Figure 3. PMN exosomes load NE extracellularly.
(A) 5 × 109 quiescent exosomes coincubated with purified NE (1μg) or left in PBS, filtered and rinsed in PBS to remove unbound NE. Exosome NE activity measured by pNA. (B)1 × 109 activated exosomes coincubated with protamine sulfate or PBS with or without α1-AT and NE activity measured by pNA. Data from A and B. shown as mean +/− SE of 4 experiments. (C-G) Model of hypothesized mechanism of NE loading upon exosomes. (C) Quiescent PMN releases exosomes (yellow spheres) constitutively as NE (blue shapes) is sequestered intracellularly in primary granules. (D) Activated PMN continues to release exosomes constitutively while degranulating, releasing NE molecules into solution which bind to exosome as it passes through locally elevated halo of degranulated NE. (E) Close-up model of activated exosome, to which multiple NE particles are bound via charge-mediated interactions. (F) Activated exosome in an environment rich in α1-AT (crescents). Steric hindrance impedes α1-AT interaction with exosomal NE, whereas free NE readily complexes with α1-AT molecules in an irreversible fashion. (G) Activated exosome after application of cationic molecules such as protamine sulfate to displace NE from exosome surface, now readily complexed and inactivated by α1-AT. See also figure S3.