Platelet proteomics on less than a drop of previously frozen, non-citrate plasma

Platelets are blood components not regularly analysed with proteomics due to the conventional wisdom that plasma for platelet research must be citrate-treated and freshly sampled to minimize artifactual changes. Information from platelets is complementary to plasma, specifically regarding immunophenotyping of patients with challenged immune systems due to infection or inflammation. We sought to develop a sample-sparing, high-throughput-compatible platelet proteomics workflow applicable to previously frozen, non-citrate platelet-rich plasma, in contrast to the field's standard, and test its efficacy by applying it to a COVID-19 cohort. We examined centrifugation of whole blood and platelet-rich plasma and volume requirements of plasma for platelet analysis. Platelet and platelet-poor plasma samples were analysed from a cohort of 79 patients, consisting of COVID-19 negative non-ICU and ICU controls and patients with COVID-19 over time. Conventional platelet count was successfully performed using flow cytometry on previously frozen plasma, showing minimal platelet aggregation and cell debris, demonstrating viability of previously frozen plasma for platelet proteomic analysis. Protein counts in platelets mostly mirrored trends in platelet count, except in severe COVID-19 patients within three days of admission to the ICU. Proteins dysregulated in this group compared to controls were enriched in terms related to platelet activation, phagosome, and efferocytosis. This agrees with prior reports using conventional platelet proteomics methods. Such similar findings suggest that the method developed here can utilize non-citrate, previously frozen plasma down to 0.5 μL per sample. This will enable platelet proteomics studies on already collected, banked plasma samples more accessible and increase biomolecular information gained.