Altered cytoskeletal integrity underlies impaired platelet shape change and defective thrombus formation in ETV6-related thrombocytopenia.

ETV6-related thrombocytopenia (ETV6-RT) is an inherited platelet disorder caused by germline ETV6 variants. Despite recent progress, the mechanisms underlying platelet dysfunction in ETV6-RT remain unclear. We investigated 12 patients from six families using functional assays, electron microscopy, quantitative proteomics and cytoskeletal imaging. Most patients exhibited mild-to-moderate thrombocytopenia with variable paediatric bleeding symptoms (median International Society on Thrombosis and Haemostasis Bleeding Assessment Tool 3, range 1-9) but consistently mild bleeding in adulthood (median 0, range 0-1). Ex vivo thrombus formation was reduced independent of platelet count. Electron microscopy revealed defective platelet shape maintenance, characterized by spheroid morphology, reduced Dmax/Dmin ratios and diminished alpha-granule pools. Flow cytometry and single-platelet total internal reflection fluorescence imaging demonstrated largely preserved calcium signalling but impaired activation-dependent shape change, dense-granule release and integrin activation. Proteomics showed reduced alpha-granule and lysosomal proteins alongside imbalanced regulators of actin remodelling and β-tubulin. Phalloidin staining confirmed impaired actin cytoskeletal remodelling with reduced lamellipodia formation, while immunofluorescence revealed abnormal β1-tubulin localization with disrupted marginal bands and reduced lysosome-associated membrane protein 1 (LAMP1) expression. Additionally, granulocyte recruitment and migration within thrombi were impaired, suggesting broader thromboinflammatory defects. These findings suggest that combined disruption of cytoskeletal integrity and granule biogenesis underlies impaired thrombus formation in ETV6-RT, providing mechanistic insight into the haemostatic defects associated with this disorder.