Comparison of Proteome Composition of Serum Enriched in Extracellular Vesicles Isolated from Polycythemia Vera Patients and Healthy Controls

Abstract

Extracellular vesicles (EVs), e.g., exosomes and microvesicles, are one of the main networks of intercellular communication. In myeloproliferative neoplasms, such as polycythemia vera (PV), excess of EVs originating from overabundant blood cells can directly contribute to thrombosis through their procoagulant activity. However, the proteomic composition of these vesicles in PV patients has not been investigated before. In this work, we examined the proteomic composition of serum EVs of PV patients in comparison to healthy controls. We processed EV-enriched serum samples using the Multiple Enzyme Filter Aided Sample Preparation approach (MED-FASP), conducted LC-MS/MS measurements on a Q-Exactive HF-X mass spectrometer, and quantitatively analyzed the absolute concentrations of identified proteins by the Total Protein Approach (TPA). Thirty-eight proteins were present at statistically significant different concentrations between PV patients’ study group and healthy controls’ group. The main protein components deregulated in PV were primarily related to excessive amounts of cells, increased platelet activation, elevated immune and inflammatory response, and high concentrations of procoagulant and angiogenic agents. Our study provides the first quantitative analysis of the serum EVs’ proteome in PV patients. This new knowledge may contribute to a better understanding of the secondary systemic effects of PV disease and further development of diagnostic or therapeutic procedures.

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…Filter aided sample preparation approach (FASP), a proteomic method for buffer exchange and protein digestion before MS analysis, and the related multiple enzyme digestion (MED) FASP have been successfully used in several EV studies, providing easy and efficient protein processing and minimal sample loss [ 123 , 124 ].…”

Exosomes and Extracellular Vesicles in Myeloid Neoplasia: The Multiple and Complex Roles Played by These “Magic Bullets”

“…Despite the fact that the excess of EVs in these patients has been thought to contribute directly to thrombosis, the hypothetic role played by proteins contained in exosomes in thrombotic events in PV patients need to be confirmed. Using enzymatic-, mass spectrometer-and absolute quantification-based approaches, Fel and colleagues detected high concentrations of procoagulant and proan-giogenic proteins in exosomes isolated from PV patients' plasma [124]. These observations were immediately confirmed by an Italian study reporting small EVs released by megakaryocytes as a biomarker of thrombopoiesis in patients affected by MF (p < 0.001) [125].…”

Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer

“…Healthy control volunteers had no family history or a personal medical history of breast cancer; however, women of a variety of ages and backgrounds were included. While all patients satisfied the inclusion criteria for the study, factors that can impact protein content, such as excessive amounts of cells in the standardised starting volume, increased platelet activation, elevated immune and inflammatory response, and high concentrations of procoagulant and angiogenic agents were not accounted for [35]. Within the human sera analysed, a significantly higher quantity of EVs was observed in the cancer patients, compared to healthy controls.…”