Engineering a Translational Human Thrombus Platform to Evaluate Clinical Therapies in Ischemic Stroke

BACKGROUND: Recanalization failure after ischemic stroke remains a major clinical challenge, partly because cerebral thrombi display marked spatial heterogeneity that is poorly reproduced by experimental models. Here, we engineered a translational human blood-based thrombus platform that recapitulates key histological features of patient-derived stroke clots. METHODS: Under controlled conditions, the platform generated reproducible thrombi with pronounced interthrombus and intrathrombus heterogeneity, including alternating red blood cell-rich and platelet-rich territories. Multiscale structural and biochemical analyses confirmed the successful replication of the clinical biphasic organization associated with resistance to rtPA (recombinant tissue-type plasminogen activator): a compact outer fibrin shell containing focal accumulations of plasminogen activator inhibitor-1, and a dense inner core further stabilized by neutrophil extracellular traps. RESULTS: Functional thrombolysis assays demonstrated that this architecture directly dictates lytic susceptibility. Specifically, artificially breaching the outer fibrin shell (halved thrombi) significantly accelerated early rtPA-mediated lysis, proving its role as an effective physical barrier. Furthermore, lytic response was inherently linked to clot composition: platelet-rich thrombi with dense plasminogen activator inhibitor-1 and neutrophil extracellular trap networks exhibited profound resistance to standard rtPA compared with red blood cell-rich clots. Supporting the platform's translational relevance, this structural resistance was successfully overcome by clinically relevant strategies, including tenecteplase and adjunctive DNAse, consistent with observations from human stroke cohorts. CONCLUSIONS: By accurately reproducing the spatial organization and differential thrombolytic responses of human cerebral thrombi, this translational framework provides a robust preclinical tool to dissect lytic resistance and evaluate next-generation precision reperfusion therapies. Furthermore, developing robust ex vivo human platforms is increasingly critical to align with the replacement, reduction, refinement ethical guidelines for animal research.