dry pellets into dedicated eppendorfs and stored at -80°C.

[0121] Proteomic analysis Proteins from isolated neutrophils were extracted in Laemmli-type buffer and 7.5pg of proteins were concentrated in a stacking band of a ID SDS-PAGE gel, followed by cysteine reduction to break disulfide bridges, alkylation to avoid disulfide bridge reformation and enzymatic digestion by the addition of trypsin solution to obtain peptides analyzed by mass spectrometry. Analyses were performed on a nanoACQUITY Ultra Performance LC system (Waters Corporation, Milford, USA) coupled to a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Massachussets, USA) with a "data-dependent" acquisition mode. Label-free quantification was performed using MaxQuant software (v.1.6.0.16. Max Planck Institute for Biochemistry, Martinsried, Germany, (Cox et aL, 2014)). Gene Ontology (GO) term enrichment analysis of differentially abundant proteins and protein networks was performed using STRING v.12 software (Szklarczyk et al., 2023). Western Blot

[0122] Proteins were extracted from patient neutrophils using RIPA lysis buffer (#10017003, Thermofisher, Waltham, USA), supplemented with protease and phosphatase inhibitors (#04693116001 and #4906845001, Roche, Switzerland). Proteins (25g) were separated by a 12% SDS-PAGE gel. (#161075, Biorad, USA) at 110V for 2 hours, then transferred by electrophoresis to a nitrocellulose membrane (#1 B23002, Thermofisher, USA). Non-specific binding sites on the membrane were blocked with 5% bovine serum albumin (BSA, Euromedex, France) diluted in Tris saline buffer (TBS, Euromedex, France) for 1 hour at room temperature. Proteins of interest were labeled with specific primary antibodies incubated overnight at 4°C.These antibodies were diluted in 1 / 1000 blocking solution: rabbit monoclonal anti-PTX3 (abl25007), mouse monoclonal anti-PAD4 (abl28086), rabbit monoclonal anti-MMP9 (ab38898), mouse monoclonal anti-NGAL (ab23477), all from Abeam. Membranes were washed three times with TBS - T (TBS with 0.1% Tween-20) and incubated for 1 h at room temperature with peroxidase-labelled secondary antibodies: anti-rabbit and anti-mouse (#70745 and #7076S, Cell Signaling Technology, USA). Immunostaining was revealed by a chemiluminescence solution. The chemiluminescence signal was recorded with iBright CL100 and analyzed using ImageStudioLive software (V.5.2.5, Li-COR Biosciences, USA). Quantitative normalization against the GAPDH reference protein was performed for each protein of interest using the corresponding specific antibody (mouse monoclonal anti-GAPDH, ab8245). Induction and inhibition of NETosis in vitro

[0123] To induce NETosis in vitro, neutrophils from healthy volunteers were isolated, as described previously. After separation from other whole blood cells, neutrophils were suspended in Hanks' Balanced Salt Solution buffer (HBSS, #P04-32505, Dominic Dutscher, France) at a concentration allowing a distribution of 400,000 cells per well. Experiments were performed in 24-well culture plates with a glass coverslip at the bottom for immunofluorescence, or a 96-well culture plate for plasmin generation.

[0124] To induce NETosis, neutrophils were incubated at 37°C with 5% CC>2 for 4 h in the presence of a bacterial strain of Escherichia coli (MOI 100), specifically chosen for its resistance to fluoroquinolones. These bacteria induce NET formation using the protein kinase C (PKC) activation pathway (Kenny et al., 2017), as does phorbol 12-myristate 13-acetate (PMA), which was used as a positive control. Neutrophils were stimulated with 50nM PMA (#P1585, Sigma-Aldrich, USA) under the same conditions as bacteria.

[0125] In addition, under certain conditions, neutrophils were incubated with bacteria or PMA and simultaneously treated with ciprofloxacin (#17850, Merck, Germany).

[0126] Immunofluorescence

[0127] After stimulation, neutrophils were fixed on the slides using 4% paraformaldehyde solution (PFA, #J19943-K2, Thermofisher, USA) for 30 minutes, then rinsed three times with PBS. Once permeabilized with 0.2% Tween20 solution for 15 minutes, the antigenic sites of the PNNs were blocked with 10% BSA solution for 3 hours. Cells were incubated with specific primary antibodies: a rabbit anti-neutrophil elastase antibody (#481001, 1 / 1000, Merck, Germany) or an anti-myeloperoxidase antibody (#MA-180878, 1 / 400, Thermofisher, USA), as well as a rabbit anti-MCM2 antibody (#3619S, 1 / 500, Cell Signaling technology, USA), overnight at 4°C. After three rinses, cells were then incubated in the dark for 1 hour at room temperature with the appropriate secondary antibodies: Alexa Fluor 488 goat anti-mouse antibody and Alexa Fluor 647 goat anti-rabbit antibody (#A11001 and #A21245 respectively, Thermofisher, USA). After three additional washes, the slides were counterstained with Hoechst 33342 solution for DNA labeling (NucBlueTM Live ReadyProbes, #R37605, Thermofisher, USA) before being mounted under a coverslip using 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 Imaged software.

[0128] In vitro measurement of NETosis

[0129] NETs were measured in neutrophils suspension after stimulation with PMA or PMA and ciprofloxacin using a homemade capture-based ELISA developed by Dr. Angles-Cano. Briefly, 1 pg / mL biotinylated anti-human neutrophil elastase (hNE) ELISA monoclonal antibody (Enzo Life Sciences, Lyon, France) were added per well on streptavidin coated 96- well plates (Roche, Paris, France). After 3 washes, samples (50 pL / well) were dispensed into each well. After lh incubation at 37°C, the plate was washed (3 times) and 50 pL per well of horseradish peroxidase-labeled anti-DNA monoclonal antibody (kit Cell Death Detection ELISAPLUS) was added prior to another lh incubation at room temperature on a shaking device. After 3 washes, the peroxidase substrate was added. The absorbance was measured for 1 hour at A405 nm in a microplate counter (VERSAmax microplate reader, Molecular Devices). The quantity of NETs was calculated according to a standard range of NET equivalents formed by fibrillar DNA coupled to neutrophil elastase.

[0130] Measurement of Neutrophils phagocytic activity

[0131] Neutrophils were stimulated with 50nM PMA and treated with ciprofloxacin in 5mL tubes. After 4 hours of stimulation, tubes were centrifuged for 15 minutes at 800g, tubes were centrifuged for 15 minutes at 800g, and neutrophils and NETs were resuspended with plasma from a heparinized blood sample from healthy volunteers. FITC-labeled opsonized Escherichia coli bacteria (Reagent B, PHAGOTESTTM, 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 addition of blocking solution and three washes, flow cytometry analysis was performed to measure the number of bacteria phagocytized per neutrophil as a function of FITC mean fluorescence intensity (MFI).

[0132] In vitro plasmin generation

[0133] Neutrophils were seeded in 96-well plates at a concentration of 4xl05, then stimulated with Escherichia coli or PMA and finally treated with ciprofloxacin as previously described. After 4h of stimulation, the plates were centrifuged for 15 min at 800g, allowing supernatants to be removed and NETs to be attached to the bottom of the plate. NETosis was confirmed by visualizing NETs with Hoechst staining and detecting neutrophil elastase activity. Next, Therasolv® urokinase was incubated at four concentrations (0, 2, 3, 4.5nM) on NETs for 1 hour at 37°C with 5% CO? to allow binding of urokinase-type plasminogen activator receptor (uPAR). After a centrifugation step to remove excess uPA, a solution containing plasminogen (0.5pM) was added to trigger plasmin generation. The kinetics of plasminogen to plasmin conversion were monitored over 2 hours by measuring the release of methyl(malonyl)hydroxy-prolylarginine-p-nitroanill69-135ide (pNA) at an absorbance of 405nm using a plate reader. Initial velocities of the reaction (Vi) were calculated using an ad hoc computer program.

[0134] Statistics

[0135] Data, expressed as mean ± standard deviation, were analyzed using GraphPad Prism8® software (GraphPad Software, Inv. CA, USA), "n" represents the number of patients or the number of experiments. Mean values were compared using the Mann-Whitney or Kruska Il- Wallis test. Statistical variance between different groups was determined by applying a two-factor analysis of variance (ANOVA) test for plasmin generation. A value of p<0.05 was considered statistically significant. All measurements were performed at least three separate times.

[0136] RESULTS

[0137] Patient characteristics

[0138] Twenty patients were included in the study, all of whom met the criteria for septic shock upon ICU admission. Based on a positive ISTH score (> 5 points), nine patients were identified as having DIG.

[0139] Patients with DIC in septic shock have a specific protein signature

[0140] Proteomic analysis revealed differences between the three groups, mainly on admission. Indeed, the combined analysis identified and quantified 1813 proteins in at least 70% of the samples and enabled us to separate the samples into three groups corresponding broadly to the three groups in our study: septic shock without DIC, septic shock with DIC and controls. This unsupervised analysis confirms that neutrophils display different protein signatures depending on the diagnosis. At day 1, 224 proteins showed differential expression in patients with DIC compared with septic shock patients without DIC. Of these proteins, 115 were more abundant and 110 less abundant in neutrophils from septic shock DIC patients. Inhibition of MCM complex activity blocks NETosis in an in vitro model of bacterial and non- bacterial stimulation

[0141] Patients with DIC associated with septic shock are characterized by greater NETosis than patients without DIC, and it has been observed that MCM complex proteins are more highly expressed in neutrophils from patients with DIC. The MCM complex plays an essential role in DNA replication in eukaryotic cells. However, the neutrophil is a hematopoietic cell in the final stage of differentiation and does not divide. We therefore hypothesized that the expression of the MCM complex in neutrophils from DIC patients reflected the induction of NETosis in these patients.

[0142] After 4 hours of stimulation with Escherichia coli, healthy neutrophils released NETs, which were visualized by fluorescence microscopy. NETs were identified by their DNA chromatin structures associated with antimicrobial myeloperoxidase granules (Figure IB); these structures were similar to those observed after stimulation with PMA (Figure 1C). MCM2 protein is present in the cytoplasm of unstimulated neutrophils (Figure 1A). Once NETs had formed following stimulation by Escherichia coli (Figure IB), MCM2 protein was still homogeneously expressed in the cytoplasm of activated neutrophils and is present on DNA filaments. Upon PMA stimulation, MCM2 is localized close to the plasma membrane of neutrophils, and on NETs (Figure 1C).

[0143] To confirm our hypothesis on the role of the MCM complex in NETosis, the impact of MCM complex inhibition by one of the known inhibitors of the MCM 2-7 complex, ciprofloxacin, has been assessed. Compared to non-stimulated neutrophils, NETs were significantly increased in Escherichia coli and PMA-stimulated neutrophils (p<0.05, Fig 2). Treatment with ciprofloxacin significantly decreased NETs formation (p<0.05, Fig 2). Thus, after 4h stimulation with PMA, the results show that NETose is inhibited by ciprofloxacin, and this response appears to be dose-dependent (Figure 3).

[0144] In addition, NETosis induced by stimulation with a fluoroquinolone-resistant strain of Escherichia coli (including ciprofloxacin) was also inhibited by 250pM ciprofloxacin (Figure 4A). These results are similar to those observed with PMA (Figure 4B). These neutrophils show multi-lobed nuclei with the presence of MPO in the cytoplasm. The distribution of MCM2 protein is also homogeneous in the cytoplasm. Our results indicate that inhibition of the MCM complex with ciprofloxacin can block NETosis induced by bacterial or non-bacterial stimuli. This inhibition opens up a promising avenue for partially inhibit NETosis and consequently immunothrombosis.

[0145] Inhibition of the MCM complex preserves neutrophil functionality

[0146] During DIG, neutrophil functionality is impaired. We studied phagocytosis and fibrinolysis in neutrophils stimulated with and without MCM complex inhibitors.

[0147] Phagocytosis capacity intact after inhibition of the MCM complex in stimulated neutrophils Neutrophils play an essential 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 coupled to the fluorochrome FITC. After a 30-minute incubation at 37°C, FITC fluorescence intensity was measured cytometrically, reflecting the number of bacteria phagocytosed by neutrophils. To assess the phagocytic capacity of neutrophils, a control tube subjected to the same experimental conditions but incubated in ice was used. Unstimulated neutrophils showed a significant increase in phagocytic activity for FITC- coupled bacteria in test tubes (incubated with bacteria at 37°C) compared with control tubes (incubated with bacteria on ice) (Figure 5A, p<0.05). After stimulation with PMA, a NETose inducer, no change in neutrophil fluorescence intensity was observed, confirming that neutrophils lose their ability to phagocytose following this stimulation (Figure 5B). However, phagocytosis activity was observed as a control for PMA-activated neutrophils in the presence of ciprofloxacin (Figure 5C, p<0.01).

[0148] These results thus show that inhibition of the MCM complex by ciprofloxacin restored the phagocytic capacity of neutrophils after their stimulation by PMA.

[0149] Plasmin production by neutrophils is preserved after inhibition of the MCM complex Patients in septic shock with DIC show a reduced capacity to generate plasmin. This reduction may be associated with the phenomenon of NETosis, due to the presence of elastase in the DNA filaments of NETs, which cleaves plasminogen into fragments, impairing their ability to generate plasmin (Cruz et aL, 2019; Delabra nche et al., 2017). This mechanism contributes to the hypofibrinolysis and microthrombi formation observed in

[0150] DIC. Plasmin generation can be assessed by an in vitro study involving the deposition of plasminogen and a chromogenic substrate, pNA, on neutrophils previously incubated with increasing doses of urokinase. When plasminogen is exposed to uPA, it is converted to plasmin, which in turn catalyzes the conversion of paranitroanilide to paranitroaniline, emitting a yellow coloration detectable by spectrophotometer. Plasmin generation was monitored for two hours using this assay.

[0151] Unstimulated neutrophils produced plasmin in proportion to the concentration of urokinase. In contrast, when neutrophils were stimulated with Escherichia coli or PMA, plasmin production was lower (Figure 6A E. coli versus unstimulated, p<0.01; Figure 6B PMA versus unstimulated, p<0.0001). Interestingly, the presence of ciprofloxacin restored plasmin production (Figure 6A E. coli + ciprofloxacin versus E. coli, p<0.0001; Figure 6B PMA + ciprofloxacin versus PMA, p<0.001).

[0152] Thus, these results suggest that inhibition of the MCM complex by ciprofloxacin would preserve neutrophil involvement in plasmin generation, thus potentially contributing to improved hypofibrinolysis and positively modulating immunothrombosis.

[0153] Accordingly, it has been demonstrated that inhibition of the MCM complex preserves phagocytosis functions and does not interfere with plasmin generation in neutrophils stimulated by a NETosis-inducing agent. Neutrophils thus participate in the elimination of microorganisms and the destruction of microthrombi by degrading fibrin clots.

[0154] SEQUENCES LISTING

[0155] SEQ. ID. N°1 - human MCM2

[0156] MAESSESFTMASSPAQRRRGNDPLTSSPGRSSRRTDALTSSPGRDLPPFEDESEGLLGTEGPLEEEEDG

[0157] EELIGDGMERDYRAI PELDAYEAEGLALDDEDVEELTASQREAAERAMRQRDREAGRGLGRMRRGLL

[0158] YDSDEEDEERPARKRRQVERATEDGEEDEEMIESIENLEDLKGHSVREWVSMAGPRLEI HHRFKNFLR

[0159] THVDSHGHNVFKERISDMCKEN RESLVVNYEDLAAREHVLAYFLPEAPAELLQIFDEAALEVVLAMYP

[0160] KYDRITN HIHVRISHLPLVEELRSLRQLHLNQLIRTSGVVTSCTGVLPQLSMVKYNCNKCNFVLGPFCQS

[0161] QNQEVKPGSCPECQSAGPFEVNMEETIYQNYQRIRIQESPGKVAAGRLPRSKDAI LLADLVDSCKPGD

[0162] EIELTGIYHN NYDGSLNTANGFPVFATVILANHVAKKDNKVAVGELTDEDVKMITSLSKDQQIGEKIFA

[0163] SIAPSIYGHEDI KRGLALALFGGEPKNPGGKHKVRGDINVLLCGDPGTAKSQFLKYIEKVSSRAIFTTGQ

[0164] GASAVGLTAYVQRH PVSREWTLEAGALVLADRGVCLI DEFDKMNDQDRTSI HEAMEQQSISISKAGI

[0165] VTSLQARCTVIAAANPIGGRYDPSLTFSENVDLTEPIISRFDI LCVVRDTVDPVQDEMLARFVVGSHVR

[0166] HHPSNKEEEGLANGSAAEPAMPNTYGVEPLPQEVLKKYIIYAKERVHPKLNQMDQDKVAKMYSDLR

[0167] KESMATGSI PITVRHI ESMIRMAEAHARIHLRDYVIEDDVNMAIRVMLESFIDTQKFSVMRSMRKTFA

[0168] RYLSFRRDN NELLLFILKQLVAEQVTYQRNRFGAQQDTIEVPEKDLVDKARQINI HN LSAFYDSELFRM

[0169] NKFSHDLKRKMILQQF

[0170] SEQ. ID. N°2 - human MCM3

[0171] MAGTVVLDDVELREAQRDYLDFLDDEEDQGIYQSKVRELISDNQYRLIVNVNDLRRKNEKRANRLLN

[0172] NAFEELVAFQRALKDFVASIDATYAKQYEEFYVGLEGSFGSKHVSPRTLTSCFLSCVVCVEGIVTKCSLV

[0173] RPKVVRSVHYCPATKKTIERRYSDLTTLVAFPSSSVYPTKDEENNPLETEYGLSVYKDHQTITIQEMPEK

[0174] APAGQLPRSVDVILDDDLVDKAKPGDRVQVVGTYRCLPGKKGGYTSGTFRTVLIACNVKQMSKDAQ

[0175] PSFSAEDIAKIKKFSKTRSKDIFDQLAKSLAPSIHGHDYVKKAI LCLLLGGVERDLENGSHI RGDI NILLIGD

[0176] PSVAKSQLLRYVLCTAPRAIPTTGRGSSGVGLTAAVTTDQETGERRLEAGAMVLADRGVVCIDEFDK

[0177] MSDMDRTAIHEVMEQGRVTIAKAGI HARLNARCSVLAAANPVYGRYDQYKTPMENIGLQDSLLSRF

[0178] DLLFIMLDQMDPEQDREISDHVLRMHRYRAPGEQDGDAMPLGSAVDI LATDDPNFSQEDQQDTQI

[0179] YEKHDNLLHGTKKKKEKMVSAAFMKKYIHVAKII KPVLTQESATYIAEEYSRLRSQDSMSSDTARTSPV

[0180] TARTLETLIRLATAHAKARMSKTVDLQDAEEAVELVQYAYFKKVLEKEKKRKKRSEDESETEDEEEKSQE

[0181] DQEQKRKRRKTRQPDAKDGDSYDPYDFSDTEEEMPQVHTPKTADSQETKESQKVELSESRLKAFKVA

[0182] LLDVFREAHAQSIGMN RLTESI NRDSEEPFSSVEIQAALSKMQDDNQVMVSEGIIFLI SEQ. ID. N°3 - human MCM4

[0183] MSSPASTPSRRGSRRGRATPAQTPRSEDARSSPSQRRRGEDSTSTGELQPMPTSPGVDLQSPAAQDV

[0184] LFSSPPQMHSSAIPLDFDVSSPLTYGTPSSRVEGTPRSGVRGTPVRQRPDLGSAQKGLQVDLQSDGAA

[0185] AEDIVASEQSLGQKLVIWGTDVNVAACKENFQRFLQRFIDPLAKEEENVGIDITEPLYMQRLGEINVIG

[0186] EPFLNVNCEHIKSFDKNLYRQLISYPQEVIPTFDMAVNEIFFDRYPDSILEHQIQVRPFNALKTKNMRNL

[0187] NPEDIDQLITISGMVIRTSQLIPEMQEAFFQCQVCAHTTRVEMDRGRIAEPSVCGRCHTTHSMALIHN

[0188] RSLFSDKQMIKLQESPEDMPAGQTPHTVILFAHNDLVDKVQPGDRVNVTGIYRAVPIRVNPRVSNVK

[0189] SVYKTHIDVIHYRKTDAKRLHGLDEEAEQKLFSEKRVELLKELSRKPDIYERLASALAPSIYEHEDIKKGILL

[0190] QLFGGTRKDFSHTGRGKFRAEINILLCGDPGTSKSQLLQYVYNLVPRGQYTSGKGSSAVGLTAYVMKD

[0191] PETRQLVLQTGALVLSDNGICCIDEFDKMNESTRSVLHEVMEQQTLSIAKAGIICQLNARTSVLAAANP

[0192] IESQWNPKKTTIENIQLPHTLLSRFDLIFLLLDPQDEAYDRRLAHHLVALYYQSEEQAEEELLDMAVLKD

[0193] YIAYAHSTIMPRLSEEASQALIEAYVDMRKIGSSRGMVSAYPRQLESLIRLAEAHAKVRLSNKVEAIDVE

[0194] EAKRLHREALKQSATDPRTGIVDISILTTGMSATSRKRKEELAEALKKLILSKGKTPALKYQQLFEDIRGQ

[0195] SDIAITKDMFEEALRALADDDFLTVTGKTVRLL

[0196] SEQ. ID. N°4 - human MCM5

[0197] MSGFDDPGIFYSDSFGGDAQADEGQARKSQLQRRFKEFLRQYRVGTDRTGFTFKYRDELKRHYNLGE

[0198] YWIEVEMEDLASFDEDLADYLYKQPAEHLQLLEEAAKEVADEVTRPRPSGEEVLQDIQVMLKSDASPS

[0199] SIRSLKSDMMSHLVKIPGIIIAASAVRAKATRISIQCRSCRNTLTNIAMRPGLEGYALPRKCNTDQAGRP

[0200] KCPLDPYFIMPDKCKCVDFQTLKLQELPDAVPHGEMPRHMQLYCDRYLCDKVVPGNRVTIMGIYSIK

[0201] KFGLTTSRGRDRVGVGIRSSYIRVLGIQVDTDGSGRSFAGAVSPQEEEEFRRLAALPNVYEVISKSIAPSI

[0202] FGGTDMKKAIACLLFGGSRKRLPDGLTRRGDINLLMLGDPGTAKSQLLKFVEKCSPIGVYTSGKGSSAA

[0203] GLTASVMRDPSSRNFIMEGGAMVLADGGVVCIDEFDKMREDDRVAIHEAMEQQTISIAKAGITTTLN

[0204] SRCSVLAAANSVFGRWDETKGEDNIDFMPTILSRFDMIFIVKDEHNEERDVMLAKHVITLHVSALTQT

[0205] QAVEGEIDLAKLKKFIAYCRVKCGPRLSAEAAEKLKNRYIIMRSGARQHERDSDRRSSIPITVRQLEAIVR

[0206] IAEALSKMKLQPFATEADVEEALRLFQVSTLDAALSGTLSGVEGFTSQEDQEMLSRIEKQLKRRFAIGS

[0207] QVSEHSIIKDFTKQKYPEHAIHKVLQLMLRRGEIQHRMQRKVLYRLK

[0208] SEQ. ID. N°5 - human MCM6

[0209] MDLAAAAEPGAGSQHLEVRDEVAEKCQKLFLDFLEEFQSSDGEIKYLQLAEELIRPERNTLVVSFVDLE

[0210] QFNQQLSTTIQEEFYRVYPYLCRALKTFVKDRKEIPLAKDFYVAFQDLPTRHKIRELTSSRIGLLTRISGQV VRTHPVHPELVSGTFLCLDCQTVIRDVEQQFKYTQPNICRNPVCANRRRFLLDTNKSRFVDFQKVRIQ

[0211] ETQAELPRGSIPRSLEVILRAEAVESAQAGDKCDFTGTLIVVPDVSKLSTPGARAETNSRVSGVDGYETE

[0212] GIRGLRALGVRDLSYRLVFLACCVAPTNPRFGGKELRDEEQTAESIKNQMTVKEWEKVFEMSQDKNL

[0213] YHNLCTSLFPTIHGNDEVKRGVLLMLFGGVPKTTGEGTSLRGDINVCIVGDPSTAKSQFLKHVEEFSPR

[0214] AVYTSGKASSAAGLTAAVVRDEESHEFVIEAGALMLADNGVCCIDEFDKMDVRDQVAIHEAMEQQ.TI

[0215] SITKAGVKATLNARTSILAAANPISGHYDRSKSLKQNINLSAPIMSRFDLFFILVDECNEVTDYAIARRIVD

[0216] LHSRIEESIDRVYSLDDIRRYLLFARQFKPKISKESEDFIVEQYKHLRQRDGSGVTKSSWRITVRQLESMIR

[0217] LSEAMARMHCCDEVQPKHVKEAFRLLNKSIIRVETPDVNLDQEEEIQMEVDEGAGGINGHADSPAPV

[0218] NGINGYNEDINQESAPKASLRLGFSEYCRISNLIVLHLRKVEEEEDESALKRSELVNWYLKEIESEIDSEEE

[0219] LINKKRIIEKVIHRLTHYDHVLIELTQAGLKGSTEGSESYEEDPYLVVNPNYLLED

[0220] SEQ. ID. N°6 - human MCM7

[0221] MALKDYALEKEKVKKFLQEFYQDDELGKKQFKYGNQLVRLAHREQVALYVDLDDVAEDDPELVDSICE

[0222] NARRYAKLFADAVQELLPQYKEREVVNKDVLDVYIEHRLMMEQRSRDPGMVRSPQNQYPAELMRR

[0223] FELYFQGPSSNKPRVIREVRADSVGKLVTVRGIVTRVSEVKPKMVVATYTCDQCGAETYQPIQSPTFM

[0224] PLIMCPSQECQTNRSGGRLYLQTRGSRFIKFQEMKMQEHSDQVPVGNIPRSITVLVEGENTRIAQPG

[0225] DHVSVTGIFLPILRTGFRQVVQGLLSETYLEAHRIVKMNKSEDDESGAGELTREELRQIAEEDFYEKLAA

[0226] SIAPEIYGHEDVKKALLLLLVGGVDQSPRGMKIRGNINICLMGDPGVAKSQLLSYIDRLAPRSQYTTGR

[0227] GSSGVGLTAAVLRDSVSGELTLEGGALVLADQGVCCIDEFDKMAEADRTAIHEVMEQQTISIAKAGILT

[0228] TLNARCSILAAANPAYGRYNPRRSLEQNIQLPAALLSRFDLLWLIQDRPDRDNDLRLAQHITYVHQHSR

[0229] QPPSQFEPLDMKLMRRYIAMCREKQPMVPESLADYITAAYVEMRREAWASKDATYTSARTLLAILRL

[0230] STALARLRMVDWEKEDVNEAIRLMEMSKDSLLGDKGQTARTQRPADVIFATVRELVSGGRSVRFSE

[0231] AEQRCVSRGFTPAQFQAALDEYEELNVWQVNASRTRITFV

Claims

CLAIMS1. Inhibitor of at least one protein of the MCM complex, for its use for the prevention and / or treatment of NETosis-induced vascular occlusion.

2. Inhibitor of at least one protein of the MCM complex for its use according to claim 1, wherein said inhibitor is selected among:- quinolones, preferably fluoroquinolones,- helicase inhibitors,- Bromodomain and Extraterminal (BET) protein inhibitors,- small molecules based on furanonaphthoquinones,- Polo-kinase 1 inhibitors,- polyphenolic compounds,- 3-hydroxy-3-methylglutatyl CoA (HMG-CoA) reductase inhibitors,- Cantharidin or derivatives thereof,- CDC7 kinase inhibitors, and- proteasome inhibitors.

3. Inhibitor of at least one protein of the MCM complex for its use according to claim 1 or claim 2, wherein said inhibitor is a fluoroquinolone of formula (I):wherein:Ri represents a hydrogen atom or NH2;R2 represents a hydrogen 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 radical;R4 represents a Ci-Ce alkyl radical; preferably a C1-C4 alkyl radical and more preferably a cyclopropyl or a ethyl radical;Rs represents a hydrogen atom or a C1-C4 alkyl radical; preferably a hydrogen atom.

4. Inhibitor of at least one protein of the MCM complex for its use according to anyone of claim 1 to 3, wherein said NETosis-induced vascular occlusion occurs in a disease associated with immunothrombosis dysregulation and is selected among septic DIC; non- septic DIC; autoimmune diseases, preferably antiphospholipid syndrome or systemic lupus erythematosus (SLE); an inflammatory disease, preferably Crohn's disease or rheumatoid arthritis; myocardial infarction, stroke, venous thromboembolism, thrombo-inclusive diseases, vaso-occlusion in sickle-cell disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis; a viral disease, preferably influenza and COVID-19, and cancer.

5. Inhibitor of at least one protein of the MCM complex for its use according to anyone of claim 1 to 4, wherein the disease associated with immunothrombosis dysregulation is DIC.

6. Inhibitor of at least one protein of the MCM complex for its use according to anyone of claims 1 to 5, wherein said inhibitor is associated with compounds directly targeting coagulation pathways, such as heparin, antithrombin, thrombomodulin.

7. Method of diagnosing NETosis-induced vaso-occlusion in a subject, comprising :- determining in biological sample, preferably a blood sample, more preferably neutrophils, from a subject the biological activity or the level of expression of at least one protein of the MCM complex;- comparing said biological activity or level of expression of the at least one protein of the MCM complex to a baseline biological activity or level of expression established from at least one healthy donor or one patient without vaso-occlusion; wherein a biological activity or an expression of the at least one protein of the MCM complex in said subject higher than said baseline level identifies the subject as having or likely to develop NETosis-induced vaso-occlusion.

8. The method of claim 7, characterised in that it is combined with:- observation of thrombotic signs characterized by disseminated microthrombosis resulting in purpuric skin lesions that can progress to ischemia and necrosis, particularly in the extremities, as well as organ dysfunction related to microthrombi, leading to visceral perfusion failure; and / or- observation of hemorrhagic clinical signs which may involve petechiae, hemorrhagic bullae, sheet bleeding from mucous membranes or puncture sites, hematuria or melena; and / or- assessment of the score of the ISTH and / or the Japanese Society of Intensive Medicine (JAAM) ; and / or- measure of the increased neutrophil DNA decompaction by neutrophil-side fluorescence light (NEUT-SFL) recorded by an automated blood cell cytometer.

9. Kit for the diagnosis of DIC comprising at least one reagent being used to determine the expression level of at least one protein of the MCM complex in neutrophils.

10. A method to evaluate the efficacy of a treatment of a NETosis-induced vasoocclusion in an animal model or a patient, comprising :- determining the biological activity or the expression of at least one protein of the MCM complex in a biological sample, preferably a blood sample, taken from the animal model or the patient before administering the treatment;- determining the expression of at least one protein of the MCM complex in said biological sample taken from the patient after administering the treatment ; and - comparing said levels of expressions.

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