2019 Describing the Archetypal Plasma Proteome of Thrombus NETs

INTRODUCTION: Neutrophil extracellular traps (NETs) represent one of the most prominent mechanisms by which immunothrombosis can facilitate, exacerbate, or stabilize pathological thrombosis in stroke. “Gold standard” quantification of NET burden classically relies on immunohistological staining for markers such as myeloperoxidase (MPO) and citrullinated histone 3 (citH3). METHODS: As part of another project, NET burden was measured in 30 retrieved stroke thrombi through immunofluorescence with MPO and citH3 and a single NET marker variable was created through principal component reduction (PC1). Samples of perioperatively collected plasma were processed through the Mass Spectrometry Resource Lab. Plasma proteomic abundance was related to thrombus NET burden and gProfiler was used as a functional analytic platform to characterize plasma pathways associated with thrombus NET burden. RESULTS: ∼2400 individual proteins were identified in patient plasma. 95 plasma proteins were associated with PC1 (|Spearman’s rho| > 0.4, p-adj < 0.05). 55 of these were also associated MPO and citH3 thrombus staining (p-adj < 0.05 for all) and thus served as candidate plasma biomarker proteins. Functional analysis of this subset of plasma proteins revealed overrepresentation of proteasome-related cellular compartments and biological processes in the plasma of patients with greater NET burden (q-value < 1E-3 for all). CONCLUSIONS: This work is the first of its kind to relate plasma proteomic markers to thrombus NET burden. Proteosome activity has previously been shown to participate in NET formation, and thus our finding of elevated proteasome-related proteins in plasma from patients with NET-rich thrombi may not only represent a new biomarker, but additionally extend uncharacterized biology relevant to NET formation either globally, or specifically in stroke.