Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults

Abstract

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.

The Platelet Releasate is Altered in Human Pregnancy

“…A) A total of 723 proteins were identified from 18 healthy pregnant (red) (26–35 weeks gestation) and 13 age‐matched non‐pregnant control (blue) platelet releasate (PR) including six proteins unique to pregnancy PR. Additionally all 277 core PR (orange) proteins were found in both pregnancy and control PR. B) Volcano plot of pregnancy versus control PR proteomes ( x ‐axis, t ‐test difference representing the difference between the mean log 2 TPA values of pregnancy to control PR; y ‐axis, the negative log transformed p ‐value) representing the proteins significantly altered in pregnancy.…”

“…Seven hundred twenty three PR proteins were identified in total (Figure A) and more extensive information including peptide sequences identified, quantitative information and comparison to published platelet proteomics datasets can be found in Table 4, Supporting Information. All 277 ‘core’ human PR proteins reproducibly identified across 32 adult human PRs by Parsons et al., were found in 717 proteins shared between our pregnant and control PR (Figure A and Table 4, Supporting Information).…”

“…Seven hundred twenty three PR proteins were identified in total (Figure A) and more extensive information including peptide sequences identified, quantitative information and comparison to published platelet proteomics datasets can be found in Table 4, Supporting Information. All 277 ‘core’ human PR proteins reproducibly identified across 32 adult human PRs by Parsons et al., were found in 717 proteins shared between our pregnant and control PR (Figure A and Table 4, Supporting Information). A further six proteins were uniquely present in pregnant PR (Figure A and Table 5, Supporting Information) including pregnancy‐specific glycoprotein (PSG) 7 (which along with PSG4 and 5 constitutes a minimal set of proteins necessary to sustain pregnancy) and human placental lactogen (CSH2), produced by trophoblasts .…”

“…Washed platelets were isolated and activated with 1 U mL –1 thrombin for 5 min under constant stirring. PR was carefully prepared as before …”

“…MS sample preparation and MS/MS analysis was performed as described . PR was solubilized and proteins precipitated overnight (95% acetone/–20 °C).…”