WO2012126531A1 - Method for diagnosing acute coronary syndrome (acs) - Google Patents

Abstract

The invention relates to a method of diagnosing the occurrence of acute coronary syndrome in a subject, comprising determining the presence of a biomarker that is indicative of the occurrence of acute coronary syndrome in an exosome sample from the subject. The exosome sample consists of exosomes that are isolated from a body fluid, a body fluid that comprises exosomes selected from serum, plasma, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva, in particular serum.

Description

METHOD FOR DIAGNOSING ACUTE CORONARY SYNDROME (ACS )

FIELD OF THE INVENTION

The present invention relates to the diagnosis of acute ischemic coronary syndromes (ACS) . The invention further relates to kits and biomarkers for use in the method .

BACKGROUND OF THE INVENTION

Patients with chest-pain are entering the emergency rooms of hospitals very frequently. Chest-pain, however, can result from many causes: gastric discomfort (e.g.

indigestion) , pulmonary distress, pulmonary embolism, dyspnea, musculoskeletal pain (pulled muscles, bruises) indigestion, pneumothorax, cardiac non-coronary conditions, and acute ischemic coronary syndrome (ACS) .

Acute coronary syndrome (ACS) is usually one of three diseases involving the coronary arteries: ST elevation myocardial infarction (30%) , non ST elevation myocardial infarction (25%) , or unstable angina (38%) . These types are named according to the appearance of the electrocardiogram (ECG/EKG) as non-ST segment elevation myocardial infarction (NSTEMI) and ST segment elevation myocardial infarction (STEMI). ACS is usually associated with coronary thrombosis.

The physician has to decide if the patient is having a life threatening ischemic ACS or not. In the case of such an ischemic cardiac event, rapid treatment by opening up the occluded coronary artery is essential to prevent further loss of myocardial tissue.

Diagnosis of ACS is often not easy. For this, cardiac biomarkers have become an essential tool to define if a patient has a myocardial necrosis related to myocardial infarction. Favorable features of biomarkers of necrosis are high concentrations in the myocardium and absence in non- myocardial tissue, release into the blood within a

convenient diagnostic time window and in proportion to the extent of myocardial injury, and quantification with

reproducible, inexpensive, rapid, and easily applied assays (cited from ACC/AHA Guidelines, Circulation 116:803-877 (2007) ) .

The cardiac troponins possess many of these features and have gained wide acceptance as the biomarkers of choice. Myocardial necrosis now is defined by an elevation of troponin above the 99th percentile of normal.

Myocardial infarction, which is necrosis related to

ischemia, is further defined by the addition of at least 1 of the following criteria: ischemic ST and T-wave changes, new left bundle-branch block, new Q waves, PCI-related marker elevation, or imaging showing a new loss of

myocardium (cited from ACC/AHA Guidelines, Circulation

116:803-877 (2007) ) .

Although troponins can be detected in blood as early as 2 to 4 h after the onset of symptoms, elevation can be delayed for up to 8 to 12 h. This timing of elevation is similar to that of Creatine Kinase-MB but persists longer, for up to 5 to 14 days.

Accurate and rapid diagnosis of ACS based on a panel of biomarkers originating from different biological pathways is essential. Therefore, an urgent need exists for markers that can add to the diagnostic accuracy of Troponins. An earlier and more accurate detection than Troponins alone will result in a more rapid treatment with subsequent reduced loss of myocardial tissue. SUMMARY OF THE INVENTION

The occlusion itself is often the result of a thrombotic event. This atherosclerotic plaque rupture brings the thrombogenic content of the plaque in contact with the blood initiating thrombosis and subsequent occlusion.

Several leucocytes including platelets are involved in this sequence of events that, after activation, release

microvesicles . Next to this, the ischemic event immediately activates endothelial cells that attract platelets that also become activated. This activation of endothelial cells and platelets is accompanied by the release of microvesicles that are secreted into the blood.

In the research leading to the invention it was contemplated that since secretion not only occurs with individual proteins like Troponin but also via vesicles containing a large number of proteins and RNA, the

components found in, on or attached to the vesicles could also be used as markers.

These vesicles are formed with a selection of lipids, protein and RNA from the secreting cell and are released as an intact vesicle. They are generally called microvesicles and have a size between 20 and 1000 nm. From these microvesicles, exosomes are the best described

particles having a size between 50 and 100 nm.

When an ACS occurs, microvesicles are secreted from several cells and tissues. The most obvious tissue is the myocardium. Apoptosis of cardiomyocytes occurs almost instantly after occluding the coronary artery and subsequent ischemia. The apoptopic cardiomyocytes secrete vesicles in the blood that are called apoptopic bodies.

In the research leading to the present invention, the expression of particular proteins associated with microvesicles , in particular exosomes, were found to be suitable biomarkers to accurately diagnose ACS.

First, proteomic analyses were performed on human plasma samples. The procedure was hampered, however, by the presence of high-abundant plasma proteins such as albumin and immune-globulins, which complicated the detection of potentially interesting low-abundant proteins. Therefore sub-fractions of plasma were investigated for the presence of proteins that may have diagnostic value for the

occurrence of an ACS.

It was then found that protein constitution in plasma exosome samples from subjects that had ACS following the moment of sampling differs from that in patients who did not have an ACS, and that this difference can be used for diagnosis of patients.

Protein secretion out of the cells can occur directly after production (constitutive pathway) or is first stored in the cell and released after a trigger (regulatory pathway) . Secretion, however, not only occurs via the above mentioned pathways with individual proteins but also occurs via vesicles containing a large number of proteins and RNA. These so-called microvesicles are formed with a selection of lipids, protein and RNA from the secreting cell and are released as an intact vesicle ranging in size between 20 and 1000 nm.

Vesicles in the size of 50-100 nm are called exosomes and the release of exosomes has been described for various cell types, including reticulocytes, B- and T- lymphocytes, dendritic cells, mast cells, platelets, macrophages and alveolar lung cells.

In several cell types, including T cells, platelets, dendritic cells and mast cells, secretion of exosomes is regulated by specific stimuli. While early studies focused on their secretion from diverse cell types in vitro,

exosomes have now been identified in body fluids such as urine, amniotic fluid, malignant ascites, broncho-alveolar lavage fluid, synovial fluid, breast milk, saliva and blood. Exosomes have a wide range of biological functions,

including immune response, antigen presentation,

intracellular communication and the transfer of RNA and proteins . DETAILED DESCRIPTION OF THE INVENTION

The present invention shows that exosomes express an array of proteins that reflect the originating host cell and that they contain valuable information regarding ongoing (patho) physiologic processes in the human body including information on the occurrence of ACS.

This surprising finding now led to the present invention, which thus provides a method for the diagnosis of ACS in a patient, based on the detection of particular proteins in, on or attached to exosomes, which proteins are herein after referred to as biomarkers or differentially present proteins. The proteins can be detected either in or on or attached to isolated exosomes and in or on exosomes that are still present in a body fluid, in particular serum.

According to the invention in principle any biomarker with diagnostic value may be used. In particular, however, specific markers were identified in/on plasma exosomes that have diagnostic value for ACS.

In one embodiment, the invention thus provides a method for the diagnosis of ACS comprising detecting a biomarker in an exosome sample or micro-vesicles of smaller or larger size from said subject, wherein said sample comprises at least one protein selected from the group of 3 proteins consisting of: Serpin F2 (IPI : IPI00879231, SWISSPRO :A2AP_HUMAN) , CD14 ( I PI : I PI 00029260 ,

SWISSPROT : CD14_HUMAN) , Cystatin C ( I PI : I PI 00032293 ,

SWISSPROT :CYTC_HUMAN) .

The IPI numbers as disclosed herein between brackets refer to the International Protein Index

(http://www.ebi.ac.uk/IPI), as indexed on December 4, 2010 followed by Swissprot database Entry name as indexed on November 30, 2010. The referenced index numbers (database accessions) as used herein include reference to fragments, isoforms and modifications thereof, hence the present invention foresees the use of fragments of the proteins as well as modifications and derivatives of the proteins disclosed herein as biomarkers in the context of the various aspects of the present invention.

According to the invention a biomarker comprises one protein or a set of multiple proteins. Such a biomarker is also identified herein as a profile or protein profile. A profile may comprise 1, 2 or 3 of the proteins Serpin F2 (IPI : IPI00879231, SWISSPROT :A2AP_HUMAN) , CD14

(IPI : IPI00029260, SWISSPROT : CD14_HUMAN) , Cystatin C

(IPI : IPI00032293, SWISSPROT : CYTC_HUMAN) in any combination, in particular CD14 and Serpin F2 or Cystatin C and Serpin F2 or CD14 and Cystatin C or CD14, Serpin F2 and Cystatin C.

The skilled person will understand that instead of detecting the complete biomarker protein, one may detect peptide fragments of said biomarker proteins which are derived from the biomarker proteins by fragmentation

thereof. The term peptide fragment as used herein refers to peptides having between 5 and 50 amino acids. These peptide fragments preferably provide a unique amino acid sequence of the protein, and are associated with the cardiovascular events as disclosed herein. The proteins and/or peptide fragment may optionally be detected as chemically modified proteins and/or peptides, such chemical modification may for instance be selected from the group consisting of glycosylation, oxidation,

(permanent) phosphorylation, reduction, myristylation, sulfation, acylation, acetylation, ADP-ribosylation,

amidation, hydroxylation, iodination, and methylation. A large number of possible protein modifications is described in the RESID database at http : //www .ebi.ac.uk/RESID (release December 2 2010) (Garavelli, J.S. (2004) The RESID Database of Protein Modifications as a resource and annotation tool; Proteomics 4: 1527-1533) and in Farriol-Mathis , N.,

Garavelli, J.S., Boeckmann, B., Duvaud, S., Gasteiger, E., Gateau, A., Veuthey, A., Bairoch, A. (2004) Annotation of post-translational modifications in the Swiss-Prot knowledge base. Proteomics 4(6): 1537-50, The skilled artisan is well aware of these modifications.

The biomarkers can be found in exosomes but also physically connected or linked to exosomes, which means both in or on their surface. When on their surface the biomarker can be either attached to the membrane, e.g. expressed on or in the membrane surface or anchored therein, or be in loose connection therewith, i.e. adhered to the exosome without being physically attached to or in the membrane. The

biomarkers may also be part of the membrane. Of the

biomarkers listed above Cystatin C is not attached to the membrane but rather adheres to it. CD14 is anchored in the membrane, and Serpin F2 has been associated with the

membrane but it is unclear how it is attached.

It was found according to the invention that the biomarkers that are attached, anchored or adhered to the exosome can also be detected in samples of body fluid, in particular in serum. In a preferred embodiment of the invention, the biomarker protein or a peptide fragment thereof is detected in, on or attached to exosomes that are preferably found in body fluids like serum, plasma or blood.

The invention will be further illustrated in the examples that follow and that are not intended to limit the invention in any way. In the Examples reference is made to the following figure:

Figure 1: Area under the curve analysis for Troponin (Trop, solid curve) measured in blood taken at intake of the patient with chest pain and Troponin plus Serpin F2

( SerpinF2_CP, dashed curve) .

EXAMPLES EXAMPLE 1

Quantitative Proteomics on human plasma exosomes with follow-up

Study population and design

The Athero-Express is a longitudinal vascular biobank study, which includes biomaterials from patients undergoing carotid and femoral end-arterectomy in two Dutch hospitals (UMC Utrecht and St. Antonius Hospital

Nieuwegein) . About 2000 patients have been included thus far. Plasma and tissue samples were obtained from all patients before (blood) or during end-arterectomy.

All patients underwent clinical follow-up 1 year after surgical intervention and filled in postal

questionnaires 1, 2 and 3 years after the operation. When patients did not respond to the questionnaire, the general practitioner was contacted by phone. Adjudication of the outcome events was done by an independent outcome event committee that was blinded to laboratory results. Two members of the committee independently assessed all endpoints. In case of disagreement, a third opinion was obtained .

The exosomal proteome

Plasma samples from 50 patients that suffered an ACS during follow up and from 50 age and sex matched control patients, without any secondary event during follow up, were pooled separately and exosomes were isolated by

ultracentrifugation . Quantitative proteomics were performed on the exosomal protein content, and allowed to compare the expression levels of the proteomes from patients that suffered events during follow up with the proteomes of control patients.

Exosomes were isolated from frozen human plasma by filter separation followed by ultracentrifugation .

Typical exosomal proteins such as CD9 and CD81 were detected in the exosome pellet using western blotting. FACS analysis with beads of defined sizes demonstrated that the pellet contains mostly particles of 50-100 nm which is in accordance with the size of exosomes.

Protein extraction and digestion

The exosome pellets collected in the Athero-Express biobank plasma were after ultracentrifugation dissolved in 40 μΐ 6% SDS in HPLC pure water. Plaque protein was, after grinding the plaque material without any blood remains to powder, also extracted with 6% SDS. Digestion and subsequent labeling, HPLC separation and mass spectrometry analysis was identical for plaque and exosome proteins. The protein content was determined by 2-D Quant Kits. After protein reduction and alkylation, the protein mixture was diluted 20 times with 50 mM triethylammonium bicarbonate (TEAB) and protein digestion was initiated by adding trypsin in a 1:40 trypsin-to-protein ratio. The protein digests were desalted using a Sep-Pak C18 cartridge and dried in a Speedvac.

These digests were labeled with iTRAQ reagents according to the manufacturer's protocol. Briefly, digested proteins were reconstituted in 30 μΐ of dissociation buffer and mixed with 70 μΐ of ethanol-suspended iTRAQ reagents (one iTRAQ reporter tag per protein sample, mass tag 114-117 Dalton) . Labeling reactions were carried out at RT for 1 hr before all the samples were mixed into a single tube and dried using a Speedvac.

Strong Cation Exchange fractionation

The combined iTRAQ labeled samples were

reconstituted with 200 μΐ buffer A (10 mM KH₂P0₄, pH 3.0, 25% v/v acetonitrile) , and loaded onto a PolySULFOETHYL A column (200 mm length x 4.6 mm ID, 200-A pore size, 5 μιη particle size) on a Shimadzu prominence HPLC system. The sample was fractionated using a gradient of 100% buffer A for 5 min, 5- 30% buffer B (10 mM KH₂P0₄, pH 3.0, 500 mM KC1 and 25% v/v acetonitrile) for 40 min, 30-100% buffer B for 5 min, and finally 100% buffer B for 5 min, at a constant flow rate of 1 ml/min for a total of 60 min. The eluted fractions were monitored through a UV detector at 214 nm wavelength.

Fractions were collected at 1-min intervals and consecutive fractions with low peak intensity were combined. Finally, a total of 20 fractions were obtained and dried in a Speedvac. Each fraction was reconstituted in 0.1%

trifluoroacetic acid and desalted. Desalted samples were dried in a Speedvac and stored at -20°C prior to mass spectrometric analysis. Mass spectrometric analysis using Q-STAR

The dried fraction was re-constituted in 100 μΐ of 0.1% formic acid. Each sample was analyzed two times using a Q-Star Elite mass spectrometer, coupled to an online

Shimadzu prominence HPLC system. For each analysis, 50 μΐ of peptide mixture was injected and separated on a home-packed nanobored C18 column with a picofrit nanospray tip (75 μιη ID x 15 cm, 5 μιη particles) . The separation was performed at a flow rate of 20 μΐ/min with a splitter of a 90 min gradient. The mass spectrometer was set to perform data acquisition in the positive ion mode, with a selected mass range of 300- 2000 m/z . Peptides with +2 to +4 charge states were selected for MS/MS and the time of summation of MS/MS events was set to 2 s . The three most abundantly charged peptides above a 5 count threshold were selected for MS/MS and dynamically excluded for 30 s with ±30 mmu mass tolerance.

Peptide quantification and protein identification were performed using ProteinPilot software v2.0.1 by

searching the combined data from the 2 runs against the International Protein Index (IPI) human database (indexed December 19, 2009) . The Paragon algorithm in ProteinPilot software was used whereby trypsin was selected as the digestion agent and cysteine modification of

methylethanethiosulfonate .

All proteins reported had an expectation value of less than 0.05 (unused score 1.3).

Quantitative proteomics were performed on exosomes from 50 patients that suffered an ACS during follow up

(Group 1) and from 50 matched control patients that did not suffer a secondary event during follow up (Group 2) . Each group was run twice in the same iTraq experiment revealing data of 2 events proteomes and 2 control proteomes. 2148 different proteins were identified in the samples, including a large number of proteins identified earlier in exosome proteomics such as CD9, CD81, Annexins, Clathrin heavy chain, Enolase 1 and many more (Olver C, Vidal M. Proteomic analysis of secreted exosomes. Subcell Biochem. 43:99-131 (2007)).

Results of Exosome proteomics

Group 1 and 2 were then compared using the

quantitative iTRAQ data. Quantitative data were available from 2 pooled events samples (Group 1 in duplo) and 2 pooled control samples (Group 2 in duplo) . Based on pilots, it was determined that a ratio of 1.2 and above means that there is significantly higher level of the protein in the event while a ratio of 0.8 and lower is a significant lower level in the event. First selection was based on proteins with identical duplo' s (both below 0.8, both above 1.2 or both between 0.8 and 1.2).

Second selection was based on proteins with lower (events/controls <0.8) or higher (events/controls >1.2) expression in group 1 vs. group 2. This revealed a list of 116 proteins.

This list of 116 proteins was uploaded and analyzed in Ingenuity Pathway Analysis software (version 8.0) . From the 116 proteins, 102 proteins were in the Ingenuity

database. This revealed that the 102 proteins are different types of proteins, including transmembrane receptors, transporters and transcription regulators, proteins that are not present in plasma. Ingenuity analysis also showed that 3 canonical pathways are significantly overrepresented in these 102 proteins:

Acute Phase Response Signaling (p= 3.5*10-11)

Coagulation System (p= 3.6*10-11) Atherosclerosis Signaling (p= 3* 10-4)

Subsequently, this group of 102 differentially

expressed proteins was complemented with a selection of plaque material derived proteins and finally narrowed down to a combined set of 34 selected exosome and plaque derived proteins for further validation in exosome samples of individual patient samples. Results of Plaque protein proteomics

Using the Athero-Express cohort, 40 carotid end- arterectomy patients were selected of which 20 had a

secondary cardiovascular event during follow-up and 20 (age, sex and time to follow-up matched) controls that did not suffer from a secondary event during follow-up.

Quantitative proteomics was performed on plaque samples as for the exosome proteomics. However, since 40 individual plaques were analyzed, four plaque extracts were run simultaneously each differently labeled by the iTraq reagent (114, 115, 116, 117 resp.) . Each run consisted of two plaque extracts of patients that suffered a

cardiovascular event and for each patient a sex and age matched control, so in total four plaque extracts in two pairs of event and control.

After the search, an excel file was generated containing the protein ID and the relative value of the two event/control pairs for each of the protein IDs.

Analysis was performed after 10 runs including 20 pairs of events with matched controls with a total of 40 patients. Using Excel 2007 with the Merge Table Add-in, a total list of protein IDs was generated. Normalization between the different runs occurred via total peptide area correction . Statistical analysis comparing events with controls using a Mann-Whitney test revealed 264 proteins that were significantly different (p<0.05) between events and controls in plaque.

Selection of exosome and plaque-derived proteins

The plaque is the origin of atherosclerotic disease leading to cardiovascular events. For this, it is very likely that plaque proteins related to future cardiovascular events can also be found in exosomes especially the plaque proteins that are related to the pathways over-represented in exosome proteins that differ between patients suffering from a cardiovascular events and healthy controls. Having established that 3 canonical pathways (acute phase,

coagulation and atherosclerosis) are over-represented in exosomes, the 264 protein data-set with differentially expressed plaque proteins between events and controls was investigated in 2 ways.

Selection was based on the presence of proteins that are related to the 3 atherosclerosis related canonical pathways and for which 2 antibodies and a recombinant protein were available.

Also from the 112 exosome-derived proteins, markers were selected based on over-representation of 3

atherosclerosis related canonical pathways and the

availability of 2 antibodies and a recombinant protein.

From the selected plaque and exosome proteins for which antibodies and recombinant protein were available, 34 proteins were chosen for Luminex bead assay development. For 17 proteins out of those 34 proteins (including Cystatin C, Serpin F2 and CD14), a reproducible and quantitative Luminex bead assay was set up that could be used for measuring the protein content in exosomes isolated from individual serum samples.

EXAMPLE 2

Verification of a selection of differentially expressed proteins in a Proof of Concept study in blood samples of individual patients (QICS study)

The Quick Identification of acute Chest pain Study (QICS)

Study objective

One of the objectives of the QICS study is the identification of sensitive predictive markers in acute chest pain patients. We will test, presentational symptoms, traditional risk factors, individual biomarkers, a profile of biomarkers, coronary calcium score, coronary

stenosis/plaque volume. Biomarkers will be retrospectively tested after defrosting of deep frozen blood taken on presentation .

Methods and Design

The Quick Identification of acute Chest pain Study

(QICS) will investigate whether a combined use of specific symptoms and signs, electrocardiography, routine and new laboratory measures, adjunctive imaging including electron beam (EBT) computed tomography (CT) and contrast multi-slice CT (MSCT) will have a high diagnostic yield for patients with acute chest pain.

All patients are investigated according a

standardized protocol in the Emergency Department. Serum and plasma are frozen for future analysis for a wide range of biomarkers at a later time point. The final diagnosis non cardiac chest pain, unstable angina, non ST elevation myocardial infarction, and ST elevation myocardial

infarction, with registration of troponin, short term outcome, and long term outcome for secondary coronary events is recorded.

Materials and Methods

Isolation and measurement

Cystatin C, Serpin F2 and CD14 were measured using Luminex multiplex technology on/in or attached to exosomes that were isolated with Exoquick™ from 250 ul of serum of individual QICS patients.

Statistical analyses

Statistical analyses were performed using the statistical software package PASW Statistics 17.0.2 (SPSS Inc, Chicago, Illinois) . Discrimination (a measure of how well the model can separate events and controls) is most often measured by the area under the receiver operating characteristic (ROC) curve, an established method for assessing biomarkers (Hlatky et al . American Heart

Association Expert Panel on Subclinical Atherosclerotic Diseases and Emerging Risk Factors and the Stroke Council. Criteria for evaluation of novel markers of cardiovascular risk: a scientific statement from the American Heart

Association. Circulation 119 (17) : 2408-16 (2009)).

ROC analyses were performed to determine the ability of the marker, in conjunction with a risk score, to

distinguish between patients with chestpain without an acute coronary syndrome and patients with chestpain that do have an acute coronary syndrome. Results

Cystatin C (235 samples) was differentially expressed in patients that had an ACS and patients that did not have an ACS when entering the emergency room with acute chest pain p-value: p= 0.003) . Serpin F2 (238 samples) showed a p-value of p< 0.001 between ACS and non-ACS while CD14 gave a p-value of 0.002 (238 samples)

The strongest marker Serpin F2 was analyzed to see if it had additional value in diagnosing acute coronary syndrome on top of Troponin levels measured at the intake of the patient.

ROC curves (Figure 1) show that the area under the curve increases from 0.835 for Troponin alone to 0.881 for Troponin plus Serpin F2. Serpin F2 plus the maximum levels of troponin measured (Tropmax) is significantly different from Tropmax alone and Serpin F2 plus High sensitive (Hs ) - Troponin is also significantly different from Hs-Troponin alone showing the added value of Serpin F2.

Area Under the Curve

The test result variable (s) : predicted probability trop has at least one tie between the positive actual state group and the negative actual state group.

a. Under the nonparametric assumption

b. Null hypothesis: true area = 0.5 SerpinF2 CP and 3 versions of troponin

SerpinF2 CP & troponin 0.881 [0.837 - <0.001

0.925]

SerpinF2 CP & tropmax 0.994 [0.988 - <0.001

1.000]

SerpinF2 CP & Hs-Troponin 0.965 [0.943 - 0.011

0.987]

Claims

1. Method of diagnosing the occurrence of acute coronary syndrome in a subject, comprising determining the presence of a biomarker that is indicative of the occurrence of acute coronary syndrome in an exosome sample from the subj ect .

2. Method as claimed in claim 1, wherein the exosome sample consists of exosomes that are isolated from a body fluid selected from serum, plasma, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva, in particular serum.

3. Method as claimed in claim 1, wherein the exosome sample consists of a body fluid that comprises exosomes and is selected from serum, plasma, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva, in particular serum.

4. Method as claimed in any one of the claims 1-3, wherein the biomarker is selected from Serpin F2, CD14, Cystatin C.

5. Method as claimed in claim 4, wherein the biomarker is any combination of two or more proteins selected from Serpin F2, CD14, Cystatin C.

6. Kit for performing the method as claimed in any one of the claims 1-5, comprising means for detecting the presence of a biomarker as defined in claim 4 or 5.

7. Kit as claimed in claim 6, wherein the means for detecting the presence of the biomarker are antibodies, antibody fragments or antibody derivates, optionally comprising a detectable label.

8. Kit as claimed in claim 6 or 7, further

comprising reagents and/or instructions for using the means for detecting a biomarker in a method as claimed in any of claims 1-5.

9. Biomarker for use in the diagnosis of acute coronary syndrome occurring in a subject, comprising a protein selected from Serpin F2, CD14, Cystatin C.

10. Biomarker as claimed in claim 9, wherein the biomarker comprises a combination of two or more proteins selected from Serpin F2, CD14, Cystatin C.

11. Biomarker as claimed in claim 9 or 10, wherein for the diagnosis of the occurrence of acute coronary syndrome in a subject the biomarker is detected in an exosome sample of the subject.

12. Biomarker as claimed in claim 11, wherein the exosome sample consists of isolated exosomes.

13. Biomarker as claimed in claim 11, wherein the exosome sample is a sample of a body fluid that comprises exosomes and is in particular serum.