Attacking the NE+ exosome with protamine sulfate effectively attenuates ECM remodeling in a COPD mouse model

Aims and Objectives: Chronic obstructive pulmonary disease (COPD) is characterized by lung extracellular matrix (ECM) remodeling which contributes to obstruction. This is driven in part by exosomes from activated neutrophils (PMNs), which express an a-1 antitrypsin (AAT) insensitive form of neutrophil elastase (NE). Protamine sulfate is a cationic compound used safely for decades in humans. In vitro, the NE upon exosomes is dissociated from the exosome surface by protamine sulfate, rendering the NE AAT-sensitive. We sought to test whether protamine sulfate would effectively prevent NE+ exosome-driven ECM remodeling in an animal COPD model. Methods: PMNs were purified from healthy human donor peripheral blood by the Ficoll gradient technique and activated with f-Met-Leu-Phe (2mM) for 30 minutes, and exosomes purified by differential ultracentrifugation. Exosomes were preincubated with PBS alone or protamine sulfate (25mM) and these were delivered together intratracheally to anesthetized female 10-12 week old A/J mice, which were sacrificed on day 7 and lung morphometry performed. Results: Activated PMN exosomes caused alveolar destruction (MLI 38.52 +/- 2.31 to 55.55 +/- 3.33mm, P= 0.03). This effect was abrogated by exosome preincubation with protamine sulfate (MLI 49.25 +/- 1.48mm, P=0.03). Conclusions: Activated PMN exosomes cause a COPD-like phenotype when given intratracheally to mice, driven in part by AAT resistant surface NE, an effect ameliorated by protamine sulfate treatment. Protamine sulfate is a promising potential therapy for COPD, which may conceivably be tailored into a disease-modifying inhaled or nebulized agent.