Abstract A090: GPVI-Driven Thromboinflammation Underlies Microcirculatory Failure after Reperfusion in Acute Ischemic Stroke

Introduction: Futile reperfusion remains a major challenge in acute ischemic stroke (AIS), where restoration of large vessel patency fails to translate into microvascular reflow. The mechanisms underlying microcirculatory failure after reperfusion and effective therapeutic targets remain elusive. Methods: We combined in vivo two-photon microscopy, immunostaining, electron microscopy, bulk and single-cell RNA sequencing, in vitro cell experiments and translational studies in both murine stroke models and AIS patients. Platelet GPVI activation was assessed in blood samples from AIS patients and mice. Functional consequences of GPVI deficiency were examined in Gp6 knockout ( Gp6 -/- ) mice subjected to transient middle cerebral artery occlusion (tMCAO). Mechanistic pathways were validated in vitro, and a novel small-molecule GPVI inhibitor (ND886) was identified and evaluated in vitro and in vivo. Results: Early and robust platelet GPVI activation was observed in AIS patients and experimental stroke models. Two-photon imaging demonstrated that neutrophil adhesion, capillary plugging, and reduced cortical microvascular flow, which were markedly attenuated after reperfusion in Gp6 -/- mice. These mice exhibited improved cerebral blood flow, reduced BBB leakage, and smaller infarct volumes. Electron microscopy revealed reduced α-granule release in Gp6 -/- platelets, consistent with attenuated activation. Bulk RNA-seq of neutrophils and single-cell transcriptomics of brain tissue showed significant downregulation of neutrophil adhesion, migration, and immune activation pathways, as well as suppressed endothelial immune activation in Gp6 -/- mice. Mechanistically, GPVI-mediated platelet activation promoted neutrophil activation via the CD14/TLR2 pathway. ND886 selectively inhibited GPVI signaling, reduced neutrophil adhesion, preserved microvascular flow, and decreased infarct size in vivo. Conclusions: In conclusion, platelet GPVI activation is a central driver of neutrophil-mediated microvascular dysfunction following reperfusion in AIS. Targeting GPVI activation and thromboinflammation with a novel small-molecule inhibitor represents a promising strategy to overcome futile reperfusion. A Phase II randomized trial (NCT 05836753) is underway to evaluate the safety and efficacy of ND886 in AIS patients receiving reperfusion therapy, with results to be presented in the Late-Breaking session.