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.