WO2017167763A1 - Methods for diagnosis of haemorrhagic atherothrombotic plaques - Google Patents

Abstract

The present invention relates to the prognosis and diagnosis of haemorrhagic atherothrombotic plaques.

Description

METHODS FOR DIAGNOSIS OF HAEMORRHAGIC

ATHEROTHROMBOTIC PLAQUES

FIELD OF THE INVENTION:

The present invention relates to the prognosis and diagnosis of haemorrhagic atherothrombotic plaques.

BACKGROUND OF THE INVENTION:

The presence of blood components within the vascular wall is a major determinant of vulnerability to rupture (Michel et al, 2012). Histological analysis of atherothrombotic carotid samples showed that plaque hemorrhage and increased intraplaque neovessels were independently related to clinical outcome and of clinical risk factors (Hellings et al., 2010). Also, different studies based on magnetic resonance imaging (MRI) suggest that plaque hemorrhage is predictive of clinical events (Saam et al, 2013). In carotid and coronary atherothrombosis, intraplaque hemorrhage is associated with an increased incidence of plaque rupture and subsequent clinical events (myocardial infarction or ischemic stroke).

In abdominal aortic aneurysm (AAA), the biologically active luminal thrombus that covers the initial atherosclerotic lesion plays an important role in AAA progression, as a source of proteases and oxidative insult. In both stenosing and dilating atherothrombosis, blood components bring proteases, associated with the fibrinolytic system (plasmin and plasminogen activators) or secreted by leukocytes (elastase, matrix metalloproteinases, etc.), but also represent an important source of oxidation (hemoglobin and pro-oxidant enzymes such as myeloperoxidase or NADPH oxidase) (Michel et al, 201 1; Leclercq et al, 2007a). In addition, red blood cell hemolysis leads to the release of hemoglobin-associated iron, but also contributes to the deposition of free cholesterol and enlargement of the necrotic/lipidic core, an additional factor of plaque vulnerability to rupture (Leclercq et al., 2007b). Polymorphonuclear neutrophils (PMNs) present in the thrombus may be activated and then release proteases such as cathepsin G and elastase, that are able to degrade different proteins of the extracellular matrix or of their environment such as free hemoglobin (Dejouvencel et al, 2010). The inventors have shown that the hemoglobin clearance via CD 163 (the scavenger receptor for hemoglogin/haptoglobin complexes) was decreased in hemorrhagic carotid plaques containing elastase (Moreno et al, 2012). The authors have also shown that hemoglobin chain beta could be cleaved by neutrophil proteases leading to the production of hemorphin 7 (Dejouvencel et al, 2010).

SUMMARY OF THE INVENTION:

The present invention relates to isolated, synthetic or recombinant atheglobin polypeptide or a function-conservative variant thereof.

The present invention also related to an antibody or an aptamer which specifically binds to the atheglobin polypeptide.

The present invention relates to a method of identifying a subject having or at risk of having or developing haemorrhagic atherothrombotic plaques, comprising a step of measuring in a biological sample obtained from said subject the level of atheglobin.

DETAILED DESCRIPTION OF THE INVENTION:

In the present invention, the inventors hypothesized that intraplaque hemorrhage and (hemo)-thrombus may represent a source of proteolytic-related biomarkers that are released by the atherothrombotic plaques. To test this hypothesis, tissue samples (carotids and AAA) were placed for 16 hours in culture medium without serum, in order to obtain the conditioned medium, containing proteins and peptides that diffused from the athero-thrombotic lesions. These samples were subsequently analyzed by SELDI-TOF MS (surface-enhanced laser desorption-ionization time of flight mass spectrometry), a method that favors detection of low molecular mass proteins and peptides. Finally, MALDI-TOF imaging MS was performed on cryosection of AAA and carotid samples for assessing, in situ, the localization of the biomarkers generated by proteolysis and identified by SELDI-TOF MS coupled with MS-MS techniques.

The inventors demonstrated that hemorrhagic samples display different profiles relative to non-hemorrhagic samples. Among the differential peptides detected, 67 of them were more abundant in hemorrhagic versus non-hemorrhagic samples (on a total of 371 peaks detected). Among these differential peaks, the m/z 3327 exhibited the highest intensity in hemorraghic compared to non-hemorrhagic carotid samples (349 ± 47 vs 160 ± 47, p=0.01). Furthermore, the m/z 3327 peak was abundantly released by the luminal layer of the thrombus of abdominal aortic aneurysm samples compared to intermediate and abluminal samples. This peptide was identified by MS-MS sequencing as a fragment of the alpha-chain of hemoglobin, corresponding to the 32 first amino acids. Polypeptide of the invention:

The present invention relates to isolated, synthetic or recombinant atheglobin polypeptide or a function-conservative variant thereof.

The term "atheglobin" refers to SEQ ID NO: 1, that corresponds to 32 amino acids of the N-terminal end of the alpha-chain of hemoglobin and that represents 3327 Da fragment of hemoglobin. SEQ ID NO: 1 for atheglobin:

VL SP ADKTN VKA A WGKVG AH AGE YG AE ALERM

In some embodiments, the polypeptide of the present invention comprises or consists of an amino acid sequence having at least 70% of identity with SEQ ID NO: 1.

According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81 ; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100%) of identity with the second amino acid sequence. Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990). In particular the polypeptide of the invention is a functional conservative variant of the polypeptide according to the invention. As used herein the term "function-conservative variant" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a "function-conservative variant" also includes a polypeptide which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent polypeptide to which it is compared. Functional properties of the polypeptide of the invention could typically be assessed in any functional assay as described in the EXAMPLE.

The polypeptides of the invention are produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. For instance, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides, by standard techniques for production of amino acid sequences. For instance, they can be synthesized using well- known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the polypeptides of the invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (polypeptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

Polypeptides of the invention can be used in an isolated (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).

In specific embodiments, it is contemplated that polypeptides according to the invention may be modified in order to improve their stability using well-known techniques. The polypeptides of the invention can be modified by the utilisation of water-soluble polymers. Pegylation is a well-established and validated approach for the modification of a range of polypeptides (Chapman, 2002). The polypeptides of the invention may be covalently linked with one or more polyethylene glycol (PEG) group(s). One skilled in the art can select a suitable molecular mass for PEG. In some embodiments, additional sites for PEGylation can be introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue. In some embodiments, additional PEGylation sites are chemically introduced by modifying amino acids on polypeptides of the invention. In some embodiments, PEGs are conjugated to the polypeptides through a linker. Suitable linkers are well known to the skilled person.

Nucleic acids, vectors and recombinant host cells:

A further object of the present invention relates to a nucleic acid sequence encoding for the polypeptide according to the invention. As used herein, a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

These nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art. Typically, said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

So, a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a polypeptide of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted. These recombinant vectors may, for example, be cloning vectors, or expression vectors.

As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

A further aspect of the invention relates to a host cell comprising a nucleic acid molecule encoding for a polypeptide according to the invention or a vector according to the invention. In particular, a subject of the present invention is a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule or vector according to the invention. A further aspect of the invention relates to a method for producing a polypeptide of the invention comprising the step consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said polypeptide; and (ii) recovering the expressed polypeptide. Aptamers and antibodies: The present invention also related to an antibody or an aptamer which specifically binds to the polypeptide of the present invention.

In some embodiments, the antibody or aptamer of the present invention specifically bind to the polypeptide which comprises or consists of a sequence having at least 70% of identity with the polypeptide of the invention.

In some embodiments, the antibody or aptamer of the present invention specifically bind to a conformational epitope of the polypeptide of the invention or specifically bind the proteolysis zone such as the N-terminal and C-terminal end of the polypeptide of the invention.

In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1 , L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.

The term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.

The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

The term "Fab' " refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.

A single chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. "dsFv" is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

Monoclonal antibodies useful in the invention may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with the appropriate antigenic forms (i.e. polypeptides of the present invention). The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.

Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Diagnostics methods:

Accordingly, the present invention relates to a method of identifying a subject having or at risk of having or developing haemorrhagic atherothrombotic plaques, comprising a step of measuring in a biological sample obtained from said subject the level of atheglobin.

As used herein, the term "subject" denotes a human subject. In a preferred embodiment of the invention, a subject according to the invention refers to any human subject afflicted with or susceptible to be afflicted with haemorrhagic atherothrombotic plaques. In a particular embodiment of the invention, a subject according to the invention refers to any human subject afflicted with haemorrhagic atherothrombotic plaques at risk of having or developing vascular event.

The term "haemorrhagic atherothrombotic plaques" has its general meaning in the art and refers to plaque haemorrhage and increased intraplaque neovessels that are related to clinical outcome and vascular events (Michel et al., 2011). The term "haemorrhagic atherothrombotic plaques" also relates to the presence of blood components (red blood cells) within the vascular wall that is a major determinant of vulnerability to rupture (1, 2). The term "haemorrhagic atherothrombotic plaques" also relates to conditions in which blood components are in presence of leukocytes and associated proteases. The term "haemorrhagic atherothrombotic plaques" also relates to intraplaque hemorrhage (IPH), carotid and coronary atherothrombosis, intraluminal thrombus (ILT), luminal thrombus, abdominal aortic aneurysm (AAA) including AAA that may be prone to rupture, haemorrhagic stroke, Hemorrhagic transformation (HT) which is a frequent complication of ischemic stroke (Zhang et al., 2014).

The term "biological sample" refers to any biological sample derived from the subject such as blood sample, plasma sample, serum sample, urine sample, saliva sample, or cerebrospinal fluid sample.

The method of the invention may further comprise a step consisting of comparing the level of atheglobin in the biological sample with a reference value, wherein detecting differential in the level of the atheglobin between the biological sample and the reference value is indicative of subject having or at risk of having or developing haemorrhagic atherothrombotic plaques.

As used herein, the "reference value" refers to a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the polypeptide level (obtained according to the method of the invention) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the polypeptide level (or ratio, or score) determined in a biological sample derived from one or more subjects having haemorrhagic atherothrombotic plaques. Furthermore, retrospective measurement of the polypeptide level (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values. In one embodiment, the reference value may correspond to the level determined in a biological sample associated with a healthy subject not afflicted with haemorrhagic atherothrombotic plaques. Accordingly, a higher level of atheglobin than the reference value is indicative of a subject having or at risk of having or developing haemorrhagic atherothrombotic plaques, and a lower or equal level of atheglobin than the reference value is indicative of a subject not having or not at risk of having or developing haemorrhagic atherothrombotic plaques.

In another embodiment, the reference value may correspond to the expression level determined in a biological sample associated with a subject afflicted with haemorrhagic atherothrombotic plaques. Accordingly, a higher or equal expression level of atheglobin than the reference value is indicative of a subject having or at risk of having or developing haemorrhagic atherothrombotic plaques, and a lower expression level of atheglobin than the reference value is indicative of a subject not having or not at risk of having or developing haemorrhagic atherothrombotic plaques.

Methods for measuring the level of a polypeptide in a biological sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for measuring the level of a polypeptide including, but not limited to, direct methods like mass spectrometry-based quantification methods, MALDI-TOF spectrometer, orbitrap, surface- enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) With or without prior fractionnation techniques such as HPLC or other type of chromatography, protein microarray methods, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, Mesoscale discovery (MSD), Luminex, ELISPOT and Enzyme Linked Immunoabsorbant Assay (ELISA).

Said direct analysis can be assessed by contacting the biological sample with a binding partner capable of selectively interacting with the polypeptide present in the biological sample. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal (e.g., a isotope-labeled, element-labeled, radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the polypeptide. In some embodiments, the binding partner may be the antibody of the invention. In another embodiment, the binding partner may be the aptamer of the invention.

The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as an isotope, a chemical element, a fluorescent molecule, a radioactive molecule, an enzyme or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as an isotope, an element, a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be produced with a specific isotope or a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited to radioactive atom for scintigraphic studies and positron emission tomography (PET) such as 1123, 1124, Inl l l, Rel86, Rel88, specific isotopes include but are not limited to 13C, 15N, 1261, 79Br, 81 Br.

The aforementioned assays generally involve the binding of the binding partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include an ELISA plate, an ELIspot plate, a bead (e.g., a cytometric bead, a magnetic bead), a microarray (e.g., a SIMS microarray), a slide or a plate. Said supports may e.g., be coated with substrates such as nitrocellulose (e. g., in glass, membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, silicon wafers.

In a particular embodiment, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said polypeptide. A biological sample containing or suspected of containing said polypeptides is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody- antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art such as Singulex, Quanterix, MSD, Bioscale, Cytof.

In one embodiment, an Enzyme-linked immunospot (ELISpot) method may be used. Typically, the biological sample is transferred to a plate which has been coated with the desired anti-polypeptide capture antibodies. Revelation is carried out with biotinylated secondary Abs and standard colorimetric or fluorimetric detection methods such as streptavidin-alkaline phosphatase and NBT-BCIP and the spots counted. In one embodiment, when multi-polypeptide level measurement is required, use of beads bearing binding partners of interest may be preferred. In a particular embodiment, the bead may be a cytometric bead for use in flow cytometry. Such beads may for example correspond to BD™ Cytometric Beads commercialized by BD Biosciences (San Jose, California) or LUMINEX® beads or ERENNA® (SINGULEX®) beads. Typically cytometric beads may be suitable for preparing a multiplexed bead assay. A multiplexed bead assay, such as, for example, the BD^(TM) Cytometric Bead Array, is a series of spectrally discrete beads that can be used to capture and quantify soluble antigens. Typically, beads are labelled with one or more spectrally distinct fluorescent dyes, and detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. A number of methods of making and using sets of distinguishable beads have been described in the literature. These include beads distinguishable by size, wherein each size bead is coated with a different target-specific antibody (see e.g. Fulwyler and McHugh, 1990, Methods in Cell Biology 33:613-629), beads with two or more fluorescent dyes at varying concentrations, wherein the beads are identified by the levels of fluorescence dyes (see e.g. European Patent No. 0 126,450), and beads distinguishably labelled with two different dyes, wherein the beads are identified by separately measuring the fluorescence intensity of each of the dyes (see e.g. U.S. patent Nos. 4,499,052 and 4,717,655). Both one-dimensional and two-dimensional arrays for the simultaneous analysis of multiple antigens by flow cytometry are available commercially. Examples of one-dimensional arrays of singly dyed beads distinguishable by the level of fluorescence intensity include the BD¹-™ Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) and Cyto-Plex^(TM) Flow Cytometry microspheres (Duke Scientific, Palo Alto, Calif). An example of a two-dimensional array of beads distinguishable by a combination of fluorescence intensity (five levels) and size (two sizes) is the QuantumPlex¹-™^ microspheres (Bangs Laboratories, Fisher, Ind.). Another example is the SIMOA™ technology (QUANTERIX™). An example of a two-dimensional array of doubly- dyed beads distinguishable by the levels of fluorescence of each of the two dyes is described in Fulton et al. (1997, Clinical Chemistry 43(9): 1749-1756). The beads may be labelled with any fluorescent compound known in the art such as e.g. FITC (FL1), PE (FL2), fluorophores for use in the blue laser (e.g. PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use in the red, violet or UV laser (e.g. Pacific blue, pacific orange). In another particular embodiment, bead is a magnetic bead for use in magnetic separation. Magnetic beads are known to those of skill in the art. Typically, the magnetic bead is preferably made of a magnetic material selected from the group consisting of metals (e.g. ferrum, cobalt and nickel), an alloy thereof and an oxide thereof. In another particular embodiment, bead is bead that is dyed and magnetized.

In another particular embodiment, beads are labeled with an isotope or a (chemical) element, and beads are identified by elemental analysis in a mass spectrometer (Cytof).

In one embodiment, protein microarray methods may be used. Typically, at least one antibody or aptamer directed against the polypeptide(s) is immobilized or grafted to an array(s), a solid or semi-solid surface(s). A biological sample containing or suspected of containing the polypeptide(s) is then labelled with at least one isotope or one element or a reactive tag or one fluorophore or one colorimetric tag that are not naturally contained in the tested biological sample. After a period of incubation of said biological sample with the array sufficient to allow the formation of antibody-antigen complexes, the array is then washed and dried. After all, quantifying said polypeptides may be achieved using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner, electrochemo luminescent scanner or any technique allowing to quantify said labels. In another embodiment, the antibody or aptamer grafted on the array is labelled.

In one embodiment, a mass spectrometry-based quantification methods may be used. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches (DeSouza and Siu, 2012). Mass spectrometry-based quantification methods may be performed using chemical labeling, metabolic labeling or proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, LTQ Orbitrap Velos, LTQ-MS/MS, a quantification based on extracted ion chromatogram EIC (progenesis LC-MS, Liquid chromatography-mass spectrometry) and then profile alignement to determine differential level of polypeptides. In one embodiment, ELISA sandwich specifically designed to measure the polypeptide of the invention may be used. The principle is a sandwich ELISA with a capturing antibody against the C-terminus and the second antibody is against the N-terminus. This sandwich ELISA gives the concentration of the polypeptide.

In some embodiments, the subject having or at risk of having or developing haemorrhagic atherothrombotic plaques produces anti-atheglobin autoantibodies specific for atheglobin polypeptide of the invention. In some embodiments, said anti-atheglobin autoantibodies specifically bind to a conformational epitope of the polypeptide of the invention or specifically bind the proteolysis zone such as the N-terminal and C-terminal end of the polypeptide of the invention.

The term "autoantibody", as used herein, has its general meaning in the art, and refers to an antibody that is produced by the immune system of a subject and that is directed against subject's own polypeptides.

In a further aspect, measuring the level of atheglobin is performed by measuring anti- atheglobin autoantibodies.

Accordingly, a further aspect of the invention relates to a method of identifying a subject having or at risk of having or developing haemorrhagic atherothrombotic plaques, comprising a step of measuring in a biological sample obtained from said subject the level of anti-atheglobin autoantibodies.

Methods for measuring the level of an autoantibody in a biological sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for measuring the level of an autoantibody including, but not limited to, direct or indirect detection in an assay of suitable format (e.g., ELISA), using atheglobin polypeptide of the invention, a conformational epitope of the polypeptide of the invention or an atheglobin proteolysis zone such as the N-terminal and C-terminal end of the polypeptide of the invention as specific atheglobin antigen. The atheglobin antigen can be provided in a purified or substantially purified form in a solution, bound to a solid support (such as beads, chips, chromatography matrices, or microtiter plates), or expressed from whole cells or a phage display system, suitable for binding and detection of anti-atheglobin autoantibodies.

In a specific embodiment, anti-atheglobin autoantibodies are detected in an enzyme- linked immunosorbent assay (ELISA) using atheglobin antigen coated to an ELISA plate. In another embodiment, anti-atheglobin autoantibodies are detected in a flow cytometry analysis using atheglobin antigen-coated beads. In still another embodiment, phage particles expressing an atheglobin antigen can be anchored, for example, to a multiwell plate via an antiphage antibody.

In a further aspect, the method of the invention can be used in a method of identifying a subject at risk of having or developing vascular events.

The term "vascular event" has its general meaning in the art and refers to adverse clinical events subsequent to vascular diseases. The term "vascular event" also refers to adverse cardiovascular events such as sudden death, myocardial infarction, angina, ischemia and other chest pain, adverse cerebrovascular events such as stroke, ischemic stroke, aneurysm and other adverse vascular events such as rupture and plaque rupture (Bolland et al, 2008; Hellings et al, 2010; Samm et al, 2013).

A further aspect of the invention relates to a method of monitoring haemorrhagic atherothrombotic plaques progression by performing the method of the invention.

A further aspect of the invention relates to a method of monitoring haemorrhagic atherothrombotic plaques treatment by performing the method of the invention.

In one embodiment, the present invention relates to a method of treating haemorrhagic atherothrombotic plaques in a subject in need thereof comprising the steps of:

(i) identifying a subject having or at risk of having or developing a haemorrhagic atherothrombotic plaques by performing the method according to the invention, and

(ii) administering to said subject a vascular disease treatment. In another embodiment, the present invention relates to a method of treating vascular event in a subject in need thereof comprising the steps of:

(i) identifying a subject afflicted with haemorrhagic atherothrombotic plaques at risk of having or developing vascular event by performing the method according to the invention, and

(ii) administering to said subject a vascular disease treatment.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). The term "vascular disease treatment" has its general meaning in the art and refers to angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, beta blockers, calcium channel blockers, acetylsalicylate, antiplatelets agents, anticlotting agents, fibrinolytic agents, LDL-cholesterol lowering drugs including statins and PCSK9 inhibitors, HDL-raising drugs such as niacin (vitamin B3 or nicotinic acid) or CETP inhibitors.

Kits:

The invention also relates to a kit for performing the methods as above described, wherein said kit comprises means for measuring the level of atheglobin that is indicative of subject having or at risk of having or developing haemorrhagic atherothrombotic plaques. In particular embodiment the kit comprises means for measuring the level of atheglobin. Typically the kit may include an antibody, or a set of antibodies as above described. In a particular embodiment, the antibody or set of antibodies are labelled as above described. In particular embodiment the kit may comprise at least one antibody directed to atheglobin. In a particular embodiment the kit comprises means for measuring the level of anti-atheglobin autoantibodies. Typically the kit may include an atheglobin antigen, or a set of atheglobin antigens as above described. The kit may also contain labels, other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: SELDI-TOF MS detection of a 3327-Da peak released preferentially by hemorrhagic carotid samples and by the luminal layer of AAA intraluminal thrombi.

A, C, Representative mass spectra showing the m/z 3327 peak in the medium conditioned by carotid samples with (n=33) or without hemorrhage (n=14) (panel A) and AAA intraluminal thrombus layers (panel B, n=9). B,D, Quantification of m/z 3327 peak intensities (arbitrary units) in carotid- and AAA intraluminal thrombus-conditioned media. * p<0.05 (Mann- Whitney). Figure 2: MALDI-TOF imaging MS and histological analysis of ILT samples

A. Bright field image of the thrombus section analyzed.

B-D. In situ distribution of m/z 3327 (B), m/z 15258 (hemoglobin alpha chain) (C) and merged images (D) in a representative AAA thrombus section after MALDI-TOF imaging MS analysis. The white arrows show areas where hemoglobin but not the 3327-Da peak was detected in the deep luminal layer of the AAA thrombus.

EXAMPLE:

Material & Methods

Carotid and AAA samples

Carotid samples (n=47) were obtained from patients undergoing endarterectomy, after giving their informed consent. This study (Biobanque en pathologie atherothrombotique carotidienne extracranienne chirurgicale) has been approved by the local Ethics committee, CCP Ambroise Pare SC-090966 (lie de France VIII).

AAA tissue samples (n=9 ITLs) were obtained from patients undergoing surgery and enrolled in the RESAA protocol (REflet Sanguin de l'evolutivite des Anevrysmes de l'Aorte abdominale). All patients gave informed written consent, and the protocol was approved by a French ethics committee (CCP Paris-Cochin, approval no 2095) (Caligiuri et al., 2006).

Carotid and AAA sample collections were declared to the French Ministry of Research (DC-2008-283).

Conditioned medium

Carotid endarterectomy samples were separated into culprit and non-culprit plaques respectively corresponding to the stenosing and the adjacent non-stenosing parts of the same samples (Duran et al, 2003). For AAA samples, the intraluminal thrombus was dissected into three parts: luminal (at the interface with the circulating blood), intermediate, and ab luminal layers. Carotid culprit and non-complicated plaque samples, as well as each layer of the AAA thrombus, were cut into small pieces (5 mm³), separately incubated (24 hours at 37°C) in a standardized volume (6 mL/g of wet tissue) of RPMI 1640 medium supplemented with antibiotics and an antimycotic. The conditioned medium was centrifuged (3000g for 10 min), and the supernatant was aliquoted and frozen at -80°C until use.

Heme assay and group attribution Heme content (considered as being proportional to hemoglobin release) was assessed by addition of formic acid to the conditioned media (v/v 30/70). OD was monitored at 405 nm, after binding of formic acid to the heme. Bovine hemoglobin was used as a standard. The hemorrhagic and non-hemorrhagic carotid groups were defined with respect to the heme concentration in the conditioned media.

Histology and immunohistochemistry

Carotid and AAA samples were embedded in paraffin and cut into 5- Dm sections. Serial sections were stained with hematoxylin/eosin to visualize cells and nuclei.

For immunofluorescence, sections were incubated for 1 h with the primary antibody, anti-elastase (monoclonal mouse anti-human, dilution 1 :50, Hycult HM2174) or anti- glycophorin A (monoclonal mouse anti-human, dilution 1 :50, Dako, M0819) at room temperature, followed by three washing steps with PBS and incubation with the appropriate secondary antibody (goat anti-mouse IgG) conjugated to Alexa Fluor 488 (Life Technologies, France) for 1 h at room temperature. DAPI (4',6'-diamidino-2-phenylindole, 100 ng/niL, Sigma Aldrich) was used for nuclear staining.

Preparation of fMLP-induced Polymorphonuclear (PMN) releasates

PMNs were obtained from EDTA venous blood after sedimentation of hemaglutinated erythrocytes by 2% dextran followed by Ficoll-Paque (GE Healthcare, France) separation of leukocytes and hypo-osmotic lysis of residual erythrocytes in the pellet. PMN were maintained in Hank's Buffered Salt Solution (Life Technologies, France). Formyl-Met-Leu- Phe (fMLP, Sigma Aldrich, France) was used as a stimulant of PMN degranulation. Briefly, fMLP (1 μΜ) was added to PMNs (10⁶ cells/mL) and incubated for 2 hours at 37°C. Then, the cell suspension was centrifuged (1500g, 5 min) and the supernatant, fMLP-induced PMN releasate, was aliquoted and frozen at -80 °C until use.

In Vitro digestion of hemoglobin by proteases

Human hemoglobin (60 μg; Sigma Aldrich, France) was incubated with fMLP- induced PMN releasates or with purified proteases: proteinase 3 (100 nM; Elastin Products), cathepsin G (250 nM; kindly provided by Dr Dominique Pidard), plasmin (60 nM ; Merck Millipore, France), elastase (250 nM; Merck Millipore, France) at pH 7.4 for 4 hours at 37°C and cathepsin D (220 nM; Sigma- Aldrich) at pH 3.2. Analysis of the fragments was performed by SELDI-TOF-MS using NP20 ProteinChip. SELDI-TOF MS analysis

Conditioned media from each layer of 9 AAA ILTs and 47 carotid samples (33 hemorragic and 14 non-hemorragic) were analyzed with CM 10 (Weak Cation Exchanger) ProteinChip Arrays (Bio-Rad Laboratories, USA). The CM 10 arrays were pre-equilibrated for 5 min in 100 binding buffer (100 mmol/L sodium acetate, pH 4) before incubation with samples. Twenty micrograms of total proteins from each sample were mixed the binding buffer (total incubation volume = 200 μί). After 90 minutes of incubation with gentle shaking, the protein arrays were washed twice with 200 of binding buffer. Finally, a last wash was performed with water and arrays were allowed to air-dry before addition of 2 x 1 μΐ_^ of a saturated solution of sinapinic acid in 10 % v/v acetronitrile and 0.1% trifluoroacetic acid. Finally, the spots were allowed to dry completely before SELDI-TOF MS analysis. The m/z values of proteins/peptides (1,000 to 500,000 Da) retained on each chromatographic surface were determined from time-of- flight measurements using a ProteinChip Reader (PCS 4000; Bio-Rad Laboratories, USA). Peak intensities were normalized by using the mean total ion current of all spectra and then analyzed by Biomarker Wizard software (Bio-Rad Laboratories, USA).

MALDI-Imaging mass spectrometry

Each sample was cryosectioned and 10 μι -ΐΐι^ sections were carefully placed onto conductive indium tin oxide-coated glass slides (Bruker Daltonics, Germany), vacuum-dried, briefly washed in 70 and 100% ethanol, dried again and directly covered with the matrix. The MALDI matrix was applied using the ImagePrep station (Bruker Daltonics, Germany). The matrix chosen was sinapinic acid at 10 mg/mL in water/acetonitrile 40:60 (v/v) with 0.2% trifluoroacetic acid. MALDI analysis was performed on an Autoflex III MALDI-TOF/TOF mass spectrometer with a Smartbeam laser using FlexControl 3.0 and Flexlmaging 2.1 software packages (Bruker Daltonics, Germany). Ions were detected in positive linear mode at a mass range of m/z 2000-20000 with a sampling rate of 0.1 GS/s. The lateral resolution (distance between raster points) was set to 200 μιη and a total of 500 laser shots were accumulated per pixel at constant laser power. A ready-made protein standard (Bruker Daltonics) was used for calibration of spectra, which was performed externally on the same target before each measurement (Le Faouder et al, 2011). For MS/MS acquisition in situ, the HCCA matrix at 7 mg/mL in water/acetonitrile 40:60 (v/v) with 0.2% trifluoroacetic acid was applied to the tissue section. Peptide identification

The m/z 3327 and m/z 2884 peaks were identified after elimination of proteins >10kDa using Centricon Plus-70, Ultracel-PL membrane lOkDa (Millipore, France). Briefly, 240 μΐ, of AAA and carodid conditioned medium samples (diluted 1 :2 in H₂0) were filtered by centrifugation at 14,000 g until all the sample was in the bottom part. The flow-through containing the peak of interest was concentrated by speed-vac and submitted to nano-HPLC before analysis by a MALDI TOF/TOF ABI 4800 + (Applied Biosystems) equipped with a YAG-200 Hz laser (355 nm). Mass spectra acquisition and processing were performed using the 4000 Series Explorer software (ABI) version 3.5.1. (Proteomic platform, Institut Claude Bernard and Institut Jacques Monod Paris, France). In addition, MS analysis in reflectron mode followed by MS/MS analysis (MALDI-TOF/TOF autoflex III) was performed on tissue sections. Generated MS/MS peaklists were submitted to an in-house Mascot (Matrix Science, Boston, MA) search engine (Database Search SwissProt, precursor tolerance: 100 ppm, MS/MS tolerance: 0.3 Da).

In situ digestion of hemoglobin by elastase

A solution of elastase (ΙμΜ) was deposited over the entire sample sections for one hour at room temperature. After the incubation, a matrix was added and the analysis by MALDI-IMS was performed.

Statistical analysis

Results are expressed as mean±sem and differences between groups were assessed by Mann-whitney tests(Prism 5, GraphPad software).

Results

SELDI-TOF MS profiles of atherothrombotic samples detect a 3327 Da fragment of hemoglobin

Medium conditioned by human carotid endarterectomy samples were analyzed by SELDI-TOF MS. Proteins and peptides were retained on a cation-exchange surface (CM 10) before TOF MS. Representative mass spectra presented in Figure 1A show that hemorrhagic samples display different profiles relative to non- hemorrhagic samples. Among the differential peptides detected, 67 of them were more abundant in hemorrhagic versus non- hemorrhagic samples (on a total of 371 peaks detected). Among these differential peaks, the m/z 3327 exhibited the highest intensity in hemorraghic compared to non-hemorrhagic carotid samples (349 ± 47 vs 160 ± 47, p=0.01) as show in Figure IB.

In parallel, we analyzed the different media conditioned by the ILT of human AAA samples. The three layers (luminal, intermediate and ab luminal) were separately incubated in culture medium and analyzed by SELDI-TOF MS. As shown in Figure 1C, the m/z 3327 peak was abundantly released by the luminal layer of the thrombus (luminal vs intermediate, p=0.01, luminal vs abluminal, p=0.01, Figure ID). This peptide, associated with the presence of hemoglobin in IPH and ILT, was identified by MS-MS sequencing as a fragment corresponding to the 32 first amino acids of the alpha-chain of hemoglobin

Identification of proteases responsible for the generation of m/z 3327

The presence of a RBC-rich clot in the vascular wall is characterized by an accumulation of different sources of proteases, including those from the fibrinolytic system and those conveyed by leukocytes (Leclercq et al, 2007a; Leclercq et al, 2007b; Fontaine et al, 2004). The proteases proteinase 3, cathepsin G, plasmin, cathepsin D (in acidic conditions) and elastase were incubated with purified human hemoglobin and then analyzed by SELDI-TOF MS. Plasmin and proteinase 3 were able to generate proteolytic fragments, but not the m z 3327 peak. Among the proteases tested, elastase, cathepsin G and cathepsin D were shown to produce the 3327-Da hemoglobin fragment, paralleled by a decrease in the two peaks corresponding to the alpha- and beta-chains of hemoglobin (15,258 and 15,998 Da respectively). Since these enzymes are mainly produced by polymorphonuclear neutrophils (PMNs), we tested the capacity of activated PMN supernatant to proteolyse hemoglobin. Our results suggest that PMNs participate in the production of the 3327-Da fragment, as confirmed by MS-MS sequencing.

MALDI-TOF imaging MS of carotid and AAA thrombus samples and immunodetection of elastase

Frozen sections of AAA thrombus and carotid samples were sprayed with a matrix (sinapinic acid or a-Cyano-4-hydroxycinnamic acid) before in situ MALDI-imaging MS analysis (Figure 2 A-D). This technique allows the detection of peptides and polypeptides contained in each pixel of the tissue section. Peak intensities corresponding to m/z 3327 and m/z 15,258 (hemoglobin alpha-chain) are shown in the reconstructed images of AAA thrombus and hemorrhagic carotid sections. In AAA thrombus samples, hemoglobin chains and the 3327-Da peptide were chiefly detected in the most luminal layer as compared to the intermediate and ab luminal layers (Figure 2B-D). However, the hemoglobin peak was observed in deep areas of the luminal layer where the 3327-Da peak was not detected (Figure 2B and D). Immunodetection of elastase and hemoglobin/hemosiderin auto-fluorescence were intense in the most luminal layer of the thrombus, corresponding to areas in which the 3327 Da peptide was particularly abundant. This area is rich in PMNs, as shown by the polylobed aspect of the nuclei relative to the intermediate and abluminal layers, almost devoid of cells.

In carotid samples, we shown by MALDI-imaging MS that the hemoglobin signal was abundant in intraplaque hemorrhage, but the 3327-Da peptide was only detected in some particular areas. Immuno-fluorescence detection of elastase was performed on adjacent serial sections and the auto-fluorescence of hemosiderin is shown in red. A positive staining for elastase was observed in areas containing both RBCs, polynuclear cells (H&E staining) and the 3327 Da peptide (detected in adjacent section analyzed by MS imaging). These results suggest that hemoglobin may represent in vivo, a substrate for elastase released by neutrophils leading to the generation of proteolytic peptides, including the 3327 Da fragment.

In situ proteolysis of hemoglobin by exogenous elastase on hemorrhagic carotid sections

In order to demonstrate the capacity of elastase to generate the m/z 3327 peptide in situ, purified neutrophil elastase was sprayed directly onto sections of hemorrhagic carotid samples before MALDI imaging analysis. Serial sections were treated or not with elastase. The degradation of hemoglobin (15,258 alpha chain) was observed in the presence of elastase, paralleled by the generation of the 3327 Da peptide.

Discussion

Atherothrombotic complications of an atheromatous plaque are characterized by the presence of blood within the lesion, including cells and plasma components, due to plaque rupture or to the leakage of neo vessels originating from the adventitia (Michel et al, 2014). Intraplaque hemorrhage (IPH) in obstructive atherothrombosis and intraluminal thrombus (ILT) in AAA represent major source of oxidative products and of proteolytic enzymes able to destabilize the vessel wall.

In the present study, we aimed at unveiling peptides diffusing from hemorrhagic atherothrombotic plaques and AAA thrombus. We chose to analyze the medium conditioned by different parts of atherothrombotic tissue by separating hemorrhagic and non-hemorrhagic carotid samples or the different layers of AAA ILTs. This medium reflects the active or passive release of potential peptides issued from a specific vascular tissue area that could be identified either by candidate-based approaches or by open strategies such as proteomics, in order to be researched in plasma, in a second step (Meilhac et al, 2007).

Different proteomic approaches have been used to address the discovery of biomarkers in human atherosclerotic samples, in particular 2D-gel analysis (Duran et al., 2003; Martin- Ventura et al, 2004). Either conditioned medium or tissue extracts were used. Tissue homogenates were used to discover osteopontin as a potential marker for primary outcome in carotid and femoral atherosclerosis patients (DE Kleijn et al., 2010). Malaud et al. performed 2D analysis on human carotid samples, comparing fibro-atheroma with hemorrhagic plaques. As expected, they found hemoglobin to be more abundant in plaques with intraplaque hemorrhage paralleled by a decreased antiprotease content (Malaud et al, 2014). In our study, most of the differential peaks identified by SELDI-TOF MS analysis were released by hemorrhagic relative to non-hemorrhagic samples. This may be due to an intense biological activity that characterizes the culprit blood-containing atherothombotic carotid samples. Also, the presence of proteases in atherothrombotic samples (Simon and Jain, 2011) may produce protein fragments that are readily detectable by SELDI-TOF MS, particularly well suited for the detection of peptides/polypeptides < 20kDa.

In AAA ILT layers, most of the biological activities are localized within the luminal part, chiefly due to the presence of RBCs, leukocytes and associated proteases. In contrast, few other peptides were found to be more abundant in the abluminal part of the thrombus, suggesting a spatio-temporal organization of the ILT.

Different proteases have been reported to be abundant in atherothrombotic lesions, being either overexpressed by vascular and inflammatory cells or conveyed by the blood in their zymogen form. Polymorphonuclear neutrophils (PMNs) represent an important source of proteases, including elastase, cathepsin G, D and proteinase 3 (Korkmaz et al, 2010). We have previously reported that elastase was one of the most powerful proteases in human carotid atherothrombotic samples, able to convert MMPs into their active forms (Leclercq et al, 2007a). Similarly, leucocyte elastase is present in large quantities in the most luminal layer of ILT (Houard et al., 2007; Fontaine et al., 2004). (Hemo)globin cleavage by cathepsin D has been described extensively, leading to the production of different fragments of both alpha and beta chains (Fruitier et al., 1998). In particular, the hemorphin family, corresponding to small hemoglobin proteolytic fragments of the beta chain including LVV-H7 beta (30-40) and VV-H7 (31-40), has been reported to be generated by cathepsin D (Fruitier et al, 1998), but also by elastase and cathepsin G (Dejouvencel et al, 2010). These hemoglobin peptides were shown to be released by AAA samples and their levels were found to be decreased in diabetic conditions, potentially due to hemoglobin glycation which induces resistance to proteolysis (Fruiter et al., 2003). The m/z 1309 (LVV-H7 30-40) was reported by Fruitier et al. after digestion by cathepsin D (Fruitier et al, 1998) in a study that investigated hemoglobin peptides produced by digestion with gingipains (Lewis et al, 1999).

The biological functions of the 1309-Da peptide (LVV-H7) have not been completely elucidated. We have shown that this hemoglobin fragment is able to moderately promote the recruitment of neutrophils (Dejouvencel et al., 2010). Other groups have shown that different peptides originating from the alpha-chain of hemoglobin display antibacterial activities; for instance, the alpha 1-23 peptide (2239 Da) was shown to limit the proliferation of Micrococcus luteus (Froidevaux et al, 2001). Lysine-gingipain (Kgp) protease from P. gingivalis has been reported to produce hemoglobin fragments (Lewis et al, 1999). In another study, Dashper et al. demonstrated that whole bacterial cells were able to generate hemoglobin peptides via both RgpA/B and Kgp proteases (Dashper et al, 2004). A fragment corresponding to the alpha chain (1-31) was described in this publication. We have recently reported that periodontal bacteria, and in particular P. gingivalis, may be present within atherothrombotic plaques (Range et al, 2014) as well as in AAA human samples (Delbosc et al, 2011). The simultaneous presence of extracellular hemoglobin in the thrombus of AAA or in hemorrhagic carotid samples, suggest that the digestion of globin by gingipains may occur in vivo.

It is noteworthy that no statistically significant difference could be observed between the non-complicated (NP) and the culprit part (CP) of the same carotid sample (data not shown). This shows that the NP from a hemorrhagic carotid sample was able to release large amounts of m/z 3327, suggesting that the macroscopic separation of the specimen did not allow us to discard microscopic intraplaque hemorrhages. Even minimal blood contamination should thus give rise to the production of m/z 3327 peak provided that neutrophil proteases (such as elastase or PR3) are present.

In the present study, we tested the capacity of neutrophils to generate hemoglobin fragments. We demonstrated that the supernatant of fMLP-activated neutrophils incubated with human hemoglobin produced a 3327 Da peptide that corresponded to the Cterm first 32 aa of the alpha-chain of hemoglobin. In vitro, elastase, cathepsin G and cathepsin D were shown to generate the m/z 3327 fragment whereas neither plasmin nor PR3 were able to produce this hemoglobin peptide. We cannot exclude that other proteases may participate in the generation of this biomarker, such as gingipains.

MALDI-TOF mass spectrometry imaging represents an in situ proteomic technique that gives complementary spatial information to that provided by SELDI-TOF MS in conditioned medium. Only a few papers have aimed at imaging atherosclerotic plaques using MALDI-TOF. Martin-Lorenzo et al. recently reported that thymosin beta4 was increased in the intimal area of the aorta in rabbits and humans (Martin-Lorenzo et al., 2015). This study and others have reported lipid changes in atheromatous versus non-atheromatous samples using MALDI-TOF imaging (Castro-Perez et al, 2014; Zaima et al, 2011) or SIMS (secondary-ion mass spectrometry) (Mas et al, 2007; Lehti et al, 2015). Here, we report for the first time MALDI-TOF imaging analysis on human vulnerable hemorrhagic plaques and thrombus of AAA. Interestingly, hemoglobin alpha and beta chains were detected in their intact forms (respectively 15,258 and 15,998 Da), corresponding to areas rich in RBCs or in free-hemoglobin. The colocalization of hemoglobin alpha-chain (15,258 Da) and the 3,327 Da peptide was not exact. In particular, hemoglobin-positive areas in which elastase was not detected, corresponded to those with low m/z 3327 intensity. This suggests that elastase (and/or other PMN proteases) may be responsible for the production of m/z 3327 in situ. This was confirmed by an in situ digestion of elastase by applying the purified enzyme onto sections of hemorrhagic carotids, which led to hemoglobin disappearance paralleled by an increased detection of the m/z 3327. In the AAA thrombus, the presence of 3327-Da-positive areas in the intermediate layer that did not contain either cells or hemoglobin, suggest that this peptide is subject to transmural, outward convection from the luminal towards the deeper layers of the ILT. It is likely that this peptide may be released directly into the bloodstream, but may also reach the residual vascular wall, as described for hemorphin 7 (Dejouvencel et al, 2010). Further studies would be needed to test its possible biological activity on the vascular cells as well as its potential as a bio marker that could reflect both the presence of hemoglobin and the protease activity associated with PMNs. For this purpose, the development of a specific antibody would be required to assess the 3327-Da globin peptide as a marker for atherothombosis.

In conclusion, our study shows that atherothombotic lesions represent a major site of proteolysis. We have described an extracellular pathway for hemoglobin catabolism, different from the canonical intracellular hemoglobin degradation pathway following endocytosis or erythrophagocytosis, that involves heme oxygenase activity leading to iron release and globin metabolism by the phagolysosome (Fredenburgh et al., 2015). Using combined proteomic approaches, a 3327 Da fragment of the hemoglobin alpha chain was found to be abundantly released by hemorrhagic carotid plaques and the ILT in AAA, potentially generated by PMN proteases. Our results also confirm the spatio-temporal organization of the ILT in AAA in which the luminal layer at the interface with the circulating blood is the most biologically active layer.

The 3327-Da peptide may thus represent a marker of vulnerability to rupture, as it reflects the presence in the vascular wall of both blood and protease activity. Its potential detection in plasma required additional studies.

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Claims

CLAIMS:

1. An isolated, synthetic or recombinant atheglobin polypeptide or a function- conservative variant thereof.

2. A nucleic acid sequence encoding for the polypeptide according to claim 1.

3. A vector comprising the nucleic acid according to claim 2.

4. A host cell comprising the nucleic acid according to claim 2 or the vector according to the claim 3.

5. An antibody or an aptamer which specifically binds to the polypeptide according to claim 1.

6. A method of identifying a subject having or at risk of having or developing haemorrhagic atherothrombotic plaques, comprising a step of measuring in a biological sample obtained from said subject the level of atheglobin.

7. The method according to claim 6 which further comprises a step consisting of comparing the level of atheglobin in the biological sample with a reference value, wherein detecting differential in the level of the atheglobin between the biological sample and the reference value is indicative of subject having or at risk of having or developing haemorrhagic atherothrombotic plaques.

8. A method of identifying a subject at risk of having or developing vascular events, comprising a step of measuring in a biological sample obtained from said subject the level of atheglobin.

9. A method of monitoring haemorrhagic atherothrombotic plaques progression by performing the method according to any of claims 6 or 7.

10. A method of monitoring haemorrhagic atherothrombotic plaques treatment by performing the method according to any of claims 6 or 7.

11. A method of treating haemorrhagic atherothrombotic plaques in a subject in need thereof comprising the steps of: (i) identifying a subject having or at risk of having or developing a haemorrhagic atherothrombotic plaques by performing the method according to any of claims 6 or 7, and

(ii) administering to said subject a vascular disease treatment.

12. A method of treating vascular event in a subject in need thereof comprising the steps of:

(i) identifying a subject afflicted with haemorrhagic atherothrombotic plaques at risk of having or developing vascular event by performing the method according to claim 8, and

(ii) administering to said subject a vascular disease treatment.