B75-13 Redox-based Post-translational Modifications in Platelet Receptor CLEC-2 and Syk-dependent Signaling Pathway in Acute Lung Injury

Oxidative stress has long been implicated in pathogenesis of acute lung injury (ALI). Our understanding of “oxidative stress” has evolved to include dysregulated redox signaling due to post-transcriptional modifications (PTMs) of cysteine amino acids such as oxidation, reduction, and glutathionylation. We recently reported that increased circulating levels of major antioxidant enzyme, extracellular superoxide dismutase (EC-SOD) protect against platelet activation in murine model of Staphylococcus aureus pneumonia. However, the mechanism of redox regulation of platelet activation in infectious pneumonia remains unknown. C-type lectin-like receptor 2 (CLEC-2) activates platelets via spleen tyrosine kinase (Syk)-dependent signaling pathway. CLEC-2 and Syk have conserved cysteines that may be susceptible to redox PTMs. We hypothesize that redox signaling in platelets occurs via redox-based PTMs in CLEC-2 and Syk-dependent signaling pathway. Washed platelets from healthy volunteers were stimulated ex vivo with PBS, reactive oxygen species (ROS), or thrombin. Immunoblotting was used to detect reduced cysteines and glutathionylation of total protein, and phosphorylation in key signaling proteins in Syk-dependent pathway. C57Bl6/J mice were inoculated intratracheally with 1x10^8 CFUs of S.aureus or PBS. Washed platelets were collected 24hrs post-infection for immunoblotting and click chemistry-based labeling of reduced cysteines followed by mass spectrometry and AlphaFold3 analysis. We report ROS-mediated activation of Syk-dependent signaling pathway, evidenced by phosphorylation of key signaling proteins phospholipaseC-γ2 (PLCγ2), linker for activation of T cells (LAT), Src-homology2 domain-containing phosphatase (SHP2), and Syk. ROS-stimulated platelets had significantly fewer reduced cysteines than PBS, indicating increased cysteine oxidation. We detected no change in glutathionylation, suggesting glutathionylation may not be the primary redox-based mechanism of platelet activation. Using redox proteomics, we identified 303 platelet proteins with redox-active cysteines. Of these proteins, 260 were oxidized and 13 were reduced in S.aureus infected mice compared to PBS. Additionally, we identified the exact redox-active cysteines. For instance, Cys99 and Cys102 in extracellular domain of CLEC-2 are oxidized in S.aureus. The extracellular location of Cys99 and Cys102 is consistent with site of action of EC-SOD. Cys99 forms hydrogen bond with Histidine 97, which is notable because histidines are three-fold more commonly found near oxidized cysteines further supporting our findings. Cys102 is structurally important because it forms a disulfide bind that is vital to structure of C-type lectin fold and ligand binding. Our data implicate CLEC-2 receptor and Syk-dependent signaling pathway in redox-based regulation of platelet activation. We identify specific redox-active cysteines that can be leveraged in future development of target-specific therapies.