Undergraduate Research Student California Polytechnic State Univ., California, United States
Introduction/Rationale: Increased intravascular coagulation is a molecular hallmark of sepsis and we showed that extracellular vesicles (EVs) from human neutrophils challenged with bacteria activate the coagulation cascade. Discovery of the EV biogenesis pathway may lead to the ability to interfere with EV production and reduce coagulation.
Methods: To investigate the mechanism of EV biogenesis, we performed proteomics on three EV subsets: those produced spontaneously, in response to fMLF, and after staphylococcal challenge. After protein abundances were normalized, we compared fold changes between subsets. We validated the proteomic results with immunoblotting. The role of nSmase was evaluated using GW4869, an nSmase inhibitor.
Results: Although the production of some neutrophil EV subsets relies on membrane curvature caused by neutral sphingomyelinase (nSmase), the production of EVs in response to staphylococcal challenge was nSmase-independent. Annexins and proteins involved in the formation of the membrane attack complex (C5b-9) were enriched in EVs produced after staphylococcal challenge.
Conclusion: Based on these results, we propose that complement-mediated bystander activation produces membrane damage, which initiates Annexin-mediated repair and EV production. To test this, we opsonized bacteria with C9-sufficient or deficient serum, and measured production of EVs by monitoring Flotillin-1. In the absence of C9, less Flotillin-1 was observed compared to the control. These data suggest a role for complement in EV production.
