Neutrophils induce P-selectin shedding from activated platelets via neutrophil elastase

Abstract Background: P-selectin (P-sel) on activated platelets plays a vital role in inflammation by mediating platelet-neutrophil interactions. However, P-sel surface expression on activated platelets is temporary, with rapid shedding into circulation as soluble P-sel (sP-sel). It is suggested that neutrophils adhesion to P-sel on activated platelets can induce P-sel shedding, however, the exact mechanism remains unclear. This study is to investigate the role of neutrophils in platelet P-sel shedding. Methods: Platelets were activated by thrombin and then incubated with whole blood, peripheral blood mononuclear cells (PBMCs), or neutrophils, with or without neutrophil elastase (NE) inhibitors, myeloperoxidase (MPO) inhibitors, or latrunculin B(LatB). Samples were collected at 0, 1, 2, and 4 hours. P-sel expression on platelets was assessed by flow cytometry and chemiluminescence immunoassay, while sP-sel was quantified by ELISA. Results: When incubated with neutrophils, P-sel expression on activated platelets was reduced (sP-sel was increased) in a time-dependent manner (P-sel-positive platelets, 70.0%→11.1%; fold decrease in CD62P/CD61, 0→0.61; fold increase in sP-sel, 0→6.94), unlike PBMCs. NE inhibitors preincubation with neutrophils could partly reverse the neutrophil-induced P-sel shedding (P-sel-positive platelets, 70.2%→43.4%; fold decrease in CD62P/CD61, 0→0.16; fold increase in sP-sel, 0→4.38), while MPO inhibitors couldn’t. The addition of LatB could also partly reversed the neutrophil-induced P-sel shedding (fold decrease in CD62P/CD61, 0→0.36; fold increase in sP-sel, 0→5.34). Conclusions: The interaction between platelets and neutrophils is mutual. Platelets promote the activation of neutrophils; and in turn, neutrophils induce P-sel shedding via neutrophil elastase, resulting in the irreversible functional downregulation of platelet P-sel-mediated interactions.