Apoptosis in Platelets from Pediatric Patients with Acute Immune Thrombocytopenic Purpura (ITP) Is Ameliorated by IV Ig.
Authors: Speer Oliver , Schmugge Markus , Azzouzi Imane , Rand Margaret L. , Kroiss Sabine 
Published: October 2019

Abstract
Interaction of auto-antibodies with platelet surface glycoproteins (GP) has been implicated in the accelerated clearance of platelets in ITP. It was recently shown, using a murine model, that the ITP induced by injection of anti-GPIIb is associated with apoptotic-like processes in platelets. These apoptotic-like processes in platelets were similar to those observed during apoptosis in nucleated cells: activation of caspase-3 (aCASP3), loss of mitochondrial inner transmembrane potential and externalisation of phosphatidylserine (PS). Administration of IVIg, that ameliorates the ITP in this model, also inhibited aCASP3 and PS externalization (Leytin et al., 2006, Br J Haematol; 133:78; Song et al., 2003, Blood; 101:3708). Whether these observations in mice are applicable to patients with ITP had not been investigated. Thus, the aim of our study was to investigate whether platelets from paediatric patients with acute ITP show an enhancement of apoptotic-like processes. Further, we examined whether the increase in platelet count in response to IVIg in these patients is accompanied by an inhibition of apoptotic-like processes in platelets. Children with clinical and laboratory diagnosis of acute ITP were enrolled in this prospective study. Severity of bleeding symptoms was assessed according to a paediatric bleeding score for ITP at the time of diagnosis and after IVIg therapy. Blood samples were obtained at these time points for measurement of platelet count and for flow cytometric analyses of platelet apoptotic-like events. Specifically, platelet aCASP3 and PS externalisation were measured in citrated platelet-rich plasma. Platelets were identified as CD42 positive events; aCASP3 was measured as % platelets with bound FLICA-FITC fluorescence; and PS exposure as % platelets with bound annexin A5-FITC fluorescence. The fraction of young, reticulated platelets (RP) was analysed using thiazole orange. All patients (median age 7.5, n = 12) presented with typical symptoms of acute ITP with a bleeding score of 2 – 3. 10 patients had platelet counts below 10’000/micro l, and 2 had platelets counts over 10’000/micro l. ITP patients had increased levels of platelets with aCASP3 (n = 11) and PS exposure (n = 9) compared with healthy control children (see Figure) and increased reticulated platelets (15.6 ± 2.7 %, n = 10; controls: 1.2 ± 0.8, n = 11). The 10 patients with platelet counts below 10,000/micro l were treated with a maximum of 3 doses of IVIg (0.4 – 0.8 g/kg/dose). All patients showed a rise in platelet counts to above 20’000/micro l and amelioration of bleeding symptoms 24 – 72 hours after IVIg administration. Concomitantly, platelet aCASP3 and PS exposure decreased (see Figure), as did RP, to 3.8 ± 1.5 % (n = 9). One patient with an initial platelet count of 18,000/micro l was not treated. This patient showed 5% positive aCASP3 platelets, 3.5 % platelets with PS exposure and 3.5% RP, similar to controls; one month later, the platelet count increased spontaneously to 60’000/micro l with 10% aCASP positive platelets, 4% platelets with PS exposure and 3 % RP. One patient was lost to follow-up. In summary, we have demonstrated enhancement of the platelet apoptotic-like processes of aCASP3 and PS externalisation in paediatric acute ITP, similar to the reported murine model. Consistent with acute ITP, platelet counts were low and levels of RP were high; after administration of IVIg, platelet counts increased whereas levels of RP decreased. IVIg administration also led to a decrease of aCASP3 and PS exposure in the patients’ platelets. Thus, amelioration of acute ITP in children by IVIg is accompanied by inhibition of enhanced apoptosis-like events in platelets.
Figure A: aCASP3 in acute ITP expressed as % platelets with FLICA-FITC fluorescence. B: PS exposure in acute ITP measured as % platelets with bound annexin A5-FITC fluorescence. After IVIg administration, fractions of aCASP3 positive platelets as well as platelets exposing PS were decreased. Figure A:. aCASP3 in acute ITP expressed as % platelets with FLICA-FITC fluorescence. B: PS exposure in acute ITP measured as % platelets with bound annexin A5-FITC fluorescence. After IVIg administration, fractions of aCASP3 positive platelets as well as platelets exposing PS were decreased.