A multi-omics approach investigating thrombolysis resistance in acute ischemic stroke thrombi

Intravenous thrombolysis outcomes in acute ischemic stroke (AIS) vary widely due to heterogeneous clot responsiveness. Defining the molecular basis of thrombolysis resistance is critical to improving treatment efficacy. Sixty-one clots from 59 AIS patients underwent ex vivo thrombolysis with rtPA and were classified as resistant, undefined, or susceptible based on 1-h lysis rates. Quantitative proteomics was performed on a subset (n = 6/group) using mass spectrometry. Whole-genome sequencing (n = 13) explored genetic associations. Clot composition was assessed using Martius Scarlet Blue staining (n = 11) and correlated with thrombolysis. At 1 h, 37 clots were resistant (0- < 25% lysis), 11 undefined (25- < 40%), and 13 susceptible (40-80%). Clot composition did not correlate significantly with thrombolysis. Proteomics identified 124 differentially expressed proteins (≥ 1.5-fold, p < 0.05), including 108 downregulated and 16 upregulated in resistant clots. Downregulated pathways included oxidative phosphorylation, neutrophil extracellular trap signaling, and integrin-mediated adhesion, while granzyme A signaling and mitochondrial dysfunction were enriched. WGS revealed no group-specific variants. Thrombolysis resistance in AIS clots is not explained by histologic composition alone but is associated with coordinated proteomic alterations in mitochondrial bioenergetics, cytoskeletal organization, and adhesion pathways, identifying mitochondrial bioenergetic failure and integrin-mediated structural remodeling as key determinants of fibrinolysis resistance and potential therapeutic targets.