Caspase Signaling Is Enhanced and Linked to Increased Eryptosis in SCD

Introduction: Patients with sickle cell disease (SCD) have increased risk of venous thromboembolism, pulmonary embolism and stroke. The disease is associated with eryptosis, a process in which stressed red blood cells (RBCs) externalize phosphatidylserine (PS), which binds platelets and contributes to clot formation and thromboinflammation. Caspase activation is implicated as a trigger via caspase-8 (C8) and Fas-signaling in the extrinsic pathway, and/or intrinsic pathway via caspase-9 (C9) and mitochondrial damage, which is described in SCD. Ultimately, caspase-3 (C3) is activated by C8 or C9 and in turn disables PS transport from the outer to inner RBC membrane, thus increasing PS exposure. We hypothesized that caspase activation in SCD is enhanced and contributes to RBC PS externalization.


Methods: We measured caspase levels in isolated RBCs from Townes Hb-SS (sickle) and Hb-AA (control) mice, using fluorometric assays (Sigma-Aldrich), and PS surface expression by flow cytometry (Annexin-Ab). We also examined the effect of caspase inhibitors in-vitro and in-vivo.


Results: HbSS RBCs had elevated C3, C8, and C9 levels compared to Hb-AA cells. Remarkably, HbSS RBCs showed a 10-fold increase in C8 levels compared to Hb-AA. In-vitro, C8 inhibition led to significant reduction in C3 activity both in Hb-AA and Hb-SS RBCs. Conversely, C9 inhibition yielded significant reduction in C3 in HbSS but not in Hb-AA RBCs. Further, in-vivo treatment with a caspase inhibitor yielded significant reduction of PS exposure in Hb-SS RBCs.


Conclusions: HbSS RBCs display increased C3 activity at baseline. The extrinsic pathway via C8 appears to have a predominant role as active C8 was greater than C9, and substantial reduction in C3 activation was observed with C8 inhibition, but not C9. Although modest, C9 is elevated in HbSS, which is likely due to RBC mitochondrial retention and damage, a known entity in SCD. Lastly, in-vivo caspase inhibition significantly reduced PS expression in HbSS, establishing a link between caspase and PS externalization in SCD. Collectively, our data shows for the first time that caspase activation contributes to eryptosis in SCD, predominantly via the extrinsic pathway. This novel understanding of caspase in eryptosis generates opportunities to evaluate the mechanisms of thromboinflammation in SCD.