Association of Platelet Desialylation and Circulating Follicular Helper T Cells in Patients With Thrombocytopenia

Abstract

Thrombocytopenia is a multifactorial condition that frequently involves concomitant defects in platelet production and clearance. The physiopathology of low platelet count in thrombocytopenia remains unclear. Sialylation on platelet membrane glycoprotein and follicular helper T cells (TFHs) are thought to be the novel platelet clearance pathways. The aim of this study was to clarify the roles of platelet desialylation and circulating TFHs in patients with immune thrombocytopenia (ITP) and non-ITP thrombocytopenia. We enrolled 190 patients with ITP and 94 patients with non-ITP related thrombocytopenia including case of aplastic anemia (AA) and myelodysplastic syndromes (MDS). One hundred and ten healthy volunteers were included as controls. We found significantly increased desialylated platelets in patients with ITP or thrombocytopenia in the context of AA and MDS. Platelet desialylation was negatively correlated with platelet count. Meanwhile, the circulating TFH levels in patients with thrombocytopenia were significantly higher than those of normal controls, and were positively correlated with desialylated platelet levels. Moreover, TFHs-related chemokine CXCL13 and apoptotic platelet levels were abnormally high in ITP patients. The upregulation of pro-apoptotic proteins and the activation of the MAPK/mTOR pathway were observed in the same cohort. These findings suggested that platelet desialylation and circulating TFHs may become the potential biomarkers for evaluating the disease process associated with thrombocytopenia in patients with ITP and non-ITP.

Keywords: CXCL13; desialylation; follicular helper T Cells (TFHs); platelet; thrombocytopenia.

Figure 1

Platelet desialylation in patients with immune thrombocytopenia (ITP) and non-ITP related thrombocytopenia. (A) ECL or RCA-I binding to platelets were examined by flow cytometry; representative dot plots from healthy control and ITP patients are shown. (B) Comparison of platelet desialylation levels between ITP patients and healthy controls. (C) ROC curve for predicting platelet desialylation in ITP patients based on ECL (left) and RCA-I (right) binding methods. (D) Correlation analysis between platelet desialylation and platelet count (<150×10⁹/L) as determined by Spearman’s test. (E) Comparison of desialylated platelet levels in ITP patients with different platelet count. (F, G) Platelet desialylation levels in patients with aplastic anemia (AA), or myelodysplastic syndrome (MDS) versus healthy controls. (H, I) Comparison of desialylated platelet levels among AA, MDS, and ITP groups. ECL, Erythrina cristagalli lectin; RCA-I, Ricinus communis agglutinin I; ROC, Receiver operating characteristic.

Figure 2

Circulating TFH levels in peripheral blood of patients with thrombocytopenia. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with ITP, and were stained with labeled antibodies. PBMCs from healthy volunteers were used as controls in parallel. (A) Representative flow cytometry plots of CD4+CXCR5+PD1+ TFHs or CD4+CXCR5+ TFHs are depicted. (B) Frequencies of CD4+CXCR5+PD1+ TFHs and CD4+CXCR5+ TFHs in ITP patients versus controls. (C) Comparison of CD4+CXCR5+ TFH levels in ITP patients with the platelet glycoprotein antibodies versus those without antibodies. (D, E) TFH levels in patients with AA, MDS versus healthy controls. (F) Frequencies of circulating TFHs among patients with AA, MDS, and ITP. TFHs, T follicular helper cells.

Figure 3

Circulating TFHs positively correlate with platelet desialylation in ITP patients. (A) Correlation between circulating CD4+CXCR5+PD1+ TFHs and ECL binding. (B) Correlation between circulating CD4+CXCR5+ TFHs and RCA-I binding. (C) Correlation between circulating CD4+CXCR5+PD1+ TFHs and RCA-I binding. Spearman’s rank correlation coefficient (r) and P-value are depicted.

Figure 4

Chemokine CXCL13 expression in patients with ITP. Plasma samples were isolated from healthy volunteers and ITP patients. The plasma concentration of CXCL13 was quantified by ELISA. ELISA, enzyme-linked immunosorbent assay.

Figure 5

Platelet apoptosis in the ITP cohort. Apoptotic platelets were stained with Annexin V-APC/7-AAD, and analyzed by flow cytometry. (A) The representative dot plots from healthy controls and ITP groups are shown. (B) Comparison of platelets undergoing apoptosis in healthy controls and patients with ITP. The P-value was calculated using the Mann-Whitney test. (C) Comparison of apoptotic platelet levels in ITP patients with different platelet count. (D) Venn diagram showing the numbers of patients presenting with increased platelet apoptosis and/or increased platelet desialylation. (E) Protein extracts from isolated platelet lysate were subjected to Western blotting using phosphorylated mTOR and MAPK, total mTOR and MAPK, pro-apoptotic Bak and Bax, anti-apoptotic Bcl-2, and GAPDH antibodies. Four representative samples from ITP patients and two representative samples from healthy volunteers are shown. The intensity of different protein bands was quantified and normalized with GAPDH. Relative intensity of p-mTOR, mTOR, p-MAPK, MAPK, Bak, Bax and Bcl-2 is depicted by the lower set of numbers.