Proteomic Profiling of Coronary Thrombus in Acute Myocardial Infarction

Ischemic heart disease is the leading contributor to the worldwide burden of disease, as assessed on the basis of disability-adjusted life-years.

ST-Elevation Myocardial Infarction (STEMI) is associated with a coronary occlusive thrombosis in 90% of patients, non-ST Elevation Myocardial Infarction (NSTEMI) only in 26% of cases. In this regard, increasing attention is given to the composition of coronary thrombus. In acute STEMI, platelet and fibrin content in the occlusive thrombus is highly dependent on ischemia time, thus one can infer that this may have a substantial impact on the efficacy of drugs or devices used for coronary reperfusion.

Among several circulating cell types - platelets, erythrocytes, monocytes, and neutrophils - as well as plasma molecules, modulating the thrombosis process, platelets constitute the major cellular component of the thrombus and their activation is essential for thrombus formation. Although the role of platelets in thrombosis has been deeply characterized, the molecular mechanisms underlying platelet activation and focal adhesion within human coronary thrombi, the composition, time changes of the occluding thrombus and its interaction with the other cells have not been clearly elucidated, mainly due to the difficult accessibility to the thrombotic material. Thrombus aspiration, although having a controversial role as an adjunctive strategy in primary percutaneous coronary intervention (PCI) for STEMI and NSTEMI, relies on the extraction of occlusive thrombi by an aspiration catheter, advanced in the coronary arterial tree and constitutes a unique opportunity for obtaining coronary thrombi from patients suffering from AMI.

Defining variable molecular pathways responsible for thrombus formation according to time lapse and pathophysiology may pave the way to newer therapeutic strategies.

Hypothesis of the study Coronary thrombus undergoes significant modification according to the pathophysiology of formation (erosion, plaque rupture or metal strut exposure) and time-lapse from symptom onset.

The exact definition the molecular composition of coronary thrombus and of its proteomic and metabolomics patterns may identify the target of newer therapeutic strategies.

Aims of the study are: to elucidate the molecular composition of coronary thrombus in AMI, to understand, at molecular level, phenotypic alteration related to thrombus formation, to highlight new potential factors involved in disease's onset or related to time of thrombus formation.

A consecutive series of patients admitted for an ACS, with either STEMI and occluded infarct-related artery or NSTEMI and visible thrombus in the culprit artery will be deemed suitable for thrombus aspiration. PCI will be performed with a standard procedure, with either femoral or radial access. All patients will be receiving acetylsalicylic acid (100 mg daily) and a P2Y12 inhibitor - clopidogrel (600 mg, then 75 mg daily), prasugrel (60 mg, then 10 mg daily) or ticagrelor (180 mg daily). After insertion of the arterial sheath, every patient will receive 70 IU/kg of heparin.

After guidewire positioning across the lesion, the thrombus aspiration system will be advanced with gentle suction and several passages will be performed through the culprit lesion.

The lyophilized thrombus from each patient enrolled will undergo to extensive molecular characterization in term of proteins, metabolites (Amino Acids and Acylcarnitines) and polar lipids.

Polar metabolites and lipids from thrombus will be extracted and analyzed following a validated methodology: 5 mg of wet thrombus will be added to a 330 μL of methanol solution containing deuterated internal standards of amino acids and carnitines and lyso-sphingomyelin 100 µg/ml as internal standard for lipidomics. These solutions will be incubated at 45 °C for 50 min and finally divided into two aliquots. The first one will be used to analyze amino acids and acyl-carnitines following our LC-MS/MS method already used in the analysis of CSF as previously reported. The second aliquot will be employed to screen phospholipids by LC-MS/MS. Data obtained will be quantified using MarkerLynx and NeoLynx software, and statistically investigated using GraphPad Prism, Simca P+ and MetaboAnalyst software.

The lyophilized thrombus will be digested by Filter Aided Sample Prep (FASP) protocol. Then, instrumental triplicates will be acquired for each sample by a platform nanoLC-ESI-QTOF-MS/MS performing label free shotgun proteomics experiments in order to identify, and simultaneously, quantify expressed proteins as already reported. The result of this study will be a list of expressed proteins during coronary thrombus in acute myocardial infarction, that may be used for bioinformatics analysis.

Data will be processed by a dedicated software (Profile Analysis, Bruker, Markerlynx). Highlighted metabolites/proteins will be identified by database and by fragmentation analysis.

Specifically for metabolomics and lipidomics, the data will be processed to obtain peak deconvolution and alignment, denoise and Total Ion Count normalization to give a table of mass pairs with associated relative intensities for all the detected peak for each sample analyzed. Then, the data matrix will be used for multivariate Analysis (PCA; PLS-DA) using SIMCA-P + 11.0 (Umetrics AB, Umeå, Sweden).

By multi-omics researches and data processing, it will be available a panel of interesting molecules as a framework for each clinical group studied. These biological compounds will be then subjected to an in-silico analysis to rebuild their functional implications in the physiology and molecular homeostasis of coronary thrombus in acute myocardial infarction. To perform such a meta-analysis of the data we will mainly use Ingenuity Pathways Analysis (IPA), (Ingenuity Systems, Mountain View, CA). Through the Pathways analysis and the Gene Ontology it will be possible to identify the metabolic pathways and the secondary genes/proteins inhibited and/or stimulated for a specific phenotype and consequently classify potential effectors molecules and/or pharmacological target.