Neutrophils Induce P-Selectin Shedding from Activated Platelets Via Neutrophil Elastase

It is suggested that neutrophil adhesion to P-selectin (P-sel) on activated platelet can induce P-sel shedding, however, the underlying mechanism remains unclear. This study is to investigate the role of neutrophil in platelet P-sel shedding. Platelets were activated by thrombin, collagen or thrombin receptor agonist peptide (TRAP). Activated platelets were co-cultured with whole blood, peripheral blood mononuclear cells or neutrophils, in the presence or absence of lipopolysaccharide, Pam3CSK4, myeloperoxidase inhibitors, neutrophil elastase (NE) inhibitors, or Latrunculin B (LatB) (inhibiting neutrophil extracellular traps (NETs) formation but not reducing protease release). Samples were collected at 0, 1, 2, and 4 hours. P-sel expression on platelets, soluble P-sel (sP-sel), and soluble NE were assessed. P-sel shedding only occurred on platelets activated by thrombin in the presence of neutrophils (P-sel-positive platelets, 70.0%→11.1%; P-sel intensity fold decrease, 0→0.61; sP-sel fold increase, 0→6.94). This shedding was partially reversed by NE inhibitors and LatB (sP-sel fold increase: 0→3.62; 0→5.18), but accelerated by MPO inhibitors (sP-sel fold increase: 0→17.59). Soluble NE was lower in co-culture of thrombin-activated platelets and neutrophils (110.78→132.75 ng/mL), compared to co-culture of thrombin-activated platelets and LatB-treated neutrophils (144.42→153.33 ng/mL), or compared to co-culture of collagen (or TRAP)-activated platelets and neutrophils (220.39→388.04 ng/mL; 287.47→385.16 ng/mL). Lipopolysaccharide and Pam3CSK4 did not change neutrophil-induced P-sel shedding. Platelet-neutrophil interaction might be mutual. Thrombin-activated platelet promotes neutrophil activation to form NETs; and in turn, neutrophil induces platelet P-sel shedding via concentrated NE in NETs, resulting in irreversible functional downregulation of platelet P-sel-mediated interactions.