Comprehensive analysis of the changes in serum proteomes are an important part of broad evaluations of the diagnosis, prognosis and therapeutic response of progressing diseases, such as autoimmune disorders, cardiovascular diseases, and cancers. With commonly used proteomic approaches, it is feasible to discover differentially expressed proteins in samples from patients that are likely to be involved in the autoimmune process or serve as biomarkers that correlate with disease or therapeutic outcomes [16, 17]. To date, only one proteomic-based study for biomarkers has been reported in ITP patients. In the study, one-dimensional gel electrophoresis was used to separate serum proteins in ITP patients, non-ITP patients with thrombocytopenia and healthy controls . Quantification of the serum proteins identified by liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis showed that the serum ceruloplasmin levels in ITP patients were statistically significantly higher than in non-ITP patients and controls. For the purpose of identifying the potential biomarkers predicting long-term response to splenectomy in ITP patients, the present study carried out a comparative proteomics analysis with 2-DE and MS on the sera from the responders, nonresponders and healthy controls. Nine protein spots that were differentially expressed by at least five-fold between the responder and nonresponder samples were further analysed with MS/MS. All of the spots were identified as Hp by the peptide sequence analysis. Although the measurement of serum ceruloplasmin levels was found to be useful for the diagnosis of ITP in the report mentioned above, we did not identify ceruloplasmin as a highly differentiated expressed protein between responders and nonresponders in this study.
Hp is an acute phase protein that is synthesised predominately in the liver by hepatocytes. The major inducer of the expression of Hp is IL-6, which is produced through the activities of the primary cytokines TNF-α and IL-1 . The serum levels of Hp remains fairly constant in any given individual; therefore, the observation of a marked change in serum Hp expression has clinical significance . Hp consists of two different polypeptide chains, α chain and β chain. There is only one type of β chain with a MW of approximately 40 kDa. However, the α chain is represented by two isoforms, α-1 and α-2, with the α-1 isoform being approximately 9 kDa and the α-2 being 16 kDa in MW . After chemical reduction of Hp, these polypeptide chains can be clearly revealed in the images of gel electrophoresis . In this study, five protein spots with MW of 38.7 - 42.2 kDa, shown in region 1 of the 2-DE images, were identified as the β chain of Hp. While three protein spots in region 2 (MW 16.9 - 17.1 kDa) and one protein spot in region 3 (MW 11.9 kDa) represented the α-2 and α-1 isoforms of Hp.
Hp has been proven to modulate both innate and adaptive immune responses in several aspects. Th1 and Th2 play a crucial role in the pathogenesis of autoimmune diseases . Studies performed both in vitro and in vivo indicate that Hp exhibits a significant modulating impact on the Th1/Th2 balance via an inhibitory effect on Th2 cytokine release and therefore promotes a dominant Th1 cellular response [23, 24]. Hp supports proliferation and functional differentiation of B and T cells as part of homeostasis and in response to antigen stimulation. Analysis of blood from Hp-deficient mice showed a significant reduction in B and T lymphocytes, and the relative number of splenic monocytes and dendritic cells appeared elevated with respect to wild-type mice . A more recent study showed that Hp-deficient mice facilitate the development of autoimmune disease of central nervous system, which is associated with the enhanced production of several inflammatory cytokines in the spinal cord . Hp is characterised by molecular heterogeneity, with three major phenotypes: Hp 1–1, Hp 2–2, and the heterozygous Hp 2–1. The Hp 2–2 genotype was associated with decreased serum concentrations of Hp. A significant over-representation of the Hp 2–2 genotype has been shown in human immune disorders, such as rheumatoid arthritis, diabetes mellitus type 2, and inflammatory bowel diseases . Taken together, these studies suggest that Hp may play an important role in the pathogenesis and clinical course of autoimmune diseases through various immunomodulatory effects. To date, very few studies in the current literature focused on the issue of the serum Hp expression in ITP patients. A recent study found that abnormally low levels of free serum Hp were present in eleven out of thirty paediatric patients with ITP . The authors considered Hp as a sensitive indicator of haemolysis and presumed that patients with low levels of serum Hp may develop autoimmune haemolytic anaemia in long-term follow-up studies. Until now, the pathophysiological roles of Hp in ITP were poorly understood.
Recent proteomic studies showed that Hp served as an important biomarker in some autoimmune diseases. Increased protein levels of Hp were found in the cerebrospinal fluid from patients with Guillain-Barré syndrome, which is an acute inflammatory autoimmune disorder in the peripheral nervous system [29, 30]. Other studies reported that elevated serum levels of Hp correlated with higher disease activity in systemic lupus erythematosus and active Behcet’s disease [31, 32]. In the present study, we studied the differentially expressed proteins in the serum of ITP patients with different responses to splenectomy using a comparative proteomics approach. Hp was identified as a highly differentially expressed protein between nonresponders and responders. Further validation by ELISA showed that the preoperative serum levels of Hp in the nonresponders were significantly lower than those in the responders and healthy controls. No statistical differences of preoperative serum Hp expression were observed between the responders and healthy controls. The results indicate that the preoperative serum levels of Hp may serve as an important predictor of long-term haematological response to splenectomy in ITP patients. Patients with lower preoperative serum levels of Hp tend to have a worse response to surgery when compared with those with higher preoperative serum levels. The ROC curve is a useful method for evaluating clinical usefulness of a biomarker. The ROC curve analysis showed that AUC of the preoperative serum Hp levels reached 0.867, which means that the preoperative serum Hp levels can be used as a potential predictive biomarker. The Youden index has been demonstrated to be a preferred means of identifying the optimal cutoff value from the ROC curve. Therefore the optimal cutoff value of the preoperative serum Hp levels (1173.80 μg/ml) was identified based on the the maximum Youden index in this study. At this cutoff value, the sensitivity was 78.4% and the specificity was 84.6%. The present study also found that the serum Hp levels did not change seven days after surgery in both responders and nonresponders. To the best of our knowledge, this is the first paper reporting a potential serum biomarker for predicting the long-term response to splenectomy in ITP patients. However, potential limitations of this study must be considered. While the removal of abundant serum proteins would facilitate the discovery and detection of less abundant proteins, including disease associated markers. It should be noted that this technique remains controversial in biomarker discovery, as it may also remove other possibly more interesting proteins bound to the abundant proteins. Since relapses of ITP may occur even two years after splenectomy, further studies with larger sample size and long-term follow-up are needed to confirm our findings. Evans syndrome which links to low serum level of Hp and poor response to splenectomy should be strictly excluded from the study.