Hypoxia-downregulated Siglec-9 glycan ligands induce abnormal activation of platelet

Abstract

Objective To investigate the expression of sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9)/E ligands in hypoxic condition and its effect on platelet activation. Methods The distribution, molecular size and glycoprotein class of Siglec-9 ligands on platelet were determined. The platelets were divided into control group and neuraminidase group [treated with α2-3, 6, 8 neuraminidase (NEU1)]. Twenty-four C57BL/6 male mice (6~8 weeks old) or cultured platelets were treated with or without hypoxia, and then the mice were divided into control 3-day group, hypoxia 3-day group, control 7-day group and hypoxia 7-day group, with 6 mice in each group. The platelets were separated and extracted from the whole blood of healthy volunteers and were placed in a hypoxia chamber, and then divided into control group, hypoxia 48-hour group, GT1b group and oseltamivir group (n=3). The expression of Siglec-E/9 ligands, NEU1, and CD62p which was activated by adenosine diphosphate (ADP), collagen and thrombin on the platelet were detected by flow cytometry. The inhibitory effect of Siglec-E/9 ligands was verified using conditional knockout mice Lyz2-cre: Siglec-Eflox/flox (male, 6~8 weeks old, n=6). Results Siglec-9 ligands are sialoglycoproteins with molecular weights of about 130×103 and are distributed broadly on the membrane of platelet. In vivo, the expression of Siglec-E ligands was decreased by 18.03% in response to hypoxia treatment (P < 0.05), and the stimulation of ADP, collagen and thrombin resulted in obviously elevated CD62p level in the platelets from the hypoxia 7-day group than those from the control 7-day group (P < 0.01). Similar Results were observed in the platelets of the hypoxia 48-hour group (P < 0.01). Compared with the platelets from wild-type mice, the platelets from the knockdown mice had significantly higher expression of CD62p after the stimulation of ADP, collagen and thrombin (P < 0.01). GT1b treatment could reverse the abnormal platelet activation induced by hypoxia (P < 0.05), and treatment of oseltamivir, NEU1 inhibitor could block the decreased expression of Siglec-9 ligand caused by hypoxia (P < 0.05). Conclusion Hypoxia can decrease the expression of Siglec-9/E ligands, reduce Siglec-9/E-mediated platelet activation, and thus lead to abnormal activation of platelets, which is associated with the increased expression of NEU1.

Keywords

• hypoxia
• platelet
• siglec-9
• glycan ligand
• neuraminidase-1