Quantifying exosomes and characterizing their tetraspanin expression in platelet-poor plasma, platelet-rich plasma and following platelet activation

Summary: Exosomes are extracellular vesicles secreted by most cells in our body following an endosomal pathway. Exosomes measure 30-150 nm in diameter and contain large number of GFs, cytokines, chemokines, lipids, metabolites and RNA. Exosomes are heterogenous based on the cellular source, quantity, size and molecular content. There is now a great focus to develop exosome-based therapy for regenerative medicine. We quantified exosomes and characterized their tetraspanin expression in clinical samples of platelet-rich plasma (PRP) used to treat patients with hair loss. We similarly analyzed exosomes in PRP following platelet activation (APRP) and in platelet-poor plasma (PPP). We hypothesized that PRP is enriched in platelet-derived exosomes while PPP, a by-product of autologous PRP preparation that is discarded, contains exosomes derived from blood lymphocytes. We selected tetraspanins, CD9, CD41, CD63 and CD81, to detect and quantify exosomes in PPP, PRP and APRP. Exocarta, an online database of exosomal contents curated based on their cellular source, highlighted CD9 and CD63 as markers on exosomes derived from both platelets and lymphocytes while CD81 as being specific to lymphocyte-derived exosomes. CD41 is a platelet maker and therefore is specific to platelet-derived exosomes. We detected differential tetraspanin expression on exosomes with a majority expressing CD41 followed by CD81 and CD63. The exosomes with CD9 expression were the fewest. There was no difference in the quantity of tetraspanin-positive exosomes between PPP, PRP and APRP, which ranged between 10¹¹-2x10¹²per ml, suggesting a lack of segregation of exosomes based on cellular source in PPP and PRP samples. There is also an increase in median size of CD9 and CD41 positive exosomes following platelet activation. We show that PRP and PPP contain exosomes derived from platelets and lymphocytes. The diversity among plasma-derived exosomes suggests their versatility and potential for varied clinical applications. Javed Shaik¹, Saketh Kollipara¹, Maria Hordinsky¹ 1. Dermatology, University of Minnesota Medical School, Minneapolis, MN, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing