Immune thrombocytopenia (ITP) is an acquired immunological disease of children and adults characterized by a transient or permanent decline platelet count. ITP is caused by peripheral destruction of platelets mediated by immunological mechanisms. Yet, the exact mechanism of immune dysfunction is often unclear. There is uncertainty as to whether the primary insult is caused by abnormal B cells, T cells or both. Moreover, reduced production of platelets or increased activation of phagocytes and apoptosis has been also proposed.
Increasing evidence supports the hypothesis of platelet apoptosis due to autoantibodies against surface platelets' proteins. In adult patients' platelets, apoptosis has been shown to be increased as measured by increased phosphatidylserine (PS) expression, loss of mitochondrial inner membrane potential (ΔΨm) and higher caspase 3 activation. Children with ITP have shown decreased in platelet apoptosis markers following immunoglobulin G intravenous transfusion.
The aim of our study, was to examine whether serum taken from children with ITP can induce apoptosis in normal platelets. We collected serum samples from 55 children with ITP on diagnosis prior to initiation of treatment. Two apoptosis markers were examined, PS exposure and mitochondrial ΔΨm of healthy donor platelets following incubation with ITP patients' sera or healthy children's sera. Nine patients' sera that were positive to both markers and two that were double negative were selected for further investigation on an antibody array of 43 apoptosis proteins and the results were compared to healthy children sera. The assay was performed on lysates of normal platelets following incubation with the tested serum samples. Four proteins were significantly elevated after incubation with the majority of ITP samples, among which HSP27 and Fas which were significantly elevated as compared to normal controls sera in 10 and 9 ITP samples, respectively. These two proteins are involved in the TNFα pathway. Serine protease HTRA2 and XIAP, were elevated in 6 ITP sera each. These proteins are involved in caspase-dependent apoptosis. Among sixteen other proteins that were significantly increased in more than 4 ITP samples, we found Bcl-2, DR6, CD40L, HSP60, IGFBP-6, TNFα, TNFβ and TRAILR-1. Interestingly, the nucleus protein, P21 that was found in a very high concentration in control samples, as compared to other measured proteins, was significantly increased following incubation with 4 ITP sera and markedly decreased in 3 other ITP sera. This pilot study enables us to focus upon new apoptotic markers that are involved in both extrinsic and intrinsic pathways. Confirmation of these preliminary results by specific ELISA assays using all 55 collected ITP sera will enable us to estimate the frequency of platelet apoptosis induced by ITP sera.