Detection of SHED CD31, Diagnosis of Atherothrombosis and Autoimmune Disorders, and Methods for Analyzing Signaling Pathways

Abstract

The present invention stems from the finding that the extracellular domain of CD31 proteins present on blood leukocytes is shed and released in the circulation as a soluble form of CD31. A method for detecting shed CD31 is further disclosed. The invention therefore relates to a method for detecting a shed ectodomain of a transmembrane protein such as CD31 and to the use of such a method as a diagnostic tool. The invention further provides methods for determining whether a candidate protein is part of a molecular complex.

Claims

1. A method for detecting a shed ectodomain of a transmembrane protein among soluble forms of said transmembrane protein in a biological sample, wherein said soluble forms include a soluble splice variant of said transmembrane protein and optionally said shed ectodomain, which comprises the steps of: a) providing a first type of bead linked to an antibody which specifically binds to an epitope located in a region that is present both on said shed ectodomain and on said splice variant (first discriminating antibody); b) providing at least one second type of bead linked to an antibody which specifically binds to an epitope located in a region that is either present on said shed ectodomain and absent from said splice variant, or present on said splice variant and absent from said shed ectodomain (second discriminating antibody); c) providing a fluorescently-labelled ligand which specifically binds to a region that is present both on said shed ectodomain and on said splice variant (signaling ligand); d) contacting said antibodies with a biological sample likely to contain said soluble fauns of said transmembrane protein; e) for each type of bead, measuring the signal obtained with said florescent label by flow cytometry; and f) comparing the signal obtained for each type of bead; wherein a difference in the signals measured at step (e) indicates that the biological sample comprises said shed ectodomain.

2. Canceled

3. The method according to claim 1, wherein: i) said soluble forms include at least three soluble fauns; ii) the discriminating antibodies are chosen in such a way as to discriminate between said soluble forms; and iii) the capture ligand or the signaling ligand specifically binds to a region that is present on all said soluble forms.

4. The method according to claim 1, wherein said capture ligand or signaling ligand is an antibody.

5. The method according to claim 1, further comprising the step of calculating the percentage and/or the amount of said soluble fauns that corresponds to said shed ectodomain.

6. The method according to claim 1, further comprising the step of calculating either the ratio of shed ectodomain to soluble forms, or the ratio of soluble splice variant to soluble forms.

7. The method according to claim 1, wherein said transmembrane protein is CD31.

8. The method according claim 1, wherein said transmembrane protein is glycoprotein VI (GPVI).

9. The method according to claim 7, wherein the first discriminating antibody specifically binds to an epitope located in the first extracellular immunoglobulin-like domain of CD31, and wherein the second discriminating antibody specifically binds to an epitope located in the sixth extracellular immunoglobulin-like domain of CD31.

10. The method according to claim 7, wherein: the first discriminating antibody specifically binds to an epitope located in the first extracellular immunoglobulin-like domain of CD31; the second discriminating antibody specifically binds to an epitope located in the sixth extracellular immunoglobulin-like domain of CD31; and the discriminating antibodies further comprise an antibody which specifically binds to an epitope located in the fifth extracellular immunoglobulin-like domain of CD31.

11. The method according to claim 7, wherein said biological sample is plasma obtained from an individual suffering from or at risk of suffering from a thrombotic or an autoimmune disorder.

12. A method for diagnosing whether an individual suffers, or is at risk of suffering, from a thrombotic or an autoimmune disorder, which comprises the step of detecting a shed ectodomain of CD31 in a biological sample of said individual, wherein the presence of said shed ectodomain of CD31 indicates that said individual suffers from or is at risk of suffering from said a thrombotic or autoimmune disorder.

13. The method of claim 12, wherein said step of detecting a shed ectodomain of CD31 is repeated at least at two different points in time in order to monitor the progression of said disorder in said individual, and/or to assess the severity of said disorder in said individual, and/or to monitor the response of said individual to a drug.

14. A method for monitoring the response of a patient to a drug, said method comprising the steps of: a) detecting shed ectodomains of CD31 in a biological sample of said patient before and after onset of a treatment of said patient with said drug; b) comparing the levels of shed ectodomains of CD31 detected at step (a); and, optionally, c) correlating a difference in said levels of shed ectodomains of CD31 with the effectiveness of the drug for treating said patient.

15. The method according to claim 12, wherein said step of detecting a shed ectodomain of CD31 comprises the steps of: a) providing a first type of bead linked to an antibody which specifically binds to an epitope located in a region that is present both on said shed ectodomain of CD31 and on a soluble splice variant of CD31 (first discriminating antibody); b) providing at least one second type of bead linked to an antibody which specifically binds to an epitope located in a region that is either present on said shed ectodomain of CD31 and absent from said soluble splice variant of CD31, or present on said soluble splice variant of CD3 1 and absent from said shed ectodomain of CD3 1 (second discriminating antibody); c) providing a fluorescently-labelled ligand which specifically binds to a region that is present both on said shed ectodomain of CD31 and on said soluble splice variant of CD31 (signaling ligand); d) contacting said antibodies with a biological sample likely to contain said soluble forms of said transmembrane protein; e) for each type of bead, measuring the signal obtained with said florescent label by flow cytometry; and f) comparing the signal obtained for each type of bead; wherein a difference in the signals measured at step (e) indicates that the biological sample comprises said shed ectodomain of CD31.

16. A diagnostic kit comprising: i) a first type of bead linked to an antibody that specifically binds to an epitope located in a region that is present both on a shed ectodomain of a transmembrane protein and on a soluble splice variant of said transmembrane protein; ii) a second type of bead linked to an antibody that specifically binds to an epitope located in a region that is either present on said shed ectodomain and absent from said soluble splice variant, or present on said soluble splice variant and absent from said shed ectodomain; and iii) a fluorescently-labelled antibody that specifically binds to an epitope located in a region that is present both on said shed ectodomain and on soluble said splice variant.

17. (canceled)

18. A method for determining whether a candidate molecule is member of a molecular complex which comprises the steps of: a) providing a bead linked to an antibody that specifically binds to a member of said molecular complex; b) contacting said bead with a biological sample containing said molecular complex; c) contacting said beads contacted with the biological sample with at least one type of fluorescently-labelled antibody that specifically binds to said candidate molecule; and d) detecting the fluorescence by flow cytometry; wherein: the detection of a signal at step (d) indicates that said candidate molecule is a member of said molecular complex; and if more than one type of fluorescently-labelled antibody is used at step (c), said antibodies are labelled with different fluorescent labels.

19. A kit for analyzing a molecular complex which comprises: i) a bead linked to an antibody that specifically binds to a member of said molecular complex; and ii) at least one type of fluorescently-labelled antibody that specifically binds to a molecule likely to be member of said molecular complex; wherein, if the kit comprises more than one fluorescently-labelled antibody, said antibodies are labelled with different fluorescent labels.

20. The method of claim 18, wherein said member of a molecular complex is CD31.

21. The kit of claim 19, wherein said member of a molecular complex is CD31.

22. The method according to claim 12, wherein said step of detecting a shed ectodomain of CD31 comprises the steps of: a) providing a bead linked to a ligand which specifically binds to a region that is present both on said shed ectodomain of CD31 and on a soluble splice variant of CD31 (capture ligand); b) providing a first type of fluorescently-labelled antibody which specifically binds to an epitope located in a region that is present both on said shed ectodomain of CD31 and on said soluble splice variant of CD31 (first discriminating antibody); c) providing at least one second type of fluorescently-labelled antibody which specifically binds to an epitope located in a region that is either present on said shed ectodomain of CD31 and absent from said soluble splice variant of CD31, or present on said soluble splice variant of CD31 and absent from said shed ectodomain of CD31 (second discriminating antibody); d) contacting said antibodies with a biological sample likely to contain said soluble forms of said transmembrane protein; e) for each fluorescent label, measuring the signal obtained with said florescent label by flow cytometry; and f) comparing the signal obtained for each fluorescent label. wherein a difference in the signals measured at step (e) indicates that the biological sample comprises said shed ectodomain of CD31.

23. The method according to claim 14, wherein said step of detecting a shed ectodomain of CD31 comprises the steps of: a) providing a first type of bead linked to an antibody which specifically binds to an epitope located in a region that is present both on said shed ectodomain of CD31 and on a soluble splice variant of CD31 (first discriminating antibody); b) providing at least one second type of bead linked to an antibody which specifically binds to an epitope located in a region that is either present on said shed ectodomain of CD31 and absent from said soluble splice variant of CD31, or present on said soluble splice variant of CD31 and absent from said shed ectodomain of CD31 (second discriminating antibody); c) providing a fluorescently-labelled ligand which specifically binds to a region that is present both on said shed ectodomain of CD31 and on said soluble splice variant of CD31 (signaling ligand); d) contacting said antibodies with a biological sample likely to contain said soluble forms of said transmembrane protein; e) for each type of bead, measuring the signal obtained with said florescent label by flow cytometry; and f) comparing the signal obtained for each type of bead; wherein a difference in the signals measured at step (e) indicates that the biological sample comprises said shed ectodomain of CD31.

24. The method according to claim 14, wherein said step of detecting a shed ectodomain of CD31 comprises the steps of: a) providing a bead linked to a ligand which specifically binds to a region that is present both on said shed ectodomain of CD31 and on a soluble splice variant of CD31 (capture ligand); b) providing a first type of fluorescently-labelled antibody which specifically binds to an epitope located in a region that is present both on said shed ectodomain of CD31 and on said soluble splice variant of CD31 (first discriminating antibody); c) providing at least one second type of fluorescently-labelled antibody which specifically binds to an epitope located in a region that is either present on said shed ectodomain of CD31 and absent from said soluble splice variant of CD31, or present on said soluble splice variant of CD31 and absent from said shed ectodomain of CD31 (second discriminating antibody); d) contacting said antibodies with a biological sample likely to contain said soluble forms of said transmembrane protein; e) for each fluorescent label, measuring the signal obtained with said florescent label by flow cytometry; and f) comparing the signal obtained for each fluorescent label. wherein a difference in the signals measured at step (e) indicates that the biological sample comprises said shed ectodomain of CD31.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0182] FIG. 1 shows a representative example of 10-color flow-cytometry analysis of human peripheral blood cells from a healthy donor. Isotype controls of antibodies anti-CD31 dom1 and anti-CD31 dom6 are shown in the insets. Lymphocytes, Monocytes and Granulocytes were gated within the FSC/SSC scatter. B (CD20 A700+) and T (CD3 PE-TR+) lymphocytes were identified and gated within the Lymphocytes and CD8+ (PerCP) and CD4+ (APC) subpopulations were gated within T lymphocytes. CD8+ and CD4+ T cells were further analyzed for the expression of HLA-DR and CD45RA and accordingly subdivided in activated (1), memory (2) and naive (3) cells. All leukocytes were positive for CD31 dom6. Lack of dom1 increased from nave (3) to memory (2) to activated (1) T cells.

[0183] FIGS. 2A and 2B show that the apparent loss of CD31 on lymphocytes is due to its extracellular shedding. a. Solubilized cell membrane-bound CD31 molecules were extracted from cultured Jurkat CD4+ T cells and coupled to fluorescent beads. The percentage of dom1-bead-bound molecules is <6% in resting conditions and >99% 5 after TCR engagement. b. Most soluble CD31 in culture supernatant ( ) of TCR-activated T cells and in human plasma ( ) consists of a single truncated fragment comprising dom1-dom5 and lacking dom6. Negligible levels of truncated CD31 lacking both dom5 and dom6 could be detected only in plasma.

[0184] FIGS. 3A and 3B show that a peptide homotypic of the residual extracellular fragment on CD31.sup.shedT induces CD31-ITIM phosphorylation. a. Proliferative response to TCR engagement of human peripheral blood mononuclear cells in the presence of increasing doses of CD31 peptide 551-574. *p<0.05 vs dose 0. b. Flow cytometry assessment of 686ITIM phosphorylation on solubilized membrane-bound CD31 from cultured Jurkat CD4+ T cells. Solubilized proteins were captured by E9-PECAM-1.2 (dom6) functional CBA beads and detection was carried out by anti-pY686 rabbit sera followed by AlexaFluor488-anti-rabbit secondary antibody. The histogram shows the Median Fluorescent Intensity (MFI)the % of the variability coefficient (CV%) of Alexafluor488 (pY686) over 2000 E9-PECAM-1.2 acquired beads. Pervan=positive control (pervanadate); CD3/CD28=anti-CD3 and anti-CD28 antibodies (1g/ml each); peptide=CD31 peptide 551-574 (100 M).

[0185] FIG. 4 illustrates the principle of the method of according to the invention.

[0186] FIG. 5 illustrates the principle of the method of according to the invention. The black box represents a discriminating antibody. The white box represents a capture or a signaling antibody.

[0187] FIG. 6A shows that shed CD31 can be used as a diagnostic and/or prognostic marker for inflammatory diseases. White boxes are indicative of a bad prognostic of the patient, and black boxes are indicative of a good prognostic of the patient. VS stands for the erythrocyte sedimentation rate. CRP stands for the C-reactive protein. sCD31 Total stands for the total level of soluble forms of CD31, measured with the method of the invention. sCD31.sup.cliv stands for the shed extodomain of CD31, measured with the method of the invention.

[0188] FIG. 6B shows that the method of the invention can be used for measuring shed ectodomains of GPVI.

BRIEF DESCRIPTION OF THE SEQUENCES

[0189] SEQ ID NO: 1 corresponds to the sequence of human CD31.

[0190] SEQ ID NOs: 2 and 3 correspond to CD31 peptides.

[0191] SEQ ID NO: 4 corresponds to a scramble peptide used as a negative control.

EXAMPLES

Example 1

Material and Methods

[0192] Assessment of CD31.sup.+ and CD31.sup.shed blood leukocytes. Ten-color flow cytometry was performed on peripheral blood leukocytes from 5 healthy individuals either in basal conditions or after overnight stimulation with soluble 1 g/ml of purified anti-CD3 antibody (R&D Systems). Ten-color flow cytometry was performed after erythrocyte hypotonic lysis (10 minutes at 37 C. 1:10 v:v in Tris 10 mM, NH.sub.4Cl 155 mM, KHCO.sub.3 10 mM, pH 7.4) on heparinized peripheral blood leukocytes from 5 healthy individuals, fixed in PBS/Formaldehyde 1%/FCS 1% for 4 minutes at 37 C. prior to processing. All experiments on human blood were approved by the International Ethical committee (see world wide web page clinicaltrials.gov; Identifier: NCT00430820). Pelleted cells were incubated for 30 minutes at room temperature and protected from light with a cocktail of fluorescent monoclonal antibodies directed to CD3 (PE-Texas Red), CD4 (PE-Cy7), CD8 (PerCP), HLA-DR (APC-Cy7), CD45RA (Pacific Blue), and CD31 (WM59, PE) from BD Biosciences and anti-CD20 (AlexaFluor700) and anti-CD31 (PECAM 1.2, FITC) from Invitrogen (1 l of each). At least 50,000 events were acquired in the lymphocyte gate using a BD LSRII equipped with 3 lasers (405, 488 and 633 nm) and analysed with BD DIVA 6.0 software.

[0193] Subtractive measurement of soluble CD31. To detect the splice variant and truncated CD31 in plasma and the culture supernatant, a cytokine bead array (CBA, BD) has been customized. Three differently functional CBA beads (A9, D5 and E9) were coupled with either one of the following purified monoclonal anti-CD31 antibodies JC70A (domain 1, DAKO), MEM-05 (domain 5, Zymed) and PECAM 1.2 (domain 6, Invitrogen). The coupled beads were then incubated with the plasma of the same 5 healthy controls or the culture supernatant and positive binding of circulating CD31 was detected by a fourth anti-CD31 monoclonal antibody, WM-59 (domains 1-2) coupled to PE (BD). The concentration of plasma CD31 including at least domain 1 (JC70A), or domains 1 to 5 (MEM-05) or all the extracellular domains 1 to 6 of CD31 (PECAM 1.2) was determined by analysing the median fluorescent intensity of the detecting antibody on 1000 gated beads on samples and serial dilutions of the same standard (recombinant, full length extracellular CD31, R&D Systems). The standard curve was obtained for each of the beads using the same known concentrations of the recombinant CD31 in order to overcome any bias due to differences in binding affinity of the diverse antibodies. The concentration in ng/ml of CD31 determined with PECAM 1.2 coupled beads (dom 1-6) was subtracted from the one obtained using MEM-05 coupled beads to obtain the amount of circulating CD31 lacking dom6 (dom 1-5). The latter was subtracted from the concentration of CD31 obtained using the JC70A-coupled beads to calculate the value of soluble CD31 lacking both dom 5 and 6 but containing at least domains 1 and 2 (dom 1-2).

[0194] Assessment of CD31-ITIM phosphorylation. Log-phase Jurkat cells (10.sup.7 cells/condition) were either left unstimulated (negative control) or incubated with pervanadate (positive control) or stimulated with anti-CD3 and anti-CD28 antibodies (R&D Systems, 1 g/ml each) in the presence or absence of peptide 551-574 (100 M), or incubated with the peptide alone during 20 minutes. Cells were then lysed with 1 ml of RIPA buffer on ice for 30 minutes, ultracentrifuged and 16 l of the supernatant was incubated with PECAM 1.2-coated Functional E9 CBA beads (BD) for 2 hours at room temperature. Beads were subsequently washed with CBA washing buffer and incubated with 2 l of undiluted rabbit anti-CD31 phospho-tyrosine 686 (pY686) sera followed by two washings and incubation with AlexaFluor488-conjugated (Fab).sub.2 fragments (1:100 in CBA whashing buffer) of goat-anti-rabbit IgG (Invitrogen). The beads (2000/condition) were finally analysed by flow cytometry in the FITC channel (530/30 nm) and data are expressed as Median fluorescence intensity (MFI)the percentage of the coefficient of variability (% CV) calculated with the DIVA 6.0 software (BD). Duplicate lysate aliquots and serial dilutions of recombinant CD31 were incubated with the PECAM 1.2-coated beads and the amount of dom1+ cell-bound CD31 was revealed using anti-CD31 WM59-R-PE (dom1) and PECAM 1.2-FITC (dom6) antibodies.

[0195] Fluorescent peptide binding. For visualisation of peptide binding to CD31.sup.+ and CD31.sup.shed CD4.sup.+ T cells, freshly purified peripheral blood leukocytes prepared as above were washed with a buffered solution containing 2 mM EDTA (to avoid endocytosis of the peptide) and incubated overnight at room temperature in a dark humidified chamber with 50 M FITC-labelled CD31 peptide 551-574 and 1:10 dilution of fluorescent monoclonal anti-CD31 (PE) and anti-CD4 (APC) antibodies (BD Biosciences) in a poly-D-Lysine coated ibidi 8-well culture chamber (Biovalley). Cells were then washed twice, nuclei counterstained with DAPI and digital images of a 0.3 m intracellular section were acquired on a Zeiss Axiovert M200 microscope (x63 immersion objective) equipped with the ApoTome and a cooled monochromatic digital camera (Zeiss).

[0196] Calcium mobilization assay. Spleen cells from C57BI/6 mice were prepared as described in Caligiuri et al. (2005 Arterioscler Thromb Vasc Biol 25:1659-1664). Cells were incubated with Fluo-3AM (Invitrogen, #F1242) as per the instructions of the manufacturer. Fluorescence of calcium-bound tracer was measured in the FITC channel on an LSRII cytometer (BD Biosciences) prior to and during 60 seconds following the addition of hamster anti-mouse CD3/CD28 monoclonal antibodies (40 g/ml each) and rat/hamster compBead (1:50) either alone or in the presence of rat anti-mouse CD31 antibody (clone 390, 10 g/ml) or in the presence of CD31 peptide 551-574 (100 M). Negative controls included rat IgG isotype control and scramble peptide. Antibodies and compBeads were from BD Biosciences.

[0197] Plasmon Surface Resonance. Homophilic binding association and dissociation constants were calculated by surface plasmon resonance (BlAcore 2000, GE). In brief, peptide 551-574 was coated at 3400 resonance units (RU) on CM5 chips according to the manufacturer's instructions. Soluble peptide 551-574 (12.5, 25, 50 and 100 M in 200 l of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween 20) was injected at 20 l/min at 25 C., on the peptide-coated channel and on an uncoated channel. Dissociation was monitored for 300 seconds. Association (kon) and dissociation (koff) constants were calculated using the BlAevaluation 3.0 Software (GE). Injection of peptide 551-574 on a channel coated with the scramble peptide yielded negligible signal.

[0198] Evaluation of immunoregulation in vitro. CD8.sup.+ T cell-mediated cytolytic activity against allogeneic mouse aortic smooth muscle cells and measurement of macrophage gelatinase (MMP-2/9) activity were performed as previously described for human cells in Caligiuri et al. (2006 Arterioscler Thromb Vasc Biol 26:618-623) using kits and reagents from Invitrogen. Briefly, primary cultures of FVB/N mouse aorta smooth muscle cells were labelled with the lipophylic tracer DIO (green) and co-cultured for 3 hours with CD8+ T cell-enriched spleen cells from C57BI/6 mice (n=3 scramble peptide and n=3 peptide 551-574, 50 M). Cytolysis was evaluated by intracellular accumulation of propidium iodide (P1). Cells were analysed by flow cytometry and the % of cytolysis was calculated by expressing the number of dead (PI+) cells among the target (DIO+) cells. Intracellular MMP-2/9 (gelatinase) activity was measured by flow cytometry in 7-day bone-marrow derived macrophages from C57BI/6 mice (n=3 scramble peptide and n=3 peptide 551-574, 50 M) three hours after the incorporation of OregonGreen gelatine (MFI). T-cell proliferation was performed using either human peripheral blood mononuclear cells of spleen cells from C57BI/6 (CD31.sup.+/+) and CD31.sup./ mice (Charles River France) as previously described (Caligiuri et al. Arterioscler Thromb Vasc Biol 25:1659-1664). Briefly, cells were plated in triplicates at 0.210.sup.6 cells/well in a U bottom 96-well plate in complete medium (RPMI 1640, 1% pyruvate, 1% glutamine, 1% penicillin-streptomicyne-fungizone, 10% decomplemented fetal calf serum, all from Invitrogen) containing 1 g/ml anti-mouse CD3/CD28 or 5 g/ml anti-human CD3 antibodies (BD) as appropriate. CD31 (551-574) and scramble peptide at 25, 50 and 100 M final concentration were deposited in the wells just before cell plating. Plated cells were cultured for 72 hours in 5% CO2 at 37 C. (.sup.3H) thymidine (0.5 Ci/well) was added for the last 16 hours and proliferation evaluated using a Tomtec harvester and analysis on a Wallac micro beta counter. Data are expressed as meanSEM of cpm in triplicates.

[0199] Evaluation of immunorequlation in vivo. Delayed type hypersensitivity (DTH) suppression was evaluated as described in the Current Protocols in Immunology (2001) 4.0.1-4.0.2 Unit 4.2. Briefly, TNCB (2-chloro-1,3,5-trinitrobenzene, Fluka #79874) was dissolved in acetone/olive oil (1:1 v/v) at a concentration of 10 mg/ml. BALB/c mice (n=6/group) were primed by painting the shaved regions of the abdomen a with a total 0.2 ml of the preparation (n=6/group). The experiment included 3 groups for peptide 551-574 (10, 50, 100 M) and 1 group treated with scramble peptide at 100 M). Five days after priming, 10 l of the TNCB-solvent mixture was painted on the right pinna, 30 minutes after subcutaneous (interscapolar) administration of the peptide 551-574 or the scramble peptide. Ear thickness increases were calculated by subtracting the thickness of the right and the left pinna of each mouse (right-left/left100), measured at 24 h with a dial caliper (Pocotest, Kroeplin Lngenmesstechnick). The measure was performed 5 times on each ear and averaged for further analysis. The immunosuppressive effect of the peptide was calculated as % suppression=(1TE/TS)100, where T=(ear thickness 24 hr after elicitation)(baseline ear thickness), E=sensitised animals, S=treated animals. Data are expressed as meanSEM.

[0200] Detection of atherosclerotic lesion size and aneurysm formation. Male 28-week old apolipoprotein E.sup./ mice (n=8-10 mice/group) from our breeding facility were maintained on a regular chow diet and kept under standard conditions. Acceleration of atherosclerosis and aneurysm formation was induced by subcutaneous angiotensin II (Sigma, #A9525) infusion (1 mg/kg/d) for 28 days using osmotic minipumps (Alzet, #2004) as previously described (Daugherty et al. J Clin Invest 105:1605-1612). All experiments were approved by our institutional Ethical committee. Atherosclerotic lesions size were measured as previously described (Caligiuri et al. Arterioscler Thromb Vasc Biol 25:1659-1664). These experiments were repeated twice with similar results.

[0201] Peptides. All experiments on human material were carried out using the human peptide sequence while the mouse equivalent was used in all mouse experiments. The sequences of the peptides are shown in the table below.

TABLE-US-00003 Human NH2-NHASSVPRSKILTVRVILAPWKK-COOH SEQIDNO:2 Mouse NH2-SSMRTSPRSSTLAVRVFLAPWKK-COOH SEQIDNO:3 Scramble NH2-SMPAVRSRFSATSLVTLKSRWPK-COOH SEQIDNO:4

Example 2

The Apparent Loss of CD31 at the Surface of Blood Lymphocytes is Due to its Shedding between the 5th and 6th Extracellular Ig-like Domains

[0202] In order to establish whether the loss CD31 was restricted to part or extended to the totality of its 6 extracellular Ig-like domains, a multicolor flow cytometry analysis of whole blood leukocytes from 5 healthy donors using two different antibodies specifically recognizing the membrane-distal and membrane-proximal Ig-like domains of the molecule was performed. To be able to discriminate between the different leukocyte populations and assess their state of maturation and activation, a panel of lineage markers as well as the expression of CD45RA and HLA-DR were simultaneously used. While the expression of CD31, as detected by a monoclonal antibody specific for the first domains of CD31 (clone WM-59, dom1-2) was recognized on nave but not on activated/memory blood T cells, all cells expressed the membrane-proximal extracellular fragment of the molecule detected by another monoclonal antibody specific for the 6.sup.th Ig-like domain of CD31 (clone PECAM 1.2, dom6), irrespective of their state of maturation/activation (FIG. 1).

[0203] Flow cell cytometry showed that T-cell receptor (TCR) engagement induces a shift of >80% of blood resting T cells from a CD31 dom1.sup.+/dom6.sup.+ to a dom1.sup./dom6.sup.+ (CD31.sup.shed) phenotype. Molecular analysis of the membrane proteins from cultured T-cell lysates demonstrated that >99% of the T cell-bound CD31 molecules drop the distal portion containing dom1 already 5 minutes after TCR stimulation in vitro (FIG. 2a). Analysis of the supernatant showed that, simultaneously, a single truncated soluble protein limited to the first 5 Ig-like domains of CD31, accumulates in the culture supernatant (FIG. 2b). Furthermore, the analysis of the plasma of the same healthy donors showed that major part of soluble CD31 in plasma was constituted of a truncated molecule comprising Ig-like domains 1 to 5 and specifically lacking the membrane-proximal 6.sup.th domain (FIG. 2b) that always remains anchored to the apparently CD31-negative (CD31 dom1.sup.) lymphocytes both in vitro and in vivo. Only a minimal fraction of soluble CD31 contained all 6 extracellular domains predicted in the previously reported (Goldberger et al. J Biol Chem 269:17183-17191) variant spliced form both in culture supernatant and in plasma (FIG. 2b). No significant other cleavage of the molecule occurs upstream of the 5.sup.th domain since the latter was virtually always present concomitantly with the first domain in the truncated soluble CD31 proteins (FIG. 2b).

[0204] Here it is demonstrated that the assumed loss of the molecule on activated/memory T lymphocytes is actually incomplete and results from shedding of CD31 between the 5th and 6th extracellular Ig-like domains. CD31 shedding occurred immediately after cell activation on T lymphocytes and was accompanied by the accumulation of the truncated molecule in the supernatant together with trace levels of the spliced variant produced by the cells. This finding was unsuspected because all commercially available tests to detect plasma CD31 use antibodies directed to CD31 domains 1 to 5, and therefore cannot discriminate between the spliced variant (containing all the 6 extracellular domains) and the truncated (domains 1 to 5) forms of CD31. The subtractive immunosorbent assay described herein is able to discriminate between the two forms of soluble CD31 and precisely quantify the proportion of each of them in the plasma. This assay showed that the major part of plasma CD31 comprises domains 1 to 5 but lacks the membrane-proximal 6th domain, which remains anchored to blood CD31 dom1-lymphocytes. Therefore, it is proposed to refer to these lymphocytes as CD31shed rather that CD31 negative cells. Previous work in vitro had indicated that CD31 shedding at an unidentified position N-terminal from the transmembrane segment of the molecule can occur in endothelial cells undergoing apoptosis (Ilan et al. 2001. Faseb J 15:362-372). For the first time, it is shown herein that shedding is responsible for the CD31 (incomplete) loss on blood lymphocytes and that the circulating CD31 consists mainly of a truncated form derived from its cleavage between the Ig-like domains 5 and 6, rather than of the secreted spliced variant form. Genetic polymorphisms for CD31 have been described, but the predictive value of soluble CD31 levels was conflicting either in atherothrombosis or other dysimmune diseases. In fact, while the amount of the spliced form can be predicted by different genetic variants, the proportion of the form resulting from protein shedding is not determined by CD31 gene polymorphism. It is proposed that the disparity between the different studies was due to fact that circulating CD31 is a mixture of the genetic variant and of the truncated form and discrimination between the two forms of CD31 was not possible. The subtractive method described herein will allow the differentiation of the prognostic value determined by genetic variants of CD31 independently of that linked to CD31 shedding.

Example 3

A Peptide Contained in the Residual Extracellular CD31 Fragment on CD31.SUP.shed .T Cells Enhances Phosphorylation of CD31-ITIM

[0205] A CD31 dom6-derived synthetic peptide corresponding to the juxta-membrane 23 aminoacids (551-574) of the ectodomain of the human molecule binds both to CD31 dom1.sup.+ and to CD31 dom1.sup. (CD31.sup.shed) CD4.sup.+ T lymphocytes ex vivo. Importantly, the binding of this peptide on T cells has functional consequences on immune cell control since it exerted dose-dependent inhibition of human peripheral blood T-cell proliferation in vitro (FIG. 3a). To assess whether the inhibitory effect of the peptide could be mediated by homophilic binding and engagement of the CD31 signaling, the level of phosphorylation of the CD31 ITIM tyrosine at position 686 (.sub.686ITIM) in cultured T cells was evaluated. Stimulation of the TCR by anti-CD3 and anti-CD28 antibodies alone or the sole presence of the peptide induced a minor increase of CD31 pY686 (FIG. 3b) but concomitant TCR-stimulation in the presence of the peptide boosted the phosphorylation the CD31 .sub.686ITIM by a factor of >23 (FIG. 3b).

Example 4

Detection of Shed CD31 in Plasma from Patients Suffering from Atherothrombosis and in Unaffected Individuals

[0206] The total amount of CD31, the amount of shed CD31 and the amount of spliced CD31 has been measured both in eleven individuals suffering from atherothrombosis and in twenty-three unaffected individuals.

[0207] The group Atherothrombosis comprised eleven individuals suffering from chest pain even at rest and presenting an abnormal coronarography.

[0208] The group No Atherothrombosis comprised twenty-three individuals. A sub-analysis was carried out on the group No Atherothrombosis, which was found to comprise: [0209] eight individuals presenting a normal coronarography and a normal carotid echodoppler in spite of chest pain; [0210] four individuals presenting a normal coronarography in spite of chest pain, but in whom atherosclerosis was detected by carotid echodoppler; and [0211] eleven individuals suffering from chest pain only on effort and presenting an abnormal coronarography (i.e. suffering from coronary atherosclerosis without thrombosis).

[0212] The total amount of CD31, the amount of shed CD31 and the amount of spliced CD31 was determined as follows.

[0213] 1. The total amount (1 l/test) of beads (E9, coupled with clone JC70A, DAKO) was transferred to a conical tube and centrifuged at 200 g for 5 minutes. The supernatant was carefully discarded and replaced with same amount of serum enhancement buffer (BD #51-9002150), and incubated at room temperature for 15 minutes.

[0214] 2. The fluorescently-labeled antibody antibody mix (PE-WM59; FITC-HC1/6; PB-PECAM1.2) was prepared, each at 1 g/ml, 1 l each/condition.

[0215] 3. 1 tube precondition was prepared, each containing 3 l of a standard dilution or a plasma sample. The reconstituted beads were centrifuged at 200 g for 5 minutes, the supernatant was discarded and the serum enhancement buffer was replaced with the fluorescently-labeled antibody mix. 3 l of this solution was distributed in each of the tubes containing the standard dilution and samples, and the solution incubated for 1 hour at 4 C. in the dark.

[0216] 4. 150 l of Washing buffer (BD #51-9003797) were added to each tube, and the signal was acquired.

[0217] As shown in the table below, the percentage of shed CD31 was higher in individuals suffering from atherothrombosis than in unaffected individuals, in spite of the fact that all individuals were suffering from chest pain.

TABLE-US-00004 CD31 Plasma Level (ng/ml) total splice shed Atherothrombosis (n = 11) 11.55 0.7 7.02 2.69 18.57 2.67 No Atherothrombosis 11.58 0.49 5.26 1.850 6.31 1.85 (N = 23) T-test Prob > F 0.9756 0.0007 0.0006

[0218] Total CD31 amounts were similar in the four groups, while the amount of shed CD31 and the amount of spliced CD31 were significantly different in each paired group comparison. Shed CD31 was increased in individuals with abnormal coronarography, with highest values in those suffering from atherothrombosis. Splice CD31 was still present in patients suffering from atherosclerosis without atherothrombosis, while it was almost undetectable in patients suffering from atherothrombosis.

[0219] These results demonstrate that high levels of CD31 soluble splice variants associated with low levels of shed CD31 indicates that the patient suffers from non specific chest pain, eventually associated with carotid plaques. A slight increase of shed CD31 levels associated with normal or reduced levels of CD31 soluble splice variants indicates that the patient suffers from atherosclerosis. An important increase of shed CD31 levels associated with undetectable amounts of CD31 soluble splice variants indicates that the patient suffers from atherothrombosis.

Example 5

Use of CBA Beads for Quantitative Assessment of Protein Association and of Phosphorylation

[0220] 8.1. Protocol Used for Assessing the CD31 ITIM-Dependent Inhibitory Pathway in T Cells

[0221] Lysis of the cells. Jurkat T cells in log phase (1010.sup.6/ml, 1 ml) were either left untouched (negative control) or incubated with Na.sub.3VO.sub.4/H.sub.2O.sub.2 (sodium ortovanadate which is a tyrosin phosphatase inhibitor, positive control for tyrosin phosphorylation). In parallel, cells were stimulated either with CD3 or with CD3+ CD31 (domain 2, WM-59 antibody) by antibody-mediated crosslinking. After 20 incubation at 37 C. in 5% CO.sub.2, the cells were lysed on ice for 30 with a RIPA buffer containing a cocktail of protease and phosphatase inhibitors. Lysates were ultragentrifuged and supernatants were used either straightforward or aliquoted and stored at 20 for further analysis.

[0222] Capture of the target protein on a solid support (1 hour at room temperature). This step follows the principle of imunoprecipitation but the signaling complex is nor denatured neither reduced, and no electrophoresis and/or blotting is carried out. As a solid support, CBA functional beads previously coupled with an antibody directed to the membrane-proximal CD31 Ig-like domain 6 (clone MBC 78.2 also called PECAM1.2) were used following the manufacturer's instructions. An aliquot of 20 l of lysate supernatant from each condition was incubated with 5 l PECAM1.2-CBA for 1 hour at room temperature.

[0223] Detection of molecules associated with CD31 and of the phosphorylation state of CD31 ITIM 686. After washing of the beads once with CBA washing buffer (100 l/tube), the beads were been aliquoted in 4 separate tubes and incubated with fluorescent antibodies directed to: [0224] CD3-FITC; [0225] CD28-PE; [0226] CD31 domain 6 (PECAM1.2 FITC) [0227] CD31 domain 2 (WM-59 PE) [0228] phosphoTyrosin (clone 4G10 FITC); [0229] phosphoSerin/Threonin (labelled with FITC) [0230] CD31 phosphotyrosin 686 (rabbit polyclonal conjugated with alexafluor 488 antirabbit secondary antibody); or [0231] SHP2-PE.

[0232] After 1 hour incubation at room temperature in the dark, beads were washed once with 100 l CBA washing buffer and more than 600 beads were acquired using an LSR II and DIVA software.

[0233] 4. Analysis. The median fluorescence in each fluorescent channel was recorded. The DIVA software also calculated the %CV (variability coefficient), which can be considered as an equivalent of error bar value for repeated measure and allow statistical comparison between samples.

[0234] 8.2. Results

[0235] The results are shown in the tables below.

TABLE-US-00005 (ng/ml) (ng/ml) % MFI MFI CD31 CD31 shed pY pSer/ dom6 dom2 CD31 (4G10) pThreo Negative unstimulated 19 18 5 485 284 control Positive Na3VO4/ 103 77 25 1205 324 pY control H2O2 T-cell CD3 crosslink 9080 83 99 2681 130 activation Antibody CD3 + CD31 2389 46 98 2312 267 CD31 crosslink treatment Peptide CD3 10962.sup.1 .sup.67.sup.2 99 .sup.3277.sup.3 329 CD31 crosslink + treatment CD31 peptide MFI CD31 MFI MFI MFI pY686 SHP-2 CD3 CD28 Negative control 1578 165 197 8165 Positive pY control 10667 193 354 3932 T-cell activation 4198 90 17876 25169 Antibody CD31 2268 149 2425 17147 treatment Peptide CD31 treatment 96632.sup.3 .sup.185.sup.4 20096.sup.5 .sup.24306.sup.5 A value in bold indicates an increase versus CD3 crosslink conditions, and values in italic indicate a decrease. .sup.1Engaged CD31 molecules oligomerize in cis. CD31 molecule being physiologically engaged as a regulatory molecule in T-cell activation, this explains the increase of CD31 domain 6 amount in T-cell stimulated samples. .sup.2If only domain 2 of CD31 was detected, one would conclude that the amount of the captured molecule was similar in all samples. Indeed, most of CD31 cis-oligomerized molecules are shed between domain 6 and 2 upon T-cell activation as show in the column % shed. .sup.3Total phosphoTyrosin is slightly decreased by CD31 antibody treatment while it is slightly increased by CD31 peptide treatment. Indeed, not only CD31 ITIMs can be phosphorylated, but also tyrosins present on other membrane proteins (e.g. CD3 and CD28) which are associated with CD31 upon T-cell activation. The differences specific for CD31 phosphoITIMs could detected by using antibodies directed to sequence-specific phosphoITIM. Not only phosphotyrosine are increased, but also phosphoserine/threonine. These aminoacid can be phosphorylated either in the CD31 cytoplasmic tail, or in one of the associated molecule. .sup.4The amount of SHP-2 associated to CD31 is increased by 50% with the antibody treatment and by 100% with the peptide treatment. .sup.5The amount of activating receptor (CD3) and the co-stimulatory molecule associated to the CD31 (CD28) are decreased by the antibody treatment, while they are increased by the CD31 peptide. This can be viewed as a counter-regulatory mechanism of the cell in the view of the dramatic increase of CD31 ITIM phosphorylation that counteracts T-cell activation.

[0236] 8.3. Conclusion

[0237] All these data were acquired starting from a very small amount of the same sample (20 l). The use of multicolor flow-cytometry warrants their analysis in a simultaneous way. The availability of a standard allows exact determination of absolute amounts of each molecule in the signaling complex.

[0238] The above experiment was performed using either FITC (FL1) or PE (FL2) conjugated antibodies. The use of non-fluorescent beads for protein capture allows the use of at least two other fluorophores for use in the blue laser (PerCP or PE-Cy7 or PE-Cy5, FL3 and APC or Cy5, FL4). The availability of additional lasers (red, violet, uv) on the cytometer further expands the capacity of this method. Up to 17 different fluorescent antibodies can be used simultaneously and therefore up to 17 different parameters (associated membrane protein, signaling molecules, phosphorylated sequences) can be detected simultaneously on the same sample, with a powerful statistical value due to the high number of beads that can be acquired.

[0239] The above method has numerous advantages compared to prior art methods. The table below compares this method with co-IP/WB, CBAFIex and CASE/Phosphlow.

TABLE-US-00006 Method Phoflow/ according to Operation IP/WB CBAFlex CASE the invention Protein extraction 30 min 30 min N/A 30 min Immunoprecipitation 1-12 h N/A N/A N/A SDS-PAGE 2 h N/A N/A N/A Blot 2 h N/A N/A N/A Fixation N/A N/A 1 h N/A Blocking 1 h N/A 1 h N/A Primary Antibody 1-18 h 1 h 1 h 1 h Washing 30 min 5 min 15 min 0-5 min Secondary Antibody 1 h N/A 1 h N/A Washing 30 min N/A 15 min N/A Detection develop. 60 min N/A 15 min N/A Data Acquisition 1 h <10 sec/samp 15 min <10 sec/samp Multi-target N/A up to 20 2 up to 20 Time 12-36 h 1.5 h 7 h 1.5 h Sample size 100 L 20-50 l 50 L 5 l Specific interactions Yes (1) N/A N/A Yes (1)

[0240] Compared to co-IP/WP, this method is quantitative, direct and much more rapid. In addition, it allows the simultaneous detection of the target protein, of phosphomolecules and of associated membrane and intracellular molecules. It also allows the simultaneous analysis of much more parameters since a Western Blot can only be rehybridized a limited number of times, whereas up to 20 antibodies can be used simultaneously in the frame of this method with the most recent generation of cytometers.

[0241] Compared to CASE/Phosphlow, or CBA Flex set, this method is much more specific since the target molecule is captured and the interactions within the molecular complex can be analyzed. In addition, it allows the simultaneous detection of the target protein, of phosphomolecules and of associated membrane and intracellular molecules.

Example 6

Optimization of the Protocol

[0242] 5 l of sample are incubated for 30-60 at 4 C. in the dark with 1 l of a mix containing functional CBA beads-immobilized monoclonal MEM-05 antibody (CD31 domain 5, Exbio, Caltag), WM-59 (CD31 domain 2) and PECAM 1.2 (also called MBC 78.2, CD31 domain 6) coupled to a fluorophore. After incubation, beads are diluted with 200 l of assay diluent buffer and acquired. The median fluorescent intensity of the PE and FITC channel on more than 1000 gated beads is analyzed on samples and serial dilutions of the standard (recombinant, full length extracellular CD31, R&D Systems). Two standard curves are obtained with each of the detecting antibodies simultaneously used with recombinant CD31 in order to overcome any bias due to differences in binding affinity of the diverse antibodies. With three discriminating antibodies directed to domains 2, 5 and 6, it is possible to discriminate full CD31 from CD31 lacking either domain 6 (6) from CD31 lacking both domain 5 and 6 (5-6).

[0243] To increase the specificity of the test, monoclonal antibodies directed to CD31 domain 1 (clone JC70A, Dako) are coupled to capture beads. Detection is based on three fluorescent antibodies directed to CD31 domain 2 (WM-59, BD), domain 5 (HC1/6, Biosource) and domain 6. Purified PECAM 1.2/MBC 78.2 is coupled with a suitable fluorophore. With current functional beads, an optimal set of fluorophores is FITC, PE and PerCP. This combination allows measurement on any basic blue-laser cytometer (FACScan, FACSCalibur, etc) available in hospital biology laboratory.

[0244] With FITC and PE as fluorophores (see example 5), the lowest detection limit is 3 ng/ml. This results in negative values of spliced CD31 in patients presenting a high risk of suffering from atherothrombosis. In contrast to this, different fluorophores may lead to an increased sensitivity. Therefore, different combinations of fluorophores and of antibodies are tested.

Example 7

Analysis of the BIOcore Cohort

[0245] A cohort of patient has been analyzed. The individuals were classified in different groups, as set forth in Example 4.

[0246] The cell analysis by cytometry confirmed that the percentage of T (CD3+) lymphocytes that display a truncated (shed) CD31 is significantly higher in patients at risk of acute coronary events (see Table 1).

TABLE-US-00007 TABLE 1 Oneway analysis of CD31shed (% of CD3 cells) by Group Level Level Difference Lower CL Upper CL p-Value ACS Norm 16.06119 10.7395 21.38292 <0.0001* ACS Carotide 15.99231 9.6004 22.38419 <0.0001* ACS SA 12.79007 7.9876 17.59257 <0.0001* SA Norm 3.27111 2.2928 8.83498 0.2462 SA Carotide 3.20223 3.3926 9.797.9 0.3377 Carotide Norm 0.06888 6.9132 7.05092 0.9844

[0247] In the above table, ACS stands for acute coronary syndromes, Carotide stands for peripheral atherosclerosis, Norm stands for normal coronary angiogram, and SA stands for stable angina.

[0248] Subpopulation analysis showed that this conclusion is valid both for CD4+ T (CD3+) cells and for CD8+ T (CD3+) cells (see Tables 2 and 3, respectively).

TABLE-US-00008 TABLE 2 Oneway analysis of CD31shed (% of CD4 cells) by Group Level Level Difference Lower CL Upper CL p-Value ACS Carotide 26.37179 18.2050 34.53856 <0.0001* ACS Norm 19.24452 12.4451 26.04397 <0.0001* ACS SA 16.43631 10.3003 22.57235 <0.0001* SA Carotide 9.93548 1.5094 18.36157 0.0213* Norm Carotide 7.12727 1.7935 16.04806 0.1161 SA Norm 2.80821 4.3006 9.91704 0.4351

TABLE-US-00009 TABLE 3 Oneway analysis of CD31shed (% of CD8 cells) by Group Level Level Difference Lower CL Upper CL p-Value ACS Carotide 8.364103 2.10954 14.61867 0.0093* ACS Norm 6.664452 1.45706 11.87185 0.0126* ACS SA 4.435567 0.26376 9.13489 0.0640 SA Carotide 3.928536 2.52463 10.38171 0.2300 SA Norm 2.228886 3.21545 7.67322 0.4186 Carotide Carotide 1.699650 5.13239 8.53169 0.6227

[0249] Plasma analysis also confirmed that the level of total soluble CD31 is not able to discriminate between the groups (see Table 4).

TABLE-US-00010 TABLE 4 Oneway analysis of total soluble CD31 (ng/ml) by Group Level Level Difference Lower CL Upper CL p-Value Norm ACS 1.017336 0.71748 2.752148 0.2474 Norm SA 0.965691 0.84806 2.779439 0.2934 Norm Carotide 0.825823 1.45023 3.101875 0.4733 Carotide ACS 0.191513 1.89216 2.275182 0.8557 Carotide SA 0.139868 2.00997 2.289702 0.8976 SA ACS 0.051645 1.51391 1.617195 0.9480

[0250] Only the method according to the invention, which allows measuring spliced CD31 and shed CD31 in plasma, can be employed for diagnosing and/or prognosing acute coronary syndromes (see Tables 5 and 6, respectively).

TABLE-US-00011 TABLE 5 Oneway analysis of spliced CD31 (ng/ml) by Group Level Level Difference Lower CL Upper CL p-Value Norm ACS 4.723988 1.97055 7.477428 0.0010* SA ACS 3.288827 0.80403 5.773619 0.0100* Carotide ACS 3.255752 0.05138 6.562888 0.0536 Norm Carotide 1.468236 2.14424 5.080714 0.4220 Norm SA 1.435162 1.44356 4.313886 0.3250 SA Carotide 0.033074 3.37908 3.445224 0.9847

TABLE-US-00012 TABLE 6 Oneway analysis of shed CD31 (ng/ml) by Group Level Level Difference Lower CL Upper CL p-Value ACS Norm 3.707532 0.87674 6.538321 0.0108* ACS SA 3.238062 0.68347 5.792657 0.0135* ACS Carotide 3.065120 0.33492 6.465159 0.0767* Carotide Norm 0.642412 3.07155 4.356373 0.7322 SA Norm 0.469470 2.49012 3.429064 0.7537 Carotide SA 0.172942 3.33506 3.680946 0.9223

[0251] In particular, the proportion of shed soluble CD31 (% of total) is very useful for distinguishing between acute coronary syndrome, stable coronary disease and peripheral atherosclerosis (see Table 7).

TABLE-US-00013 TABLE 7 Oneway analysis of shed CD31 (% of Total soluble forms of CD31) by Group Level Level Difference Lower CL Upper CL p-Value ACS Norm 22.00424 8.4993 35.50915 0.0017* ACS Carotide 18.24801 2.0274 34.46865 0.0278* ACS SA 18.06073 5.8735 30.24799 0.0041* SA Norm 3.94351 10.1759 18.06290 0.5808 Carotide Norm 3.75623 13.9620 21.47449 0.6750 SA Carotide 0.18729 16.5484 16.92299 0.9823

[0252] Importantly, it was also found that the measure in the plasma of patients of spliced and of shed CD31, and not that of total CD31, allows predicting recurrence of Major Adverse Cardiovascular Events (MACE, such as death, fatal and non fatal myocardial infarction).

TABLE-US-00014 TABLE 8 1-way Test, ChiSquare Approximation Oneway analysis of: ChiSquare DF Prob > ChiSq Total CD31 (ng/ml) by MACE 0.1564 1 0.6925 Spliced CD31 (ng/ml) by MACE 5.0064 1 0.0253* Shed CD31 (ng/ml) by MACE 4.4985 1 0.0339*

Example 8

Shed CD31 as a Diagnostic and/or Prognostic Marker of Inflammatory Diseases

[0253] It was investigated whether the measure of soluble spliced and shed CD31 using the method of the invention could help evaluating the risk of treatment failure in patients with chronic inflammatory diseases.

[0254] Data from 73 patients affected either by rheumatoid arthritis or spondyloarthritis were collected.

[0255] It was found that plasma levels of CD31 were associated both with the patient's outcome and with a positive response to biotherapy (see FIG. 6A). This cannot be predicted by the currently used biomarkers (ESR and CRP).

[0256] The above patients suffered from very severe inflammation. In such cases, the level of shed CD31 may increase of several folds above the baseline levels (even above 1000 ng/ml), and consequently lead to a significant increase of total soluble CD31. Therefore, not only shed CD31 but also total soluble CD31 can be used as a biomarker for inflammation when the inflammation is very severe.

[0257] However, differentiating shed CD31 form total soluble CD31 is required in patients in which the inflammatory state is less obvious.

Example 9

Measure of the Plasma Levels of Soluble GPVI

[0258] The method according to the invention was adapted for measuring plasma levels of soluble GPVI. The soluble form of GPVI is cleaved from platelets upon activation and which could evaluate the occurring of thrombus formation in patients at risk.

[0259] A reproducible method for measuring shed GPVI in the plasma using (i) an antibody for capture (coupled to the CBA beads); and (ii) a fluorescently labeled natural ligand of GPVI (convulxin) for the detection has been set up. The antibody for capture was either clone No. 3j24.2 or clone No. 9012.2. These two monoclonal antibodies are described in patent application No. PCT/US2000/018152, published as WO/2001/000810. These antibodies specifically recognize epitopes located on the ectodomain of GPVI.

[0260] No current ELISA can achieve the sensitivity and precision of this new method (see FIG. 6B). Standard curves can be obtained both with recombinant GPVI dimers and with recombinant GPVI monomers. This finding is valuable since GPVI dimerizes upon platelet activation, just before being cleaved. After shedding it circulates both as a dimer and as a monomer, and the function and/or diagnostic value might differ depending on the dimerization status.

[0261] The method according to the invention thus allows measuring dimers and monomers shed GPVI in the plasma of individuals.

[0262] Specifically detecting the shed form of GPVI can be carried out as follows. GPVI spliced form comprises the cytoplasmic tail and can therefore be detected using specific antibodies recognizing the cytoplasmic tail such as those raised against a maltose-binding protein (MBP)GPVI cytoplasmic tail fusion protein (Suzuki-Inoue et al. J Biol Chem. 2002 277:21561-6.). On the other hand, the shed form of GPVI comprises the complete ectodomain. Therefore, the method of the invention is performed either using specific antibodies (anti-tail and anti ectodomain) on separate beads for the capture and fluorescently labeled convulxin for the detection, or vice versa (capture by convulxin-coupled beads and detection by differently labeled fluorescent anti-tail and anti-ectodomain antibodies).