Prolonged isolated thrombocytopenia (PIT) represents a significant complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is associated with an adverse patient prognosis and higher transplant-related mortality owing to a higher risk of infection events, severe (grades 3 to 4) acute graft-versus-host disease (GVHD) and chronic GVHD. PIT is defined as a peripheral platelet count less than 100×109/L without sustained anemia or leukopenia for more than 3 months after allo-HSCT (Zhang X, et al. Biol Blood Marrow Transplant, 2011). However, the underlying mechanisms remain unclear. Different kinds of functional glycoproteins are expressed on the platelet surface, with sialic acid residues at the end of their glycan. Desialylation of platelet glycoproteins has been found to be associated with accelerated platelet clearance in refrigerated platelets (Gerard Jansen AJ, et al. Blood 2012). Platelet-specific glycoprotein GPIbα,the functional subunit of the von Willebrand factor receptor, was the majorly desialylated glycoprotein; NEU1, one of the four human sialidases, was the enzyme that catalyzed GPIbα desialylation. However, few studies have focused on this mechanism in patients suffering PIT after allo-HSCT. In this study, we hypothesized that desialylation on platelet surfaces is associated with PIT after allo-HSCT. The mechanisms participating in this process may include GPIbα clustering, platelet apoptosis and phagocytosis by macrophages.
Patients and methods:
Blood samples were collected 90 days after allo-HSCT from 70 patients with PIT. Samples from post-transplantation patients who have normal platelet counts were taken as controls. Sialylation and desialylation were measured by detecting specific lectins via flow cytometry. Human sialidase expression was determined by immunofluorescence, flow cytometry and reverse transcription PCR. Platelet apoptosis markers were measured by flow cytometry, and macrophages stimulated from THP-1 cells were used in the phagocytosis assay.
We tested sialic acid residues and the desialylation markers, including β-galactose and β-N-Acetyl glucosamine, on the platelet surface, and found that platelets from PIT patients had significantly higher desialylation levels. Serum sialic acid levels were measured, and the results showed higher levels in PIT patients. Further, NEU1 was found to be over-expressed on the surface of platelets from PIT patients, and was found to be the enzyme that catalyzed the platelet surface desialylation. To further reveal the mechanism that lead to PIT, we proved that GPIbα was the desialylated glycoprotein on platelets from PIT patients. We found that GPIbα desialylation and clustering in PIT patients induced changes in the expression of Bcl-2 family protein, as a 2-fold increase in active Bax expression and a similar decrease in Bcl-XL expression were observed. Depolarization of the inner mitochondria membrane was augmented in desialylated platelets from PIT patients, indicating increased platelet apoptosis. Moreover, macrophages stimulated from the THP-1 cell line preferred to phagocytose desialylated platelets from PIT patients in vitro; this process could be blocked by the sialidase inhibitor, DANA. In the in vitrostudy, we found that dexamethasone led to a 32% decrease in phagocytosis, whereas oseltamivir, an antiviral medicine that can block sialidase from influenza virus, could also partially function on human sialidase and protect 43% of platelets from phagocytosis.
In conclusion, our results demonstrate that desialylation played a role in the mechanism of prolonged isolated thrombocytopenia after allo-HSCT, most likely through platelet apoptosis induction and increased phagocytosis by macrophages in the peripheral circulation. Dexamethasone and oseltamivir could decrease platelet apoptosis and inhibit platelet phagocytosis in vitro, implying a novel potential method for treating PIT after allo-HSCT.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.