Stroke emboli from patients with atrial fibrillation enriched with neutrophil extracellular traps.

Background: Recent literature has demonstrated remarkable heterogeneity in the composition of acute ischemic stroke (AIS) emboli, which may impact susceptibility to therapy. Objectives: In this study, we explored differences in proteomic composition of retrieved embolic material from patients with stroke with and without atrial fibrillation (AF) (AF+ and AF-, respectively). Methods: The full proteome of retrieved thromboembolic material from 24 patients with AIS was obtained by mass spectrometry. Known marker proteins were assigned groups representing broad classes of embolus components: red blood cells, platelets, neutrophils, eosinophils, histones, complement, and other clotting-associated proteins (eg, fibrinogen). Relative protein abundances were compared between AF+ and AF- samples. Functional implications of differences were explored with gene set enrichment analysis and Gene Ontology enrichment analysis and visualization tool. Results: One hundred sixty-six proteins were differentially expressed between AF+ and AF- specimens. Eight out of the 15 neutrophil proteins (P < .05; fold change, >2) and 4 of the 14 histone proteins were significantly enriched in AF+ emboli (P < .05; fold change, >2). Gene set enrichment analysis revealed a significant representation of proteins from published neutrophil extracellular trap (NET) proteomic gene sets. The most significantly represented functional Gene Ontology pathways in patients with AF involved neutrophil activation and degranulation (P < 1 × 10-7). Conclusion: The present analysis suggests enrichment of NETs in emboli of patients with stroke and AF. NETs are a significant though understudied structural component of thrombi. This work suggests not only unique stroke biology in AF but also potential therapeutic targets for AIS in this population.

Histone content, and thus DNA content, is associated with differential in vitro lysis of acute ischemic stroke clots.

BACKGROUND: Fibrin, von Willebrand factor, and extracellular DNA from neutrophil extracellular traps all contribute to acute ischemic stroke thrombus integrity. OBJECTIVES: In this study, we explored how the proteomic composition of retrieved thromboemboli relates to susceptibility to lysis with distinct thrombolytics. METHODS: Twenty-six retrieved stroke thromboemboli were portioned into 4 segments, with each subjected to 1 hour of in vitro lysis at 37 °C in 1 of 4 solutions: tissue plasminogen activator (tPA), tPA + von Willebrand factor-cleaving ADAMTS-13, tPA + DNA-cleaving deoxyribonuclease (DNase) I, and all 3 enzymes. Lysis, characterized by the percent change in prelysis and postlysis weight, was compared across the solutions and related to the corresponding abundance of proteins identified on mass spectrometry for each of the thromboemboli used in lysis. RESULTS: Solutions containing DNase resulted in approximately 3-fold greater thrombolysis than that with the standard-of-care tPA solution (post hoc Tukey, P < .01 for all). DNA content was directly related to lysis in solutions containing DNase (Spearman's ρ > 0.39 and P < .05 for all significant histones) and inversely related to lysis in solutions without DNase (Spearman's ρ < -0.40 and P < .05 for all significant histones). Functional analysis suggests distinct pathways associated with susceptibility to thrombolysis with tPA (platelet-mediated) or DNase (innate immune system-mediated). CONCLUSION: This study demonstrates synergy of DNase and tPA in thrombolysis of stroke emboli and points to DNase as a potential adjunct to our currently limited selection of thrombolytics in treating acute ischemic stroke.

CT hyperdense cerebral artery sign reflects distinct proteomic composition in acute ischemic stroke thrombus.

BACKGROUND: Hyperdense cerebral artery sign (HCAS) is an imaging biomarker in acute ischemic stroke (AIS) that has been shown to be associated with various clinical outcomes and stroke etiology. While prior studies have correlated HCAS with histopathological composition of cerebral thrombus, it is unknown whether and to what extent HCAS is also associated with distinct clot protein composition. METHODS: Thromboembolic material from 24 patients with AIS were retrieved via mechanical thrombectomy and evaluated with mass spectrometry in order to characterize their proteomic composition. Presence (+) or absence (-) of HCAS on preintervention non-contrast head CT was then determined and correlated with thrombus protein signature with abundance of individual proteins calculated as a function HCAS status. RESULTS: 24 clots with 1797 distinct proteins in total were identified. 14 patients were HCAS(+) and 10 were HCAS(-). HCAS(+) were most significantly differentially abundant in actin cytoskeletal protein (P=0.002, Z=2.82), bleomycin hydrolase (P=0.007, Z=2.44), arachidonate 12-lipoxygenase (P=0.004, Z=2.60), and lysophospholipase D (P=0.007, Z=2.44), among other proteins; HCAS(-) clots were differentially enriched in soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (P=0.0009, Z=3.11), tyrosine-protein kinase Fyn (P=0.002, Z=2.84), and several complement proteins (P<0.05, Z>1.71 for all), among numerous other proteins. Additionally, HCAS(-) thrombi were enriched in biological processes involved with plasma lipoprotein and protein-lipid remodeling/assembling, and lipoprotein metabolic processes (P<0.001), as well as cellular components including mitochondria (P<0.001). CONCLUSIONS: HCAS is reflective of distinct proteomic composition in AIS thrombus. These findings suggest that imaging can be used to identify mechanisms of clot formation or maintenance at the protein level, and might inform future research on thrombus biology and imaging characterization.