Detection of platelet-derived shed CD31

1. A method for detecting a platelet-derived shed ectodomain of a CD31 protein, a lymphocyte-derived shed ectodomain of a CD31 protein, and a soluble splice variant of said CD31 protein in a biological sample, wherein said platelet-derived shed ectodomain of a CD31 protein is a CD31 fragment (i) comprising the 1 st to 5 th extracellular immunoglobulin (lg)-like domains and the whole or a part of the 6 th extracellular lg-like domain of said full-length CD31 protein, said part of the 6 th extracellular lg-like domain comprising an amino acid sequence corresponding to residues 494 to 501 of human CD31 protein and (ii) not comprising the intracellular domain of said full-length protein, the method comprising:

a) providing a capture antibody wherein said capture antibody specifically binds to an epitope located in a region that is present on said platelet-derived shed ectodomain, on said lymphocyte-derived shed ectodomain and on said splice variant;

b) providing a first discriminating antibody which specifically binds to an epitope located in a region that is present on said platelet-derived shed ectodomain and said splice variant and absent from said lymphocyte-derived shed ectodomain;

c) providing a second discriminating antibody which specifically binds to an epitope located in a region that is present on said splice variant and absent from said platelet-derived shed ectodomain and from said lymphocyte-derived shed ectodomain;

d) providing a third discriminating antibody which specifically binds to an epitope located in a region that is present on said platelet-derived shed ectodomain, on said lymphocyte-derived shed ectodomain and on said splice variant;

wherein said discriminating antibodies are labeled with a detectable label;

e) contacting said antibodies with the biological sample;

f) measuring the signal obtained from the label associated with each discriminating antibody;

wherein the capture antibody and the third discriminating antibody bind within the region defined by the 1 st , 2 nd , 3 rd , 4 th and 5 th extracellular domains of said CD31, the first discriminating antibody binds within the 6th Ig-like extracellular domain of said CD31, and the second discriminating antibody binds to the intracellular domain of said CD31.

2. A method of detection of platelet aggregation in a subject, said method comprising detection of a CD31 protein fragment comprising at least a part of the 6 th extracellular immunoglobulin-like domain of said full-length CD31protein and not comprising the intracellular domain of said full-length protein in a biological sample from a subject, wherein the step of detecting said CD31 protein fragment is performed according to the method as defined in claim 1 .

3. A method of diagnosis of thrombosis or an inflammatory condition, said method comprising detection of a CD31 protein fragment comprising at least a part of the 6 th extracellular immunoglobulin-like domain of said full-length CD31 protein and not comprising the intracellular domain of said full-length protein in a biological sample from a subject, wherein the step of detecting said CD31 protein fragment is performed according to the method as defined in claim 1 .

4. A method for monitoring the response of a patient to a drug, said method comprising the steps of:

a) detecting a CD31 protein fragment comprising at least a part of the 6 th extracellular immunoglobulin-like domain of said full-length CD31 protein and not comprising the intracellular domain of said full-length protein in a biological sample of said patient before and after onset of a treatment of said patient with said drug, wherein the step of detecting said CD31 protein fragment is performed according to the method as defined in claim 1 ; and

b) comparing the levels of the CD31 fragments detected at step (a).

5. A diagnostic kit suitable to perform the method of claim 1 comprising:

a) at least one antibody which specifically binds to an epitope located in a region that is present on said lymphocyte-derived shed ectodomain, on platelet-derived shed ectodomains, and on said splice variant,

b) at least one antibody which specifically binds to an epitope located in a region that is present on said platelet-derived shed ectodomain and said splice variant, but absent from said lymphocyte-derived shed ectodomain;

c) at least one antibody which specifically binds to an epitope located in a region present in said splice variant, but absent from said platelet-derived shed ectodomain and said lymphocyte-derived shed ectodomain; and

d) at least one antibody which specifically binds to an epitope located in a region that is present on said platelet-derived shed ectodomain, on said lymphocyte-derived shed ectodomain and on said splice variant,

wherein said at least one antibody of a), b), and c) are labeled with a detectable label.

6. The diagnostic kit according to claim 5 , said kit comprising:

a) a bead linked to an antibody that specifically binds to an epitope located in a region that is present in the 1 st , 2 nd , 3 rd , 4 th or 5 th like extracellular domain of CD31;

b) a first labelled antibody that specifically binds to an epitope located in the 6 th extracellular immunoglobulin-like domain of CD31;

c) a second labelled antibody that specifically binds to an epitope located in a region that is present in the 1 st , 2 nd , 3 rd , 4 th or 5 th Ig-like extracellular domain of CD31; and

d) a third labelled antibody that specifically binds to an epitope located in a region that is present in the intracellular domain or the juxtamembrane domain of said CD31.

7. The method according to claim 1 , wherein said capture antibody is immobilized on a solid support.

8. The method according to claim 1 , wherein said discriminating antibodies are labeled with a fluorescent label.

9. The method according to claim 1 , wherein the signal in step f) is measured by flow cytometry.

10. The method according to claim 1 , further comprising the step of calculating the percentage and/or the amount of said soluble forms that corresponds to said platelet-derived shed ectodomain, and/or the step of calculating either the ratio of platelet-derived shed ectodomain to soluble forms, or the ratio of soluble splice variant to soluble forms.

11. The method according to claim 4 , further comprising a step c) of correlating a difference in said levels of CD31 fragments with the effectiveness of the drug for treating said patient.

12. A method for selecting a suitable treatment for an individual suffering from a thrombotic or inflammatory disorder, wherein said method comprises:

a) detecting a platelet-derived shed ectodomain of CD31 protein, which is a CD31fragment comprising at least a part of the 6 th extracellular immunoglobulin-like domain of said full-length CD31 protein and not comprising the intracellular domain of said full-length protein, in a biological sample from said individual, wherein the step of detecting said CD31 protein fragment is performed according to the method as defined in claim 1 , and

b) deducing thereof a suitable treatment for said individual.

13. The method according to claim 1 , wherein said biological sample is plasma, blood or urine.

14. The method according to claim 1 , wherein said biological sample is obtained from an individual suffering from or at risk of suffering from a thrombotic or an autoimmune disorder.