Abstract 16584: Spleen Tyrosine Kinase Inhibition Interferes With Platelet NLRP3 Inflammasome Activation in Sickle Cell Mice Leading to Reduced Platelet Aggregation

Abstract

Introduction: The NOD-like receptor protein 3 (NLRP3) inflammasome is a critical inflammatory mechanism that has recently been found to be upregulated in platelets in sickle cell disease (SCD). Fostamatinib is a prodrug of the Spleen Tyrosine Kinase (Syk) inhibitor R406 that targets platelets and is approved for the treatment of immune thrombocytopenia. The effect of Syk inhibition on platelet NLRP3 inflammasome activation in SCD is unexplored. Methods: We used a SCD mouse model, the Townes strain, which expresses human sickle hemoglobin. NLRP3 inflammasome activation was assessed in isolated platelets with fluorescent labeled inhibitor of caspase-1 (FLICA) assay and immunofluorescence staining of intraplatelet NLRP3 and the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) coupled with confocal laser scanning microscopy. Platelet aggregation and ATP secretion were studied with whole blood impedance aggregometry and luminescence. Isolated platelets or blood samples were treated with the Syk inhibitor R406 and/or the NLRP3 activator nigericin where indicated. Collagen was used as platelet agonist. Results: R406 markedly decreased platelet caspase-1 activation and NLRP3:ASC speck formation found in platelets from SCD mice (RFUs: 228 ± 25.3 vs. 450.2 ± 63, mean ± SD, R406 vs. vehicle, p<0.001). R406 had no effect in platelets from control mice, where only low levels of caspase-1 activity and NLRP3:ASC specks were detected in platelets. Platelet aggregation and ATP secretion were elevated in SCD mice, which was abrogated by R406 (AUC: aggregation, 39 ± 2.6 vs. 65.8 ± 4.7, mean ± SD, R406 vs. vehicle, p<0.001; ATP, 7.2 ± 1.5 vs. 13.9 ± 1.6, mean ± SD, R406 vs. vehicle, p<0.001). Strikingly, the NLRP3 activator nigericin reversed the inhibitory effect of R406 on platelet aggregation (44 ± 11.5% increase, p<0.001) and ATP secretion (48.9 ± 22% increase, p<0.01) in SCD mice, suggesting a functionally active link in platelets between Syk and NLRP3. Conclusion: Syk inhibition with R406 impairs NLRP3 inflammasome activation in platelets from SCD mice and interferes with platelet aggregation and ATP secretion in an NLRP3-dependent manner. Targeting Syk and NLRP3 in platelets might be a useful therapeutic approach in SCD.