Deciphering glycomics and neuroproteomic alterations in experimental traumatic brain injury: Comparative analysis of aspirin and clopidogrel treatment

Abstract

As populations age, the number of patients sustaining traumatic brain injury (TBI) and concomitantly receiving preinjury antiplatelet therapy such as aspirin (ASA) and clopidogrel (CLOP) is rising. These drugs have been linked with unfavorable clinical outcomes following TBI, where the exact mechanism(s) involved are still unknown. In this novel work, we aimed to identify and compare the altered proteome profile imposed by ASA and CLOP when administered alone or in combination, prior to experimental TBI. Furthermore, we assessed differential glycosylation PTM patterns following experimental controlled cortical impact model of TBI, ASA, CLOP, and ASA + CLOP. Ipsilateral cortical brain tissues were harvested 48 h postinjury and were analyzed using an advanced neuroproteomics LC-MS/MS platform to assess proteomic and glycoproteins alterations. Of interest, differential proteins pertaining to each group (22 in TBI, 41 in TBI + ASA, 44 in TBI + CLOP, and 34 in TBI + ASA + CLOP) were revealed. Advanced bioinformatics/systems biology and clustering analyses were performed to evaluate biological networks and protein interaction maps illustrating molecular pathways involved in the experimental conditions. Results have indicated that proteins involved in neuroprotective cellular pathways were upregulated in the ASA and CLOP groups when given separately. However, ASA + CLOP administration revealed enrichment in biological pathways relevant to inflammation and proinjury mechanisms. Moreover, results showed differential upregulation of glycoproteins levels in the sialylated N-glycans PTMs that can be implicated in pathological changes. Omics data obtained have provided molecular insights of the underlying mechanisms that can be translated into clinical bedside settings.

Keywords: Aspirin; Clopidogrel; Neuroproteomics; PTMs; TBI.

Figure 1

Heat map representation of the neuroproteomics analysis of the differentially expressed proteins among the ASA, CLOP, ASACLOP, and TBI groups, in comparison to control. Hierarchical clustering analysis of protein expression profiles. (A) Hierarchical clustering analysis of the differentially expressed proteins between ASA and the Ctrl groups. (B) Hierarchical clustering analysis of the differentially expressed proteins between CLOP and the Ctrl groups. (C) Hierarchical clustering analysis of the differentially expressed proteins between ASACLOP and the Ctrl groups. (D) Hierarchical clustering analysis of the differentially expressed proteins between TBI and the Ctrl groups. Each column represents one sample, and each horizontal line refers to a protein. Color legend is on the bottom-right of the figure. Red indicates proteins with a greater expression relative to the geometrical means; green indicates proteins with a lower expression relative to the geometrical means.

Figure 2

Venn diagram analysis depicting shared and unique protein hits among the experimental groups. Venn diagrams showing the number of common and unique proteins detected in the four different groups of study: ASA versus Ctrl, CLOP versus Ctrl, ASACLOP versus CTRL, and TBI versus Ctrl. (A) The common and unique upregulated proteins. Statistically significant unique proteins were identified including 16 proteins (ASA versus CTRL); 19 proteins (CLOP versus CTRL); 12 proteins (ASACLOP versus CTRL); and 16 proteins (TBI versus CTRL). (B) The common and unique downregulated proteins. Statistically significant unique proteins were identified including: 14 proteins (ASA versus CTRL); 10 proteins (CLOP versus CTRL); 13 proteins (ASACLOP versus CTRL); and five proteins (TBI versus CTRL). This work was done using “the InteractiVenn” software.

Figure 3

Principal component analysis (PCA) corresponding to glycomics changes and protein data among the experimental groups. (A) PCA corresponding to glycomics changes. Data points of Ctrl (green rectangle), ASA (red diamond), CLOP (purple triangle), TBI (blue circle), and combination of ASA and CLOP (white square) are shown. The number following the hyphen indicates the replicate number. (B) PCA corresponding to protein data. Data points of Ctrl (blue triangle), ASA (blue circle), CLOP (red square), TBI (green diamond), and combination of ASA and CLOP (white triangle) are shown.