Neutrophil α-defensin-1 is present in human stroke thrombi and induces NETosis in vitro

Ischemic stroke thrombi contain complex immunothrombotic components, including neutrophil extracellular traps (NETs), which contribute to thrombus stability and resistance to fibrinolysis. However, the endogenous triggers of NETosis within sterile stroke thrombi remain poorly understood. Neutrophil α-defensins are cationic peptides with emerging pro-thrombotic roles, but their presence and function in human stroke clots have not been fully characterized. This study investigated whether α-defensin-1 is present in ischemic stroke thrombi and whether it can induce NETosis.

Thrombi were collected from 6 patients undergoing mechanical thrombectomy for acute ischemic stroke and analyzed using immunofluorescence to assess α-defensin-1 localization and NET markers. Human neutrophils isolated from healthy donors were stimulated in vitro with α-defensin-1, phorbol 12-myristate 13-acetate, or vehicle control. NETosis was quantified as the percentage of citrullinated histone H3-positive nuclei using standardized threshold-based ImageJ analysis.

α-Defensin-1 was detected in all analyzed thrombi, predominantly in extracellular regions enriched with neutrophils and less abundant in erythrocyte-rich areas. NETs were a prominent feature of thrombi, as demonstrated by citrullinated histone H3 staining. In vitro, exposure to recombinant α-defensin-1 significantly increased NETosis at 10 µg/mL compared with unstimulated controls, whereas lower concentrations showed no significant effect. These findings indicate that α-defensin-1 is a structural component of stroke thrombi and induces NETosis in vitro.

α-Defensin-1 may be an underappreciated component of stroke thrombi that induces NETosis in vitro, suggesting a link between neutrophil degranulation and thrombus composition.