Cathepsin G is a strong platelet agonist released by neutrophils.
The present studies were undertaken to characterize a serine protease released by N-formyl-L-Met-L-Leu-L-Phe (fMet-Leu-Phe)-stimulated neutrophils that rapidly induces platelet calcium mobilization, secretion and aggregation. The biological activity associated with this protease was unaffected by leupeptin, was only weakly diminished by N-p-tosyl-L-Lys-chloromethane, but was strongly inhibited by alpha 1-antitrypsin, soyabean trypsin inhibitor, N-tosyl-L-Phe-chloromethane and benzoyloxycarbonyl-Gly-Leu-Phe-chloromethane (Z-Gly-Leu-PheCH2Cl). These observations indicated that the biological activity of neutrophil supernatants could be attributed to a chymotrypsin-like enzyme such as cathepsin G. Furthermore, platelet aggregation and 5-hydroxytryptamine release induced by cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils were found to be blocked by antiserum to cathepsin G in a concentration-dependent manner but were unaffected by antiserum to elastase. The biological activity present in neutrophil supernatants co-purified with enzymic activity for cathepsin G during sequential Aprotinin-Sepharose affinity chromatography and carboxymethyl-Sephadex chromatography. SDS/polyacrylamide-gel electrophoresis of the reduced, purified protein, demonstrated three polypeptides with apparent Mr values of 31,500, 29,000 and 28,000 and four polypeptides were resolved on acid-gel electrophoresis. Purified cathepsin G from neutrophils cross-reacted with anti-(cathepsin G) serum in a double immunodiffusion assay and elicited platelet calcium mobilization, 5-hydroxytryptamine secretion and aggregation. Calcium mobilization and secretion induced by low concentrations of cathepsin G were partially dependent on arachidonic acid metabolites and ADP, while stimulation by higher enzyme concentrations was independent of amplification pathways, indicating that cathepsin G is a strong platelet agonist. These results suggest that pathological processes which stimulate neutrophils and release cathepsin G can in turn result in the recruitment and activation of platelets.
Selak
M A
MA
Department of Pharmacology, Temple University Medical School, Philadelphia, PA 19140.
Chignard
M
M
Smith
J B
JB
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood; D056649:Cathepsin G; D002403:Cathepsins / Q000097:blood* / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D066298:In Vitro Techniques; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010974:Platelet Aggregation / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases; D012702:Serotonin Antagonists / Q000494:pharmacology
D007106:Immune Sera; D011480:Protease Inhibitors; D012702:Serotonin Antagonists; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D002118:Calcium