Hypotheses and aims: Enhanced platelet-monocyte aggregate formation and platelet-induced tissue factor (TF) expression on monocytes are central drivers of immunothrombosis. Besides pro-coagulant pathways (induction of TF), platelet-monocyte interaction also orchestrates inflammation. Increased levels of platelet-monocyte complexes and an expansion of CD16+ monocytes have been reported in severe COVID-19, which is in line with previous findings from our group demonstrating that activated platelets induce a shift from classical monocytes (CM) towards CD16+ intermediate (IM) and non-classical monocytes (NCM). There is strong evidence that this also applies for platelet-derived extracellular vesicles (pEVs), whereas the effect of red blood cell-derived EVs (rbcEVs) on monocyte subset distribution has not been assessed so far. We suggest that both, pEVs and rbcEVs can shift monocyte subsets in whole blood towards IM and NCM, resulting in dysregulated amplification of monocyte and platelet activation.
Beyond triggering immunothrombosis, the shift of monocyte subsets induced by pEVs and rbcEVs could also play a role in transfusion-related inflammatory reactions. Storage of packed red blood cells and platelet concentrates is associated with an increased release of EVs, which are co-administered during transfusion and may induce transfusion-related immunomodulation. Observational studies suggest that transfusing red blood cells after longer storage could be harmful, especially for critically ill patients. We hypothesize that EVs isolated from fresh vs. stored platelet concentrates and packed red blood cells differ in their ability to induce CD16+ monocytes. We will further test whether storage of red blood cells under hypoxic conditions, which has been shown to improve post-transfusion recovery, has an impact on the immunomodulating effect of rbcEVs.
Methods: To assess the effect of pEVs on monocyte subset distribution, pEVs will be enriched from septic plasma by differential centrifugation and will be co-cultured with primary human monocytes. The relative abundance of classical, intermediate, and non-classical monocytes will be characterized by flow cytometry based on the expression of CD14, CD16, CCR2, CX3CR1, and CCR5. In addition, monocyte TF expression will be determined by flow cytometry. EVs will be characterized using nanoparticle tracking analysis and flow cytometry (CD41 and CD235a as platelet and red blood cell markers,
Annexin V as marker for the exposure of phosphatidylserine on the EV surface, P-selectin and integrin αIIbβ3 expression). To study the potential of EVs from blood products to induce a shift towards pro-inflammatory monocytes, primary human monocytes will be co-cultured with pEVs isolated from platelet concentrates (fresh vs. 5d storage) or with rbcEVs isolated from packed red blood cells (fresh vs. 28d storage; normoxic vs. hypoxic storage). Blood products will be provided by the Austrian Red Cross.
Main supervisor: V. Weber (University for Continuing Education Krems)
Proposed mobility: E.I. Buzás, Budapest (EV characterization in blood products)
