Activation of Caspases-8 and -9 Occurs In Platelets From Children with Immune, but Not Chemotherapy-Related, Thrombocytopenia.

Abstract 3700

Apoptotic-like processes in platelets are similar to those observed during apoptosis in the cytoplasm of nucleated cells: activation of caspase-8, caspase-9, and caspase-3, loss of mitochondrial inner membrane potential, and externalization of phosphatidylserine (PS) (Leytin et al, Br J Haematol 2006; Lopez et al, J Thromb Haemost 2009). We recently showed that platelets in pediatric primary immune thrombocytopenia (ITP) have activated caspase-3 (aCASP3) and externalized PS, both of which were reduced after IVIg administration (Speer et al, Blood 2008;112: 3417). To gain a more complete understanding of the apoptosis that occurs in ITP platelets, in the present study, we investigated whether caspase-8 and caspase-9 are also activated in platelets from children with ITP, and examined whether the increase in platelet count in response to IVIg is associated with a decrease in activated caspase-8 (aCASP8) and -9 (aCASP9) in platelets, as was observed for aCASP3. In addition, we measured the mitochondrial membrane potential in platelets before and after IVIg therapy. Children with primary ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a pediatric bleeding score for ITP at the time of diagnosis. Blood samples were obtained at the time of diagnosis and after IVIg therapy for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. In citrated platelet-rich plasma, platelets were identified as CD42 positive events; aCASP8 and aCASP9 were measured as % platelets with bound FITC-fluorescent-labeled inhibitors of activated caspases; and mitochondrial membrane potential was measured as mean fluorescence intensity of the membrane potential sensitive fluorescent tetramethylrhodamine ethyl ester (TMRE). All patients (median age 5.4 yrs, n = 8) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3 and had platelet counts < 20×10⁹/L. Results from ITP patients were compared with 2 control groups, healthy children (platelet counts: 266–348 × 10⁹/L, median age 6.8 yrs, n = 7) and children with thrombocytopenia as a result of chemotherapy for malignancies (cTP) (platelet counts: 3–51 × 10⁹/L, median age 10.2 yrs, n = 7). ITP patients had significantly higher proportions of platelets with aCASP8 (17.5±5.1%) and aCASP9 (16.9±5.8%) compared with both healthy children (aCASP8 1.0±0.3%; aCASP9 1.1±0.3%) and children with cTP, (aCASP8 2.2±0.4%; aCASP9 1.9±0.4%) (p<0.01-0.05). In contrast, a loss of mitochondrial membrane potential was not observed in platelets from ITP patients at baseline, in healthy controls, or cTP. All ITP patients were treated with a maximum of 3 doses of IVIg (0.4 – 0.8 g/kg/dose) and showed a rise in platelet counts to > 20 × 10⁹/L and amelioration of bleeding symptoms by 24 – 72 hours after IVIg administration. Concomitantly, the fractions of platelets with aCASP8 and aCASP9, decreased towards control values (ITP patients after IVIg: aCASP8 7.8±5.3%; aCASP9 6.9±2.1; p=0.5 for both compared to controls). Again no change in mitochondrial potential was observed after IVIg. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP8 and aCASP9 specifically in pediatric primary ITP, which were not observed in cTP. However, the platelet mitochondrial membrane potential was unchanged in ITP (before and after IVIg) and did not differ compared cTP and healthy children. Consistent with primary ITP, the patients' platelet counts were low and increased with IVIg administration. In parallel, IVIg led to a decrease of aCASP8 and aCASP9 in the patients' platelets. Together with our previously reported results (Speer et al, Blood 2008;112: 3417), we show that apoptotic events in platelets such as activation of caspases-8, -9, and -3 and PS exposure are increased specifically in ITP but not in cTP, and are decreased after IVIg treatment. As we detected no loss of the mitochondrial membrane potential in platelets from ITP patients, it may be that apoptotic processes in these platelets are not activated by mitochondrial signaling, but rather via an extrinsic signaling cascade including caspase-8, leading to the activation of caspase-3 and caspase-9. However, the complete signaling pathway leading to caspase-8 activation in platelets of pediatric ITP remains to be elucidated.