Proteomics of human platelet lysates and insight from animal studies on platelet protein diffusion to hippocampus upon intranasal administration

Abstract

Human platelet lysates (HPLs) from allogeneic platelet concentrates (PCs) are biomaterials, which are rich in various trophic factors, increasingly used in regenerative medicine and biotherapy. Understanding how preparation methods influence the HPL protein profile, biological function, and clinical outcomes is crucial. Our study sheds light on the proteomes and functionality of different HPLs, with the aim of advancing their scientifically grounded clinical applications. To achieve this, PCs suspended in plasma underwent three distinct processing methods, resulting in seven HPL types. We used three characterization techniques: label-free proteomics and tandem mass tag (TMT)-based quantitative proteomics, both before and after the immunodepletion of abundant plasma proteins. Bioinformatic tools assessed the proteome, and western blotting validated our quantitative proteomics data. Subsequent pre-clinical studies with fluorescent labeling and label-free proteomics were used as a proof of concept for brain diffusion. Our findings revealed 1441 proteins detected using the label-free method, 952 proteins from the TMT experiment before and after depletion, and 1114 proteins from the subsequent TMT experiment on depleted HPLs. Most detected proteins were cytoplasmic, playing key roles in catalysis, hemostasis, and immune responses. Notably, the processing methodologies significantly influenced HPL compositions, their canonical pathways, and, consequently, their functionality. Each HPL exhibited specific abundant proteins, providing valuable insight for tailored clinical applications. Immunoblotting results for selected proteins corroborated our quantitative proteomics data. The diffusion and differential effects to the hippocampus of a neuroprotective HPL administered intranasally to mice were demonstrated. This proteomics study advances our understanding of HPLs, suggesting ways to standardize and customize their production for better clinical efficacy in regenerative medicine and biotherapy. Proteomic analyses also offered objective evidence that HPPL, upon intranasal delivery, not only effectively diffuses to the hippocampus but also alters protein expression in mice, bolstering its potential as a treatment for memory impairments.

FIG. 1.

(a) Total proteins (mg/ml) in human platelet lysates (HPLs). Results are expressed as the mean ± SD (N = 4, biological replicates). (^*p < 0.05, ^***p < 0.001) ns, not significant; (b) Venn diagram of the total number of identified proteins using label-free vs those from the TMT-labeled techniques; and from the current study using a label-free technique vs a study by Huang et al.

FIG. 2.

Categorization of human platelet lysates (HPLs) by (a) subcellular localization and (b) general function and a corresponding list of growth factors and cytokines. Data are presented as percentages of the total number of identified proteins in a given group per total number of proteins recognized by the IPA. (c) Categorization of HPLs by gene ontology (GO)-biological processes, GO-cellular components, and GO-molecular functions using DAVID. Data are presented as significance −log10 (BH-adjusted p value) of the prediction term.

FIG. 3.

Principal component analysis (PCA) plot showing a grouping of different human platelet lysates (HPLs) based on their protein expressions.

FIG. 4.

(a) Venn diagram of the number of quantified proteins in human platelet lysates (HPLs); and (b) heatmap of quantified protein in HPLs and categorization of their respective clusters by gene ontology-biological processes. Presented as significance −log10(BH-adjusted p value) of the prediction term.

FIG. 5.

Major canonical pathways discovered by the Ingenuity pathway analysis (IPA) and the degrees of activation of proteins with significant changes in abundances at each processing step. The pathways are given in statistically significant (p value) and activation z-scores (a z-score of > 0 suggests activation, whereas a z-score of < 0 shows inhibition; NA, not available).

FIG. 6.

Western blot results of proteins in human platelet lysates (HPLs) and their respective quantification results from proteomics including original- and depleted-HPLs from TMT-proteomics (1) and depleted-HPLs from TMT-proteomics (2).

FIG. 7.

(a) Heatmap of quantification of trophic proteins found in depleted human platelet lysate (HPL) proteomes of the TMT-based experiment (2) and positive regulatory proteins involved in complement and coagulation cascades found in original HPL proteomes of the TMT-based experiment (1); (b) fluorescent labeling of HPPL in sagittal cross section mice brain of control and HPPL-treated group; (c) Venn diagram of exclusive proteins (EPs) identified using the Homo sapiens database and up-regulated proteins (URPs) identified using both the Homo sapiens and Mus musculus databases in HPPL-treated mice group; (d) correlation heatmap between mice hippocampus proteomes; (e) volcano plot of differential-expressed proteins (DEPs) in HPPL-treated mice group; and (f) protein–protein interaction analysis of DEPs.

FIG. 8.

(a) Human platelet lysates (HPLs) preparation scheme and (b) tandem mass tag (TMT)-based LC-MS/MS scheme for original human platelet lysates (HPLs) and depleted-HPLs. Abbreviations: Freeze-thawed platelet lysate (FTPL), serum-converted platelet lysate (SCPL), heat-treated SCPL (HSCPL), platelet pellet lysate (PPL), heat-treated PPL (HPPL), filtered HPPL: 0.2–0.1 μm (MHPPL), and Planova 20 N (NHPPL).