Cathepsin G-dependent platelet stimulation by activated polymorphonuclear leukocytes and its inhibition by antiproteinases: role of P-selectin-mediated cell-cell adhesion.
Human PMN stimulated by fMLP are able to activate coincubated, autologous platelets. Cathepsin G, a neutral serine protease stored in the azurophilic granules of PMN, is the major platelet activator in this system. We previously proposed that shear-induced close PMN-platelet contact creates the conditions for which cathepsin G activity on platelets is protected against antiproteinases. The aim of this study was to investigate the adhesive mechanisms, possibly creating between PMN and platelet membranes the microenvironment in which cathepsin G, discharged from stimulated PMN onto adherent platelets, is protected against antiproteinases. Microscopic examination showed that under conditions of high shear, 71.3% +/- 6.1% of PMN were associated to platelets forming small clumps. This percentage decreased to 10% +/- 2% and 13% +/- 4%, respectively, in the presence of an inhibitory antibody to P-selectin or 20 mmol/L mannose-1-phosphate and to 10.8% +/- 3.7% when cells were not stirred. Similarly, PMN pretreatment with neuraminidase abolished PMN binding to platelets. These results indicate that P-selectin mediates PMN-platelet adhesion occurring before PMN stimulation. Prevention of PMN-platelet contact significantly potentiated the inhibitory effect of alpha 1-protease inhibitor on subsequent cathepsin G-induced platelet serotonin release. Because anti-P-selectin antibody, mannose-1-phosphate, and neuraminidase treatment of PMN did not modify PMN-induced platelet activation in the absence of antiproteinases, it is suggested that P-selectin-mediated PMN-platelet adhesion results in the formation of a sequestered microenvironment between cell membranes, in which higher amounts of antiproteinases are required to prevent the activity of released cathepsin G. These data add a new functional role to P-selectin-mediated PMN-platelet adhesion that could be important in vivo because of the presence of antiproteinases in plasma.
Evangelista
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V
Giulio Bizzozero Laboratory of Platelet and Leucocyte Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Negri Sud, Santa Maria Imbaro, Italy.
Piccardoni
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P
White
J G
JG
de Gaetano
G
G
Cerletti
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C
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000502:physiology*; D002240:Carbohydrate Sequence; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology*; D002448:Cell Adhesion; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology*; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D019007:P-Selectin; D015539:Platelet Activation / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D012397:Rosette Formation; D012697:Serine Endopeptidases; D013917:Thrombin / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D009842:Oligopeptides; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D000515:alpha 1-Antitrypsin; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D013917:Thrombin