Extracellular vesicles (EVs) are derived from the membrane of platelets and released in the circulation upon activation or injury. Analogous to the parent cell, platelet derived EVs play an important role in hemostasis and immune responses by transfer of bioactive cargo from the parent cells. Platelet activation and release of EVs increases in several pathological inflammatory diseases, such as sepsis. We have previously reported that the M1 protein released from the bacterial pathogen Streptococcus pyogenes directly mediates platelet activation.
Researchers from Lund University isolated EVs from these pathogen-activated platelets using acoustic trapping and their inflammation phenotype was characterized using quantitative mass spectrometry-based proteomics and cell-based models of inflammation. The researchers determined that M1 protein mediated release of platelet derived EVs that contained the M1 protein. The isolated EVs derived from pathogen-activated platelets contained a similar protein cargo to those from physiologically activated platelets (thrombin), and included platelet membrane proteins, granule proteins and cytoskeletal proteins, coagulation factors and immune mediators. Immunomodulatory cargo, complement proteins and IgG3, were significantly enriched in EVs isolated from M1 protein-stimulated platelets. Acoustically enriched EVs were functionally intact and exhibited proinflammatory effects on addition to blood, including platelet-neutrophil complex formation, neutrophil activation, and cytokine release. Collectively, these findings reveal novel aspects of pathogen-mediated platelet activation during invasive streptococcal infection.
Schematic illustration of the sample processing
Whole blood was collected from five healthy donors, from which platelet-rich plasma (PRP) was prepared. Platelets in PRP were stimulated with one of three stimulants to release EVs. The platelets were then centrifuged, and EVs from the platelet-poor plasma were isolated using acoustic trapping. The isolated EVs were analyzed using mass spectrometry, high-sensitivity flow cytometry, transmission electron microscopy (TEM), immunoblots, and stimulation of whole blood.