SCD-induced upregulation of the platelet NLRP3 inflammasome depends on HMGB1/TLR4 and BTK. (A) HMGB1 plasma levels are elevated in SCD patients in steady state (SS) compared with healthy controls. (B) HMGB1 plasma levels are elevated in SCD patients in crisis compared with the same patients in SS. (C) HMGB1 plasma levels in healthy subjects and SCD patients in SS and crisis correlate with plasma-induced platelet caspase-1 activity. (D) A neutralizing anti-HMGB1 antibody (HMGB1 AB) or the HMGB1 inhibitor glycyrrhizin inhibits platelet caspase-1 activity induced by PPP from SCD patients in SS and crisis and has no effect in the presence of PPP from healthy controls. (E) Preincubation of platelets with the TLR4 inhibitor TAK242 inhibits platelet caspase-1 activity induced by PPP from SCD patients in SS and crisis. Preincubation of platelets with the BTK inhibitor ibrutinib or a combination of TAK242, MCC950, and ibrutinib inhibits platelet caspase-1 activity in the presence of PPP from healthy and SCD subjects. Data are shown as median with IQR and minimum and maximum values (A,D-E), before-after plot (B), and scatter plot with linear regression (C). Statistical tests used: Mann-Whitney U test (A), Wilcoxon signed-rank test (B), Spearman rank correlation, ρ = correlation coefficient (C), and Friedman test with Dunn multiple comparisons (D-E).