Pathogenic Role of SERPINB3-positive Neutrophils in Reinforcing Thrombus Stiffening during Ischemic Stroke

The contribution of immune cells to thrombus architecture and mechanical properties in acute ischemic stroke (AIS) remains poorly understood. Using 3D imaging and multiplex staining, we mapped immune cells in human stroke thrombi and identified neutrophils as the dominant population. Analysis of 19 thrombi confirmed their positive correlation with collagen, increased stiffness, and poorer clinical outcomes. To preserve the spatial context, we developed a laser capture-based proteomic workflow and analyzed thrombus neutrophils from 34 patients with AIS stratified by 90-day outcomes, followed by validation in an independent cohort of 22 patients. Proteomic analysis revealed SERPINB3 as a neutrophil-enriched protein strongly correlated with poor prognosis. In murine models of FeCl₃-induced carotid artery thrombosis and middle cerebral artery occlusion, experiments using wild-type, neutrophil-depleted, and Serpinb3a knockout mice demonstrated that neutrophil-derived SERPINB3 promotes early thrombus formation, enhances collagen deposition, and contributes to progressive thrombus stiffening. Mechanistically, SERPINB3 secreted by neutrophils amplifies thrombus stiffness through upregulation of TGFβ1, neutrophil extracellular traps, and COL1A1. Targeted SERPINB3 knockdown delayed vascular occlusion, improved thrombolysis efficiency, and resulted in better neurological recovery. Collectively, these findings identify a neutrophil-driven mechanism underlying thrombus stiffening and establish SERPINB3 as both a prognostic biomarker and a promising therapeutic target in AIS. This project has been registered with the Chinese Clinical Trial Registration Platform (https://www.chictr.org.cn/index.html) and has successfully passed the review process (Registration Number: ChiCTR2300077911).