cGAS integrates Toll-like receptor signaling in platelets to promote thromboinflammation

Thromboinflammation is driven by innate immune signaling and promotes tissue injury during sepsis. Activation of platelets by bacterial products and alarmins amplifies inflammation, coagulation, and thrombotic dysfunction. However, the mechanisms by which these diverse inflammatory signals converge to elicit common platelet responses remain largely undefined. Mitochondria are key integrators of cellular stress responses and can activate innate immune pathways through the release of mitochondrial DNA and engagement of the DNA sensor cGAS. We therefore hypothesized that stimulation of innate immune receptors generates endogenous cGAS ligands within platelets, providing a unifying mechanism by which distinct inflammatory cues converge on cGAS-STING signaling. Here, we demonstrate that activation of TLR4, TLR1/2, and TLR5 engages a platelet-intrinsic mtDNA-cGAS-STING axis that promotes platelet activation. In a model of endotoxemia, genetic deletion of cGAS reduced platelet activation, thrombocytopenia, pyroptosis and the early systemic inflammatory response. Platelet transfer experiments further established that platelet-intrinsic cGAS is sufficient to amplify inflammatory responses in vivo. Mechanistically, we demonstrated that cytosolic accumulation of mtDNA and activation of cGAS-STING signaling pathway in platelets are dependent on mitochondrial calcium uniporter (MCU) and correlated with the loss of mitochondrial membrane potential and the production of mitochondrial reactive oxygen species. In contrast, canonical platelet agonists activated platelets independently of cGAS and failed to induce substantial cytosolic mtDNA accumulation. Importantly, this signaling architecture was conserved in human platelets, where TLR1/2-mediated activation required MCU- and STING-dependent signaling, whereas responses to thrombin receptor-activating peptide and collagen-related peptide remained largely unaffected. Together, these findings identify cGAS as an intracellular signal integration node that allows diverse innate immune receptors to converge on a common platelet activation pathway through sensing of mitochondria-derived DNA, allowing a rapid platelet response to microbial products.