Neutrophil cathepsin G modulates platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion.
1. Close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of P-selectin expression on platelets by agonists such as thrombin and neutrophil-derived cathepsin G being critical in mediating platelet-neutrophil adhesion. This study compared the effects of thrombin and cathepsin G on platelet P-selectin expression and on P-selectin-mediated platelet-neutrophil adhesion. 2. Washed platelets and platelet-neutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) were incubated with either the supernatant of activated neutrophils, purified cathepsin G or thrombin. Platelet P-selectin expression and platelet adhesion to neutrophils was quantified by flow fluorocytometric analysis. 3. The supernatant from activated neutrophils stimulated platelet P-selectin expression comparable to that produced by purified cathepsin G or thrombin. P-selectin expression induced by both activated neutrophil supernatant and purified cathepsin G was completely inhibited by alpha 1-antichymotrypsin, a specific inhibitor of cathepsin G. Unlike thrombin, which induced maximum platelet P-selectin expression by 10 min, sustained to 120 min, cathepsin G induced an initial large increase in platelet P-selectin expression, followed by a progressive reduction over 30-60 min to baseline levels. 4. Co-incubation of neutrophils with thrombin-stimulated platelets resulted in a significant increase in P-selectin-mediated platelet-neutrophil adhesion, which was completely inhibited by preincubation of neutrophils with anti-sialyl Lewis(x) monoclonal antibody. Thrombin produced maximum platelet-neutrophil adhesion by 10 min which remained stable over 120 min. In contrast, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation. Addition of cathepsin G to thrombin-stimulated platelets caused a progressive reduction over 30-60 min to baseline levels of platelet-neutrophil adhesion. 5. Neutrophil-derived cathepsin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion. Therefore, cathepsin G may modulate thrombin-mediated platelet-neutrophil adhesive interactions in inflammation and thrombosis.
Ilton
M K
MK
Department of Medicine, University of Western Australia, Nedlands, Australia.
Taylor
M L
ML
Misso
N L
NL
Thompson
P J
PJ
Hung
J
J
D000328:Adult; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism* / Q000502:physiology; D015539:Platelet Activation; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D000514:alpha 1-Antichymotrypsin / Q000494:pharmacology
D017077:Culture Media, Conditioned; D019007:P-Selectin; D015842:Serine Proteinase Inhibitors; D000514:alpha 1-Antichymotrypsin; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D013917:Thrombin