title: Intracellular Ca2+ rise in human platelets induced by polymorphonuclear-leucocyte-derived cathepsin G.
abstract: Cathepsin G, a serine protease released by polymorphonuclear-leucocyte azurophilic granules upon stimulation, activates human platelets, inducing an increase in intra-platelet Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (50-200 nM). The [Ca2+]i rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca2+ transient. Higher (100-200 nM) cathepsin G concentrations induced a monophasic increase in intracellular Ca2+. Acetylsalicylic acid, nordihydroguaiaretic acid and ketanserin did not affect platelet activation by cathepsin G, whereas the ADP-scavenger system phosphocreatine/creatine kinase significantly decreased Ca2+ mobilization, platelet aggregation and 5-hydroxytryptamine secretion by cathepsin G. Preventing cathepsin G-induced platelet aggregation with the synthetic peptide RGDSP (Arg-Gly-Asp-Ser-Pro) did not significantly affect cathepsin G-induced Ca2+ transients. Ni2+ (4 mM), a bivalent-cation-channel inhibitor, decreased the cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either decreasing Ni2+ or increasing cathepsin G concentration. Preventing Ca2+ influx across the plasma membrane with 4 mM-EGTA totally abolished Ca2+ transients. However, EGTA also strongly decreased catalytic activity of cathepsin G, which is essential for platelet activation. Evidence of a rapid and sustained bivalent-cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 s or 3 min after cathepsin G. No accumulation of InsP3 could be detected when platelets were stimulated with cathepsin G. All these data indicate that cathepsin G induces a [Ca2+]i increase mainly through an influx across the plasma membrane. This massive Ca2+ entry is probably due to opening of receptor-operated channels and is amplified by endogenous ADP release.
authors: Bizzozero Laboratory of Platelet and Leucocyte Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Santa Maria Imbaro, Italy.
authors: M
authors: M
authors: Molino
authors: M
authors: M
authors: Di Lallo
authors: G
authors: G
authors: de Gaetano
authors: C
authors: C
authors: Cerletti
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000737:chemistry* / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000097:blood*; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology*; D002413:Cations, Divalent / Q000097:blood; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000097:blood; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D009532:Nickel / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000097:blood; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology; D012697:Serine Endopeptidases
chemical_list: D002413:Cations, Divalent; D005456:Fluorescent Dyes; D010716:Phosphatidylinositols; C049925:fura-2-am; D008345:Manganese; D009532:Nickel; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D002118:Calcium; D016257:Fura-2