Proteomic Analysis of Cardioembolic and Large Artery Atherosclerotic Clots Using Reverse Phase Protein Array Technology Reveals Key Cellular Interactions Within Clot Microenvironments

Abstract

Thrombus characteristics are dependent on clot composition, but identification of the etiology based on histological analysis has proved inconclusive. Identification of proteomic signatures may help to differentiate between clots of different etiologies such as cardioembolic, large artery atherosclerotic, and other known etiologies, information that could enhance an individualized medicine approach to secondary stroke prevention. In this study, total protein extracts from cardioembolic (n=25) and large artery atherosclerotic (n=23) thrombus specimens were arrayed in quadruplicate on nitrocellulose slides and immunostained for 31 proteins using a Dako Autostainer (Agilent Technologies, Inc., Santa Clara, USA). We quantified 31 proteins involved in platelet and/or endothelial function, inflammation, oxidative stress, and metabolism. Pathway analysis showed more heterogeneity and protein network interactions in the cardioembolic clots but no specific correlations with clot etiology. Reverse-phase protein arrays are a powerful tool for assessing cellular interactions within the clot microenvironment and may enhance understanding of clot formation and origination. This tool could be further explored to help in identifying stroke etiology in large vessel occlusion patients with embolic stroke of an undetermined source.

Gene expression profiles of ischemic stroke clots retrieved by mechanical thrombectomy are associated with disease etiology

“…Proteins encoded by key DEGs identified in our study have also been found to be differentially expressed in proteomic studies of stroke thrombi 23–27. For example, protein levels of apolipoprotein E (APOE), which had increased gene expression in CE clots, were also found to be higher in CE on liquid chromatography coupled to tandem mass spectrometry analysis by Dargazanli et al 26.…”

Advancing Stroke Research on Cerebral Thrombi with Omic Technologies

“…Abbasi et al recently compared 25 CE vs. 23 LAA thrombi using proteomic techniques and applying pathway analyses and reverse-phase protein arrays to assess cellular interactions within the clots [ 36 ]. The analysis revealed strong interactions between PPAR-gamma, arginase-1, CD63, CD234, PKCαβ Thr 638/641, and VWF in CE clots, indicating that platelet signaling dominates in CE vs. LAA thrombi.…”

Thrombus composition and thrombolysis resistance in stroke

“…Considering the importance of thrombus composition and structure in IVT resistance, identifying factors that influence these parameters may help better predict response to IVT. The determinants are most likely multifactorial, encompassing stroke etiology (eg, cardioembolic vs large artery atherosclerosis) [ 131 , 132 ] and underlying conditions (eg, cancer) [ 133 ], risk factors, comorbidities, and inflammation, all of which factors can be interrelated. With respect to etiology, there is converging evidence that AIS thrombi from cardioembolic origin have higher leukocyte, DNA, and NET content as compared with AIS thrombi linked to large artery atherosclerosis [ 82 , 131 , 134 , 135 ].…”

“…The determinants are most likely multifactorial, encompassing stroke etiology (eg, cardioembolic vs large artery atherosclerosis) [ 131 , 132 ] and underlying conditions (eg, cancer) [ 133 ], risk factors, comorbidities, and inflammation, all of which factors can be interrelated. With respect to etiology, there is converging evidence that AIS thrombi from cardioembolic origin have higher leukocyte, DNA, and NET content as compared with AIS thrombi linked to large artery atherosclerosis [ 82 , 131 , 134 , 135 ]. Whether the higher DNA and NET content of AIS thrombi from cardioembolic origin translates into higher resistance to IVT remains to be determined.…”