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Background Pleural infection is a complex disease with significant morbidity and mortality. Pleural fluid drainage and administration of antibiotics are the first line treatment. Intrapleural fibrinolytic deficiency leads to accumulation of fibrin deposits which segregate the pleural cavity and hamper fluid drainage. Patients with septations receive intrapleural fibrinolytic treatment (IFT, tissue plasminogen activator and DNase) to improve drainage. Citrullination of fibrin by Peptidylarginine deiminase 4 (PAD4), a key enzyme that initiates NETosis by citrullinating histones, has been suggested to lead to formation of denser fibrin clot, resulting in increased resistance to lysis.Aim To assess the level of intrapleural citrullinated fibrin and its effect on the fibrinolytic potential ex vivo with clinical samples obtained from pleural infection patients prospectively at baseline and post-IFT.Results We found an increase ( p<0.0001) in soluble fibrin after IET, confirming that the treatment acts to increase the cleaved fibrous tissue. Significant decrease in the percentage of citrullinated fibrin was observed in the pleural cavity of pleural infection patients after IFT (p<0.0001), suggesting citrullinated fibrin contributes to fibrinolytic deficiency. A tendency of negative correlation (r= -0.5351, p= 0.073) was found between citrullinated fibrin and D-dimers, suggesting citrullinated fibrin could lead to fibrinolytic deficiency. Subsequent ex vivo experiments showed an increase in the level of D-dimers in the presence of the PAD4 inhibitor GSK484, tPA and Dnase (p= 0.0003), confirming the contribution of citrullinated fibrin to fibrinolytic deficiency. Patients with higher intrapleural level of citrullinated fibrin were also found to be more likely to develop septations (p= 0.0078). Moreover, the baseline intrapleural level of soluble fibrin was found to be potentially higher in patients who responded well to first line antimicrobial treatment in comparison to patients who proceeded to acquire IFT (p= 0.0965).Conclusion Our data suggests that the citrullination of fibrin contributes to fibrinolytic deficiency and hence the formation of fibrous septations. Our data also suggests that PAD4 inhibitor could potentially improve pleural fluid drainage.