Proteomic Profiling of Coronary Thrombus in Acute Myocardial Infarction

Proteomic and Lipidomic Profiling of Coronary Thrombus in Acute Myocardial Infarction

ST-elevation myocardial infarction (STEMI) is mostly caused by the rupture or the erosion of a vulnerable atherosclerotic plaque, initiating with intraluminal thrombosis and resulting in total occlusion of the coronary artery. Thrombus formation is a complex and dynamic process involving flow, blood cells and several plasma proteins, and it has not been clearly elucidated. To define - through proteomic approach - the composition of occluding thrombus and its time changes in patients with STEMI, trying to identify novel biomarkers of coronary thrombosis.

Study Overview

• Status: Unknown
• Conditions: Acute Myocardial Infarction; STEMI - ST Elevation Myocardial Infarction
• Intervention / Treatment: Other: Time from the onset of symptoms to PCI

Detailed Description

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.

• Study Type: Observational
• Enrollment (Anticipated): 50

Contacts and Locations

• Study Contact: Name: Marco Zimarino, MD, PhD; Phone Number: +39 3476045261; Email: m.zimarino@unich.it
• Study Contact Backup: Name: Francesco Radico, MD; Phone Number: +39 3474943605; Email: francescoradico@hotmail.it

Study Locations

• Italy
  • Chieti, Italy
    • Recruiting
    • Santissima Annunziata Hospital
    • Contact: Marco Zimarino, MD, PhD; Phone Number: +39 3476045261; Email: m.zimarino@unich.it

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

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.

Description

Inclusion Criteria:

• patients with STEMI and occluded infarct-related artery or NSTEMI and visible thrombus deemed suitable for thrombus aspiration

Exclusion Criteria:

• thrombus aspiration not performed or unsuccessfull

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Elapsed time of onset-of-pain-to-PCI <3 hours; Patients with AMI undergoing percutaneous coronary intervention (PCI) prior to 3 hours from the onset of symptoms	Other: Time from the onset of symptoms to PCI; Patients will be divided in two groups according to the time from the onset of symptoms to PCI
Elapsed time of onset-of-pain-to-PCI >3 hours; Patients with AMI undergoing percutaneous coronary intervention (PCI) after at least 3 hours from the onset of symptoms	Other: Time from the onset of symptoms to PCI; Patients will be divided in two groups according to the time from the onset of symptoms to PCI

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change through time in thrombus proteomic composition	Compare concentrations of different protein types in the supernatant of thrombus from patients with STEMI<3hs vs <3hs, through proteomic approaches	within 24 hours

Collaborators and Investigators

• Sponsor: G. d'Annunzio University
• Collaborators: Catholic University of the Sacred Heart; Azienda Sanitaria Locale n. 2 - Lanciano Vasto Chieti
• Investigators: Principal Investigator: Marco Zimarino, MD, PhD, G. D'Annunzio University. Institute of Cardiology; Study Chair: Damiana Pieragostino, Department of Medical, Oral and Biotechnological Sciences, G. d'Annunzio University - Chieti

Publications and helpful links

General Publications

• Pieragostino D, D'Alessandro M, di Ioia M, Rossi C, Zucchelli M, Urbani A, Di Ilio C, Lugaresi A, Sacchetta P, Del Boccio P. An integrated metabolomics approach for the research of new cerebrospinal fluid biomarkers of multiple sclerosis. Mol Biosyst. 2015 Jun;11(6):1563-72. doi: 10.1039/c4mb00700j.
• Del Boccio P, Pieragostino D, Di Ioia M, Petrucci F, Lugaresi A, De Luca G, Gambi D, Onofrj M, Di Ilio C, Sacchetta P, Urbani A. Lipidomic investigations for the characterization of circulating serum lipids in multiple sclerosis. J Proteomics. 2011 Nov 18;74(12):2826-36. doi: 10.1016/j.jprot.2011.06.023. Epub 2011 Jul 4.
• Zimarino M, Angeramo F, Prasad A, Ruggieri B, Malatesta S, Prati F, Buttitta F, De Caterina R. Reduction of atherothrombotic burden before stent deployment in non-ST elevation acute coronary syndromes: Reduction of myocardial necrosis achieved with nose-dive manual thrombus aspiration (REMNANT) trial. A volumetric intravascular ultrasound study. Catheter Cardiovasc Interv. 2016 Nov;88(5):716-725. doi: 10.1002/ccd.26301. Epub 2015 Nov 3.

Study record dates

Study Major Dates

• Study Start (Actual): January 1, 2017
• Primary Completion (Anticipated): December 31, 2019
• Study Completion (Anticipated): December 31, 2019

Study Registration Dates

• First Submitted: November 3, 2018
• First Submitted That Met QC Criteria: November 3, 2018
• First Posted (Actual): November 6, 2018

Study Record Updates

• Last Update Posted (Actual): April 18, 2019
• Last Update Submitted That Met QC Criteria: April 16, 2019
• Last Verified: April 1, 2019

More Information

Terms related to this study

• Keywords: Proteomics; Lipidomics; Thrombus aspiration; Coronary thrombus
• Additional Relevant MeSH Terms: Ischemia; Pathologic Processes; Necrosis; Myocardial Ischemia; Heart Diseases; Cardiovascular Diseases; Vascular Diseases; Embolism and Thrombosis; Myocardial Infarction; Infarction; ST Elevation Myocardial Infarction; Thrombosis
• Other Study ID Numbers: PROTEOTHR18

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No