The NLRP3 Inflammasome in Platelets Is Upregulated in Sickle Cell Disease and Promotes Platelet Aggregation and In Vitro Thrombosis

Sickle cell disease (SCD) is a severe monogenic disorder characterized by chronic hemolysis and abnormal coagulation and inflammation. Activated platelets play a critical role at the interphase of thrombosis and inflammation in the disease context. Sensing of free heme, a degradation byproduct of hemoglobin oxidation during hemolysis, involves the pattern recognition receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3), which typically forms multiprotein inflammasome complexes and controls caspase-1 activity and cleavage of IL-1β not only in immune cells but also platelets. We have shown recently that the NLRP3 inflammasome in platelets controls platelet activation, aggregation, and in vitro thrombus formation, which was dependent on platelet bruton's tyrosine kinase (BTK). The role of the platelet NLRP3 inflammasome and BTK in SCD, however, is unexplored.

Platelets were isolated from humanized SCD mice (Townes), which express human sickle hemoglobin, and patients with SCD in steady state and acute vasoocclusive crisis. Platelet NLRP3 activity, as monitored by caspase 1 activation (assessed by fluorescent labeled inhibitor of caspase-1, FLICA), and cleavage and secretion of IL1β (assessed by Western blot and ELISA), was significantly elevated in SCD patients in steady state as compared to healthy subjects (FLICA: mean 340.3 ± 30.2 SD vs. mean 262.2 ± 33.9 SD, p<0.001), and further upregulated in SCD patients in crisis (FLICA: mean 390.3 ± 34.0 SD vs. mean 340.3 ± 30.2 SD, p<0.05). NLRP3 activity in platelets was also significantly increased in homozygous SCD mice, as compared to heterozygous and control mice (FLICA: mean 165.1 ± 32.4 SD vs. mean 86.7 ± 20.5 SD, p<0.001), which was significantly reversed by the heme scavenger hemopexin or pharmacological inhibition of the NLRP3 inflammasome/BTK pathway. Moreover, incubation of platelets with heme induced intraplatelet colocalization of NLRP3 and the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), as evaluated with immunofluorescence staining coupled with confocal laser scanning microscopy, indicating the assembly of the NLRP3 inflammasome in platelets upon exposure to heme. We studied the functional effects of platelet NLRP3 inflammasome upregulation in SCD, and found that collagen-induced platelet aggregation was significantly elevated in homozygous SCD mice as compared to control animals (area under the curve, AUC: mean 65.25 ± 7.99 SD vs. mean 40.60 ± 7.92 SD, p<0.05). In SCD patients in acute crisis, aggregation of circulating platelets was significantly upregulated as compared to patients in steady state (AUC: mean 113.70 ± 16.26 SD vs. mean 78.53 ± 1.93 SD, p<0.05). Moreover, platelet aggregation in steady state patients was significantly elevated as compared to healthy subjects (AUC: 78.53 ± 1.93 SD vs. mean 30.87 ± 4.34 SD, p<0.01). The upregulated platelet aggregation responses in homozygous SCD mice and SCD patients in acute crisis were dependent on heme/NLRP3/BTK pathway, as evaluated by hemopexin (mice: 69.3 ± 5.7% inhibition; patients: 61.6 ± 5.2% inhibition), the NLRP3 inhibitor MCC950 (mice: 75.7 ± 3.9% inhibition; patients: 72.1 ± 3.8% inhibition), and the BTK inhibitor CGI1746 (mice: 65.9 ± 3.6% inhibition; patients: 67.1 ± 4.1% inhibition) (p<0.01). Similar results were obtained with a collagen-coated flow chamber system.

Our data show that the platelet NLRP3 inflammasome is upregulated in SCD via heme, which results in elevated platelet aggregation and in vitro thombus formation, and is potentially targetable using BTK inhibitors for affected patients.