Mass Spectrometry-Based Proteomic Profiling of Thrombotic Material Obtained by Endovascular Thrombectomy in Patients with Ischemic Stroke

Abstract

Thrombotic material retrieved from acute ischemic stroke (AIS) patients represents a valuable source of biological information. In this study, we have developed a clinical proteomics workflow to characterize the protein cargo of thrombi derived from AIS patients. To analyze the thrombus proteome in a large-scale format, we developed a workflow that combines the isolation of thrombus by endovascular thrombectomy and peptide chromatographic fractionation coupled to mass-spectrometry. Using this workflow, we have characterized a specific proteomic expression profile derived from four AIS patients included in this study. Around 1600 protein species were unambiguously identified in the analyzed material. Functional bioinformatics analyses were performed, emphasizing a clustering of proteins with immunological functions as well as cardiopathy-related proteins with blood-cell dependent functions and peripheral vascular processes. In addition, we established a reference proteomic fingerprint of 341 proteins commonly detected in all patients. Protein interactome network of this subproteome revealed protein clusters involved in the interaction of fibronectin with 14-3-3 proteins, TGFβ signaling, and TCP complex network. Taken together, our data contributes to the repertoire of the human thrombus proteome, serving as a reference library to increase our knowledge about the molecular basis of thrombus derived from AIS patients, paving the way toward the establishment of a quantitative approach necessary to detect and characterize potential novel biomarkers in the stroke field.

Increased Immunosignals of Collagen IV and Fibronectin Indicate Ischemic Consequences for the Neurovascular Matrix Adhesion Zone in Various Animal Models and Human Stroke Tissue

“…In an in vitro experiment, the soluble fibronectin was further found to impact the formation of fibrin with more and more fibrin matrices in case of increasing fibronectin concentrations (Ramanathan and Karuri, 2014). Circulating fibronectin is thus believed to have a pivotal role on clot formations (To and Midwood, 2011), which is supported by detected fibronectin in proteomic analyses of thrombi that were evacuated by endovascular treatment in human stroke (Muñoz et al, 2018). Additional data on circulating fibronectin recently emerged from an experimental study by Dhanesha et al (2019): by applying a filament-and clot-based stroke model these authors were able to demonstrate that mice deficient for the fibronectin containing the extra domain A exhibited a decreased thrombo-inflammatory response.…”

Proteomics Analysis of Brain Tissue in a Rat Model of Ischemic Stroke in the Acute Phase

“…However, due to the existence of the BBB, potential indexes from peripheral blood do not tend to truly respond to the pathophysiological process in the brain during IS. Thus, besides blood protein measurement, there is an increasing need to search for potential biomarkers from brain tissues (Ning et al, 2011;Chen et al, 2015;Lind et al, 2015;Smith and Gerszten, 2017;Muñoz et al, 2018;Simats et al, 2018). To address this concern, we used iTRAQ-labeled proteomics combined with LC-MS/MS and bioinformatics to screen multiple biomarkers and pathways in this study.…”

“…To date, several studies exploring potential biomarkers following IS have been published (Zhou et al, 2017;Makris et al, 2018). However, a lack of biomarkers with sufficient bioinformatics prediction hinders the clinical prognosis (Muñoz et al, 2018). Moreover, these studies merely focus on peripheral blood-based biomarkers, which provide little help for discovering novel therapeutic targets and elucidating sophisticated molecular mechanisms in brain (Rai et al, 2002;Laskowitz et al, 2009;Devaux, 2017;Misra et al, 2017).…”

Insights from thrombi retrieved in stroke due to large vessel occlusion

“…34,38,39,43,45,49 Thrombus composition analysis unveiling pathogenic feature: bacterial signatures, endothelial cells, calcifications, vascular wall components, valve embolisms. 30,45,48,49,50,52,53,56 Correlation studies between thrombus composition analysis, etiology and imaging to predict thrombus stroke etiology even when the thrombus is not extracted. 100 Assessing thrombus age.…”